β-Hydroxy- A nd β-Aminophosphonate Acyclonucleosides as Potent Inhibitors of Plasmodium falciparum Growth

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β‑Hydroxy- and β‑Aminophosphonate Acyclonucleosides as Potent

Inhibitors of Plasmodium falciparum Growth

́

Thomas Cheviet, Sharon Wein, Gabriel Bourchenin, Manon Lagacherie, Christian Perigaud,

Rachel Cerdan,* and Suzanne Peyrottes *

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ABSTRACT: Malaria is an infectious disease caused by a parasite of the genus

Plasmodium, and the emergence of parasites resistant to all current antimalarial drugs

highlights the urgency of having new classes of molecules. We developed an eﬀective

method for the synthesis of a series of β-modiﬁed acyclonucleoside phosphonate

(ANP) derivatives, using commercially available and inexpensive materials (i.e.,

aspartic acid and purine heterocycles). Their biological evaluation in cell culture

experiments and SAR revealed that the compounds’ eﬀectiveness depends on the

presence of a hydroxyl group, the chain length (four carbons), and the nature of the

nucleobase (guanine). The most active derivative inhibits the growth of Plasmodium

falciparum in vitro in the nanomolar range (IC₅₀= 74 nM) with high selectivity index

(SI > 1350). This compound also showed remarkable in vivo activity in P. berghei-

infected mice (ED₅₀∼ 0.5 mg/kg) when administered by the ip route and is,

although less eﬃcient, still active via the oral route. It is the ﬁrst ANP derivative with

such potent antimalarial activity and therefore has considerable potential for development as a new antimalarial drug.

have been made to develop new drugs with novel mechanisms of

action.^1c,3We have recently reviewed the literature on inhibitors

of the purine salvage pathway and highlighted two main families

of potential drugs (Figure 1),⁴namely, immucillin derivatives

and acyclonucleoside phosphonates (ANPs), which target the

purine nucleoside phosphorylase (PNP)⁵and the hypoxan-

thine−guanine−xanthine phosphoryl transferase (HGXPRT),⁶

respectively. Several generations of immucillin have been

studied as transition state inhibitors of PNP and have exhibited

potent antimalarial activities in vitro and in vivo.⁷Some have

already been the subject of preclinical studies,^7bbut no further

development has been reported to date. In addition, ANPs are

also promising as a class of antimalarial agents because they

inhibit HGXPRT.⁸This enzyme is crucial for the synthesis of

inosine 5′-monophosphate (IMP) from hypoxanthine, the

central and unique precursor for all purine-containing

nucleotides required during plasmodial nucleic acid biosyn-

thesis. These compounds were designed on the basis of the

crystal structure of 2-(phosphonoethoxy)ethylguanine (PEEG,

Figure 1, X = O) in complex with HsHGPRT⁹and incorporate a

purine base (preferably guanine or hypoxanthine), a ﬁve atoms

INTRODUCTION

■

Drug discovery is mainly based on the complementarity of

target-based (protein screening) and phenotypic (whole cell

screening) approaches, and both strategies have been used to

identify novel antimalarials.¹

Cell-based phenotypic screening has proven to be a valuable

approach for drug candidate identiﬁcation, and it is used for

decades to identify new chemotypes. One of its limitations is

that it fails to provide information on the molecular target and/

or the mechanism of action. On the contrary, target-based

approaches involve the screening of a library of compounds

against a deﬁned protein followed by structural optimization of a

hit-compound for potency and selectivity. In the case of parasitic

diseases, there is a rather limited number of validated molecular

targets if we consider the strict deﬁnition that a validated target is

associated with a registered drug, for which the corresponding

mode of action has been proven. As example, several proteins

from the purine and pyrimidine synthesis pathways have been

proposed as chemotherapeutic targets for various parasites.²

Special attention was given to the purine salvage pathway as

many parasitic protozoa lack the de novo pathway for the

biosynthesis of nucleoside 5′-monophosphates. Indeed, they

rely exclusively on the enzymes from the purine salvage pathway

and related transporters for the supply of purines and purine-

containing nucleotides. In the case of malaria, one of the most

important human parasitic diseases and a major cause of

mortality worldwide, parasite resistance to traditional antima-

larial drugs is now a serious concern. Thus, increasing eﬀorts

Received: January 23, 2020

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A

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Figure 1. Examples of immucilin,^7bANP (X = O,⁹NH,¹¹S,¹²CH₂¹³), and aza-C-ANP¹⁴derivatives described in the literature⁴as potent inhibitors of

PNP and HG(X)PRT.

linker (preferably including an heteroatom), and the phospho-

nate group. The selectivity of these series of ANPs is usually

modest with the exception of the Aza-C-ANPs. In addition, due

to the anionic nature of ANPs at physiological pH, these

compounds generally require conversion to neutral prodrugs to

observe eﬀective biological activity in cell culture exper-

iments.^8b,10This observation points to the fact that drugs

must be able to enter the targeted cell, in this case the infected

red blood cell, in order to block parasite processes.

RESULTS AND DISCUSSION

■

Chemistry. We focused speciﬁcally on the synthesis of α/β-

hydroxyphosphonate derivatives as structural analogues of

hydroxycarboxylic acids¹⁵(the latter are involved in various

biological processes). Thus, we describe a limited number of

regio- and stereoselective synthetic approaches to obtain these

derivatives as pure enantiomers under mild conditions.¹⁶We

previously reported on the synthesis of β-hydroxyphosphonate

ribonucleoside derivatives using the eﬃcient ring-opening

reaction of epoxides with phosphorus nucleophile, catalyzed

by BF₃·etherate.¹⁷In this case, chirality at the β-position

(relative to the phosphorus atom) is controlled and originated

from the naturally occurring chiral pool. Similarly, our general

approach to synthesis (Scheme 1) involved the use of ^D- or L-

aspartic acid or (R)- or (S)-glycidol, as cheap and/or naturally

occurring chiral starting materials. Thus, propyl derivatives

(compounds 1−3, Figure 2, n = zero, namely, C3 series) may be

obtained by the direct coupling of glycidol with the appropriate

nucleobase precursors, followed by a ring-opening reaction. For

butyl derivatives (compounds 4−10, 12, and 13, Figure 2, n = 1,

namely, C4 series), the required epoxide or aziridine should be

obtained in a few steps, through intramolecular nucleophilic

substitution from aspartic acid, in accordance with previously

described procedures.¹⁸To avoid side reactions during the

coupling steps, exocyclic functions of the purines were protected

either by a tert-butyloxycarbonyl (Boc) group or a methoxy

group (for 6-oxopurines).

Herein, we describe the synthesis of a new series of ANPs

(Figure 2). Their antimalarial potency was evaluated by a

Figure 2. Structures of the studied compounds.

phenotypic approach against P. falciparum (in vitro) and P.

berghei (in vivo) and revealed diﬀerent properties from those of

immucilins and aza-C-ANP.

Thus, Boc protected adenine, 2-amino-6-O-methylpurine and

2,6-diaminopurine (obtained beforehand using previously

reported protocols¹⁹) were coupled either with (R)- or (S)-

Scheme 1. Proposed Retrosynthesis

B

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a

Scheme 2. Synthetic Route to C3-β-Hydroxy-ANPs Containing Adenine, Guanine, and 2,6-Diaminopurine as Nucleobases

a

Reagents and conditions: (i) bis(Boc)adenine or bis(Boc)-2-amino-6-O-methylpurine or tetra(Boc)-2,6-diaminopurine, PPh₃, DEAD, THF, 0 °C

to rt, 24 h; (ii) diethyl phosphite, N,O-BSA, CH₂Cl₂, reﬂux, 3−4 h, then addition of 14−16 in CH₂Cl₂, −60°C; (iii) BF₃·Et₂O, −60 °C to rt,

overnight; (iv) triﬂuoroacetic acid in CH₂Cl₂, 0 °C to rt, 5 h; (v) TMSBr in CH₃CN, rt, 1−2 d, then H₂O, 0 °C to rt, 1 h.

a

Scheme 3. Synthetic Route to C4-β-Hydroxy-ANPs Containing Adenine, Guanine, and 2,6-Diaminopurine as Nucleobases

a

Reagents and conditions: (i) bis(Boc)adenine or bis(Boc)-2-amino-6-O-methylpurine or tetra(Boc)-2,6-diaminopurine, PPh₃, DEAD, THF, 0 °C

to rt, 24 h; (ii) diethyl phosphite, N,O-BSA, CH₂Cl₂, reﬂux, 3−4 h; 21−23 were added in CH₂Cl₂, −60°C; (iii) BF₃·Et₂O, −60 °C to rt, overnight;

(iv) triﬂuoroacetic acid in CH₂Cl₂, 0 °C to rt, 5 h; (v) TMSBr in CH₃CN, rt, 1−2 d, then H₂O, 0 °C to rt, 1 h.

glycidol under Mitsunobu conditions²⁰and yielded the

enantiomeric pairs of epoxides 14−16 (Scheme 2). Then, the

ring-opening step was carried out in the presence of an excess of

silylated diethyl phosphite and BF₃·Et₂O and generated fully

protected β-hydroxyphosphonate derivatives 17−19. Removal

of the various protecting groups was performed using a standard

protocol with triﬂuoroacetic acid and then bromotrimethylsi-

lane. The resulting phosphonic acids were eventually converted

into their sodium salts, compounds 1−3, by percolation onto

ion-exchange resin (Dowex Na⁺form).

We then envisaged obtaining the corresponding β-hydrox-

yphosphonate derivatives, including a butyl chain, compounds

4−6 (Figure 2). The synthesis required the (R)- and (S)-2-

hydroxyethyloxirane 20 (Scheme 3), which were prepared in

three steps starting from ^D- or L-aspartic acid according to

previously reported procedures.^18aIsolation of epoxide 20 was

problematic (due to its volatility and solubility in water).

Therefore, we decided to use it as a crude after ﬁltration of the

reaction media. The solution of 20 in dichloromethane was

added directly in the Mitsunobu coupling step with the required

nucleobases, providing the enantiomeric pairs of epoxides 21−

23. The above-mentioned steps (ring-opening reaction and

removal of protecting groups) were performed on these

substrates and yielded isolated intermediates 24−26 and then

the desired ﬁnal compounds 4−6 as sodium salts.

As xanthine and hypoxanthine derivatives are important

substrates during the biosynthesis of P. falciparum nucleic acids

(through the salvage pathway), we chose to synthesize these

analogues in the C4 series (compounds 7 and 8, Figure 2) from

derivatives 24 and 25 (Scheme 4). Brieﬂy, acidic treatment

allowed the removal of Boc protecting groups and yielded

intermediates 27 and 28 quantitatively, which were then

subjected to a diazotization reaction.¹³Thus, β-hydroxyphosph-

onate derivatives 29 and 30, including hypoxanthine and 2-oxo-

6-O-methylpurine, were converted into their phosphonic acids

(7 and 8 isolated as sodium salts) with trimethylsilyl bromide.

C

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The guanine derivatives 11a and 11b were obtained by

adapting the existing procedure for 11a^13,21and using an

alternative route for 11b²²(cf. Supporting Information for

details).

Scheme 4. Synthetic Route to C4-β-Hydroxy-ANPs

Containing Xanthine and Hypoxanthine as Nucleobases

a

To access the β-aminophosphonate in the C4-series (Scheme

6), we developed an original synthetic route. The preparation of

enantiomerically pure N-Boc-aziridine 35(S) or 35(R) was

previously reported in a few steps from ^L- or D-aspartic acid,

respectively.^18dBrieﬂy, quantitative synthesis of aspartic acid

dimethyl esters was carried out using SOCl₂in methanol,

followed by the introduction of a tert-butyloxycarbonyl

protecting group on the amine. The subsequent reduction of

the diesters in the presence of NaBH₄and CaCl₂yielded the

corresponding (S)- or (R)-N-Boc-amino diols.^18bThe latter

compounds were converted to the 1,4-ditosylated intermediates.

In situ this led to the intramolecular cyclization in the presence of

potassium hydroxide and provided the enantiomeric pair of N-

Boc-aziridine 35 in 45% overall yields (over ﬁve steps).^18dThen,

nucleophilic displacement of the remaining tosyl group by the

appropriate purine precursors was accomplished in the presence

of potassium carbonate and led to the isolation of the N-9-

regioisomers 36 and 37 (sole products).

a

Reagents and conditions: (i) triﬂuoroacetic acid 20% in CH₂Cl₂, 0

°C to rt, 45 min; (ii) NaNO₂, H₂O, AcOH 1 M, 70 °C, 1 h; (iii)

TMSBr in CH₃CN, rt, 1−2 d, then TEAB, 0 °C to rt, 1 h.

We showed that compounds with (R)-stereochemistry and

four-carbon linker had the best activities (see in vitro data

section, Table 1). We thus extended our SAR study to non-

natural purines (Figure 2, compounds 9 and 10), incorporating

2-amino- and 6-amino-2-ﬂuoropurines.

The ring-opening of activated aziridines was somewhat

puzzling. It is highly dependent on both the nature of the

protecting groups on the aziridine ring and the phosphorus

nucleophilic species (data not shown). Best conditions involved

the use of a Boc group and the addition of the lithium phosphite

generated in situ from diethyl phosphite and LiHMDS at low

temperature. After overnight stirring, satisfactory yields of the

desired β-aminophosphonate derivatives 38 and 39 were

isolated and we observed concomitant and partial removal of

the Boc group on the exocyclic amino group of the nucleobases.

Final removal of the remaining Boc groups was performed by

treating derivatives 38 and 39 with triﬂuoroacetic acid in

dichloromethane yielding compounds 40 and 41. On hydrolysis

of the diethyl phosphonate moiety (using TMSBr in DMF), this

led to the expected derivatives 12 and 13.

Compounds 9 and 10 were prepared following the same

synthetic approach as described for derivatives 4−6 (Scheme 5).

Compound 20(R) was coupled under Mitsunobu conditions to

protected N-(Boc)₂-2-aminopurine and N-(Boc)₂-6-amino-2-

ﬂuoropurine in order to produce the corresponding epoxides 31

and 32. The ring-opening reaction yielded the expected β-

hydroxyphosphonate derivatives 33 and 34. The concomitant

removal of Boc and diethyl phosphoester groups yielded the

targeted phosphonic acids 9(R) and 10(R) as sodium salts.

We then probed the essentiality of the hydroxyl group at the

β-position relative to the phosphorus atom by either removing it

(Figure 2, compounds 11a and 11b including a butyl or butenyl

linker) or replacing it by an amino group (Figure 2, compounds

12 and 13, corresponding to β-aminophosphonate derivatives).

Enantiomeric Excess Characterization. ³¹P NMR spec-

troscopy was used in combination with a chiral solvating agent

a

Scheme 5. Synthetic Route to C4-β-Hydroxy-ANPs Containing 2-Amino- and 2-Fluoro-6-aminopurines as Nucleobases

a

Reagents and conditions: (i) bis(Boc)-2-amino-purine or bis(Boc)-6-amino-2-ﬂuoropurine, PPh₃, DEAD, THF, 0 °C to rt, 24 h; (ii) diethyl

phosphite, N,O-BSA, CH₂Cl₂, reﬂux, 3−4 h; 31 or 32 was added in CH₂Cl₂, −60 °C; (iii) BF₃.Et₂O, −60 °C to rt, overnight; (iv) TMSBr in

CH₃CN, rt, 1−2 d, then H₂O, 0 °C to rt, 1 h.

D

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a

Scheme 6. Synthetic Route to C4-β-Amino-ANPs Containing Guanine and 2,6-Diaminopurine as Nucleobases

a

Reagents and conditions: (i) bis(Boc)-2-amino-6-O-methylpurine or tetra(Boc)-2,6-diaminopurine, K₂CO₃, DMF, rt, 3 d; (ii) diethyl phosphite,

LiHMDS, THF, −78 °C, 15 min, then 36 or 37 was cannulated in THF, −78 °C to rt, then rt overnight; (iii) triﬂuoroacetic acid in CH₂Cl₂, 0 °C

to rt, 5 h; (iv) TMSBr in DMF, rt, 1−2 d, then TEAB, 0 °C to rt, 1 h.

Figure 3. Separation of pair of enantiomers of two families of ANPs by ³¹P NMR: (A) comparison of ³¹P NMR of enantiomers of β-

hydroxyphosphonates; (B) comparison of ³¹P NMR of enantiomers of β-aminophosphonates. Characteristics of α-cyclodextrin are drawn from the

literature.²⁴

(CSA) to stereodiscriminate a pair of isomers and check the

enantiopurity of our compounds. This simple rapid approach

requires a small amount of compound. It has already been

reported for chiral α-amino and α-hydroxyphosphonic acids

using α-cyclodextrin as CSA.²³Thus, several factors (temper-

ature, pD, etc..) were tested to determine the best conditions for

our derivatives based on β-hydroxyphosphonates (Supporting

Information Figures S1 −S3). The best separation was observed

at T = 15 °C, the lowest tested temperature. This may be

because it slows the conformational exchange rate of both

enantiomers inside the cyclodextrin cavity and, thus, enhances

their diﬀerentiation. The optimal pD was slightly diﬀerent

(always under basic conditions), depending on the studied

compounds (β-amino and β-hydroxyphosphonates). This

suggests that the ionization of the hydroxyl and amino group

plays an important role in the complexation of the compound

inside the cavity. The two enantiomers of each compound were

tested separately and showed only one peak at a speciﬁc δ, at the

same pD (Figure 3). The procedure made it possible to

diﬀerentiate the two peaks from a racemic mixture. Thus, we

demonstrate the enantiopurity of our ﬁnal compounds and the

reliability of our synthesis.

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Table 1. Comparative Eﬀects of ANP Derivatives on the Growth of P. falciparum (3D7 Strain) and Erythroblast (K562) Human

a

Cell Line

b

IC₅₀(μM)

c

compd

1(R)

1(S)

2(R)

2(S)

3(R)

3(S)

4(R)

4(S)

5(R)

5(S)

6(R)

6(S)

7(R)

7(S)

base (X, Y)

chain length (n)

C3 (n = 0)

polar group

OH

P. falciparum

K562

>1000

SI

adenine (X = NH₂, Y = H)

>1000

595

327 97

510

>1000

0.074 0.007

6.1 1.1

48

>1000

590

NA

>1.7

>2.6

>2

NA

>1351

164

>21

NA

>1

>16

>38

>17

NA

1807

>1000

25.3 3.3

guanine (X = OH, Y = NH₂)

2,6-diaminopurine (X = Y= NH₂)

adenine (X = NH₂, Y = H)

C3 (n = 0)

C4 (n = 1)

OH

guanine (X = OH, Y = NH₂)

2,6-diaminopurine (X = Y= NH₂)

hypoxanthine (X = OH, Y = H)

xanthine (X = OH, Y = OH)

8(R)

8(S)

9(R)

10(R)

11a

910

2-aminopurine(X = H, Y= NH₂)

2-ﬂuoro-6-aminopurine (X = NH₂, Y = F)

guanine

C4 (n = 1)

C4

OH

X = Y = CH₂

X = Y = CH

NH₂

67 11

26.3 5.3

74 8.6

≫100

>100

≫100

0.014 0.002

11b

12(R)

12(S)

13(R)

13(S)

chloroquine

guanine (X = OH, Y = NH₂)

C4

2,6-diaminopurine (X = Y = NH₂)

NH₂

a

b

Values for X, Y, and n are related to formulas shown in Figure 2. Values are the mean of at least three independent experiments performed in

c

duplicate ( SEM). For other values, the mean of two independent experiments (each performed in duplicate) and diﬀering by <30% are given. SI

(selectivity index) is the ratio of IC₅₀value for K562 cells to the IC₅₀value for P. falciparum. NA: not applicable. Parasite growth was assessed after

48 h of contact with the compound by measuring the incorporation of [³H] hypoxanthine into nucleic acids from 48 to 66 h.²⁵

In Vitro Antimalarial Activities. The ability of our novel

derivatives to interfere with parasite growth in cell culture was

monitored by adding the compounds to a P. falciparum-infected

erythrocyte suspension for a complete 48 h parasite cycle before

assessing parasite viability (Table 1). The C3-series analogues

1−3 showed low antimalarial activities with IC₅₀higher than 300

μM for both R and S enantiomers, irrespective of the nature of

the nucleobase. In this series, the most potent analogue is the

2,6-diaminopurine. In the C4-series with a hydroxyl group in the

β-position relative to the phosphorus atom, we tested both

enantiomers (compounds 4−8) and diﬀerent nucleobases

(compounds 4−10). The best activity was always observed for

the R-enantiomer (Table 1) when the analogue was active

(compounds 5, 6, and 8). Concerning the nature of the

nucleobase, the guanine derivatives 5(R) and 5(S) were the

most interesting compounds with an IC₅₀value of 74 nM and 6.1

μM, respectively. Compounds 6(R), 9(R), 10(R) exhibited

modest antiplasmodial activities with IC₅₀values between 26

and 67 μM. The use of hypoxanthine (compounds 7, both R and

S isomers) and xanthine (compounds 8, both R and S isomers)

resulted in total loss of the activity indicating the important role

of the amino substitution at position 2.

antiplasmodial activity, at least 1000-fold (in the μM range)

compared to compound 5(R) (in the nM range).

In order to assess the eﬀect of the analogues on the

development of P. falciparum parasites, an infected-erythrocyte

culture treated with 5(R) was followed over 69 h. Thin blood

smears showed that treated parasites developed normally for 48

h until the end of the cycle at the schizont stage (Figure 4A).

However, only few new parasites were detected at the following

cycle when treated with 2 μM compound 5(R), and they died

before 69 h. Immunoﬂuorescence assays on mature parasites at

late schizont stage showed that the daughter cells developed

normally in the presence of compound 5(R) and that the DNA

content of the newly formed merozoites seemed unaﬀected by

the treatment (Figure 4B). This suggests that the mode of action

of compound 5(R) could be diﬀerent from other classes of ANPs

that have been described to target DNA synthesis (Keough et

al.^9,26). This hypothesis is supported by the comparison of our

data with the literature. It has been reported that both guanine

and hypoxanthine ANP derivatives equally inhibited recombi-

nant Pf HGXPRT.^12,13In contrast, our guanine derivatives

(compounds 5) have an antimalarial activity between nM and

low μM range, whereas our hypoxanthine derivatives (com-

pounds 7) were not active at 1 mM. Additionally, Cesnek et al.¹³

showed that the guanine derivative corresponding to our

compound 11a has no aﬃnity for Pf HGXPRT with K_i> 100

μM. In our experiments, this compound 11a showed a modest

inhibition of P. falciparum growth in vitro with an IC₅₀value of

74 μM. Altogether, these data suggest that our series of

Finally, concerning the nature of the polar group in the β-

position relative to the phosphorus atom, the hydroxyl group

remained the best option (Table 1). Its replacement by an NH₂

group (compounds 12 and 13, with IC₅₀values of >100 μM) or

removal (compounds 11a,b) led to a signiﬁcant decrease of the

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Figure 4. Eﬀect of compound 5(R) on the development of P. falciparum. A tightly synchronized culture of P. falciparum at ring stage was treated or not

with 2 μM 5(R). (A) Follow-up of parasite development on Giemsa-stained smears. Parasitemia is indicated for each parasite stage of the culture.

Treatment with 5(R) showed no morphological eﬀect until the late schizont stage (48 h). At time 69 h, all parasites visible at time 48 h have

disappeared. (B) Immunoﬂuorescence assay on infected erythrocytes at time 45 h, at segmented schizont stage. Formation of merozoites membrane

was visualized by labeling the internal membrane complex (α-mTIP) and the membrane surface (α-MSP1). The labeling of nuclei with Hoechst

showed that all merozoites, in control and treated parasites, possessed a nucleus.

analogues have a diﬀerent mode of action from other classes of

ANPs and a target that is unlikely to be the Pf HGXPRT.

We then evaluated the compounds for toxicity on human

blood cell lines (K562 cells) and calculated selectivity indexes

(SI) (Table 1). None of the derivatives inhibited human blood

cell growth below a concentration of 1 mM indicating a high

selectivity for infected red blood cells. All ANPs tested are of

anionic character which generally limits their passive diﬀusion

across biological membranes, explaining their low toxicity

toward human cell lines. Our data showed that the ANPs

developed in this study aﬀected parasite growth and were

therefore likely able to speciﬁcally enter into infected red blood

cells. This could be due to the presence of channels transporting

negatively charged molecules on the infected erythrocyte

membrane that are induced by the parasite.²⁷

Altogether, these data revealed a unique antimalarial

pharmacophore and the compound 5(R) shows considerable

potential with an antimalarial activity of 74 nM and a SI higher

than 1350.

Evaluation of Compounds 5(R) and 5(S) on the Drug-

Resistant Strains of P. falciparum. Compounds 5(R) and

5(S) were selected for further studies with the multidrug-

resistant W2 strain and the chloroquine-resistant FcM29 strain

of P. falciparum (Table 2). Both derivatives showed equivalent

potency in the three strains 3D7, W2, and FcM29.

Table 2. Comparative Eﬀects of Derivatives 5(R) and 5(S) on

the Growth of P. falciparum Strains, Sensitive (3D7) or

Resistant (FcM29 and W2) to Chloroquine

a

IC₅₀(μM)

compd

5(R)

5(S)

3D7

FcM29

W2

0.074 0.007

6.1 1.1

0.03 0.003

4.5 1.3

0.045 0.006

5.6 1.4

chloroquine

0.016 0.001

0.117 0.010

0.131 0.022

a

Values ( SEM) are the mean of at least three independent

experiments performed in duplicate.

potent eﬃcacy in vivo with an ED₅₀value of 0.56 mg/kg (Figure

5A). Parasites were not detected after a treatment with 30 mg/kg

at day 5, but we observed a parasite recrudescence for two mice

after day 11 (6.9% and 16.7% of parasitemia) and for one mouse

after day 14 (1.7% of parasitemia). In parallel, we tested the

eﬀect of a single dose of 30 mg/kg administered at day 1. This

treatment showed a decrease of the parasitemia by 95% as

compared to the controls. The eﬃcacy of the derivative may

enable the dosage regimens to be modiﬁed for further

development. The activity was also evaluated by oral

administration after 4 days of treatment. In these conditions,

the ED₅₀was found to be 30 mg/kg indicating a low

bioavailability likely due to the polarity and anionic character

of the compound (Figure 5B).

Antimalarial Activity in P. berghei Infected Mice.

According to the results obtained in vitro, compound 5(R)

was evaluated in an in vivo assay using P. berghei-infected mice

(Figure 5). After 4 days of treatment by intraperitoneal (ip)

route, the parasitemia was quantiﬁed at day 5 and the dose

required to decrease the parasitemia by 50% (ED₅₀) was

determined. In these conditions, the derivative 5(R) showed a

In comparison, artesunate, an artemisinin derivative, showed

an ED₅₀value of ∼0.1 mg/kg by ip route (Figure 5C) and a

treatment of 27 mg/kg did not allow a complete cure. This

suggests that the derivative 5(R) showed a potent activity

comparable to this widely used antimalarial drug.

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Figure 5. In vivo antimalarial activity of 5(R) and artesunate in P. berghei-infected mice. 5(R) antimalarial activity was determined after intraperitoneal

(ip) (A) or oral (po) (B) administration. Mice were treated with one daily dose for 4 consecutive days from day 1 or with a single dose of 30 mg/kg on

day 1 after infection. (C) Artesunate antimalarial activity after intraperitoneal administration. Mice were treated with one daily dose for 4 consecutive

days from day 1. In all cases, parasitemia was determined at day 5. In all cases, controls received vehicle only.

polarimeter. Speciﬁc rotations [α]^Dare given in deg cm³g⁻¹dm⁻¹.

Purity was determined by high performance liquid chromatography

We reported and tested 24 derivatives, including 22 novel acyclic

(HPLC). Purity of all ﬁnal compounds was 95% or higher. The

CONCLUSION

20

■

nucleoside phosphonates, as potential inhibitors of P. falciparum

growth. Synthesis of β-hydroxyphosphonates with diﬀerent

instrument was a Waters HPLC system (separation module 2695, 996

photodiode array detector 2996). The column was a Waters Symmetry

chain lengths (three to four carbon atoms) bearing various

nucleobases was performed. An original synthetic pathway to β-

aminophosphonates was developed and constitutes a new

method for accessing compounds of this class with therapeutic

potential. Biological assays of phosphonic acid compounds (as

sodium salts) on cell cultures revealed that the compounds’

eﬀectiveness depends on the hydroxyl group, the chain length,

and the nature of the base. Of all compounds, derivative 5(R)

appears to have the most suitable characteristics (i.e., guanine as

nucleobase, a butyl chain, a hydroxy group in the β-position and

with R stereochemistry). It shows remarkable in vitro activity

against P. falciparum infected red blood cells (IC₅₀= 74 nM),

and it has a high selectivity index (SI > 1350). No toxicity on

human cell lines was found. The derivative 5(R) also showed a

considerable activity in vivo (ED₅₀∼ 0.5 mg/kg by ip route) in

the same range as the currently used artesunate. To our

knowledge, this is the ﬁrst report on the antiplasmodial activity

(in vitro and in vivo) of an acyclonucleoside phosphonate

derivative. Given these encouraging results, we will pursue our

research on this family of β-substituted ANPs to improve oral

bioavailability using a prodrug strategy and to decipher the

mechanism of action of the compound 5(R) which is likely

diﬀerent from the other ANPs.

Shield (50 mm × 4.6 mm, 3.5 μm) RP-18-column. The elution solvents

were water containing 0.1% (v/v) of TFA (solvent A) and acetonitrile

containing 0.1% (v/v) TFA (solvent B). A linear gradient was applied

from 0% to 100% of solvent B in 7 or 15 min with a ﬂow rate of 1 mL/

min.

General Procedure A. Preparation of Epoxide Derivatives in the

C3 Series. To a solution of commercially available (R)- or (S)-glycidol

(1.1 equiv) in freshly distilled tetrahydrofuran (1.5 mL/mmol of

nucleobase) were added under argon atmosphere triphenylphosphine

(1.2 equiv) and the nucleobase (1 equiv). The reaction mixture was

cooled to 0 °C, and diethyl azodicarboxylate (1.2 equiv) was added

dropwise. After 10 min, the reaction mixture was allowed to warm to

room temperature and stirred overnight. The reaction mixture was

concentrated under vacuum, dissolved in a small amount of diethyl

ether, and stored at 4 °C overnight. The triphenylphosphine oxide

precipitate was ﬁltrated oﬀ and washed with cold diethyl ether. The

ﬁltrate was then concentrated under vacuum and the crude puriﬁed by

ﬂash chromatography on silica gel, using either an isocratic of AcOEt or

CH₂Cl₂/MeOH (0 to 5%) to isolate the desired product.

N,N-6-Bis(tert-butoxycarbonyl)-N-9-(2R-(oxiran-2-yl)methyl)-

adenine (14(R)). Compound 14(R) (1.86 g) was obtained in 87% yield

as an oil, according to procedure A, from (R)-glycidol (362 μL). R_f=

0.41 in AcOEt. [α]_D²⁰= +22.5° (c 1.02, MeOH). ¹H NMR (DMSO-

d₆): δ = 8.86 (s, 1H, H₋₂), 8.60 (s, 1H, H₋₈), 4.67 (dd, 1H, ²J = 15.0 Hz,

³J = 2.7 Hz, CH₂N), 4.40 (dd, 1H, ²J = 15.0 Hz, ³J = 6.0 Hz, CH₂N),

3.47 (m, 1H, CHO), 2.83 (m, 1H), 1.37 (s, 18H, CH₃). ¹³C NMR

(CDCl₃): δ = 152.4 (CH₋₂), 145.1 (CH₋₈), 128.6 (C₋₅), 83.9

(C(CH₃)₃), 49.9 (CHO), 45.5 (OCH₂), 45.4 (CH₂N), 27.9 (CH₃).

MS (ESI) m/z 392.2 [M + H]⁺. HRMS: calcd C₁₈H₂₆N₅O₅[M + H]⁺

392.1934, obs 392.1921.

N,N-6-Bis(tert-butoxycarbonyl)-N-9-(2S-(oxiran-2-yl)methyl)-

adenine (14(S)). Compound 14(S) (1.01 g) was obtained in 86% yield

as a solid, according to procedure A, from (S)-glycidol (181 μL). R_f=

0.41 in AcOEt. [α]_D²⁰= −16.8° (c 1.01, MeOH). ¹H NMR (DMSO-

d₆): δ = 8.86 (s, 1H, H₋₂), 8.60 (s, 1H, H₋₈), 4.65 (dd, 1H, ²J = 15.0 Hz,

³J = 2.7 Hz, CH₂N), 4.40 (dd, 1H, ²J = 15.0 Hz, ³J = 6.0 Hz, CH₂N),

EXPERIMENTAL SECTION

■

Chemistry. General Information. Solvents were dried by

standard procedures. Tetrahydrofuran (THF) was freshly distilled

from Na under argon and in the presence of benzophenone. All reagents

and chemicals were obtained from commercial sources. Thin layer

chromatography (TLC) was performed on precoated aluminum sheets

of silica gel 60 F₂₅₄(Merck, no. 5554). Product spots on TLC plate were

visualized with a UV lamp (254 nm). Flash chromatography on normal

phase (silica gel) and on reversed phase (C18AQ) was performed on

Biotage or Interchim Flash Chromatography Systems, respectively.

NMR spectra were recorded on Bruker Avance 500 (¹H at 500 MHz,

¹³C at 125.8 MHz) or 300 (¹H at 300 MHz, ¹³C at 125.8 MHz)

spectrometer with TMS as internal standard or referenced to the

3.47 (m, 1H, CHO), 2.83 (m, 1H), 1.38 (s, 18H, CH₃). ¹³C NMR

(DMSO-d₆): δ = 153.3 (C₋₄), 151.6 (CH₋₂), 150.0, 149.1 (C₋₆, C

O), 147.0 (CH₋₈), 127.5 (C₋₅), 83.3 (C(CH₃)₃), 49.4 (CHO), 45.1

(OCH₂), 45.0 (CH₂N), 27.3 (CH₃). MS (ESI) m/z 392.3 [M + H]⁺.

HRMS: calcd C₁₈H₂₆N₅O₅[M + H]⁺392.1934, obs 392.1941.

N,N-2-Bis(tert-butoxycarbonyl)-6-methoxy-N-9-(2R-(oxiran-2-

yl)methyl)-2-aminopurine (15(R)). Compound 15(R) (1.72 g) was

obtained in 74% yield as a solid, according to procedure A, from (R)-

glycidol (336 μL). R_f= 0.2 in AcOEt. [α]_D²⁰= +20.5° (c 1.05, MeOH).

¹H NMR (DMSO-d₆): δ = 8.39 (s, 1H, H₋₈), 4.54−4.34 (m, 2H,

residual solvent signal (CDCl₃, DMSO-d₆or D₂O) for H and ¹³C.

1

H₃PO₄was used as external standard for ³¹P NMR, and corresponding

spectra were recorded with proton decoupling at ambient temperature.

Chemical shifts (δ) are quoted in parts per million (ppm). Mass spectra

(ESI) and high-resolution mass spectroscopy (HR-MS) results were

recorded in the negative and/or positive-ion mode using a SYNAPT G2

mass spectrometer. Optical rotation was determined by a Kruss P8000

̈

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CH₂N), 4.07 (s, 3H, OCH₃), 3.40 (m, 1H, CHO), 2.81 (m, 1H,

OCH₂), 2.50 (m, 1H partially covered by DMSO, OCH₂), 1.39 (s,

18H, CH₃). ¹³C NMR (DMSO-d₆): δ = 160.6 (C₋₆), 152.7 (C₋₄),

151.0 (C₋₂), 150.3 (CO), 144.9 (CH₋₈), 118.7 (C₋₅), 82.6

(C(CH₃)₃), 54.3 (OCH₃), 49.3 (CHOH), 44.8 (OCH₂), 44.7

(CH₂N), 27.3 (CH₃). MS (ESI) m/z 422.2 [M + H]⁺. HRMS: calcd

C₁₉H₂₈N₅O₆[M + H]⁺422.2040, obs 422.2034.

N,N-2-Bis(tert-butoxycarbonyl)-6-methoxy-N-9-(2S-(oxiran-2-yl)-

methyl)-2-aminopurine (15(S)). Compound 15(S) (1.75 g) was

obtained in 76% yield as a solid, according to procedure A, from (S)-

glycidol (332 μL). R_f= 0.2 in AcOEt. [α]_D²⁰= −22.1° (c 0.98, MeOH).

¹H NMR (DMSO-d₆): δ = 8.39 (s, 1H, H₋₈), 4.54−4.33 (m, 2H,

CH₂N), 4.07 (s, 3H, OCH₃), 3.41 (m, 1H, CHO), 2.81 (m, 1H,

OCH₂), 2.50 (m, 1H partially covered by DMSO, OCH₂), 1.39 (s,

18H, CH₃). ¹³C NMR (DMSO-d₆): δ = 160.6 (C₋₆), 152.7 (C₋₄),

151.0 (C₋₂), 150.3 (CO), 144.9 (CH₋₈), 118.7 (C₋₅), 82.7

(C(CH₃)₃), 54.3 (OCH₃), 49.3 (CHOH), 44.9 (OCH₂), 44.7

(CH₂N), 27.3 (CH₃). MS (ESI) m/z 422.2 [M + H]⁺. HRMS: calcd

C₁₉H₂₈N₅O₆[M + H]⁺422.2040, obs 422.2047.

N,N-2,6-Tetra(tert-butoxycarbonyl)-N-9-(2R-(oxiran-2-yl)-

methyl)-2,6-diaminopurine (16(R)). Compound 16(R) (1.40 g) was

obtained in 84% yield as a solid, according to procedure A, from (R)-

glycidol (165 μL). R_f= 0.55 in AcOEt. [α]_D²⁰= +22.2° (c 1.01, MeOH).

¹H NMR (DMSO-d₆): δ = 8.66 (s, 1H, H₋₈), 4.67−4.36 (m, 2H,

4.48−4.39 (m, 2H, CH₂N), 2.93−2.90 (m, 1H, CHO), 2.72−2.70 (m,

1H, CH₂CH), 2.43−2.41 (m, 1H, OCH₂CH), 2.38−2.32 (m, 1H,

CH₂CH₂N), 1.95−1.88 (m, 1H, CH₂CH₂N), 1.41 (s, 18H, CH₃). ¹³

C

NMR (CDCl₃) δ 153.4 (C₋₄), 152.1 (CH₋₂), 150.5 (C₋₆), 150.4 (C

O), 144.9 (CH₋₈), 128.9 (C₋₅), 83.8 (C(CH₃)₃), 49.4 (CHOH), 46.8

(OCH₂CH), 41.4 (s, CH₂N), 32.7 (CH₂CH₂N), 27.8 (CH₃). HRMS

(TOF ESI > 0) found: 406.2055. Calculated for C₁₉H₂₈N₅O₅: 406.2050

(M + H)⁺.

N,N-6-Bis(tert-butoxycarbonyl)-N-9-(2S-(oxiran-2-yl)ethyl)-

adenine (21(S)). Compound 21(S) (1 g) was obtained in 63% yield,

according to procedure B, from 2-(S)-bromo-1,4-butanediol (1.32 g).

R_f= 0.50 in CH₂Cl₂/MeOH (9/1, v/v). [α]_D²⁰−20.98 (c 0.4, CH₂Cl₂).

¹H NMR (CDCl₃) δ 8.83 (s, 1H, H₋₂), 8.10 (s, 1H, H₋₈), 4.49−4.40

(m, 2H, CH₂N), 2.93−2.91 (m, 1H, CHO), 2.73−2.71 (m, 1H,

OCH₂CH), 2.43−2.42 (m, 1H, OCH₂CH), 2.39−2.32 (m, 1H,

CH₂CH₂N), 1.95−1.88 (m, 1H, CH₂CH₂N), 1.41 (s, 18H, CH₃). ¹³

C

NMR (CDCl₃) δ 153.4 (C₋₄), 152.1 (C₋₂), 150.5 (C₋₆), 150.4 (C

O), 144.9 (C₋₈), 128.9 (C₋₅), 83.8 (C(CH₃)₃), 49.4 (CHOH), 46.8

(OCH₂CH), 41.4 (CH₂N), 32.7 (CH₂CH₂N), 27.8 (CH₃). HRMS

(TOF ESI > 0) found: 406.2055. Calculated for C₁₉H₂₈N₅O₅: 406.2050

(M + H)⁺.

N,N-2-Bis(tert-butoxycarbonyl)-6-methoxy-N-9-(2R-(oxiran-2-

yl)ethyl)-2-aminopurine (22(R)). Compound 22(R) (1.57 g) was

obtained in 66% yield, according to procedure B, from 2-(R)-bromo-

1,4-butanediol (1.85 g). R_f= 0.52 in CH₂Cl₂/MeOH (95/5, v/v).

[α]_D²⁰+20.86° (c 0.9, CH₂Cl₂). ¹H NMR (DMSO-d₆) δ 8.45 (s, 1H,

H₋₈), 4.39−4.35 (m, 2H, CH₂N), 4.06 (s, 3H, OCH₃), 2.94−2.93 (m,

1H, CHO), 2.60−2.57 (m, 1H, OCH₂CH), 2.29−2.27 (m, 1H,

OCH₂), 2.10−2.09 (m, 1H, CH₂CH₂N), 1.98−1.95 (m, 1H,

CH₂CH₂N), 1.43 (s, 18H, CH₃). ¹³C NMR(DMSO-d₆) δ 160.3

(C₋₆), 152.4 (C₋₄), 150.5 (C₋₂), 150.2 (CO), 144.7 (CH₋₈), 118.8

(C₋₅), 82.4 (C(CH₃)₃), 54.1 (OCH₃), 49.0 (CHOH), 45.1

(OCH₂CH), 40.6 (CH₂N), 31.9 (CH₂CH₂N), 27.1 (CH₃). HRMS

CH₂N), 3.45 (m, 1H, CHO), 2.83 (m, 1H, OCH₂), 2.44 (m, 1H,

OCH₂), 1.36 (s, 36H, CH₃). ¹³C NMR (DMSO-d₆): δ = 154.0 (C₋₄),

151.0, 150.2 (C₋₆, C₋₂), 148.0, 149.5, 149.6, 150.2 (CO), 125.9

(CH₋₈), 83.4, 82.8 (C(CH₃)₃), 49.2 (CHOH), 44.7 (OCH₂, CH₂N),

27.2, 27.0 (CH₃). MS (ESI) m/z 607.3 [M + H]⁺. HRMS: calcd

C₂₈H₄₃N₆O₉[M + H]⁺607.3092, obs 607.3088.

N,N-2,6-Tetra(tert-butoxycarbonyl)-N-9-(2S-(oxiran-2-yl)-

methyl)-2,6-diaminopurine (16(S)). Compound 16(S) (2.31 g) was

obtained in 84% yield as a solid, according to procedure A, from (S)-

glycidol (275 μL). R_f= 0.42 in AcOEt/petroleum ether (6/4, v/v).

[α]_D²⁰= −21.8° (c 1.0, MeOH). ¹H NMR (DMSO-d₆): δ = 8.66 (s, 1H,

H-8), 4.70−4.36 (m, 2H, CH₂N), 3.45 (m, 1H, CHO), 2.83 (m, 1H,

OCH₂), 2.44 (m, 1H, OCH₂), 1.36 (s, 36H, CH₃). ¹³C NMR (DMSO-

d₆): δ = 154.0 (C-4), 151.0, 150.2 (C-6, C-2), 148.0, 149.5, 149.6, 150.2

(CO), 125.9 (CH-8), 83.4, 82.8 (C(CH₃)₃), 49.2 (CHOH), 44.7

(OCH₂, CH₂N), 27.2, 27.0 (CH₃). MS (ESI) m/z 607.3 [M + H]⁺.

HRMS: calcd C₂₈H₄₃N₆O₉[M + H]⁺607.3092, obs 607.3082.

General Procedure B. Preparation of Epoxide Derivatives in the

C4 Series. To a solution of enantiomerically pure 2-bromo-1,4-

butanediol²⁸(2 equiv) in freshly distilled dichloromethane (6.5 mL/

mmol of nucleobase) was added under argon atmosphere anhydrous

cesium carbonate (Cs₂CO₃, 3.6 equiv). The reaction mixture was

stirred at room temperature and followed by TLC (CH₂Cl₂/MeOH

95/5 v/v, with sulfuric acid revelation) until completion. After 24 h, the

suspension was ﬁltered and the precipitate washed with 3 mL of

anhydrous dichloromethane. The ﬁltrate was then transferred to a

round-bottom ﬂask ﬂushed with argon, and anhydrous tetrahydrofuran

(3.8 mL/mmol of nucleobase), triphenylphosphine (1.2 equiv), and the

nucleobase (1 equiv) were successively added. The reaction mixture

was cooled to 0 °C, and diethyl azodicarboxylate (1.2 equiv) was added

dropwise. After 10 min, the reaction mixture was allowed to warm to

room temperature and stirred for 24 h with TLC monitoring (CH₂Cl₂/

MeOH 95/5 v/v). The reaction mixture was concentrated under

vacuum, dissolved in a small amount of diethyl ether, and stored at 4 °C

overnight. The triphenylphosphine oxide precipitate was removed by

ﬁltration and washed with cold diethyl ether. The ﬁltrate was then

concentrated under vacuum and the crude puriﬁed by ﬂash

chromatography on silica gel, using CHCl₃/AcOEt (0 to 100%) to

remove the remaining triphenylphosphine oxide, and then CH₂Cl₂/

MeOH (0 to 10%) to isolate the desired product, which was obtained as

a pale yellow oil.

(TOF ESI > 0) found: 436.2199. Calculated for C₂₀H₃₀N₅O₆: 436.2196

(M + H)⁺.

N,N-2-Bis(tert-butoxycarbonyl)-6-methoxy-N-9-(2S-(oxiran-2-yl)-

ethyl)-2-aminopurine (22(S)). Compound 22(S) (787 mg) was

obtained in 66% yield, according to procedure B, from 2-(S)-bromo-

1,4-butanediol (926 mg). R_f= 0.52 in CH₂Cl₂/MeOH (95/5, v/v).

[α]_D²⁰−22.53° (c 1.4, CH₂Cl₂). ¹H NMR (CDCl₃) δ 7.96 (s, 1H, H₋₈),

4.43−4.35 (m, 2H, CH₂N), 4.14 (s, 3H, OCH₃), 2.93−2.90 (m, 1H,

CHO), 2.73−2.72 (m, 1H, OCH₂CH), 2.41−2.40 (m, 1H, OCH₂CH),

2.32−2.26 (m, 1H, CH₂CH₂N), 1.97−1.92 (m, 1H, CH₂CH₂N), 1.43

(s, 18H, CH₃). ¹³C NMR (CDCl₃) δ 161.6 (C₋₆), 152.8 (C₋₄), 152.1

(C₋₂), 151.1 (CO), 143.0 (CH₋₈), 120.2 (C₋₅), 83.2 (C(CH₃)₃),

54.7 (OCH₃), 49.4 (CHOH), 46.9 (OCH₂CH), 41.4 (CH₂N), 32.9

(CH₂CH₂N), 28.0 (CH₃). HRMS (TOF ESI > 0) found: 436.2200.

Calculated for C₂₀H₃₀N₅O₆: 436.2196 (M + H)⁺.

N,N-2,6-Tetra(tert-butoxycarbonyl)-N-9-(2R-(oxiran-2-yl)ethyl)-

2,6-diaminopurine (23(R)). Compound 23(R) (625 mg) was obtained

in 90% yield, according to procedure B, from 2-(R)-bromo-1,4-

20

butanediol (376 mg). R_f= 0.34 in CH₂Cl₂/MeOH (95/5, v/v). [α]_D

+20.5° (c 0.9, CH₂Cl₂). ¹H NMR (DMSO-d₆): δ = 8.72 (s, 1H, H₋₈),

4.45 (t, 2H, CH₂N), 2.93−2.96 (m, 1H, CHO), 2.56 (m, 1H, CH₂CH),

2.27 (m, 1H, OCH₂CH), 2.20−2.13 (m, 1H, CH₂CH₂N), 2.16−1.95

(m, 1H, CH₂CH₂N), 1.35 (s, 36H, CH₃). ¹³C NMR (DMSO-d₆) δ

153.9 (C₋₄), 150.7 (C₋₆), 148.0, 149.4, 149.5, 150.3 (CO), 126.3

(C₋₅), 82.8, 83.4 (C(CH₃)₃), 49.2 (CHOH), 45.1 (OCH₂CH), 41.4

(CH₂N), 31.9 (CH₂CH₂N), 27.2, 27.0 (CH₃). MS (ESI) m/z 621.3 [M

+ H]⁺; 643.3 [M + Na]⁺; 1263.6 [M + 2Na]⁺.

N,N-2,6-Tetra(tert-butoxycarbonyl)-N-9-(2S-(oxiran-2-yl)ethyl)-

2,6-diaminopurine (23(S)). Compound 23(S) (3.01 g) was obtained in

89% yield, according to procedure B, from 2-(S)-bromo-1,4-butanediol

20

(1.84 g). R_f= 0.23 in CH₂Cl₂/MeOH (95/5, v/v). [α]_D−19.8° (c

0.85, CH₂Cl₂). ¹H NMR (DMSO-d₆): δ = 8.71 (s, 1H, H₋₈), 4.44 (t,

2H, CH₂N), 2.94 (m, 1H, CHO), 2.56 (m, 1H, CH₂CH), 2.26 (m, 1H,

OCH₂CH), 2.20−2.13 (m, 1H, CH₂CH₂N), 2.04−1.97 (m, 1H,

CH₂CH₂N), 1.35 (s, 36H, CH₃). ¹³C NMR (DMSO-d₆) δ 153.9 (C₋₄),

150.7 (C₋₆), 148.0, 149.4, 149.5, 150.2 (CO), 126.3 (C₋₅), 82.8,

N,N-6-Bis(tert-butoxycarbonyl)-N-9-(2R-(oxiran-2-yl)ethyl)-

adenine (21(R)). Compound 21(R) (637 mg) was obtained in 53%

yield, according to procedure B, from 2-(R)-bromo-1,4-butanediol (1

20

g). R_f= 0.50 in CH₂Cl₂/MeOH (9/1, v/v). [α]_D= +18.52° (c 0.8,

1

CH₂Cl₂). H NMR (CDCl₃) δ 8.82 (s, 1H, H₋₂), 8.09 (s, 1H, H₋₈),

I

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83.4 (C(CH₃)₃), 49.1 (CHOH), 45.1 (OCH₂CH), 41.1 (CH₂N), 31.9

(CH₂CH₂N), 27.2, 27.0 (CH₃). MS (ESI) m/z 621.33 [M + H]⁺;

Diethyl (R)-(3-(2-(Bis(tert-butoxycarbonyl)amino)-6-methoxy-

9H-purin-9-yl)-2-hydroxypropyl)phosphonate (18(R)). Compound

18(R) (107 mg) was obtained in 41% yield, according to procedure C,

643.31 [M + Na]⁺.

20

from 15(R) (200 mg). R_f= 0.16 in CH₂Cl₂/MeOH (95/5, v/v). [α]_D

N,N-2-Bis(tert-butoxycarbonyl)-N-9-(2R-(oxiran-2-yl)ethyl)-2-

aminopurine (31(R)). Compound 31(R) (1.90 g) was obtained in 71%

yield, according to procedure B, from 2-(R)-bromo-1,4-butanediol (2.2

+8° (c 1.0, MeOH). ¹H NMR (DMSO-d₆): δ 8.30 (s, 1H, H₋₈), 5.41 (d,

1H, J = 5.1 Hz, OH), 4.39 (m, 1H, CHOH), 4.05 (m, 9H, CH₂N,

OCH₃, OCH₂CH₃), 1.98 (m, 2H, CH₂P), 1.38 (s, 18H, CH₃), 1.22

20

g). R_f= 0.40 in CH₂Cl₂/MeOH (95/5, v/v). [α]_D+20.80° (c 0.9,

1

CH₂Cl₂). H NMR (CDCl₃) δ 9.12 (s, 1H, H₋₆), 8.16 (s, 1H, H₋₈),

(6H, OCH₂CH₃). ¹³C NMR (DMSO-d₆): δ 160.5 (C₋₆), 152.8 (C₋₄),

150.3 (CO), 145.5 (CH₋₈), 118.8 (C₋₅), 82.5 (C(CH₃)₃), 72.7

(CHOH), 64.0, 61.1 (OCH₂CH₃), 54.2 (OCH₃), 27.3 (CH_{3, Boc}), 16.0,

16.1 (OCH₂CH₃). ³¹P NMR (DMSO-d₆): 28.1. MS (ESI) m/z 560.3

[M + H]⁺. HRMS: calcd C₂₃H₃₉N₅O₉P [M + H]⁺560.2485, obs

560.2481.

4.45 (m, 2H, CH₂N), 2.94 (m, 1H, CHO), 2.76 (m, 1H, OCH₂CH),

2.45 (m, 1H, OCH₂CH), 2.42−2.30 (m, 1H, CH₂CH₂N), 2.05−1.93

(m, 1H, CH₂CH₂N), 1.43 (s, 18H, CH₃). ¹³C NMR (CDCl₃) δ 149.6

(CO), 146.2 (CH₋₈), 83.3 (C(CH₃)₃), 49.3 (CHOH), 46.8

(OCH₂CH), 41.2 (CH₂N), 32.6 (CH₂CH₂N), 29.7, 27.9 (CH₃). MS

(ESI) m/z 406.21 [M + H]⁺.

Diethyl (S)-(3-(2-(Bis(tert-butoxycarbonyl)amino)-6-methoxy-

9H-purin-9-yl)-2-hydroxypropyl)phosphonate (18(S)). Compound

18(S) (1.01 g) was obtained in 43% yield, according to procedure C,

N,N-6-Bis(tert-butoxycarbonyl)-N-9-(2R-(oxiran-2-yl)ethyl)-2-ﬂu-

oroadenine (32(R)). Compound 32(R) (725 mg) was obtained in 50%

yield, according to procedure B, from 2-(R)-bromo-1,4-butanediol

(1.16 g). R_f= 0.43 in CH₂Cl₂/MeOH (9/1, v/v). [α]_D²⁰= +18.25° (c

20

from 15(S) (1.75 g). R_f= 0.16 in CH₂Cl₂/MeOH (95/5, v/v). [α]_D

−6.8° (c 0.95, MeOH). ¹H NMR (CDCl₃): δ = 8.08 (s, 1H, H₋₈), 5.23

(m, 1H, OH), 4.72 (m, 1H, CHOH), 4.40−4.20 (m, 2H, CH₂N),

4.10−3.95 (m, 7H, OCH₃, OCH₂CH₃), 2.00−1.67 (2m, 2H, CH₂P),

1

0.8, CH₂Cl₂). H NMR (CDCl₃) δ 8.07 (s, 1H, H₋₈), 4.39 (m, 2H,

CH₂N), 2.96−2.92 (m, 1H, CHO), 2.76 (m, 1H, CH₂CH), 2.47 (m,

1H, OCH₂CH), 2.42−2.27 (m, 1H, CH₂CH₂N), 1.96−1.85 (m, 1H,

CH₂CH₂N), 1.46 (s, 18H, CH₃). ¹³C NMR (CDCl₃) δ 157.7 (C₋₄),

156.0 (C₋₆), 154.1, 154.0 (C₋₂), 151.0, 151.1 (CO), 144.9 (C₋₈),

126.0, 125.9 (C₋₅), 83.3 (C(CH₃)₃), 48.2 (CHOH), 45.8 (OCH₂CH),

40.5 (CH₂N), 31.4 (CH₂CH₂N), 26.7 (CH₃). MS (ESI) m/z 424.2.

1.37 (2s, 18H, CH₃), 1.23 (m, 6H, OCH₂CH₃). ¹³C NMR (CDCl₃): δ

161.4 (C₋₆), 152.9 (C₋₄), 151.7 (C₋₂), 150.9 (CO), 144.5 (CH₋₈),

119.5 (C_−5,), 83.1 (C(CH₃)₃), 65.3 (CHOH), 62.2, 62.1 (OCH₂CH₃),

54.6 (OCH₃), 49.9, 49.7 (NCH₂), 31.5, 30.1 (PCH₂), 27.8 (CH₃),

16.3, 16.2 (OCH₂CH₃). ³¹P NMR (DMSO-d₆): 28.1. MS (ESI) m/z

560.3 [M + H]⁺; 582.23 [M + Na]⁺.

General Procedure C. Ring-Opening Reaction Leading to β-

Hydroxyphosphonates Derivatives. To a solution of diethyl phosphite

(6 equiv) in freshly distilled dichloromethane (3 mL/mmol of epoxide)

was added under argon N,O-BSA (6.4 equiv). The reaction mixture was

stirred and reﬂuxed for 4 h. Reaction progress was followed by ³¹P NMR

to check the disappearance of diethyl phosphite signal (δ ∼ 7 ppm),

while silylated diethyl phosphite appeared (δ ∼ 127 ppm). The reaction

mixture was allowed to reach to room temperature and then cooled to

−60 °C. A solution of the epoxide (1 equiv) in freshly distilled

dichloromethane (6.6 mL/mmol of epoxide) was added dropwise.

Then, boron triﬂuoride diethyl etherate (6 equiv) was added dropwise,

and the reaction mixture was stirred for 3 h and then allowed to warm to

room temperature overnight. Volatiles were removed under reduced

pressure; the residue was dissolved in ethyl acetate and washed with

water. The organic layer was dried over magnesium sulfate and

concentrated under reduced pressure. Puriﬁcation by ﬂash chromatog-

raphy on silica gel using CH₂Cl₂/MeOH (0 to 10%) gave rise to the

desired product as a white solid.

Diethyl (R)-(3-(2,6-(Tetra(tert-butoxycarbonyl)diamino)-9H-

purin-9-yl)-2-hydroxypropyl)phosphonate (19(R)). Compound

19(R) (482 mg) was obtained in 36% yield, according to procedure

C, from 16(R) (1.08 g). R_f= 0.18 in CH₂Cl₂/MeOH (95/5, v/v).

[α]_D²⁰+11.8° (c 1.2, MeOH). ¹H NMR (DMSO-d₆): δ = 8.56 (s, 1H,

H₋₈), 5.51 (1H, OH), 4.47 (m, 1H, CHOH), 4.21 (m, 2H, CH₂N),

3.99 (m, 4H, OCH₂CH₃), 2.00 (m, 2H, CH₂P), 1.36 (s, 36H, CH₃),

1.22 (pt, 6H, OCH₂CH₃). ¹³C NMR (DMSO-d₆): δ 154.0 (C₋₄), 150.7

(C₋₂), 150.2, 149.6, 149.3, 148.5 (C₋₆, CO), 126.1 (C_−5,), 83.4, 82.7

(C(CH₃)₃), 64.0 (CHOH), 61.1, 60.9 (OCH₂CH₃), 32.1 (CH₂N),

30.3 (CH₂P), 27.0, 27.2 (CH₃), 16.0, 16.1 (OCH₂CH₃). ³¹P NMR

(DMSO-d₆): 27.9. MS (ESI) m/z 745.3 [M + H]⁺. HRMS: calcd

C₃₂H₅₄N₆O₁₂P [M + H]⁺745.3537, obs 745.3543.

Diethyl (S)-(3-(2,6-(Tetra(tert-butoxycarbonyl)diamino)-9H-

purin-9-yl)-2-hydroxypropyl)phosphonate (19(S)). Compound

19(S) (1.76 g) was obtained in 66% yield, according to procedure C,

20

from 16(S) (2.18 g). R_f= 0.21 in CH₂Cl₂/MeOH (95/5, v/v). [α]_D

−8° (c 1.0, MeOH). ¹H NMR (DMSO-d₆): δ = 8.56 (s, 1H, H₋₈), 5.52

(d, 1H, J = 5.1 Hz, OH), 4.47 (m, 1H, CHOH), 4.20 (m, 2H, CH₂N),

3.99 (m, 4H, OCH₂CH₃), 2.00 (m, 2H, CH₂P), 1.36 (s, 36H, CH₃),

Diethyl (R)-(3-(6-(Bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)-

2-hydroxypropyl)phosphonate (17(R)). Compound 17(R) (1.37 g)

was obtained in 62% yield, according to procedure C, from 14(R) (1.62

20

g). R_f= 0.42 in CH₂Cl₂/MeOH (90/10, v/v). [α]_D+7.8° (c 1.02,

1.23 (pt, 6H, OCH₂CH₃). ¹³C NMR (DMSO-d₆): δ 154.0 (C₋₄), 150.7

(C₋₂), 150.2, 149.6, 149.3, 148.5 (C₋₆, CO), 126.1 (C₋₅), 83.4, 82.7

(C(CH₃)₃), 63.9 (CHOH), 61.1, 60.9 (OCH₂CH₃), 32.1 (CH₂N),

30.3 (CH₂P), 27.1, 27.2 (CH₃), 16.0, 16.1 (OCH₂CH₃). ³¹P NMR

(DMSO-d₆): 27.9. MS (ESI) m/z 745.3 [M + H]⁺. HRMS: calcd

C₃₂H₅₄N₆O₁₂P [M + H]⁺745.3537, obs 745.3531.

1

MeOH). H NMR (DMSO-d₆): δ = 8.83 (s, 1H, H₋₂), 8.52 (s, 1H,

H₋₈), 5.50 (d, 1H, OH), 4.46 (m, 1H, CHOH), 4.24 (m, 2H, CH₂N),

3.90−4.04 (m, 4H, OCH₂CH₃), 1.84−2.08 (m, 2H, CH₂P), 1.38 (s,

18H, CH₃), 1.22 (t, 6H, J = 6.9 Hz, 3H, OCH₂CH₃). ¹³C NMR

(CDCl₃) δ 153.7 (C₋₄), 152.0 (C₋₂), 150.6 (C₋₆), 150.4 (CO),

146.4 (C₋₈), 128.7 (C₋₅), 84.0 (C(CH₃)₃), 65.5 (CHOH), 62.5, 62.4

(OCH₂CH₃), 49.9, 49.7 (CH₂N), 31.6, 30.2 (CH₂P), 27.9 (CH₃),

16.5, 16.4 (OCH₂CH₃). ³¹P NMR (DMSO-d₆): δ = 28.1. MS (ESI) m/

z 530.3 [M + H]⁺. HRMS: calcd C₂₂H₃₇N₅O₈P [M + H]⁺530.2380, obs

530.2391.

Diethyl (R)-(4-(6-(Bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)-

2-hydroxybutyl)phosphonate (24(R)). Compound 24(R) (886 mg)

was obtained in 52% yield, according to procedure C, from 21(R) (1.28

20

g). R_f= 0.5 in CH₂Cl₂/MeOH (9/1, v/v). [α]_D+17.05° (c 0.09,

1

CH₂Cl₂). H NMR(CDCl₃) δ 8.85 (s, 1H, H₋₂), 8.13 (s, 1H, H₋₈),

4.54−4.44 (m, 2H, CH₂N), 4.09 (m, 4H, OCH₂CH₃), 3.95−3.84 (m,

1H, CHOH), 2.18, 2.01 (2m, 2H, CH₂CH₂N), 1.93 (m, 2H, CH₂P),

1.46 (s, 18H, CH₃), 1.33−1.28 (m, 6H, OCH₂CH₃). ¹³C NMR

(CDCl₃) δ 153.6 (C₋₄), 152.1 (C₋₂), 150.7 (C₋₆), 150.5 (CO),

145.5 (C₋₈), 129.0 (C₋₅), 83.9 (C(CH₃)₃), 63.4 (CHOH), 62.2

(OCH₂CH₃), 40.9 (CH₂N), 38.0−37.8 (d, J = 17.5 Hz, CH₂CH₂N),

34.1, 33.0 (CH₂P), 28.0 (CH₃), 16.6, 16.5 (OCH₂CH₃). ³¹P NMR

(CDCl₃) δ 29.2 (s). HRMS (TOF ESI > 0) found: 544.2534.

Calculated for C₂₃H₃₉N₅O₈P: 544.2536 (M + H)⁺.

Diethyl (S)-(4-(6-(Bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)-

2-hydroxybutyl)phosphonate (24(S)). Compound 24(S) (746 mg)

was obtained in 54% yield, according to procedure C, from 21(S) (1 g).

R_f= 0.5 in CH₂Cl₂/MeOH (9/1, v/v). [α]_D²⁰−19.3° (c 0.3, CH₂Cl₂).

Diethyl (S)-(3-(6-(Bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)-

2-hydroxypropyl)phosphonate (17(S)). Compound 17(S) (0.95 g)

was obtained in 64% yield, according to procedure C, from 14(S) (1.14

20

g). R_f= 0.42 in CH₂Cl₂/MeOH (90/10, v/v). [α]_D−6.9° (c 1.01,

1

MeOH). H NMR (DMSO-d₆): δ = 8.83 (s, 1H, H₋₂), 8.51 (s, 1H,

H₋₈), 5.48 (d, 1H, OH), 4.46 (m, 1H, CHOH), 4.24 (m, 2H, CH₂N),

4.01 (m, 4H, OCH₂CH₃), 1.85−2.24 (m, 2H, CH₂P), 1.39 (s, 18H,

CH₃), 1.23 (m, 6H, OCH₂CH₃). ¹³C NMR (CDCl₃) δ 153.8 (C₋₄),

152.1 (C₋₂), 150.7 (C₋₆), 150.6 (CO), 146.5 (C₋₈), 128.8 (C₋₅),

87.0 (C(CH₃)₃), 65.6 (CHOH), 62.6, 62.5 (OCH₂CH₃), 50.0, 49.8

(CH₂N), 31.7, 30.3 (CH₂P), 28.0 (CH₃), 16.7, 16.6 (OCH₂CH₃). ³¹

P

NMR (DMSO-d₆): δ = 28.1. MS (ESI) m/z 530.3 [M + H]⁺. HRMS:

calcd C₂₂H₃₇N₅O₈P [M + H]⁺530.2380, obs 530.2375.

J

https://dx.doi.org/10.1021/acs.jmedchem.0c00131

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

¹H NMR(CDCl₃) δ 8.85 (s, 1H, H₋₂), 8.13 (s, 1H, H₋₈), 4.55−4.43

(m, 2H, CH₂N), 4.20 (s, 1H, OH), 4.15−4.05 (m, 4H, CH₂CH₃),

3.96−3.85 (m, 1H, CHOH), 2.22−1.97 (2m, 2H, CH₂CH₂N), 1.97−

1.90 (m, 2H, CH₂P), 1.46 (s, 18H, CH₃), 1.31 (6H, CH₃CH₂). ¹³C

NMR (CDCl₃) δ 153.6 (C₋₄), 152.1 (C₋₂), 150.7 (C₋₆, CO), 145.5

(C₋₈), 129.0 (C₋₅), 83.9 (C(CH₃)₃), 63.4 (CHOH), 62.2

(OCH₂CH₃), 40.9 (CH₂N), 37.9 (CH₂CH₂N), 34.1, 33.0 (CH₂P),

28.0 (CH₃), 16.5, 16.6 (CH₃CH₂). ³¹P NMR (CDCl₃) δ 29.2 (s).

HRMSTOF ESI+ found: 544.2540. Calculated for C₂₃H₃₉N₅O₈P:

544.2536 (M + H)⁺.

(CDCl₃) δ 153.1 (C₋₄), 151.4 (C₋₂), 149.8 (CO), 147.2 (C₋₈), 83.6

(C(CH₃)₃), 63.4 (CHOH), 62.4 (OCH₂CH₃), 40.9 (CH₂N), 37.9−

37.8 (CH₂CH₂N), 34.5, 33.1 (CH₂P), 28.2 (CH₃), 16.7, 16.6

(OCH₂CH₃). ³¹P NMR (CDCl₃) δ 29.0 (s). MS (ESI) m/z 566.24

[M + Na]⁺.

Diethyl (R)-(4-(6-(Bis(tert-butoxycarbonyl)amino)-9H-2-ﬂuoro-

purin-9-yl)-2-hydroxybutyl)phosphonate (34(R)). Compound

34(R) (830 mg) was obtained in 89% yield, according to procedure

C, from 32(R) (703 mg). R_f= 0.25 in CH₂Cl₂/MeOH (95/5, v/v). ¹H

NMR(CDCl₃) δ 8.35 (s, 1H, H₋₈), 4.45 (m, 2H, CH₂N), 4.12−4.05

(m, 4H, OCH₂CH₃), 3.95 (m, 1H, CHOH), 2.20−1.90 (2m, 4H,

Diethyl (R)-(4-(2-(Bis(tert-butoxycarbonyl)amino)-6-methoxy-

9H-purin-9-yl)-2-hydroxybutyl)phosphonate (25(R)). Compound

25(R) (761 mg) was obtained in 37% yield, according to procedure

CH₂CH₂N, CH₂P), 1.49 (s, 18H, CH₃), 1.30 (m, 6H, OCH₂CH₃). ¹³

C

NMR (CDCl₃) δ 155.8 (C₋₆), 154.1 (C₋₄), 150.9 (C₋₂), 149.0 (C

O), 144.8 (C₋₈), 83.4 (C(CH₃)₃), 62.4 (CHOH), 61.2 (OCH₂CH₃),

40.1 (CH₂N), 36.4, 36.3 (CH₂CH₂N), 33.2, 31.8 (CH₂P), 26.8 (CH₃),

15.4, 15.3 (OCH₂CH₃). ³¹P NMR (CDCl₃) δ 29.1 (s). MS (ESI) m/z

562.25 [M + H]⁺.

General Procedure D. Removal of Boc Protecting Groups. To a

stirred solution of β-hydroxyphosphonate (1 equiv) in anhydrous

dichloromethane (14 mL/mmol) under argon and at room temper-

ature was added dropwise a solution of triﬂuoroacetic acid in anhydrous

dichloromethane (19 mL/mmol, 3/7, v/v). The reaction mixture was

stirred at room temperature for 5 h. Volatiles were removed under

reduced pressure, and the crude was puriﬁed by ﬂash chromatography

on silica gel using CH₂Cl₂/MeOH (0 to 10%). The desired compound

was obtained as a white foam.

20

C, from 22(R) (1.57 g). R_f= 0.60 in CH₂Cl₂/MeOH (9/1, v/v). [α]_D

+15.24° (c 0.1, CH₂Cl₂). ¹H NMR(CDCl₃) δ 7.97 (s, 1H, H₋₈), 4.50−

4.29 (2m, 2H, CH₂N), 4.14 (s, 3H, OCH₃), 4.13−4.00 (m, 4H,

OCH₂CH₃), 3.83 (m, 1H, CHOH), 2.17−1.88 (2m, 4H, CH₂CH₂N,

CH₂P), 1.46 (s, 18H, CH₃), 1.29 (t, J = 7.1 Hz, 6H, OCH₂CH₃). ¹³

C

NMR (CDCl₃) δ 161.6 (C₋₆), 153.0 (C₋₄), 152.0 (C₋₂), 151.1 (C

O), 143.5 (CH₋₈), 120.0 (C₋₅), 83.3 (C(CH₃)₃), 63.1 (CHOH), 62.2

(OCH₂CH₃), 54.7 (OCH₃), 40.8 (CH₂N), 38.2, 38.1 (CH₂CH₂N),

34.3, 33.2 (CH₂P), 28.1 (CH₃), 16.8 (OCH₂CH₃). ³¹P NMR (CDCl₃)

δ 29 (s). HRMS (TOF ESI > 0) found: 574.2642. Calculated for

C₂₄H₄₁N₅O₉P: 574.2642 (M + H)⁺.

Diethyl (S)-(4-(2-(Bis(tert-butoxycarbonyl)amino)-6-methoxy-

9H-purin-9-yl)-2-hydroxybutyl)phosphonate (25(S)). Compound

25(S) (742 mg) was obtained in 45% yield, according to procedure

Diethyl (R)-(4-(6-Amino-9H-purin-9-yl)-2-hydroxybutyl)-

phosphonate (27(R)). The compound 27(R) (530 mg) was obtained

in quantitative yield, according to procedure D, from 24(R) (840 mg).

R_f= 0.42 in CH₂Cl₂/MeOH (90/10, v/v). [α]_D²⁰+15° (c 0.2, CH₂Cl₂).

¹H NMR(CDCl₃) δ 8.31 (s, 1H, H₋₂), 7.91 (s, 1H, H₋₈), 6.87 (s, 2H,

20

C, from 22(S) (1.25 g). R_f= 0.53 in CH₂Cl₂/MeOH (9/1, v/v). [α]_D

−18.2° (c 0.1, CH₂Cl₂). ¹H NMR (CDCl₃) δ 7.97 (s, 1H, H₋₈), 4.50−

4.30 (2m, 2H, CH₂N), 4.15 (s, 1H, OCH₃), 4.13−4.03 (m, 4H,

OCH₂CH₃), 3.88−3.78 (m, 1H, CHOH), 2.18−1.85 (2m, 4H,

CH₂CH₂N, CH₂P), 1.46 (s, 18H, CH₃), 1.29 (t, 6H, OCH₂CH₃).

¹³C NMR (CDCl₃) δ 161.6 (C₋₆), 153.0 (C₋₄), 152.0 (C₋₂), 151.1

(CO), 143.5 (CH₋₈), 120.0 (C₋₅) 83.3 (C(CH₃)₃), 63.1 (CHOH),

62.2, 62.1 (OCH₂CH₃), 54.7 (OCH₃), 40.8 (CH₂N), 38.2, 38.1

NH₂), 4.50−4.32 (2m, 2H, CH₂N), 4.13−4.03 (m, 4H, CH₂CH₃),

3.90−3.85 (m, 1H, CHOH), 2.18−2.12 (m, 1H, CH₂CH₂N), 2.02−

1.88 (m, 3H, CH₂CH₂N, CH₂P), 1.31−1.27 (m, 6H, CH₃CH₂). ¹³C

NMR (CDCl₃) δ 154.5 (C₋₆), 150.3 (C₋₂), 149.9 (C₋₄), 142.0

(CH₋₈), 119.5 (C₋₅), 63.2 (CHOH), 62.3−62.1 (CH₂CH₃), 40.9

(CH₂N), 38.2, 38.1 (CH₂CH₂N), 34.3, 33.0 (CH₂P), 16.5 (CH₃CH₂).

³¹P NMR (CDCl₃) δ 28.7 (s). HRMS (TOF ESI > 0) found: 344.1487.

(CH₂CH₂N), 34.3, 33.2 (CH₂P), 28.0 (CH₃), 16.5 (OCH₂CH₃). ³¹

P

NMR(CDCl₃) δ 29 (s). HRMS (TOF ESI > 0) found: 574.2640.

Calculated for C₂₄H₄₁N₅O₉P: (M + H)⁺.

Calculated for C₁₃H₂₃N₅O₄P: 344.1488 (M + H)⁺.

Diethyl (R)-(4-(2,6-(Tetra(tert-butoxycarbonyl)diamino)-9H-

purin-9-yl)-2-hydroxybutyl)phosphonate (26(R)). Compound

26(R) (2.04 g) was obtained in 70% yield, according to procedure C,

Diethyl (S)-(4-(6-Amino-9H-purin-9-yl)-2-hydroxybutyl)-

phosphonate (27(S)). The compound 27(S) (127 mg) was obtained

in quantitative yield, according to procedure D, from 24(S) (200 mg).

1

from 23(R) (2.38 g). R_f= 0.42 in CH₂Cl₂/MeOH (90/10, v/v). H

20

R_f= 0.39 in CH₂Cl₂/MeOH (90/10, v/v). [α]_D−16.2° (c 0.1,

NMR (DMSO-d₆): δ = 8.67 (s, 1H, H₋₈), 5.11 (d, 1H, OH), 4.36 (m,

1H, CHOH), 3.96−3.75 (2m, 6H, CH₂N, OCH₂CH₃), 2.10−1.90 (m,

1

CH₂Cl₂). H NMR (CDCl₃) δ 8.31 (s, 1H, H₋₂), 7.91 (s, 1H, H₋₈),

2H, CH₂P), 1.37 (2s, 36H, CH₃), 1.18 (6H, OCH₂CH₃). ¹³C NMR

(DMSO-d₆) δ 153.8 (C₋₄), 150.7 (C₋₂), 150.3, 149.6, 149.4 (CO),

147.9 (CH₋₈), 126.2 (C₋₅), 83.3, 82.8 (C(CH₃)₃), 63.1 (CHOH),

60.9, 60.8 (OCH₂CH₃), 34.4 (CH₂N), 32.6 (CH₂P), 27.2, 27.0 (CH₃),

16.1, 16.0 (OCH₂CH₃). ³¹P NMR (DMSO-d₆): 28.4. MS (ESI) m/z

759.37 [M + H]⁺. HRMS: calcd C₃₃H₅₆N₆O₁₂P [M + H]⁺759.3694,

obs 759.3687.

6.87 (s, 2H, NH₂), 4.51−4.31 (2m, 2H, CH₂N), 4.15−4.00 (m, 4H,

CH₂CH₃), 3.96−3.83 (m, 1H, CHOH), 2.20−1.98 (m, 1H,

CH₂CH₂N), 1.96−1.89 (m, 2H, CH₂P), 1.29 (m, CH₃CH₂). ¹³C

NMR (CDCl₃) δ 154.5 (C₋₆), 150.3 (C₋₂), 149.9 (C₋₄), 142.0

(CH₋₈), 119.5 (C₋₅), 63.2 (CHOH), 62.3, 62.2 (CH₂CH₃), 40.9

(CH₂N), 38.2 (CH₂CH₂N), 34.3, 33.0 (CH₂P), 16.5 (CH₃CH₂). ³¹

P

NMR (CDCl₃) δ 28.7 (s). HRMS (TOF ESI > 0) found: 344.1490.

Calculated for C₁₃H₂₃N₅O₄P: 344.1488 (M + H)⁺.

Diethyl (S)-(4-(2,6-(Tetra(tert-butoxycarbonyl)diamino)-9H-

purin-9-yl)-2-hydroxybutyl)phosphonate (26(S)). Compound 26(S)

(1.57 g) was obtained in 70% yield, according to procedure C, from

Diethyl (R)-(4-(2-Amino-6-methoxy-9H-purin-9-yl)-2-

hydroxybutyl)phosphonate (28(R)). Compound 28(R) (470 mg)

was obtained in quantitative yield, according to procedure D, from

1

23(S) (1.83 g). R_f= 0.24 in CH₂Cl₂/MeOH (95/5, v/v). H NMR

20

(DMSO-d₆): δ = 8.67 (s, 1H, H₋₈), 5.11 (d, 1H, OH), 4.36 (m, 1H,

CHOH), 4.0−3.80 (2m, 6H, CH₂N, OCH₂CH₃), 2.20−1.85 (2m, 4H,

OCH₂CH₃, CH₂P), 1.36 (2s, 36H, CH₃), 1.18 (m, 6H, OCH₂CH₃).

¹³C NMR (DMSO-d₆) δ 153.8 (C₋₄), 150.7 (C₋₂), 150.3, 149.6, 149.4

(CO), 147.9 (CH₋₈), 126.2 (C₋₅), 83.3, 82.8 (C(CH₃)₃), 63.1

(CHOH), 60.9, 60.8 (OCH₂CH₃), 34.4 (CH₂N), 32.6 (CH₂P), 27.2,

27.0 (CH₃), 16.1, 16.0 (OCH₂CH₃). ³¹P NMR (DMSO-d₆): 28.4. MS

(ESI) m/z 759.37 [M + H]⁺.

25(R) (720 mg). R_f= 0.50 in CH₂Cl₂/MeOH (90/10, v/v). [α]_D

+20.95° (c 0.5, CH₂Cl₂). ¹H NMR(CDCl₃) δ 7.81 (s, 1H, H₋₈), 4.43−

4.26 (m, 2H, CH₂N), 4.13 (s, 3H, OCH₃), 4.12−4.05 (m, 4H,

CH₂CH₃), 4.0−3.92 (m, 1H, CHOH), 2.14−1.88 (m, 4H, CH₂CH₂N,

CH₂P), 1.30 (m, 6H, CH₃CH₂). ¹³C NMR (CDCl₃) δ 162.6 (C₋₆),

157.8 (C₋₂), 149.3 (C₋₄), 140.2 (CH₋₈), 114.7 (C₋₅), 63.4 (CHOH),

62.4, 62.3 (CH₂CH₃), 55.1 (OCH₃), 41.4 (CH₂N), 38.1, 38.0

(CH₂CH₂N), 33.9, 32.8 (CH₂P), 16.5 (CH₃CH₂). ³¹P NMR(CDCl₃)

Diethyl (R)-(4-(2-(Bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)-

2-hydroxybutyl)phosphonate (33(R)). Compound 33(R) (598 mg)

was obtained in 73% yield, according to procedure C, from 31(R) (609

δ 29.1 (s). HRMS (TOF ESI > 0) found: 374.1589. Calculated for

C₁₄H₂₅N₅OP: 374.1593 (M + H)⁺.

Diethyl (S)-(4-(2-Amino-6-methoxy-9H-purin-9-yl)-2-

hydroxybutyl)phosphonate (28(S)). Compound 28(S) (280 mg)

was obtained in quantitative yield, according to procedure D, from

1

mg). R_f= 0.25 in CH₂Cl₂/MeOH (95/5, v/v). H NMR(CDCl₃) δ

9.10 (s, 1H, H₋₆), 8.23 (s, 1H, H₋₈), 4.46 (m, 2H, CH₂N), 4.09 (m, 4H,

OCH₂CH₃), 3.86 (m, 1H, CHOH), 2.17−1.85 (2m, 4H, CH₂CH₂N,

CH₂P), 1.45 (s, 18H, CH₃), 1.29 (m, 6H, OCH₂CH₃). ¹³C NMR

20

25(R) (430 mg). R_f= 0.50 in CH₂Cl₂/MeOH (90/10, v/v). [α]_D

−17.62° (c 1.0, CH₂Cl₂). ¹H NMR (CDCl₃) δ 7.81 (s, 1H, H₋₈), 4.42−

K

https://dx.doi.org/10.1021/acs.jmedchem.0c00131

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

4.25 (m, 2H, CH₂N), 4.13 (s, 3H, OCH₃), 4.12−4.05 (m, 4H,

CH₂CH₃), 4.00−3.92 (m, 1H, CHOH), 2.13−1.92 (m, 4H,

CH₂CH₂N, CH₂P), 1.31 (m, 6H, CH₃CH₂). ¹³C NMR (CDCl₃) δ

162.6 (C₋₆), 157.8 (C₋₂), 149.3 (C₋₄), 140.2 (CH₋₈), 114.7 (C₋₅),

63.4 (CHOH), 62.4, 62.3 (CH₂CH₃), 55.1 (OCH₃), 41.4 (CH₂N),

General Procedure F. Removal of the Diethyl Phosphonate

Groups. To a stirred solution of diethyl-β-hydroxyphosphonate (1

equiv) in anhydrous acetonitrile (20 mL/mmol), cooled at 0 °C, was

added dropwise bromotrimethylsilane (6.6 equiv). The reaction

mixture was stirred at room temperature until completion of the

reaction was indicated by TLC monitoring (isopropanol/water/

ammoniac 7/2/1 v/v/v). When in situ removal of Boc groups was

required, water was added to the reaction mixture and stirring was

pursued 1 h at rt. Then, triethylammonium bicarbonate solution

(TEAB 1M) was added to the reaction mixture until pH = 7, and the

volatiles were evaporated under high vacuum and the aqueous residue

was freeze-dried. The resulting lyophilizate was puriﬁed by ﬂash

chromatography silica gel on C18 reverse phase using water/MeOH

gradient (0 to 100%) and freeze-dried, giving rise to the desired

phosphonic acid. Eventually, the latter was dissolved in a small amount

of water and percolated through a Dowex resin column (Na⁺form) to

aﬀord after freeze-drying the corresponding compound as sodium salt.

(R)-(3-(6-Amino-9H-purin-9-yl)-2-hydroxypropyl)phosphonic

Acid (1(R)). Compound 1(R) (216 mg) was obtained in 45% yield,

according to procedure F, from 17(R) (805 mg). R_f= 0.66 in iPrOH/

38.1, 38.0 (CH₂CH₂N), 33.9, 32.8 (CH₂P), 16.5, 16.4 (CH₃CH₂). ³¹

P

NMR (CDCl₃) δ 29.2 (s). HRMS (TOF ESI > 0) found: 374.1597.

Calculated for C₁₄H₂₅N₅O₅P: 374.1593 (M + H)⁺.

General Procedure E. Preparation of Oxopurines β-Hydrox-

yphosphonate Derivatives. A solution of β-hydroxyphosphonate (1

equiv) in acetic acid 1 M (15 mL/mmol) was heated at 65 °C. Sodium

nitrite (15 equiv) dissolved in water (2.22 mL/mmol of NaNO₂) was

added portionwise over a 30 min period. The reaction mixture was

stirred for 2 h at 65 °C, then cooled to 0 °C and quenched by addition of

aqueous sodium 1 M until pH = 7. Volatiles were removed under

reduced pressure and the crude was puriﬁed by ﬂash chromatography

silica gel on C18 reverse phase using water/MeOH gradient (0 to 100%

MeOH) to aﬀord the product as a white solid after freeze-drying.

Diethyl (R)-(2-Hydroxy-4-(6-oxo-1,6-dihydro-9H-purin-9-yl)-

butyl)phosphonate (29(R)). Compound 29(R) (156 mg) was

obtained in 66% yield, according to procedure E, from 27(R) (236

mg). R_f= 0.30 in CH₂Cl₂/MeOH (90/10, v/v). [α]_D²⁰+14.77° (c 1.2,

1

H₂O/NH₄OHc (7/4/2, v/v/v). Purity (HPLC) > 96%. H NMR

(D₂O): δ = 8.00 (s, 1H, H₋₂), 7.96 (s, 1H, H₋₈), 4.38−4.21 (m, 2H,

CH₂N), 4.16−4.05 (m, 1H, CHOH), 1.95−1.83 (m, 2H, CH₂P). ¹³

C

1

CH₂Cl₂). H NMR (CDCl₃) δ 8.14 (s, 1H, H₋₂), 7.91 (s, 1H, H₋₈),

NMR (D₂O) δ 154.8 (C₋₆), 151.7 (CH₋₂), 148.4 (C₋₄), 142.7

(CH₋₈), 117.7 (C₋₅), 66.2 (CHOH), 49.9 (CH₂N), 34.1, 32.4

(CH₂P). ³¹P NMR (D₂O): δ = 19.8. MS (ESI) m/z 272.06 [M − H]⁻.

HRMS: calcd C₈H₁₁N₅O₄P [M − H]⁻272.0549, obs 272.0551.

(S)-(3-(6-Amino-9H-purin-9-yl)-2-hydroxypropyl)phosphonic

Acid (1(S)). Compound 1(S) (256 mg) was obtained in 47% yield,

according to procedure F, from 17(S) (898 mg). R_f= 0.54 in iPrOH/

4.36 (m, 2H, CH₂N), 4.11−4.08 (m, 4H, CH₂CH₃), 3.94 (m, 1H,

CHOH), 2.18−1.93 (2m, 4H, CH₂CH₂N, CH₂P), 1.30 (m, 6H,

CH₃CH₂). ¹³C NMR (CDCl₃) δ 159.2 (C₋₆), 149.2 (C₋₄), 145.4,

145.3 (CH₋₂) 140.9 (CH₋₈), 124.7 (C₋₅), 63.2 (CHOH), 62.3−62.1

(CH₂CH₃), 40.9 (CH₂N), 38.4, 38.3 (CH₂CH₂N), 34.3, 33.2 (CH₂P),

16.5 (CH₃CH₂). ³¹P NMR (D₂O) δ 29.4 (s). HRMS (TOF ESI > 0)

found: 345.1326. Calculated for C₁₃H₂₂N₄O₅P: 345.1328 (M + H)⁺.

Diethyl (S)-(2-Hydroxy-4-(6-oxo-1,6-dihydro-9H-purin-9-yl)-

butyl)phosphonate (29(S)). Compound 29(S) (250 mg) was obtained

in 83% yield, according to procedure E, from 27(S) (300 mg). R_f= 0.32

in CH₂Cl₂/MeOH (90/10, v/v). [α]_D²⁰−16.53° (c 0.5, CH₂Cl₂). ¹H

NMR (D₂O) δ 8.22 (s, 1H, H₋₂), 8.14 (s, 1H, H₋₈), 4.43−4.40 (m, 2H,

CH₂N), 4.09−4.0 (m, 4H, CH₂CH₃), 3.86−3.80 (m, 1H, CHOH),

1

H₂O/NH₄OHc (7/4/2, v/v/v). Purity (HPLC) > 99%. H NMR

(D₂O): δ = 8.02 (s, 1H, H₋₂), 8.00 (s, 1H, H₋₈), 4.40−4.21 (m, 2H,

CH₂N), 4.17−4.06 (m, 1H, CHOH), 1.97−1.77 (2m, 2H, CH₂P). ¹³

C

NMR (D₂O) δ 155.0 (C₋₆), 151.9 (CH₋₂), 148.6 (C₋₄), 142.8

(CH₋₈), 117.8 (C₋₅), 66.3 (CHOH), 50.0, 49.8 (CH₂N), 34.1, 32.4

(CH₂P). ³¹P NMR (D₂O): δ = 19.7. MS (ESI) m/z 318.04 [M + H]⁺;

296.05 [M − Na + 2H]⁺; 274.07 [M − 2Na + 3H]⁺. HRMS: calcd

C₈H₁₁N₅O₄Na₂P [M + H]⁺318.0344, obs 318.0349.

2.27−2.23 (m, 1H, CH₂CH₂N), 2.20−2.14 (m, 2H, CH₂P), 2.08−2.00

(m, 1H, CH₂CH₂N), 1.23 (m, 6H, CH₃CH₂). ¹³C NMR (D₂O) δ 146.1

(CH₋₂), 142.3 (CH₋₈), 63.4 (CHOH), 63.3, 63.2 (CH₂CH₃), 41.0

(CH₂N), 37.0, 36.9 (CH₂CH₂N), 33.2, 31.8 (CH₂P), 15.5, 15.4

(CH₃CH₂). ³¹P NMR(D₂O) δ 31 (s). HRMS (TOF ESI > 0) found:

(R)-(3-(2-Amino-6-oxo-9H-purin-9-yl)-2-hydroxypropyl)-

phosphonic Acid (2(R)). Compound 2(R) (370 mg) was obtained in

68% yield, according to procedure F, from 18(R) (878 mg). R_f= 0.44 in

1

iPrOH/H₂O/NH₄OHc (7/4/2, v/v/v). Purity (HPLC) > 99%. H

345.1329. Calculated for C₁₃H₂₂N₄O₅P: 345.1328 (M + H)⁺.

NMR (D₂O): δ = 7.76 (s, 1H, H₋₈), 4.33−4.17 (m, 2H, CH₂N), 4.05−

3.93 (m, 1H, CHOH), 2.00−1.80 (m, 2H, CH₂P). ¹³C NMR (D₂O) δ

158.8 (C₋₆), 153.6 (C₋₄), 140.6 (CH₋₈), 115.7 (C₋₅), 66.2 (CHOH),

49.7, 49.6 (CH₂N), 33.9, 32.6 (CH₂P). ³¹P NMR (D₂O): δ = 20.1. MS

(ESI) m/z 288.05 [M − H]⁻, 577.11 [2M − H]⁻. HRMS: calcd

C₈H₁₁N₅O₅P [M − H]⁻288.0498, obs 288.0498.

Diethyl (R)-(2-Hydroxy-4-(6-methoxy-2-oxo-2,3-dihydro-9H-

purin-9-yl)butyl)phosphonate (30(R)). Compound 30(R) (231 mg)

was obtained in 49% yield, according to procedure E, from 28(R) (469

mg). R_f= 0.40 in CH₂Cl₂/MeOH (90/10, v/v). [α]_D²⁰+19.6° (c 0.6,

1

CH₂Cl₂). H NMR (D₂O) δ 7.79 (s, 1H, H₋₈), 4.24−4.20 (m, 2H,

CH₂N), 4.04 (s, 3H, OCH₃), 4.06−3.97 (m, 4H, CH₂CH₃), 3.84−3.66

(m, 1H, CHOH), 2.24−2.17 (m, 1H, CH₂CH₂N), 2.17−2.11 (m, 2H,

CH₂P), 1.98−1.91 (m, 1H, CH₂CH₂N), 1.21−1.17 (m, 6H, CH₃CH₂).

¹³C NMR (D₂O) δ 166.8 (C₋₆), 162.0 (C₋₂), 154.3 (C₋₄), 140.1

(CH₋₈), 112.4 (C₋₅), 63.2−63.1 (CH₂CH₃, CHOH), 53.8 (OCH₃),

39.8 (CH₂N), 37.0, 36.9 (CH₂CH₂N), 32.9, 31.8 (CH₂P), 15.4, 15.3

(CH₃CH₂). ³¹P NMR (D₂O) δ 31.2 (s). HRMS (TOF ESI > 0) found:

(S)-(3-(2-Amino-6-oxo-9H-purin-9-yl)-2-hydroxypropyl)-

phosphonic Acid (2(S)). Compound 2(S) (176 mg) was obtained in

70% yield, according to procedure F, from 18(S) (421 mg). R_f= 0.47 in

1

iPrOH/H₂O/NH₄OHc (7/4/2, v/v/v). Purity (HPLC) > 96%. H

NMR (D₂O): δ = 7.76 (s, 1H, H₋₈), 4.32−4.19 (m, 2H, CH₂N), 4.05−

3.93 (m, 1H, CHOH), 1.97−1.78 (m, 2H, CH₂P). ¹³C NMR (D₂O) δ

158.8 (C₋₆), 153.5 (C₋₄), 151.6 (C₋₂), 140.6 (CH₋₈), 115.6 (C₋₅),

66.2 (CHOH), 49.7, 49.5 (CH₂N), 34.1, 32.3 (CH₂P). ³¹P NMR

(D₂O): δ = 19.9. MS (ESI) m/z 290.07 [M − 2Na + 3H]⁺, 312.05 [M −

Na + 2H]⁺; 334.03 [M + H]⁺. HRMS: calcd C₈H₁₁N₅O₅Na₂P [M +

H]⁺334.0293, obs 334.0302.

375.1431. Calculated for C₁₄H₂₄N₄O₆P: 375.1433 (M + H)⁺.

Diethyl (S)-(2-Hydroxy-4-(6-methoxy-2-oxo-2,3-dihydro-9H-

purin-9-yl)butyl)phosphonate (30(S)). Compound 30(S) (70 mg)

was obtained in 53% yield, according to procedure E, from 28(S) (130

mg). R_f= 0.40 in CH₂Cl₂/MeOH (90/10, v/v). [α]_D²⁰−20.20° (c 1.1,

(R)-(3-(2,6-Diamino-9H-purin-9-yl)-2-hydroxypropyl)phosphonic

Acid (3(R)). Compound 3(R) (206 mg) was obtained in 64% yield,

according to procedure F, from 19(R) (719 mg). R_f= 0.54 in iPrOH/

1

CH₂Cl₂). H NMR (D₂O) δ 7.79 (s, 1H, H₋₈), 4.25−4.20 (m, 2H,

CH₂N), 4.04 (s, 3H, OCH₃), 4.07−3.97 (m, 4H, CH₂CH₃), 3.84−3.46

(m, 1H, CHOH), 2.20−2.11 (m, 3H, CH₂CH₂N, CH₂P), 1.98−1.91

(m, 1H, CH₂CH₂N), 1.21−1.16 (m, 6H, CH₃CH₂). ¹³C NMR (D₂O)

δ 166.9 (C₋₆), 162.0 (C₋₂), 154.4 (C₋₄), 140.1 (CH₋₈), 112.4 (C₋₅),

63.2, 63.1 (CH₂CH₃, CHOH), 53.8 (OCH₃), 39.7 (CH₂N), 37.0, 36.9

(CH₂CH₂N), 32.9, 31.8 (CH₂P), 15.4, 15.3 (s, CH₃CH₂). ³¹P NMR

(D₂O) δ 31.2 (s). HRMS (TOF ESI > 0) found: 375.1434. Calculated

for C₁₄H₂₄N₄O₆P: 375.1433 (M + H)⁺

1

H₂O/NH₄OHc (7/4/2, v/v/v). Purity (HPLC) > 95%. H NMR

(D₂O): δ = 7.78 (s, 1H, H₋₈), 4.32−4.20 (m, 2H, CH₂N), 4.10−3.96

(m, 1H, CHOH), 1.87−1.67 (m, H, CH₂P). ¹³C NMR (D₂O) δ 159.5

(C₋₆), 155.7 (C₋₄), 140.6 (CH₋₈), 66.6 (CHOH), 49.6, 49.4 (CH₂N),

34.2, 32.5 (CH₂P). ³¹P NMR (D₂O): δ = 18.7. MS (ESI) m/z 289.08

[M − 2Na + 3H]⁺, 311.06 [M − Na + 2H]⁺, 333.05 [M + H]⁺. HRMS:

calcd C₈H₁₂N₆O₄Na₂P [M + H]⁺333.0453, obs 333.0460.

L

https://dx.doi.org/10.1021/acs.jmedchem.0c00131

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(S)-(3-(2,6-Diamino-9H-purin-9-yl)-2-hydroxypropyl)phosphonic

Acid (3(S)). Compound 3(S) (705 mg) was obtained in 61% yield,

according to procedure F, from 19(S) (193 mg). R_f= 0.56 in iPrOH/

(S)-(4-(2,6-Diamino-9H-purin-9-yl)-2-hydroxybutyl)phosphonic

Acid (6(S)). Compound 6(S) (220 mg) was obtained in 70% yield,

according to procedure F, from 26(S) (693 mg). R_f= 0.20 in iPrOH/

1

H₂O/NH₄OHc (7/4/2, v/v/v). Purity (HPLC) > 98%. H NMR

H₂O/NH₄OHc (7/2/1, v/v/v). Purity (HPLC) > 99%. H NMR

(D₂O): δ = 7.80 (s, 1H, H₋₈), 4.28−4.17 (m, 2H, CH₂N), 4.03−3.93

(m, 1H, CHOH), 1.80−1.54 (m, H, CH₂P). ¹³C NMR (D₂O) δ 159.7

(C₋₆), 155.8 (C₋₄), 151.0 (C₋₂), 140.6 (CH₋₈), 112.7 (C₋₅), 67.1

(CHOH), 49.8, 49.6 (CH₂N), 34.4, 32.8 (CH₂P). ³¹P NMR (D₂O): δ

= 17.5. MS (ESI) m/z 289.08 [M − 2Na + 3H]⁺, 311.06 [M − Na +

2H]⁺, 333.05 [M + H]⁺, 355.03 [M + Na]⁺. HRMS: calcd

C₈H₁₂N₆O₄Na₂P [M + H]⁺333.0453, obs 333.0450.

(D₂O): 7.77 (s, 1H, H₋₈), 4.17−4.06 (m, 2H, CH₂N), 4.0−3.90 (m,

1H, CHOH), 2.20−2.08 (m, 1H, CH₂CH₂N), 1.98−1.73 (m, 3H,

CH₂CH₂N, CH₂P). ¹³C NMR (D₂O) δ 159.7 (C₋₆), 155.9 (C₋₄),

151.0 (CH₋₂), 140.3 (CH₋₈), 112.9 (C₋₅), 65.4 (CHOH), 40.3

(CH₂N), 37.0, 36.9 (CH₂CH₂N), 36.4, 35.5 (CH₂P). ³¹P NMR

(D₂O): 20.2. MS (ESI) m/z 303.12 [M + H]⁺. HRMS: calcd

C₉H₁₆N₆O₄P [M + H]⁺303.0971, obs 303.0971.

(R)-(4-(6-Amino-9H-purin-9-yl)-2-hydroxybutyl)phosphonic Acid

(4(R)). Compound 4(R) (216 mg) was obtained in 95% yield, according

to procedure F, from 24(R) (358 mg). R_f= 0.21 in iPrOH/H₂O/

NH₄OHc (7/2/1, v/v/v). Purity (HPLC) > 99%. ¹H NMR (D₂O): δ =

7.99 (s, 1H, H₋₂), 7.96 (s, 1H, H₋₈), 4.26−4.16 (m, 2H, CH₂N), 4.00−

3.87 (m, 1H, CHOH), 2.24−2.11 (m, 1H, CH₂CH₂N), 1.99−1.79 (m,

(R)-(2-Hydroxy-4-(6-oxo-1,6-dihydro-9H-purin-9-yl)butyl)-

phosphonic Acid Disodic Salt (7(R)). Compound 7(R) (169 mg) was

obtained in 70% yield, according to procedure F, from 29(R) (250 mg).

R_f= 0.19 in iPrOH/H₂O/NH₄OHc (7/2/1, v/v/v). Purity (HPLC) >

20

1

99%. [α]_D+16.28° (c 6.45, H₂O). H NMR(D₂O) δ 8.19 (s, 1H,

H₋₂), 8.16 (s, 1H, H₋₈), 4.40−4.37 (t, J = 7.3 Hz, 2H, CH₂N), 4.00−

3.93 (m, 1H, CHOH), 2.28−2.21 (m, 1H, CH₂CH₂N), 2.03−1.99 (m,

1H, CH₂CH₂N), 1.81−1.76 (m, 2H, CH₂P). ¹³C NMR (D₂O) δ 158.7

(C₋₆), 148.9 (C₋₄), 145.5 (CH₋₂), 142.4 (CH₋₈), 123.3 (C₋₅), 65.6

(CHOH), 41.3 (CH₂N), 37.2, 37.1 (CH₂CH₂N), 36.5, 35.5 (CH₂P).

³¹P NMR(D₂O) δ 19.8 (s). HRMS (TOF ESI < 0) found: 287.0547.

3H, CH₂CH₂N, CH₂P). ¹³C NMR (D₂O) δ 154.9 (C₋₆), 151.8 (C₋₄),

148.3 (CH₋₂), 142.2 (CH₋₈), 117.9 (C₋₅), 65.5 (CHOH), 40.9

(CH₂N), 37.0, 36.9 (CH₂CH₂N), 36.7, 35.5 (CH₂P). ³¹P NMR

(D₂O): δ = 20.7. MS (ESI) m/z 332.02 [M + H]⁺; 310.04 [M − Na +

2H]⁺; 288.07 [M − 2Na + 3H]⁺. HRMS: calcd C₉H₁₅N₅O₄P [M − 2Na

+ 3H]⁺288.0862, obs 288.0867.

Calculated for C₉H₁₂N₄O₅P: 287.0545 (M − H)⁻.

(S)-(4-(6-Amino-9H-purin-9-yl)-2-hydroxybutyl)phosphonic Acid

(4(S)). Compound 4(R) (167 mg) was obtained in 69% yield, according

to procedure F, from 24(S) (398 mg). R_f= 0.26 in iPrOH/H₂O/

NH₄OHc (7/2/1, v/v/v). Purity (HPLC) > 95%. ¹H NMR (D₂O): δ =

7.98 (s, 1H, H₋₂), 7.96 (s, 1H, H₋₈), 4.25−4.15 (m, 2H, CH₂N), 3.98−

3.87 (m, 1H, CHOH), 2.22−2.11 (m, 1H, CH₂CH₂N), 1.98−1.80 (m,

(S)-(2-Hydroxy-4-(6-oxo-1,6-dihydro-9H-purin-9-yl)butyl)-

phosphonic Acid Disodic Salt (7(S)). Compound 7(S) (77 mg) was

obtained in 52% yield, according to procedure F, from 29(S) (155 mg).

R_f= 0.19 in iPrOH/H₂O/NH₄OHc (7/2/1, v/v/v). Purity (HPLC) >

20

1

99%. [α]_D−14.19° (c 4.65, H₂O). H NMR (D₂O) δ 8.19 (s, 1H,

H₋₂), 8.16 (s, 1H, H₋₈), 4.41−4.38 (t, J = 7.2 Hz, 2H, CH₂N), 3.98−

3.92 (m, 1H, CHOH), 2.30−2.24 (m, 1H, CH₂CH₂N), 2.05−1.99 (m,

1H, CH₂CH₂N), 1.86−1.81 (dd, J = 17.3, 6.6 Hz, 2H, CH₂P). ¹³C

NMR(D₂O) δ 158.7 (C₋₆), 148.9 (C₋₄), 145.4 (CH₋₂), 142.4 (CH₋₈),

123.3 (C₋₅), 65.4 (CHOH), 41.3 (CH₂N), 37.1, 37.0 (CH₂CH₂N),

3H, CH₂CH₂N, CH₂P). ¹³C NMR (D₂O) δ 154.9 (C₋₆), 151.8 (C₋₄),

148.3 (CH₋₂), 142.2 (CH₋₈), 117.9 (C₋₅), 65.5 (CHOH), 40.9

(CH₂N), 37.0, 36.9 (CH₂CH₂N), 36.7, 35.5 (CH₂P). ³¹P NMR

(D₂O): δ = 20.6. MS (ESI) m/z 310.04 [M + H]⁺; 288.07 [M − Na +

2H]⁺. HRMS: calcd C₉H₁₅N₅O₄P [M − Na + 2H]⁺288.0862, obs

288.0860.

36.5, 35.5 (CH₂P). ³¹P NMR(D₂O) δ 20.2. HRMS (TOF ESI > 0)

found: 289.0703. Calculated for C₉H₁₄N₄O₅P: 289.0702 (M + H)⁺.

(R)-(4-(2,6-Dioxo-1,2,3,6-tetrahydro-9H-purin-9-yl)-2-

hydroxybutyl)phosphonic Acid Disodic Salt (8(R)). Compound 8(R)

(90 mg) was obtained in 46% yield, according to procedure F, from

30(R) (206 mg). R_f= 0.17 in iPrOH/H₂O/NH₄OHc (7/2/1, v/v/v).

Purity (HPLC) > 99%. [α]_D²⁰+17.5° (c 4.4, H₂O). ¹H NMR(D₂O) δ

7.79 (s, 1H, CH₋₈), 4.25−4.21 (t, J = 7.3 Hz, 2H, CH₂N), 4.01−3.94

(m, 1H, CHOH), 2.27−2.20 (m, 1H, CH₂CH₂N), 2.00−1.84 (m, 3H,

(R)-(4-(2-Amino-6-oxo-9H-purin-9-yl)-2-hydroxybutyl)-

phosphonic Acid (5(R)). Compound 5(R) (165.6 mg) was obtained in

68% yield, according to procedure F, from 25(R) (404 mg). R_f= 0.60 in

20

iPrOH/H₂O/NH₄OHc (7/4/2, v/v/v). Purity (HPLC) > 95%. [α]_D

+7° (c 0.0097, H₂O). ¹H NMR (D₂O): δ = 7.75 (s, 1H, H₋₈), 4.17−

4.05 (m, 2H, CH₂N), 3.98−3.87 (m, 1H, CHOH), 2.21−2.08 (m, 1H,

CH₂CH₂N), 1.96−1.77 (m, 3H, CH₂CH₂N, CH₂P). ¹³C NMR (D₂O)

δ 158.7 (C₋₆), 153.5 (C₋₄), 151.3 (CH₋₂), 140.7 (CH₋₈), 115.7 (C₋₅),

65.4 (CHOH), 40.6 (CH₂N), 37.1, 37.0 (CH₂CH₂N), 36.7, 35.4

CH₂CH₂N, CH₂P). ¹³C NMR (D₂O) δ 160.0 (C₋₆), 153.5 (C₋₂),

143.2 (C₋₄), 139.1 (CH₋₈), 115.2 (C₋₅), 65.1 (CHOH), 41.6 (CH₂N),

36.5 (CH₂CH₂N), 36.4, 35.4 (CH₂P). ³¹P NMR (D₂O) δ 20.4. HRMS

(TOF ESI < 0) found: 303.0492. Calculated for C₉H₁₂N₄O₆P:

303.0494 (M + H)⁺.

(CH₂P); ³¹P NMR (D₂O): δ = 20.5. MS (ESI) m/z 304.08 [M − Na +

2H]⁺, 326.06 [M + H]⁺. HRMS: calcd C₉H₁₅N₅O₅P [M + H]⁺

304.0811, obs 304.0808.

(S)-(4-(2-Amino-6-oxo-9H-purin-9-yl)-2-hydroxybutyl)-

phosphonic Acid (5(S)). Compound 5(S) (171 mg) was obtained in

51% yield, according to procedure F, from 25(S) (558 mg). R_f= 0.10 in

(S)-(4-(2,6-Dioxo-1,2,3,6-tetrahydro-9H-purin-9-yl)-2-

hydroxybutyl)phosphonic Acid Disodic Salt (8(S)). Compound 8(S)

(97 mg) was obtained in 40% yield, according to procedure F, from

30(S) (261 mg). R_f= 0.15 in iPrOH/H₂O/NH₄OHc (7/2/1, v/v/v).

Purity (HPLC) > 98%. [α]_D²⁰−17.58° (c 4.95, H₂O). ¹H NMR (D₂O)

δ 7.78 (s, 1H, CH₋₈), 4.23−4.20 (t, J = 7.3 Hz, 2H, CH₂N), 4.01−3.94

(m, 1H, CHOH), 2.26−2.19 (m, 1H, CH₂CH₂N), 1.98−1.85 (m, 3H,

1

iPrOH/H₂O/NH₄OHc (7/2/1, v/v/v). Purity (HPLC) > 99%. H

NMR (D₂O): δ = 7.77 (s, 1H, H₋₈), 4.15−4.05 (m, 2H, CH₂N), 3.98−

3.87 (m, 1H, CHOH), 2.23−2.09 (m, 1H, CH₂CH₂N), 1.96−1.80 (m,

3H, CH₂CH₂N, CH₂P). ¹³C NMR (D₂O) δ 158.7 (C₋₆), 153.5 (C₋₄),

151.3 (CH₋₂), 140.0 (CH₋₈), 115.7 (C₋₅), 65.2 (CHOH), 40.5

(CH₂N), 37.0, 36.9 (CH₂CH₂N), 36.8, 35.0 (CH₂P). ³¹P NMR

CH₂CH₂N, CH₂P). ¹³C NMR (D₂O) δ 160.2 (C₋₆), 154.5 (C₋₂),

144.9 (C₋₄), 139.2 (C₋₈), 115.1 (C₋₅), 65.1 (CHOH), 41.3 (CH₂N),

36.6, 36.5 (CH₂CH₂N), 36.4, 35.4 (CH₂P). ³¹P NMR (D₂O) δ 20.4.

HRMS (TOF ESI > 0) found: 349.0291. Calculated for

C₉H₁₂N₄O₆Na₂P: 349.0290 (M + H)⁺.

(D₂O): δ = 21.0. MS (ESI) m/z 348.04 [M + H]⁺, 326.06 [M − Na +

2H]⁺, 304.08 [M − 2Na + 3H]⁺. HRMS: calcd C₉H₁₅N₅O₅P [M − 2Na

+ 3H]⁺304.0811, obs 304.0813.

(R)-(4-(2,6-Diamino-9H-purin-9-yl)-2-hydroxybutyl)phosphonic

Acid (6(R)). Compound 6(R) (72 mg) was obtained in 31% yield,

according to procedure F, from 26(R) (501 mg). R_f= 0.19 in iPrOH/

(R)-(4-(2-Amino-9H-purin-9-yl)-2-hydroxybutyl)phosphonic Acid

(9(R)). Compound 9(R) (190 mg) was obtained in 40% yield, according

to procedure F. R_f= 0.32 in iPrOH/H₂O/NH₄OHc (7/2/1, v/v/v).

Purity (HPLC) > 99%. [α]_D²⁰+2.8° (c 0.0124, H₂O). ¹H NMR (D₂O)

δ 8.64 (s, 1H, H₋₆), 8.18 (s, 1H, H₋₈), 4.32 (m, 2H, CH₂N), 4.00−3.95

(m, 1H, CHOH), 2.28−2.26 (m, 1H, CH₂CH₂N), 2.11−1.85 (2m, 3H,

1

H₂O/NH₄OHc (7/2/1, v/v/v). Purity (HPLC) > 99%. H NMR

(D₂O): δ = 7.76 (s, 1H, H-8), 4.14−4.05 (m, 2H, CH₂N), 3.97−3.90

(m, 1H, CHOH), 2.19−2.07 (m, 1H, CH₂CH₂N), 1.96−1.75 (m, 3H,

CH₂CH₂N, CH₂P). ¹³C NMR (D₂O) δ 159.7 (C-6), 156.5 (C-4),

155.8 (CH-2), 140.2 (CH-8), 115.2 (C-5), 65.6 (CHOH), 40.4

(CH₂N), 37.1, 37.0 (CH₂CH₂N), 36.6, 35.4 (CH₂P). ³¹P NMR

(DMSO-d₆): 20.2. MS (ESI) m/z 303.10 [M + H]⁺. HRMS: calcd

C₉H₁₆N₆O₄P [M + H]⁺303.0968, obs 303.0971.

CH₂CH₂N, CH₂P). ¹³C NMR (D₂O) δ 158.9 (C₋₆), 152.9 (C₋₂),

147.5 (C₋₄), 145.2 (CH₋₈), 126.5 (C₋₅), 65.3 (CHOH), 40.3 (CH₂N),

36.6, 36.5 (CH₂CH₂N), 36.5, 35.3 (CH₂P). ³¹P NMR (D₂O): δ = 20.8.

MS (ESI) m/z 286.07 [M − H]⁻, 573.15 [2M − H]⁻. HRMS: calcd

C₉H₁₅N₅O₄P [M + H]⁺288.0862, obs 288.0861.

M

https://dx.doi.org/10.1021/acs.jmedchem.0c00131

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(R)-(4-(6-Amino-2-ﬂuoro-9H-purin-9-yl)-2-hydroxybutyl)-

phosphonic Acid (10(R)). Compound 10(R) (144 mg) was obtained in

26% yield, according to procedure F. R_f= 0.39 in iPrOH/H₂O/

NH₄OH_c(7/2/1, v/v/v). Purity (HPLC) > 96%. [α]_D²⁰+3.5 (c 0.007,

H₂O). ¹H NMR δ 7.85 (s, 1H, H₋₈), 4.15 (m, 2H, CH₂N), 3.97−3.93

(m, 1H, CHOH), 2.21−2.17 (m, 1H, CH₂CH₂N), 1.96−1.80 (m, 3H,

(R)-tert-Butyl-(2-(2,6-diamino-di(N-tert-butyloxycarbonyl)-N-9-

purine)ethyl)aziridine-N-carboxylate (37(R)). Compound 37(R) (1

g) was obtained in 87% yield, according to procedure G, from 35(R)

20

(600 mg). R_f= 0.3 in hexane/AcOEt (5/5, v/v). [α]_D−41.5° (c 1,

1

CH₂Cl₂). H NMR (CDCl₃) δ 8.33 (s, 1H, H₈), 4.53−4.41 (m, 2H,

CH₂N), 2.38−2.29 (m, 3H, CH₂CH₂N, CHN), 1.91 (d, J = 3.3 Hz, 1H,

CHCH₂N), 1.75−1.68 (m, 1H, CH₂CH₂N), 1.47 (s, 9H, CH₃), 1.42

CH₂CH₂N, CH₂P). ¹³C NMR(D₂O) δ 157.2 (C₋₆), 153.1 (C₋₂), 150.8

(C₋₄), 141.6 (CH₋₈), 141.5 (C₋₅), 65.2 (CHOH), 40.3 (CH₂N), 36.8,

36.7 (CH₂CH₂N), 36.7, 35.4 (CH₂P). ³¹P NMR (DMSO-d₆): 20.7. MS

(s, 36H, CH₃). ¹³C NMR(CDCl₃) δ 162.3 (CO), 154.3 (C₋₄), 151.1

(C₋₂), 151.0 (CO), 150.3 (C₋₆), 146.3 (C₋₈), 127.6 (C₋₅), 83.8,

83.3, 81.9 (C(CH₃)₃), 42.53 (CH₂N), 34.9 (CHN), 32.7 (CH₂CH₂N),

31.9 (CHCH₂N), 28.1, 28.0, 27.8 (C(CH₃)₃). HRMS (TOF ESI > 0)

found: 720.3937. Calculated for C₃₄H₅₄N₇O₁₀: 720.3932 (M + H)⁺.

(S)-tert-Butyl-(2-(2,6-diamino-di(N-tert-butyloxycarbonyl)-N-9-

purine)ethyl)aziridine-1-carboxylate (37(S)). Compound 37(S) (1.07

g) was obtained in 93% yield, according to procedure G, from 35(S)

(ESI) m/z 326.2 [M − H]⁻.

N-Boc tosylated aziridines 35(R) and 35(S) were prepared in 5 steps

following reported procedures.^18d,29

(R)-tert-Butyl-(2-(2-tosyloxy)ethyl)aziridine-N-carboxylate

(35(R)). R_f= 0.3 in hexane/AcOEt (5/5, v/v). ¹H NMR (CDCl₃) δ 7.80

(d, J = 8.2 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 4.23−4.14 (m, 2H,

CH₂O), 2.45 (s, 3H, CH₃), 2.42−2.39 (m, 1H, CHN), 2.26 (d, J = 7

Hz, 1H, CH₂N), 1.92 (d, J = 3.7 Hz, 1H, CH₂N), 1.90−1.84 (m, 1H,

CH₂CH₂O), 1.78−1.73 (m, 1H, CH₂CH₂O), 1.43 (s, 9H, CH₃).

20

(600 mg). R_f= 0.3 in hexane/AcOEt (5/5, v/v). [α]_D+40.6° (c 1,

1

CH₂Cl₂). H NMR (CDCl₃) δ 8.33 (s, 1H, H8), 4.55−4.39 (m, 2H,

CH₂N), 2.39−2.25 (m, 3H, CH₂CH₂N, CHN), 1.91 (d, J = 3.3 Hz, 1H,

CHCH₂N), 1.76−1.67 (m, 1H, CH₂CH₂N), 1.47 (s, 9H, CH₃), 1.42

HRMS (TOF ESI > 0) found: 364.1194. Calculated for

(s, 36H, CH₃). ¹³C NMR (CDCl₃) δ 162.2 (CO), 154.3 (C-4), 152.1

(C-2), 151.1, 151.0 (CO), 150.3 (C-6), 146.3 (C-8), 127.6 (C-5),

83.8, 83.3, 81.9 (C(CH₃)₃), 42.5 (CH₂N), 35.0 (CHN), 32.7

(CH₂CH₂N), 32.0 (CHCH₂N), 28.1, 28.0, 27.9 (C(CH₃)₃). HRMS

(TOF ESI > 0) found: 720.3928. Calculated for C₃₄H₅₄N₇O₁₀:

720.3932 (M + H)⁺.

C₁₆H₂₃N₅O₆SNa: 364.1195 (M + Na)⁺.

(S)-tert-Butyl-(2-(2-tosyloxy)ethyl)aziridine-N-carboxylate(35(S)).

R_f= 0.3 in hexane/AcOEt (5/5, v/v). ¹H NMR (CDCl₃) δ 7.80 (d, J =

8.2 Hz, 1H, H), 7.35 (d, J = 8.2 Hz, 1H, H), 4.23−4.14 (m, 2H, CH₂O),

2.45 (s, 3H, CH₃), 2.43−2.38 (m, 1H, CHN), 2.26 (d, J = 6.5 Hz, 1H,

CH₂N), 1.92 (d, J = 3.4 Hz, 1H, CH₂N), 1.90−1.84 (m, 1H,

CH₂CH₂O), 1.79−1.72 (m, 1H, CH₂CH₂O), 1.42 (s, 9H, CH₃).

General Procedure H. Preparation of β-Aminophosphonates

38(R) or 38(S) from N-Boc Aziridine Derivatives 36(R) and 36(S).

Diethyl phosphite (5 equiv) was dissolved in anhydrous THF (1 mL/

mmol DEP) under argon atmosphere and cooled at −78 °C. LiHMDS

(5 equiv) was added dropwise to the mixture. The reaction mixture was

kept under stirring at −78 °C for 15 min. Then, the solution of aziridine

36(R) or 36(S) (1 equiv) in anhydrous THF (2.8 mL/mmol aziridine)

was added dropwise. After 5 min, the cooled bath was removed and the

mixture slowly warmed to room temperature. The reaction was stirred

overnight and followed by TLC monitoring (CH₂Cl₂/MeOH 95/5 v/

v). After 24 h, the mixture was quenched by addition of saturated

aqueous solution of NH₄Cl, until dissolution of the previously formed

salts. The resulting solution was extracted twice by ethyl acetate and the

organic layers were gathered, dried over MgSO_4,concentrated under

vacuum, and ﬁnally puriﬁed on silica gel by ﬂash chromatography

(dichloromethane/methanol gradient up to 10% methanol) to aﬀord

the expected N-Boc β-aminophosphonate 38(R) or 38(S) as a white

foam.

(R)-2-Amino-(N-tert-butoxycarbonyl)-6-methoxy-N-9-(3-amino-

4-diethylphosphonobutyl)purine (38(R)). Compound 38(R) (915

mg) was obtained in 62% yield, according to procedure H, from 36(R)

(1.37 g). R_f= 0.4 in CH₂Cl₂/MeOH 95/5 (v/v). [α]_D²⁰−20.8° (c 1,

CH₂Cl₂). ¹H NMR (CDCl₃) δ 7.82 (s, 1H, H₈), 7.33 (s, 1H, NH), 6.26

(d, J = 7.4 Hz, 1H, CHNH), 4.33−4.28 (m, 1H, CH₂N), 4.23−4.17 (m,

1H, CH₂N), 4.11 (s, 3H, OCH₃), 4.06−4.0 (m, 4H, CH₂CH₃), 3.78−

3.66 (m, 1H, CHNH), 2.41−2.29 (m, 2H, CH₂P, CH₂CH₂N), 2.09−

1.99 (m, 2H, CH₂P, CH₂CH₂N), 1.54 (s, 9H, CH₃), 1.44 (s, 9H, CH₃),

1.28−1.22 (m, 6H, CH₂CH₃). ¹³C NMR (CDCl₃) δ 161.5 (C₋₆), 155.8

(CO), 153.4 (C₋₄), 152.4 (C₋₂), 150.7 (CO), 141.7 (C₋₈), 117.9

(C₋₅), 81.3, 79.3 (C(CH₃)₃), 61.8 (CH₂CH₃), 54.4 (OCH_3,), 44.7

(CHNH), 41.0 (CH₂N), 35.5 (CH₂CH₂N), 31.0, 30.0 (CH₂P), 28.6,

28.4 (C(CH₃)₃), 16.5 (CH₂CH₃). ³¹P NMR(CDCl₃) δ 28.6 (s).

HRMS (TOF ESI > 0) found: 342.1377. Calculated for C₁₆H₂₄N₅O₆S:

342.1375 (M + H)⁺.

General Procedure G. Preparation of N-Boc Purinyl Aziridines

Derivatives. To a solution of protected purine nucleobase (1 equiv) in

anhydrous DMF (1.5 mL/mmol), and under argon atmosphere, was

added anhydrous K₂CO₃(2.6 equiv), and the mixture was heated at 60

°C for 1 h. The mixture was then cooled at room temperature, and a

solution of N-Boc tosylated aziridine 35 (1.1 equiv) in anhydrous DMF

(1.5 mL/mmol) was added dropwise over a 30 min period. The

reaction mixture was kept under stirring for 3 days at room temperature

with TLC monitoring (hexane/ethyl acetate, 5/5, v/v). Then, the

volatiles were evaporated under high vacuum, and the resulting residue

was dissolved in ethyl acetate, sonicated, and the undissolved salts were

ﬁltered on a sintered glass (porosity 4). The ﬁltrate was then evaporated

and puriﬁed on silica gel by ﬂash chromatography (petroleum ether/

ethyl acetate gradient) to aﬀord the N-Boc purinyl aziridine as a white

foam.

(R)-tert-Butyl-(2-(2-amino-di(N-tert-butyloxycarbonyl)-6-me-

thoxy-N-9-purine)ethyl)aziridine-N-carboxylate (36(R)). Compound

36(R) (983 mg) was obtained in 69% yield, according to procedure G,

from 35(R) (1 g). R_f= 0.3 in hexane/AcOEt (5/5, v/v). [α]_D²⁰−38.2 (c

1, CH₂Cl₂). ¹H NMR (CDCl₃) δ 8.14 (s, 1H, H₈), 4.49−4.36 (m, 2H,

CH₂N), 4.15 (s, 3H, CH₃O), 2.37−2.25 (m, 4H, CHN, CH₂CH₂N,

CHCH₂N), 1.90 (d, J = 3.5 Hz, 1H, CHCH₂N), 1.75−1.68 (m, 1H,

CH₂CH₂N), 1.47 (s, 9H, CH₃), 1.44 (s, 18H, CH₃). ¹³C NMR

(CDCl₃) δ 162.2 (CO), 161.6 (C₋₆), 152.8 (C₋₄), 152.0 (C₋₂),

151.2 (CO)_,143.7 (C_₈), 120.3 (C₋₅), 83.2, 81.9 (C(CH₃)₃), 54.7

(OCH₃), 42.4 (CH₂N), 35.0 (CHN), 32.8 (CH₂CH₂N), 31.9

(CHCH₂N), 28.1 (C(CH₃)₃). HRMS (TOF ESI > 0) found:

535.2880. Calculated for C₂₅H₃₉N₆O₇: 535.2880 (M + H)⁺.

(S)-tert-Butyl-(2-(2-amino-di(N-tert-butyloxycarbonyl)-6-me-

thoxy-N-9-purine)ethyl)aziridine-N-carboxylate (36(S)). Compound

36(S) (893 mg) was obtained in 44% yield, according to procedure G,

from 35(S) (1.3 g). R_f= 0.3 in hexane/AcOEt (5/5, v/v). [α]_D²⁰+38.8°

(c 1, CH₂Cl₂). ¹H NMR (CDCl₃) δ 8.13 (s, 1H, H₈), 4.48−4.35 (m,

2H, CH₂N), 4.14 (s, 3H, CH₃O), 2.35−2.23 (m, 4H, CHN, CHCH₂N,

CH₂CH₂N), 1.88 (d, J = 3.5 Hz, 1H, CHCH₂N), 1.75−1.68 (m, 1H,

CH₂CH₂N), 1.46 (s, 9H, CH₃), 1.43 (s, 18H, CH₃). ¹³C NMR

(CDCl₃) δ 162.2 (CO), 161.6 (C₋₆), 152.8 (C₋₄), 152.0 (C₋₂),

151.1 (CO), 143.7 (C₋₈), 120.3 (C₋₅), 83.1, 81.9 (C(CH₃)₃), 54.6

(OCH₃), 42.4 (CH₂N), 35.0 (CHN), 32.8 (CH₂CH₂N), 31.9

(CHCH₂N), 28.0 (C(CH₃)₃). HRMS (TOF ESI > 0) found:

535.2882. Calculated for C₂₅H₃₉N₆O₇: 535.2880 (M + H)⁺.

HRMS (TOF ESI > 0) found: 573.2800. Calculated for C₂₄H₄₂N₆O₈P:

573.2802 (M + H)⁺.

(S)-2-Amino-(N-tert-butoxycarbonyl)-6-methoxy-N-9-(3-amino-

4-diethylphosphonobutyl)purine (38(S)). Compound 38(S) (84 mg)

was obtained in 40% yield, according to procedure H, from 36(S) (193

20

mg). R_f= 0.4 in CH₂Cl₂/MeOH 95/5 (v/v) [α]_D+21.2° (c 1,

CH₂Cl₂). ¹H NMR (CDCl₃) δ 7.82 (s, 1H, H8), 7.32 (s, 1H, NH), 6.26

(d, J = 7.4 Hz, 1H, CHNH), 4.33−4.28 (m, 1H, CH₂N), 4.23−4.17 (m,

1H, CH₂N), 4.11 (s, 3H, OCH₃), 4.07−4.00 (m, 4H, CH₂CH₃), 3.76−

3.67 (m, 1H, CHNH), 2.41−2.29 (m, 2H, CH₂P, CH₂CH₂N), 2.12−

1.99 (m, 2H, CH₂P, CH₂CH₂N), 1.54 (s, 9H, CH₃), 1.44 (s, 9H, CH₃),

1.28−1.22 (m, 6H, CH₂CH₃). ¹³C NMR (CDCl₃) δ 161.5 (C-6), 155.8

(CO), 153.4 (C-4), 152.4 (C-2), 150.6 (CO), 141.7 (C-8), 118.0

N

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Journal of Medicinal Chemistry

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Article

(C-5), 81.3, 79.3 (C(CH₃)₃), 61.8 (CH₂CH₃), 54.4 (OCH₃), 44.7

(CHNH), 41.0 (CH₂N), 35.5 (CH₂CH₂N), 31.0, 30.1 (CH₂P), 28.6,

28.4 (C(CH₃)₃), 16.5 (CH₂CH₃). ³¹P NMR (CDCl₃) δ 28.6 (s).

HRMS (TOF ESI > 0) found: 573.2808. Calculated for C₂₄H₄₂O₈N₆P:

573.2802 (M + H)⁺.

(C₋₄), 140.7 (CH₋₈), 114.7 (C₋₅), 64.1 (OCH₂CH₃), 54.4 (OCH₃),

46.2 (CHNH), 40.1 (CH₂N), 35.4, 35.3 (CH₂CH₂N), 30.0, 28.8

(CH₂P), 16.6 (OCH₂CH₃). ³¹P NMR (MeOD) δ 26.0 (s). HRMS

(TOF ESI > 0) found: 373.1761. Calculated for C₁₄H₂₆N₆O₄P:

373.1753 (M + H)⁺.

General Procedure I. Preparation of β-Aminophosphonates

39(R) or 39(S) from N-Boc Aziridine Derivatives 37(R) and 37(S).

Diethyl phosphite (3 equiv) was dissolved in anhydrous THF (1 mL/

mmol DEP) under argon atmosphere and cooled at −78 °C. LiHMDS

(3 equiv) was added dropwise to the mixture. The mixture was kept

under stirring at −78 °C for 15 min. Then, a solution of aziridine 37(R)

or 37(S) (1 equiv) in anhydrous THF (2.8 mL/mmol of aziridine) was

added dropwise. After 5 min, the cooled bath was removed and the

mixture slowly warmed to room temperature. The reaction was stirred

overnight followed by TLC monitoring (CH₂Cl₂/MeOH 95/5 v/v).

After 24 h, the mixture was quenched by addition of saturated aqueous

solution of NH₄Cl until dissolution of the previously formed salts. The

resulting solution was extracted twice by ethyl acetate and the organic

layers were gathered, dried over MgSO_4,concentrated under vacuum,

and ﬁnally puriﬁed on silica gel by ﬂash chromatography (dichloro-

methane/methanol gradient up to 10% methanol) to aﬀord the

expected N-Boc β-aminophosphonate 39(R) or 39(S) as a white foam.

(R)-2,6-Diamino-(N-tert-butoxycarbonyl)-N-9-(3-amino-4-

diethylphosphonobutyl)purine (39(R)). Compound 39(R) (1.47 g)

was obtained in 76% yield, according to procedure I, from 37(R) (2.13

Diethyl (S)-(4-(2-Amino-6-methoxy-9H-purin-9-yl)-2-

aminobutyl)phosphonate (40(S)). Compound 40(S) (290 mg) was

obtained in quantitative yield, according to procedure D′, from 38(S)

(447 mg). R_f= 0.25 in CH₂Cl₂/MeOH (90/10, v/v). [α]_D²⁰+13.5° (c

1, MeOH). ¹H NMR(MeOD) δ 7.92 (s, 1H, H₈), 4.39−4.23 (2m, 2H,

CH₂N), 4.13−4.07 (m, 4H, CH₂CH₃), 4.06 (s, 3H, OCH₃), 3.42−3.35

(m, 1H, CHNH₂), 2.44−2.18 (m, 4H, CH₂CH₂N, CH₂P), 1.28 (m,

6H, CH₂CH₃). ¹³C NMR (MeOD) δ 162.9 (C₋₆), 162.1 (C₋₂), 140.7

(CH₋₈), 64.1 (CH₂CH₃), 54.4 (OCH₃), 46.2 (CHNH), 40.1 (CH₂N),

35.4, 35.3 (CH₂CH₂N), 29.9, 28.8 (CH₂P), 16.6 (CH₂CH₃). ³¹P

NMR(MeOD) δ 26.0 (s). HRMS (TOF ESI > 0) found: 373.1756.

Calculated for C₁₄H₂₆N₆O₄P: 373.1753 (M + H)⁺.

Diethyl (R)-(4-(2,6-Diamino-9H-purin-9-yl)-2-aminobutyl)-

phosphonate (41(R)). Compound 41(R) (663 mg) was obtained in

quantitative yield, according to procedure D′, from 39(R) (1.47 g). R_f=

0.2 in CH₂Cl₂/MeOH (90/10, v/v). [α]_D²⁰−12.8° (c 1, MeOH). ¹H

NMR (MeOD) δ 7.90 (s, 1H, H₈), 4.35−4.22 (m, 2H, CH₂N), 4.15−

4.07 (m, 4H, CH₂CH₃), 3.46−3.38 (m, 1H, CHNH₂), 2.43−2.19 (m,

4H, CH₂CH₂N, CH₂P), 1.30 (m, 6H, CH₂CH₃). ¹³C NMR (MeOD) δ

154.8 (C₋₂), 153.2 (C₋₄), 141.0 (CH₋₈), 117.0 (C₋₆), 113.2 (C₋₅),

64.2 (CH₂CH₃), 46.1 (CHNH), 40.1 (CH₂N), 35.2 (CH₂CH₂N),

29.9, 28.7 (CH₂P), 16.6 (CH₂CH₃). ³¹P NMR(MeOD) δ 26.0 (s).

20

g). R_f= 0.4 in CH₂Cl₂/MeOH 95/5 (v/v)). [α]_D−20.9° (c 1,

CH₂Cl₂). ¹H NMR (CDCl₃) δ 7.97 (s, 1H, H₈), 7.87 (s, 1H, NH), 7.74

(s, 1H, NH), 6.30 (d, J = 7.6 Hz, 1H, CHNH), 4.34−4.18 (2m, 2H,

CH₂N), 4.07−3.99 (m, 4H, CH₂CH₃), 3.75−3.65 (m, 1H, CHNH),

2.42−2.25 (m, 2H, CH₂CH₂N, CH₂P), 2.15−2.00 (m, 2H, CH₂CH₂N,

CH₂P), 1.53 (s, 9H, CH₃), 1.51 (s, 9H, CH₃), 1.44 (s, 9H, CH₃), 1.28−

1.20 (m, 6H, CH₂CH₃). ¹³C NMR (CDCl₃) δ 153.0 (C₋₄), 152.6

(C₋₂), 150.8, 150.2 (CO), 149.8 (C₋₆), 142.1 (C₋₈), 118.2 (C₋₅),

82.3, 81.0, 79.3 (C(CH₃)₃), 61.8 (CH₂CH₃), 44.6 (CHNH), 41.0

(CH₂N), 35.5, 35.4 (CH₂CH₂N), 31.1, 30.0 (CH₂P), 28.6, 28.3

(C(CH₃)₃), 16.5 (CH₂CH₃). ³¹P NMR(CDCl₃) δ 28.54 (s). HRMS

HRMS (TOF ESI > 0) found: 358.1761. Calculated for C₁₃H₂₅N₇O₃P:

358.1756 (M + H)⁺.

Diethyl (S)-(4-(2,6-Diamino-9H-purin-9-yl)-2-aminobutyl)-

phosphonate (41(S)). Compound 41(S) (685 mg) was obtained in

quantitative yield, according to procedure D′, from 39 (S) (1.58 g). R_f=

0.2 in CH₂Cl₂/MeOH (90/10, v/v). [α]_D²⁰+12.4° (c 1, MeOH). ¹H

NMR (MeOD) δ 7.86 (s, 1H, H8), 4.34−4.22 (2m, 2H, CH₂N), 4.14−

4.07 (m, 4H, CH₂CH₃), 3.44−3.36 (m, 1H, CHNH₂), 2.44−2.17 (m,

4H, CH₂CH₂N, CH₂P), 1.29 (m, 6H, CH₂CH₃). ¹³C NMR (MeOD) δ

(TOF ESI > 0) found: 658.3333. Calculated for C₂₈H₄₉N₇O₉P:

658.3329 (M + H)⁺.

155.9 (C-2), 153.1 (C-4), 140.4 (CH-8), 117.2 (C-6), 113.4 (C-5),

64.3, 64.1 (CH₂CH₃), 46.1 (CHNH), 40.0 (CH₂N), 35.3

(CH₂CH₂N), 29.9, 29.0 (CH₂P), 16.6 (CH₂CH₃). ³¹P NMR

(MeOD) δ 26.0 (s). HRMS (TOF ESI > 0) found: 358.1758.

Calculated for C₁₃H₂₅N₇O₃P: 358.1767 (M + H)⁺.

(S)-2,6-Diamino-(N-tert-butoxycarbonyl)-N-9-(3-amino-4-

diethylphosphonobutyl)purine (39(S)). Compound 39(S) (1.58 g)

was obtained in 78% yield, according to procedure I, from 37(S) (2.22

g). R_f= 0.4 in CH₂Cl₂/MeOH 95/5 (v/v). [α]_D²⁰+20.1° (c 1, CH₂Cl₂).

¹H NMR (CDCl₃) δ 8.04 (s, 1H, H8), 7.87 (s, 1H, NH), 7.81 (s, 1H,

General Procedure F′. Removal of the Diethyl Phosphonate

Groups. To a stirred solution of diethyl-β-aminophosphonate (1 equiv)

in anhydrous dimethylformamide (20 mL/mmol), cooled at 0 °C, was

added dropwise bromotrimethylsilane (6.6 equiv). The reaction

mixture was stirred at room temperature until completion of the

reaction was indicated by TLC monitoring (isopropanol/water/

ammoniac 7/2/1 v/v/v). When in situ removal of Boc groups was

required, water was added to the reaction mixture and stirring was

pursued 1 h at rt. Then, triethylammonium bicarbonate solution

(TEAB 1 M) was added to the reaction mixture until pH = 7. The

volatiles were evaporated under high vacuum, and the aqueous residue

was freeze-dried. The resulting lyophilizate was puriﬁed by ﬂash

chromatography silica gel on C18 reverse phase using water/MeOH

gradient (0 to 100%) and freeze-dried, giving rise to the desired

phosphonic acid. Eventually, the latter was dissolved in a small amount

of water and percolated through a Dowex resin column (Na⁺form) to

aﬀord after freeze-drying the corresponding compound as sodium salt.

(R)-(4-(2-Amino-6-oxo-9H-purin-9-yl)-2-aminobutyl)phosphonic

Acid (12(R)). Compound 12(R) (520 mg) was obtained in 82% yield,

according to procedure F′, from 40(R) (679 mg). R_f= 0.15 in iPrOH/

H₂O/NH₄OH_c(7/2/1, v/v/v). Purity (HPLC) > 98%. [α]_D²⁰−8.8° (c

0.1, H₂O). ¹H NMR (D₂O) δ 7.82 (s, 1H, H₈), 4.21 (t, J = 7.4 Hz, 2H,

CH₂N), 3.47−3.37 (m, 1H, CHNH₂), 2.23−2.17 (m, 2H, CH₂CH₂N),

1.89−1.79 (m, 1H, CH₂P), 1.67−1.54 (m, 1H, CH₂P). ¹³C NMR

(D₂O) δ 158.9 (C₋₆), 153.8 (C₋₂), 151.5 (C₋₄), 139.7 (CH₋₈), 115.8

(C₋₅), 46.6 (CHNH₂), 39.6 (CH₂N), 34.7, 34.6 (CH₂CH₂N), 31.5,

NH), 6.32 (m, 1H, CHNH), 4.32−4.18 (2 m, 2H, CH₂N), 4.03−4.00

(m, 4H, CH₂CH₃), 3.75−3.64 (m, 1H, CHNH), 2.41−1.98 (2m, 4H,

CH₂CH₂N, CH₂P), 1.52 (s, 9H, CH₃), 1.50 (s, 9H, CH₃), 1.43 (s, 9H,

CH₃), 1.27−1.20 (m, 6H, CH₂CH₃). ¹³C NMR (CDCl₃) δ 155.8 (C-

4), 152.6 (C-2), 150.8, 150.2 (CO), 149.8 (C-6), 142.1 (C-8), 118.2

(C-5), 82.3, 81.0, 79.3 (C(CH₃)₃), 61.7 (CH₂CH₃), 44.6 (CHNH),

41.0 (CH₂N), 35.5, 35.4 (CH₂CH₂N), 31.0, 29.9 (CH₂P), 28.6, 28.3,

28.2 (C(CH₃)₃, 16.5 (CH₂CH₃). ³¹P NMR (CDCl₃) δ 28.5 (s). HRMS

(TOF ESI > 0) found: 658.3339. Calculated for C₂₈H₄₉N₇O₉P:

658.3329 (M + H)⁺.

General Procedure D′. Removal of Boc Protecting Groups. To a

stirred solution of β-aminophosphonate (1 equiv) in anhydrous

dichloromethane (14 mL/mmol) under argon, and at room temper-

ature, was added dropwise a solution of triﬂuoroacetic acid in

anhydrous dichloromethane (19 mL/mmol, 3/7, v/v). The reaction

mixture was stirred at room temperature for 5 h. Volatiles were removed

under reduced pressure, and the crude was puriﬁed by ﬂash

chromatography on silica gel using CH₂Cl₂/MeOH (0 to 10%). The

desired compound was obtained as a white foam.

Diethyl (R)-(4-(2-Amino-6-methoxy-9H-purin-9-yl)-2-

aminobutyl)phosphonate (40(R)). Compound 40(R) (594 mg) was

obtained in quantitative yield, according to procedure D′, from 38(R)

(915 mg). R_f= 0.25 in CH₂Cl₂/MeOH (90/10, v/v). [α]_D²⁰−12.9° (c

1, MeOH). ¹H NMR (MeOD) δ 7.91 (s, 1H, H₈), 4.40−4.25 (m, 2H,

CH₂N), 4.14−4.07 (m, 4H, CH₂CH₃), 4.06 (s, 1H, OCH₃), 3.43−3.36

30.7 (CH₂P). ³¹P NMR(D₂O) δ 16.36. HRMS (TOF ESI > 0) found:

(m, 1H, CHNH₂), 2.45−2.18 (m, 4H, CH₂CH₂N, CH₂P), 1.27 (m,

6H, CH₂CH₃). ¹³C NMR (MeOD) δ 162.9 (C₋₆), 162.0 (C₋₂), 155.1

347.0611. Calculated for C₉H₁₄N₆O₄PNa₂: 347.0610 (M + H)⁺.

O

https://dx.doi.org/10.1021/acs.jmedchem.0c00131

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Article

(S)-(4-(2-Amino-6-oxo-9H-purin-9-yl)-2-aminobutyl)phosphonic

Acid (12(S)). Compound 12(S) (162 mg) was obtained in 64% yield,

according to procedure F′, from 40(S) (270 mg). R_f= 0.15 in iPrOH/

H₂O/NH₄OH_c(7/2/1, v/v/v). Purity (HPLC) > 98%. [α]_D²⁰+9.7° (c

Biological Experiments. In Vitro Antimalarial Activity. Drug

eﬀects on in vitro P. falciparum growth (3D7, W2, and FcM29 strains)

were measured in microtiter plates according to a modiﬁed Desjardins

test.^25,31P. falciparum infected erythrocyte suspension (1.5% ﬁnal

hematocrit and 0.6% parasitemia) was grown in complete medium

(Hepes-buﬀered RPMI 1640 + 10% AB human serum) with or without

ANP analogues. The compounds were dissolved in RPMI 1640 and

then further diluted in culture medium. Parasite growth was assessed by

measuring the incorporation of [³H]hypoxanthine into nucleic acids as

previously described.²⁵Suspensions of P. falciparum infected

erythrocytes were incubated with various concentrations of analogues

during 48 h. Then 30 μL of [³H]hypoxanthine (0.5 μCi/well) was

added for an additional 18 h period. The reactions were stopped by

freezing at −80 °C. Cells were lysed by thawing, and the parasite

macromolecules including nucleic acids were recovered by harvesting

the lysate on glass-ﬁber ﬁlter plates (Uniﬁlter 96 GF/C, PerkinElmer)

using a FilterMate cell harvester (Packard Instruments). The

radioactivity was counted on a TopCount microplate scintillation

counter (Packard Instruments). Radioactivity background was

obtained from incubation of noninfected erythrocytes under the same

conditions, and the value obtained was deduced. Results were expressed

as IC₅₀, which is the drug concentration leading to 50% inhibition of

parasite growth. A nonlinear regression model (sigmoidal dose−

response/variable slope; GraphPad Prism 6) was used to calculate the

IC₅₀values.

In Vitro Toxicity against Erythroblast K562 Cell Line. K-562

cells were cultured in complete medium (RPMI 1640 complemented

with 10% fetal calf serum in 96-well microplates (8000 cells per well)

and incubated 24 h at 37 °C and under 5% CO₂in the presence of

various concentrations of tested analogues as above. After 24 h

incubation at 37 °C, 30 μL of complete medium containing 0.5 μCi

[³H]thymidine was added to each well for an additional 6 h period. The

reactions were stopped by freezing at −80 °C. Cell macromolecules

including nucleic acids were recovered as above. Radioactivity

background was obtained from the incubation of culture medium alone.

Eﬀect of Compounds 5 on P. falciparum Development. Drug

eﬀects on parasite development were determined on a tightly

synchronized culture at ring stage. Infected red blood cells were

treated or not with 2 μM 5(R) during 69 h at 2.5% hematocrit and 1−

2% parasitemia. Culture medium with or without drug was changed

every 24 h during the entire treatment period. Thin blood smears were

realized every 6 h to determine the parasitemia and the morphological

changes of parasites. Determination of parasitemia and morphological

observations were done on smears ﬁxed in methanol and stained with

Diﬀ-Quick (pH 7.2; Dade Behring, France). Immunoﬂuorescence

assays (IFAs) were performed on smeared infected erythrocytes ﬁxed

with 4% formaldehyde in phosphate saline buﬀer (PBS) for 30 min at

room temperature, quenched with 0.1 M glycine (4 min) in PBS, and

permeabilized with 0.1% (v/v) Triton X-100 in PBS (5 min). Smears

were then blocked in PBS containing 1.5% BSA during 30 min and then

incubated with primary antibodies (α-mTIP (myosin Tail Domain

Interacting Protein)³²or mouse α-MSP1(Merozoite Surface Protein

1)³³diluted in PBS containing 0.15% BSA for 1 h. After three washes,

appropriate secondary antibodies were added for 1 h before staining of

nuclei with Hoechst (5 min). Observations were performed with a Zeiss

Axioimager epiﬂuorescence microscope equipped with a Zeiss Axiocam

MRmCCD camera at Montpellier RIO imaging facility. Images were

processed using Zen Blue 2.3 (Zeiss) for optical sectioning, luminosity,

and contrast adjustment.

1

0.1, H₂O). H NMR (D₂O) δ 7.77 (s, 1H, H₈), 4.22−4.09 (m, 2H,

CH₂N), 3.49−3.36 (m, 1H, CHNH₂), 2.22−2.18 (m, 2H, CH₂CH₂N),

1.91−1.85 (m, 1H, CH₂P), 1.70−1.64 (m, 1H, CH₂P). ¹³C NMR

(D₂O) δ 159.0 (C₋₆), 153.9 (C₋₂), 151.6 (C₋₄), 139.8 (C₋₈), 115.8

(C₋₅), 46.6 (CHNH₂), 39.7 (CH₂N), 34.7, 34.6 (CH₂CH₂N), 31.5,

30.7 (CH₂P). ³¹P NMR (D₂O) δ 16.3. HRMS (TOF ESI > 0) found:

347.0605. Calculated for C₉H₁₄N₆O₄PNa₂: 347.0610 (M + H)⁺.

(R)-(4-(2,6-Diamino-9H-purin-9-yl)-2-aminobutyl)phosphonic

Acid (13(R)). Compound 13(R) (471 mg) was obtained in 73% yield,

according to procedure F′, from 41(R) (663 mg). R_f= 0.15 in iPrOH/

H₂O/NH₄OH_c(7/2/1, v/v/v). Purity (HPLC) > 97%. [α]_D²⁰−8.0° (c

1

0.1, H₂O). H NMR (D₂O) δ 7.87 (s, 1H, H₈), 4.23−4.14 (m, 2H,

CH₂N), 3.35−3.28 (m, 1H, CHNH₂), 2.17−2.04 (m, 2H, CH₂CH₂N),

1.80−1.73 (m, 1H, CH₂P), 1.60−1.50 (m, 1H, CH₂P). ¹³C NMR

(D₂O) δ 156.1 (C₋₂), 151.0 (C₋₄), 140.2 (CH₋₈), 117.3 (C₋₆), 113.0

(C₋₅), 45.3 (CHNH₂), 40.8 (CH₂N), 38.1, 38.0 (CH₂CH₂N), 37.0,

36.1 (CH₂P). ³¹P NMR(D₂O) δ 17.2. HRMS (TOF ESI < 0) found:

346.0770. Calculated for C₉H₁₅N₇O₃PNa₂: 346.0769 (M + H)⁺.

(S)-(4-(2,6-Diamino-9H-purin-9-yl)-2-aminobutyl)phosphonic

Acid (13(S)). Compound 13(S) (435 mg) was obtained in 71% yield,

according to procedure F′, from 41(S) (630 mg). R_f= 0.15 in iPrOH/

H₂O/NH₄OH_c(7/2/1, v/v/v). Purity (HPLC) > 96%. [α]_D²⁰+8.9° (c

1

0.1, H₂O). H NMR (D₂O) δ 7.89 (s, 1H, H₈), 4.19−4.10 (m, 2H,

CH₂N), 3.16−3.09 (m, 1H, CHNH₂), 2.04−1.98 (m, 1H, CH₂CH₂N),

1.88−1.84 (m, 1H, CH₂CH₂N), 1.67−1.61 (m, 1H, CH₂P), 1.48−1.41

(m, 1H, CH₂P). ¹³C NMR (D₂O) δ 156.1 (C₋₂), 151.0 (C₋₄), 140.1

(C₋₈), 117.3 (C₋₆), 113.0 (C₋₅), 45.3 (CHNH₂), 40.8 (CH₂N), 38.1,

38.0 (CH₂CH₂N), 37.0, 36.1 (CH₂P). ³¹P NMR (D₂O) δ 17.2. HRMS

(TOF ESI > 0) found: 346.0769. Calculated for C₉H₁₅N₇O₃PNa₂:

346.0769 (M + H)⁺.

N-9-[4-Phosphonic acid butyl]guanine (11a).^13,21,30Compound

11a was obtained in 58% yield according to literature. R_f= 0.18

(iPrOH/H₂O/NH₄OH 7/2/1). Purity (HPLC) > 98%. ¹H NMR

(D₂O): δ = 1.3 (t, 9H), 1.55−1.66 (m, 4H), 1.84−1.95 (m, 2H), 3.20

(m, 6H), 4.07 (m, 2H), 7.84 (s, 1H). ¹³C NMR (D₂O): δ = 20.22,

20.25, 26.7, 27.7 (CH₂), 30.2, 30.3, (CH₂P), 43.2, 46.6 (CH₂N), 115.8

(C₋₅), 140.1 (C₋₈), 151.4 (C₋₄), 153.6 (C₋₂), 158.8(C₋₆). ³¹P NMR

(D₂O): 25.7. MS (ESI) m/z 286.07 [M − HNEt₃]⁻; 288.09 [M + 2H-

HNEt₃]⁺. HRMS: calcd C₉H₁₅N₅O₄P [M + 2H − HNEt₃]⁺288.0862,

obs 288.0864.

(Z)-(4-(2-Amino-6-hydroxy-9H-purin-9-yl)but-1-en-1-yl)-

phosphonic Acid Disodic Salt (11b). The diethyl-(Z)-(4-(2-amino-6-

methoxy-9H-purin-9-yl)-but-1-en-1-yl)phosphonate (261 mg, 1 equiv)

was dissolved under argon atmosphere in anhydrous DMF (20 mL/

mmol) at 0 °C. TMSBr (6.6 equiv) was added dropwise to the solution,

and the mixture was kept at 0 °C for 5 min, then allowed to slowly warm

to room temperature and stirred for 3 days. The reaction progress was

followed by TLC monitoring (isopropanol/water/ammoniac 7/2/1 v/

v/v). The reaction was quenched by addition of a triethylbutylammo-

nium solution (1 M, pH 7). The volatiles were removed under vacuum,

and the resulting aqueous solution was freeze-dried. The crude was

puriﬁed on reverse phase ﬂash chromatography (water/methanol

gradient, 0 to 100% methanol), leading to the phosphonate

triethylammonium salts as a white powder. The compound was

percolated through a Na⁺Dowex resin, and after freeze-drying of the

require fractions the sodium salts were obtained as a white lyophilizate.

The desired compound (130 mg) was obtained in 54% yield. R_f= 0.18

(iPrOH/H₂O/NH₄OH 7/2/1). Purity (HPLC) > 96%. ¹H NMR (500

MHz, D₂O) δ 7.82 (s, 1H, H₋₈), 6.11 (ddt, J = 45.8, 13.0, 7.4 Hz, 1H,

CHCH₂), 5.83 (ddt, J = 17.7, 13.1, 1.6 Hz, 1H, CHP), 4.15 (t, J = 7.1

Hz, 2H, CH₂N), 2.93 (qdd, J = 7.0, 2.8, 1.5 Hz, 2H, CH₂CH). ¹³C

NMR (126 MHz, D₂O) δ 158.9 (C₋₆), 153.5 (C₋₂), 151.4 (C₋₅), 141.1

(CHCH₂), 140.1 (CH₋₈), 126.3 (d, J = 171.7 Hz, CHP), 115.9 (C₋₄),

42.8 (CH₂N), 30.2 (d, J = 21.8 Hz, 1C, CH₂CH). ³¹P NMR (202 MHz,

D₂O) δ 10.5. HMRS TOF ESI+ found: 286.0708. Calculated for

C₉H₁₃N₅O₄P: 286.0705 (M + H)⁺.

In Vivo Antimalarial Activity. Female Swiss NMRI mice (Charles

River Laboratories, France) were infected on day 0 by intravenous

injection into the caudal vein of 10⁷P. berghei ANKA-infected

erythrocytes (clone 2.34). Mice were treated once a day for four

consecutive days from day 1 by ip or oral route. Compound 5(R) was

dissolved in 0.9% NaCl or water (100 μL per mouse and 3 mice per

group) for ip or oral administration, respectively. Artesunate was

dissolved in DMSO (100 μL per mouse and 3 mice per group). Doses

were 0, 0.1, 0.3, 1, 3, 10, and 30 mg/kg for compound 5(R) and 0, 0.3, 1,

3, 9, and 27 mg/kg for artesunate by ip. By oral route, doses were 0, 1, 3,

10, and 30 mg/kg for compound 5(R). Control (untreated) group

P

https://dx.doi.org/10.1021/acs.jmedchem.0c00131

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

received only 0.9% NaCl or water. On day 5, parasitemia was counted

on Giemsa-stained thin blood smears and the dose leading to the

reduction of 50% of the parasitemia (ED₅₀) was determined.

Parasitemia was followed for 30 days.

The experiments done here conformed to the national and European

regulations and to French laws (EU Directive No. 86/609 modiﬁed by

the Directive 2010/63 regarding the Protection of Animals used for

Experimental and Other Scientiﬁc Purposes). The animal studies were

performed at the “Centre d’Elevage et de Conditionnement

ments. S.W. and R.C. designed the biological studies. S.W.

performed the biological experiments. All authors have given

approval to the ﬁnal version of the manuscript.

Funding

This work is part of the research project “NAN-Pal” supported

́

by the Region Occitanie and SATT AxLR.

Notes

The authors declare no competing ﬁnancial interest.

̀

Experimental des Modeles Animaux”, Montpellier, under Permission

No. E 34-172-23 (University of Montpellier) after approval by the

Animal Experimenting Commission (Project No. 01230.02).

ACKNOWLEDGMENTS

■

This work is part of the research project “NAN-Pal” supported

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00131.

■

́

by the Region Occitanie and SATT AxLR. We thank the Centre

sı

̀

d’Elevage et de Conditionnement Experimental des Modeles

Animaux (University of Montpellier, France) for providing

access to their facilities for animal studies, as well as the imaging

facility Montpellier Ressources Imagerie, member of the

national infrastructure France-Bioimaging supported by the

French National Research Agency (ANR-10-INBS-04, “Invest-

ments for the future”). We thank the technical excellence of

Mathilde Gens and Natacha Venturini.

Synthesis information and spectral data (NMR, MS and

HPLC) for ﬁnal compounds and additional ﬁgures for

NMR studies (PDF)

Spectral data (NMR) for intermediates (PDF)

Molecular formula strings and some data for ﬁnal

compounds (CSV)

ABBREVIATIONS USED

■

AcOH, acetic acid; ANP, acyclonucleosides phosphonate; Boc,

tert-butyloxycarbonyl; NO-BSA, N,O-bis(trimethyl)-

silylacetamide; CSA, chiral solvating agent; DEAD, diethylazo-

dicarboxylate; DEP, diethyl phosphite; DMF, N,N-dimethylfor-

mamide; DMSO, dimethyl sulfoxide; ED₅₀, dose required to

decrease parasitemia by 50%; ESI, electrospray ionization; H-

Asp-OH, aspartic acid; HGXPRT, hypoxanthine−guanine−

xanthine phosphoryl transferase; HPLC, high performance

liquid chromatography; HR-MS, high resolution mass spec-

trometry; IC₅₀, concentration required for 50% of inhibition of

the parasite growth; IMP, inosine 5′-monophosphate; ip,

intraperitoneal; LiHMDS, lithium hexamethyldisilazane;

MeOH, methanol; NMR, nuclear magnetic resonance; pD,

pH in deuterated media; Pf , Plasmodium falciparum; PNP,

purine nucleoside phosphorylase; RP, reverse phase chromatog-

raphy; rt, room temperature; SI, selectivity index; TEAB,

triethylammonium bicarbonate; TFA, triﬂuoroacetic acid; THF,

tetrahydrofuran; TLC, thin layer chromatography; TMSBr,

trimethylsilyl bromide; UV, ultraviolet

AUTHOR INFORMATION

■

Corresponding Authors

Rachel Cerdan − Laboratory of Pathogen Host Interactions

(LPHI), UMR 5235 UM-CNRS, Universite de Montpellier,

́

34095 Montpellier, France; Email: rachel.cerdan@

umontpellier.fr

Suzanne Peyrottes − Team Nucleosides & Phosphorylated

Eﬀectors, Institute for Biomolecules Max Mousseron (IBMM),

UMR 5247 UM-CNRS-ENSCM, Universite de Montpellier,

34095 Montpellier, France; orcid.org/0000-0003-1705-

0576; Email: suzanne.peyrottes@umontpellier.fr

́

Authors

Thomas Cheviet − Team Nucleosides & Phosphorylated

Eﬀectors, Institute for Biomolecules Max Mousseron (IBMM),

UMR 5247 UM-CNRS-ENSCM, Universite de Montpellier,

34095 Montpellier, France

́

Sharon Wein − Laboratory of Pathogen Host Interactions

́

(LPHI), UMR 5235 UM-CNRS, Universite de Montpellier,

34095 Montpellier, France

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J. Med. Chem. XXXX, XXX, XXX−XXX

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β-Hydroxy- A nd β-Aminophosphonate Acyclonucleosides as Potent Inhibitors of Plasmodium falciparum Growth

DOI: 10.1021/acs.jmedchem.0c00131

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