Structure-activity relationships of β-hydroxyphosphonate nucleoside analogues as cytosolic 5′-nucleotidase II potential inhibitors: Synthesis, in vitro evaluation and molecular modeling studies

European Journal of Medicinal Chemistry 77 (2014) 18e37

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European Journal of Medicinal Chemistry

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Original article

Structureeactivity relationships of b-hydroxyphosphonate nucleoside

analogues as cytosolic 5⁰-nucleotidase II potential inhibitors:

Synthesis, in vitro evaluation and molecular modeling studies

Maïa Meurillon ^a, Zsuzsanna Marton ^b, Audrey Hospital ^a, Lars Petter Jordheim ^c,

Jérôme Béjaud ^a, Corinne Lionne ^b, Charles Dumontet ^c, Christian Périgaud ^a,

,

Laurent Chaloin ^b, Suzanne Peyrottes ^a

*

^aInstitut des biomolécules Max Mousseron (IBMM), UMR 5247 CNRS e UM1 e UM2, Université Montpellier 2, cc1705, Place Eugène Bataillon,

34095 Montpellier cedex 5, France

^bCentre d’études d’agents pathogènes et biotechnologies pour la santé (CPBS), UMR 5236 CNRS e UM1 e UM2, 1919 route de Mende,

34293 Montpellier cedex 5, France

^cUniversité de Lyon 1, INSERM U1052 CNRS UMR 5286, Centre de Recherche en Cancérologie de Lyon (CRCL), Centre Léon Bérard, 69000 Lyon, France

a r t i c l e i n f o

a b s t r a c t

The cytosolic 5⁰-nucleotidase II (cN-II) has been proposed as an attractive molecular target for the

development of novel drugs circumventing resistance to cytotoxic nucleoside analogues currently used

for treating leukemia and other malignant hemopathies. In the present work, synthesis of b-hydrox-

yphosphonate nucleoside analogues incorporating modiﬁcations either on the sugar residue or the

nucleobase, and their in vitro evaluation towards the puriﬁed enzyme were carried out in order to

determine their potency towards the inhibition of cN-II. In addition to the biochemical investigations,

molecular modeling studies revealed important structural features for binding afﬁnities towards the

target enzyme.

Article history:

Received 22 July 2013

Received in revised form

10 January 2014

Accepted 22 February 2014

Available online 24 February 2014

Keywords:

Nucleotides

Enzyme inhibitors

Phosphonate

Leukemia

1. Introduction

obtain these effects, the phosphorylated forms (nucleotides) must

compete with endogenous nucleotides. While much effort has been

Enzymes are attractive biological targets for small-molecule

drug discovery, especially those involved in the nucleic acid

biosynthesis. Polymerases are key enzymes in these processes and

they have been extensively studied as targets for the treatment of

viral infections and cancers [1]. Thus, nucleos(t)ide analogues are

widely used as therapeutic agents because they mimic physiolog-

ical metabolites and interfere with key steps in viral particle pro-

duction and/or in cancer cell proliferation, respectively [2]. To

carried out for the identiﬁcation of novel analogues that are

intrinsically virotoxic or cytotoxic, very little is known regarding

the relationship between the size of the pools of endogenous

nucleos(t)ides and the therapeutic effect of these drugs. It is ex-

pected that their biological effect is directly related to the ability of

unnatural nucleotides to compete with the physiological ones,

thereby emphasizing the importance of mechanisms regulating

nucleotide pools [3,4]. Although the individual steps involved in

nucleoside transport and metabolism have been dissected and the

majority of the related proteins identiﬁed, the overall regulation of

nucleos(t)ide pools in mammalian cells remains unclear. Thus,

intracellular monophosphorylation of nucleoside analogues is

balanced by a family of enzymes called 5⁰-nucleotidases (5⁰-NTs: EC

3.1.3.5), which catalyze the hydrolysis of deoxyribo- and ribonu-

cleoside 5⁰-monophosphates into the corresponding nucleosides

and inorganic phosphate [3,5,6]. Among the eight members of this

family, our interest for the cytosolic 5⁰-nucleotidase II (cN-II, EC

3.1.3.5) arose from the fact that its expression level is of crucial

Abbreviations: AML, acute myeloid leukemia; cN-II, cytosolic 5⁰-nucleotidase II;

DMAP, 4-(dimethylamino)pyridine; DMF, N,N-dimethylformamide; DMSO, dime-

thylsulfoxide; IMP, inosine 5⁰-monophosphate; 5-NTs, 5⁰-nucleotidases; RNA,

ribonucleic acid; TMSBr, trimethylsilyl bromide; TMSCl, trimethylsilyl chloride;

TPPS, 4,4⁰,4⁰⁰,4⁰⁰-(porphine-5,10,15,20-tetrayl)tetrakis benzenesulfonic acid; Ts,

p-toluenesulfonyl.

* Corresponding author.

E-mail addresses: suzanne.peyrottes@univ-montp2.fr, peyrottes@univ-montp2.fr

(S. Peyrottes).

http://dx.doi.org/10.1016/j.ejmech.2014.02.055

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

19

interest for patients treated with nucleoside analogue-based

chemotherapy [7,8]. Indeed, a high level of expression of cN-II

mRNA in blasts is predictive of worse outcome in patients

receiving cytarabine-based regimens (a well-known nucleoside

analogue used to treat acute myeloid leukemia, AML) [9,10]. In

addition, the inhibition of cN-II expression by short hairpin RNA

was associated with the induction of apoptosis in human astrocy-

toma cells, suggesting that cN-II could be a therapeutic target in

brain tumors [11]. Finally, recent reports showed that hyperactive

mutated cN-II in relapsed children with acute lymphoblastic leu-

kemia, and treated with the antimetabolite 6-mercaptopurine, was

associated with a worse survival [12,13].

In light of the various structural, functional and regulatory

properties of this enzyme, we envisaged cN-II as an attractive target

for developing different types of inhibitors that could interfere with

protein function or regulation [14,15]. It must be highlighted that

cN-II is called high-K_mnucleotidase indicating that K_mvalue for its

favorite substrate, IMP (inosine 5⁰-monophosphate) is in the

millimolar range. Therefore, potential competitive inhibitors are

expected to exhibit K_ivalues in the same range. In addition, cN-II

acts as a tetramer (dimer of dimer) and possesses two regulatory

or effector sites. The effector site 1 is located closed to the IMP

binding site at the interface between two monomers and regulates

the enzyme activity (for instance, in presence of ATP, the k_catis

increased and K_mis lowered for IMP [16]. Several attempts to target

this site have been performed to ﬁnd other type of inhibitors (such

as non-competitive ones) but yet not successful. Our previous

studies led to the selection of a ﬁrst series of 5⁰-monophosphate

compounds 17e18), (ii) opening of the sugar ring (Fig. 1, com-

pounds 19e22).

As we previously reported the crucial role of a hydrophobic

clamp around the nucleobase for substrate recognition, that is

promoted by three protein residues Phe157, His209 and Tyr210

(Fig. 2) [15], we performed further modeling studies of compounds

2 and 4 within the active site of the enzyme. It revealed that a large

hydrophobic pocket was available near the nucleobase and there-

fore, we envisaged a second series of derivatives including non-

natural pyrimidines. The volume of this cavity has been esti-

mated to be w1200 Å³(Fig. 2) allowing various kind of modiﬁca-

tions at the C5 position of uracil and cytosine. Thus, we decided to

append lipophilic or aromatic substituents on the nucleobase

(Fig. 1, compounds 23aee and 24aee), that should allow additional

P

e

P

and/or hydrophobic interactions between the ligand and the

protein residues (especially Phe157 and farther Phe354, see Fig. 2).

We have synthesized and evaluated a series of thirty-two

b

-

hydroxyphosphonate derivatives as potential cN-II ligands to

determine their activity against cN-II and possible mechanisms of

inhibition. In addition, molecular modeling and docking studies

were performed to identify the interaction proﬁle of these com-

pounds, as well as key structural parameters responsible for their

properties.

2. Results and discussion

2.1. Chemistry

nucleoside analogues, such as

b-(S)-hydroxyphosphonate de-

Synthesis of altrofuranoside derivatives 5e8 and acyclophosph-

onates 19e22 was initiated from the previously obtained 1-[6-

deoxy-6-diethylphosphono-b-D-allofuranosyl] uracil 1 (Scheme 1)

rivatives (compounds 2 and 4, Fig. 1), able to interfere with the

hydrolysis of IMP by puriﬁed recombinant cN-II [15]. Thus, these

two derivatives were considered as a starting point for further

optimization and we elaborated several computer-based strategies

to improve their efﬁciency. First, we investigated the effect of the

following sugar modiﬁcations: (i) inversion of the C2⁰and C3⁰-

conﬁgurations (Fig. 1, compounds 5e16) or of the C5⁰stereo-

[17]. Inversionof the C2⁰conﬁgurationwas envisaged through awell-

known two-steps sequence [18] involving the formation of the 2,2⁰-

anhydro intermediate 29 andsubsequentopening of the nucleobase-

sugar ring. Thus, diethylphosphoester analogue 5 was reacted with

trimethylsilyl bromide affording the corresponding phosphonic acid

(as sodium salt after percolation on a Dowex ion-exchange resin) 6 in

low yield, due to puriﬁcation losses. From intermediate 5, the

chemistry i.e. the (R)-isomer of the b-hydroxyphosphonate (Fig. 1,

Fig. 2. Surface representation of the IMP binding site (PDB: 2XCW) in presence of the

cytosine derivative 4 obtained by molecular docking (starting point for optimization)

and showing a large cavity (delineated by the three residues F157, H209 and Y210)

surrounding the nucleobase. The magnesium ion is depicted as a green sphere and

secondary elements of cN-II are shown in gray. The contacts between derivative 4 and

ꢀ

the hydrophobic clamp are indicated in A. (For interpretation of the references to color

Fig. 1. Structure of the studied compounds.

in this ﬁgure legend, the reader is referred to the web version of this article.)

20

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

Scheme 1. Synthesis of

b

-D-altrofuranoside derivatives 5e8 and acyclophosphonates 19e22. Reagents & Conditions : a) Diphenylcarbonate, DMF then Na₂CO₃; b) NaOH, MeOH/

water; c) TMSBr, DMF then Dowex exchange (Na^þform); d) 1) N-Methyl pyrrolidine, TMSCl, (CF₃CO)₂O, CH₃CN then 4-nitrophenol 2) NH₄OH, dioxan; e) NaIO₄, dioxan/water then

NaBH₄.

cytosine analogue 7 was generated using a previously published

procedure [19,20] involving activation of the 4-keto group by 4-

nitrophenol in presence of N-methyl pyrrolidine, trimethylsilyl

chloride and triﬂuoroacetic anhydride, and then treatment with

ammoniacal solution. Removal of the phosphonate protecting

groups and isolation of derivative 8 was carried out using the same

procedure followed for 6.

exchange afforded acyclic analogues 20 and 22 in 54% and 24%

yields, respectively.

Synthesis of the -hydroxyphosphonate nucleoside analogues

b

with -gluco- and b-D-mannofuranoside conﬁgurations (Fig. 1,

b-D

compounds 9e12 and 13e16, respectively) were obtained using a

similar ex-chiral pool pathway as previously reported for the cor-

responding

b-D-allofuranoside derivatives [17]. This approach

Scission of the 2⁰,3⁰-bond of allofuranoside derivative 1 by so-

dium periodate oxidation followed by sodium borohydride reduc-

tion of the dialdehyde intermediate (not isolated) resulted in the

formation of the acyclic analogue 19 in quantitative yield [21]. The

latter was converted to the corresponding cytosine derivative 21 by

using the nitrophenylationeaminolysis procedure previously

described for compound 7. Finally, the ethyl phosphonate protect-

ing groups of compounds 19 and 21 were eliminated using TMSBr

and puriﬁcation by reverse phase chromatography and ionic

involved the preparation of an appropriate phosphonylated sugar

intermediate 36 (Scheme 2) and further glycosylation reaction with

the nucleobase. Thus, commercially available diacetone D-glucose

was converted into 3-benzylated derivative 30, which upon acidic

treatment provided the corresponding 5,6-diol 31 in 93% yield.

Then, selective tosylation of the primary alcohol was carried out,

followed by in situ nucleophilic substitution of this good leaving

group by iodine using sodium iodide in acetone, and compound 33

was obtained in 70% yield over the two steps.

Scheme 2. Synthetic pathways for intermediate 36. Reagents & Conditions: a) BnBr, KOH, DMSO; b) AcOH aq; c) TsCl, DMAP, pyridine; d) NaI, acetone; e) P(OEt)₃; f) BzCl, pyridine; g)

Ac₂O, H₂SO_4cat, AcOH.

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

21

The diethylphosphonate group was installed through

a

pyrimidines. As the substrate cavity was larger than the space

occupied by the natural nucleobase of the previously studied li-

gands (Fig. 2), we decided to introduce aromatic and hydrophobic

substituents in the C5 position of both compounds 2 and 4. Access

to such a kind of derivatives may readily be achieved using either

Sonogashira [23] or Suzuki [24] coupling reactions, that could be

MichaeliseArbuzov reaction leading to derivative 34, which was

ﬁrst benzoylated on position 5 and then peracetylated to afford 36

(Scheme 2), this two-step procedure being necessary to obtain the

required intermediate in 83% yield.

Compound 36 was successively engaged in glycosylation reac-

tion with either uracil or N-4-benzoylcytosine (Scheme 3), in

presence of an excess of tin(IV) chloride as previously reported [17],

and phosphonate nucleoside derivatives 37 and 38 were isolated in

73% and 65% yields, respectively. Removal of sugar protecting

groups was performed in one step from compound 37 using hy-

drogenation in presence of ammonium formate, leading to uracil

derivative 9. Whereas for cytosine analogue 38, two steps were

required due to the presence of the benzoyl group on the nucleo-

base, which necessitate the use of methanolic ammonia. Finally,

phosphonic acids 10 and 12 were obtained using the same proce-

dure as described above.

carried out from the corresponding

b-hydroxyphosphonate C5-

iodo-nucleoside intermediate. Furthermore, this synthetic

approach allows to introduce an aromatic group either directly in

the C5 position or through a two-methylene spacer that can be rigid

(triple bond) or somehow ﬂexible (double bond). Thus, treatment

of fully protected b-hydroxyphosphonate analogues 41 [17] with ICl

in dichloromethane afforded the corresponding protected 5-

iodouracil derivative 43 [25], whereas I₂/HIO₃in acetic acid and

tetrachloromethane [26] was used for cytosine derivative 42 and

gave rise to the intermediate 44 (Scheme 4). Removal of the sugar

protecting groups under basic conditions afforded compounds 45

and 46 in 84% and 97% yields, respectively. Under Sonogashira

conditions [23,27], reaction of the latter ones with n-butyl, tert-

butyl- or phenyl-acetylene, in the presence of Et₃N, CuI and

Pd(PPh₃)₂, produced derivatives 47aec and 48aec, respectively.

Following removal of the phosphonate protecting groups with

TMSBr, the corresponding 5-(n-butyl, or tert-butyl, or phenyl)-

ethynyl-nucleoside phosphonic acids 23aec and 24aec were ob-

tained, as their sodium salts, in yields ranging from 23% to 95%.

After the successful introduction of alkynyl groups at the C5

position, we extended the study to other hydrophobic phenyl and

(E)-phenylethenyl derivatives. Synthesis of the corresponding

compounds was accomplished using a Suzuki coupling reaction

from intermediates 45 and 46 [24]. Thus, treatment of these

compounds with phenyl and (E)-1-(2-phenyl)ethylene boronic

acids in presence of TPPS and PdCl₄Na₂led to the formation of the

required C5-substituted derivatives 49d,e and 50d,e in yields on a

range of 25e59%. Final step of deprotection of phosphonic acid

Synthesis of phosphonate nucleoside analogues 13 and 14

(Scheme 3) including D-mannose as sugar residue was performed

with a two-steps oxydationereduction procedure. First, selective

elimination of the 2⁰-acetyl group of compound 37 was carried out

in presence of aqueous hydrazine [22]. Thus, oxidation of the sec-

ondary alcohol of intermediate 39 was achieved using the Desse

Martin periodinane reagent. The 2⁰-keto-intermediate was not

isolated but directly reduced in presence of sodium borohydride,

affording derivative 40 in 55% yields over the two steps. Due to the

steric hindrance of the b-face of the nucleoside derivative, a com-

plete stereoselectivity of the reaction was observed. The ﬁnal

deprotection steps were carried out, leading successively to com-

pounds 13 and 14 in the uracil series. The corresponding cytosine

analogues 15 and 16 were obtained as reported above after nitro-

phenylationeaminolysis and deprotection steps, respectively.

The second part of our work was devoted to the synthesis of

hydroxyphosphonate nucleoside analogues incorporating modiﬁed

b-

Scheme 3. Synthesis of b-D-gluco- and b-D-mannofuranoside derivatives, 9e12 and 13e16. Reagents & Conditions : a) Uracil or N-4-benzoylcytosine, N,O-bis(trimethylsilyl)acet-

amide, CH₃CN then SnCl₄; b) NH₄CO₂H, Pd/C, MeOH from 37 to 1) BCl₃, CH₂Cl₂at ꢀ78 ^ꢁC, 2) NH₃sat, MeOH from 38; c) TMSBr, DMF then Dowex exchange (Na^þ); d) NH₂NH₂aq,

AcOH, pyridine; e) 1) Dess Martin reagent, CH₂Cl₂, 2) NaBH₄, CH₂Cl₂; f) BCl₃, CH₂Cl₂at ꢀ78 ^ꢁC; g) 1) N-Methyl pyrrolidine, TMSCl, (CF₃CO)₂O, CH₃CN then 4-nitrophenol 2) NH₄OH,

dioxan.

22

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

Scheme 4. Synthesis of C5-substituted derivatives 23aee and 24aee. Reagents & Conditions : a) Icl, CH₂Cl₂from 41; I₂, HIO₃, AcOH, CCl₄from 42; b) NH₃/MeOH; c) Alkynes,

Pd(PPh₃)₄, CuI, NEt₃, DMF; d) TMSBr, DMF then Dowex exchange (Na^þform); e) TPPS, PdNa₂Cl₄, KOH, boronic acids, H₂O/DMF.

functionality was performed as previously and targeted derivatives

23d,e, 24d,e were isolated as their sodium salts.

and 16 was weaker (48% and 35%, respectively) than the corre-

sponding allofuranose derivative 4 (85% at 1 mM). Interestingly, the

activity of compound 8 (altrofuranose) was slightly better than

compound 4, with K_ivalues of 0.98 and 1.74 mM respectively,

despite the inversion of the C2⁰-conﬁguration.

2.2. Nucleotidase activity inhibition assays

Surprisingly, the inversion of the C5⁰-conﬁguration (compounds

17 and 18) was also detrimental for the protein interaction as well

as the scission of the C2⁰eC3⁰bond (compounds 19e22), and all

those compounds showed a percentage of inhibition of the nucle-

otidase activity below 45%.

The potential inhibitory effect of the

b-hydroxyphosphonate

derivatives was evaluated using an enzyme activity assay with the

recombinant puriﬁed cN-II and IMP as substrate (Tables 1 and 2).

Best inhibitors, for which inhibition parameters are shown in bold

characters in Tables 1 and 2, were selected using the following

thresholds: inhibition at 1 mM > 50% or K_i< 2.5 mM. Fittings and

double reciprocal representations are shown in Fig. S1. As observed

in our previous studies [15], the inhibition was very weak (<20%,

Supporting Information) for derivatives bearing phosphonoester

groups such as compounds 1, 3, 5, 7, 9, 19 and 21. This result sup-

ported that interactions between the two oxygens of the phos-

phonate functionality and the magnesium ion present in the active

site are required for the enzyme inhibition.

In general, the cytosine-based analogues were more efﬁcient

cN-II inhibitors than their uracil-based counterparts. The parent

compounds 2 and 4 exhibited more than 70% of inhibition of the

nucleotidase activity at 1 mM. Their kinetics evaluation corrobo-

rated the inhibition observed with the rapid screening assay with K_i

values of 2.08 and 1.74 mM, respectively.

We then evaluated the effect of substituents at the C5 position

(Table 2). For several compounds, including phenyl-ethynyl or

ethenyl groups, the inhibition could not be measured using the

rapid assay as the green malachite reagent interfered with this type

of compounds. Consequently, kinetic studies were performed

allowing the determination of the inhibition constants for most of

the compounds and a few of them exhibited an inhibition below

45% (23a, 23e and 24a,b). Among this series of derivatives, marked

differences were observed between uracil- and cytosine-based

analogues. Within the uracil-based series, the presence of hin-

dered ethynyl groups (i.e. tert-butyl and phenyl) seemed to improve

the afﬁnity of the related derivatives for cN-II as compared to the

reference compound 2. Indeed, derivatives 23b, 23c and 23e were

able to inhibit the nucleotidase activity with K_iranging from 2.18 to

6 mM. In contrast, for cytosine-based compounds, almost all

modiﬁcations were detrimental when including an ethynyl spacer

(compounds 24aec). Whereas, derivative 24d (where the phenyl

group is directly attached to the C5 position) exhibited a K_iof 2 mM

similar to the reference compound 4. Only, compound 24e (with a

phenyl-ethenyl group) exhibited a lower K_ivalue (1.14 mM) than

the other compounds. It should be noted that this constitutes an

When comparing compounds within the uracil series (Table 1),

all derivatives 6 (altrofuranose), 10 (glucofuranose) and 14 (man-

nofuranose) showed a much lower activity than compound 2

(allofuranose). A similar tendency of the effect of sugar modiﬁca-

tions was also observed in the cytosine series. The modiﬁcation of

the C3⁰-stereocenter (either to glucofuranose or to mannofuranose)

seemed to be less advantageous as the inhibition of compounds 12

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

23

Table 1

Evaluation of the

modiﬁcation as cN-II inhibitors.

improvement in terms of inhibition as this derivative was found to

be more active than its parent compound 4 and as efﬁcient as de-

rivative 8 (altrofuranose series).

b-hydroxyphosphonate derivatives incorporating sugar-

Generic structure

Compound #

Inhibition

at 1 mM

(%)

K_i(mM)

2.3. Molecular modeling studies

Molecular docking of the studied derivatives was carried out to

deﬁne the essential structural elements leading to enzyme inhibi-

tion and compared to the ones already reported for parent com-

pounds 2 and 4 [15]. Brieﬂy, key residues of cN-II are involved in the

binding of the b-hydroxyphosphonate nucleoside analogues such

as: (i) Phe157, His209 and Tyr210 acting as tweezers around the

nucleobase, (ii) Glu161, Arg202 and Asn158 binding either the oxo

or the amino group of the pyrimidine bases, (iii) Asp54 and Asn250

bridging the b-hydroxyl group and ﬁnally the negatively charged

oxygens of the phosphonate group interacting with the magnesium

ion present in the active site.

Z]Y]H, X]W]OH, B]U

B]C

X]Z]H, W]Y]OH, B]U

B]C

Y]W]H, Z]X]OH, B]U

B]C

X]W]H, Z]Y]OH, B]U

X]W]H, Z]Y]OH, B]C

2

4

6

72 ꢂ 6

85 ꢂ 5

25 ꢂ 1

80 ꢂ 10

25 ꢂ 5

48 ꢂ 5

25 ꢂ 5

35 ꢂ 2

2.08 ꢂ 0.63

1.74 ꢂ 1.06

>100

8

0.98 ꢂ 0.24

10

12

14

16

n.d.

8.90 ꢂ 3.34

>50

11.1 ꢂ 10.9

When comparing the docking poses of compounds within the

uracil series (Fig. 3A), we clearly observed that for compounds 6

and 10 the

than for 2 and 14, thus weakening the hydrogen bonding with

Asp54 and Asn250. For compounds 2 and 14, the -hydroxyl group

b-hydroxyl group is oriented in the opposite direction

b

is located at the same place but the distance between the C4-

ꢀ

oxygen of the nucleobase and Arg202 increased (i.e. 3.18 A vs.

ꢀ

4.43 A, Fig. 3B) and a stronger interaction is favored for compound

2. These two observations corroborated the decreased efﬁciency of

all sugar-modiﬁed uracil derivatives.

B]U

B]C

17

18

43 ꢂ 6

42 ꢂ 8

n.d.

Concerning the cytosine-based derivatives (Fig. 3C and D), a

speciﬁc interaction can be observed between Ser251 and the 3⁰-

hydroxyl group of the sugar moiety, especially for compound 8.

Similarly, an electrostatic interaction between Lys215 and the 2⁰-

hydroxyl group may highlight the potential role of Lys215 in sta-

bilizing the cis orientated 2⁰,3⁰-diol of parent compound 4 (Fig. 3C).

Few structural features may explain the observed difference in

terms of efﬁciency between compounds 4 and 8. All interactions

were found to be stronger for derivatives 8 compared to 4 according

to the distances measured between the 2⁰- and the 3⁰-hydroxyl

groups and either Ser251 or Lys215, in addition these interactions

are strengthened by a better stacking with His209 for the analogue

8 (Fig. 3C). We hypothesized that the inversion of the C2⁰-conﬁg-

uration led to a change in the sugar puckering with concomitant

rotation of the nucleobase around the glycosidic bound, thus

moving this closer to His209. When comparing compounds 8 and

16, both compounds were found to superimpose except the 3⁰-

hydroxyl group, owing to the inversion of the C3⁰-conﬁguration

(Fig. 3D). This observation highlighted the importance of the sta-

bilization of the sugar counterpart through the interaction of the 3⁰-

hydroxyl group with Ser251. Finally, due to the increased ﬂexibility

of the acyclic derivative 22, the nucleobase (cytosine) is oriented in

a very different way than for the corresponding parent analogue 4

(Fig. 4A) weakening all interactions, except the hydrogen bond

between the 2⁰-hydroxyl and Lys215 that is preserved for both

compounds.

Then, we examined the effect of the nucleobase modiﬁcations

introduced in order to ﬁll in the protein cavity observed in the vi-

cinity of the substrate. Compound 23a (including an n-butyl group)

is accommodated by the protein without any steric hindrance, but

the end of the cavity being more polar (presence of Thr155 and

Asn158 residues) it probably resulted in less favored contacts

(Fig. 4B and C). In contrast, the compounds 23b and 23c (including a

shorter tail as a tert-butyl and phenyl groups, respectively) inter-

acted only with the hydrophobic part of the cavity (Fig. 4C and D).

The length of the “tail” anchored to the C5-position of the nucle-

obase must be ﬁne-tuned in order to ﬁll perfectly the hydrophobic

part of the cavity without extending too much, and avoiding to

B]U

B]C

20

22

20 ꢂ 4

45 ꢂ 5

n.d.

4.06 ꢂ 1.32

Bold value highlights the compounds of interest.

Table 2

Evaluation of the

modiﬁcations as cN-II inhibitors.

b-hydroxyphosphonate derivatives incorporating nucleobase-

Generic structure

Compound # clogP^a

Inhibition K_i(mM)

at 1 mM

(%)

X]OH, R]H

R]C^C-nBu

R]C^C-tBu

R]C^C-Ph

R]Ph

R]C]C-Ph

X]NH₂, R]H

R]C^C-nBu

R]C^C-tBu

R]C^C-Ph

R]Ph

2

23a

23b

23c

23d

23e

4

24a

24b

24c

24d

24e

L3.36 72 ꢂ 6

2.08 ꢂ 0.63

ꢀ0.97 45 ꢂ 3

n.d.

ꢀ1.21 n.d.^b

5.94 ꢂ 3.64

2.18 ꢂ 0.51

n.d.

3.36 ꢂ 1.74

1.74 ꢂ 1.06

n.d.

7.33 ꢂ 2.28

8.85 ꢂ 5.50

2.01 ꢂ 0.54

1.14 ꢂ 0.42

ꢀ1.68 n.d.^b

ꢀ1.31 24 ꢂ 7

ꢀ0.73 n.d.^b

L3.20 85 ꢂ 5

ꢀ0.81 40 ꢂ 10

ꢀ1.05 46 ꢂ 20

ꢀ1.52 n.d.^b

L1.16 60 ꢂ 10

R]C]C-Ph

ꢀ0.57 n.d.^b

Bold value highlights the compounds of interest.

a

clogP values were calculated using the Mol Inspiration software program.

Precipitate in presence of the chemical reagent used (green malachite).

b

24

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

Fig. 3. (A) Docking poses obtained by docking uracil-based compounds onto cN-II enzyme; namely compounds 2 (cyan), 6 (green), 10 (pink) and 14 (yellow), the black arrow

highlights the rocking motion of the -hydroxyl group. (B) Difference in the binding modes of compounds 2 (cyan) and 14 (yellow) and resulting interactions with R202 (weaker

b

interaction with 14). (C) Predicted binding for cytosine-based compounds 4 (blue) and 8 (orange) showing stronger interactions of compound 8 with protein residues S251 and

K215. (D) Comparison of the interactions of with S251 for analogues 8 (orange) and 16 (gray). Secondary structure of cN-II is shown as cartoon representation and phosphonate

analogues in sticks. The active site Mg^2þion is depicted as a green sphere. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version

of this article.)

reach the polar environment present further down the cavity. In

this respect, interactions of compounds 23ced were carefully

studied, as various positions of the aromatic substituent were

envisaged by introducing the phenyl group directly on the C5-

position and through a double or a triple bond. Interestingly, a

nice superimposition of the aromatic ring for compounds 23c and

23e was observed (Fig. 4D), ﬁtting the overall cavity and corrobo-

rating the improved efﬁcacy of such derivatives compared to other

uracil-based compounds.

As cytosine-based compounds were generally found to be more

efﬁcient in terms of enzyme inhibition than the uracil ones, the C5-

substitution of the nucleobase was performed with the same

groups and the resulting compounds 24aee were also analyzed by

docking. The compounds 24a (40% of inhibition) and 24c (K_ivalue

of 8.85 mM) modestly inhibited cN-II activity and seemed to behave

like 23e, showing the extension of the tail close to the polar end of

the cavity (Fig. 5A). In contrast, the derivative 24b exhibited a K_i

value of 7.33 mM, despite occupying only the apolar part of the

binding pocket as observed for 23b (K_ivalue of 5.94 mM). This

feature was not found with a phenyl-ethenyl group and binding

modes of the uracil-based compound 23e compared to the cytosine

analogue 24e were clearly different, conﬁrming that the nucleobase

location is also driven by the amino group of cytosine.

Remarkably, the maximum size of an “extended” binding group

for the nucleobase was accomplished with compound 24e (Fig. 5B)

that is shown to be able to interact with another part of the binding

pocket (without exiting). However, two alternatives binding poses

were observed for this derivative either shifted to the left or the

right side of the central groove connecting the active site with the

effector site 1 (Fig. 6). Each binding mode was representative as a

cluster of poses observed for both orientations and resulting

docking scores were almost identical. Interestingly, the most

important interactions with the protein were preserved (with

Asp206 and Lys215). Since it was impossible to conclude about the

most probable binding pose, a molecular dynamics simulation was

carried out using as starting point the energy minimized docking

poses. As shown in Fig. 6 (panels AeC) from snapshots of the

simulation, one conformation (blue one) of 24e exhibited higher

mobility than the other one (red). The root-mean square deviation

(RMSD) of all 24e atoms conﬁrmed this result as shown in Fig. 6D.

Indeed, the RMSD for one conformer (blue) increased continuously

with temperature without coming back to its basal level while it

ꢀ

was smaller (less than 1 A) for the other conformer (red). In addi-

tion, the high mobility of the blue conformer was also observed by

monitoring the distance between one residue of the protein (Pro

293) and the benzyl group of 24e. According to these simulations,

the derivative 24e should bind preferentially in the right side of

central cavity. This result is in agreement with other derivatives

bearing a phenyl group and that localize in these groove. Additional

simulations included ﬁve rounds of heating and cooling (simulated

annealing) provided very similar RMSD curves for the most mobile

conformer of this compound (Fig. S3). Because of the two different

binding poses predicted for this particular compound, we checked

Interestingly, within the cytosine-based series the smallest base

modiﬁcation (compound 24d including a phenyl group directly

attached to the C5-position) appears to be advantageous for a

tighter binding (Fig. 5B). Indeed, the phenyl group was found to ﬁt

the small hydrophobic cavity without overlapping to the polar side.

This last result corroborates the in vitro inhibition observed with

this compound (K_iabout 2 mM).

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

25

Fig. 4. (A) Comparison of binding poses obtained with compounds 4 (furanose ring) and 22 (acyclic). The arrow shows the ﬂip motion of the nucleobase for compound 22. (B)

Binding mode obtained with nucleobase-modiﬁed derivatives 23a and 23b. The parent compound 2 (cyan) and ATP (bound to effector site 1) are shown in stick model. (C) Same

derivatives as in (B) showing the hydrophobic pocket and including F157 (blue circle) and polar residues T155, N158 and H352 belonging to the same cavity (red circle). (D) Binding

poses of derivatives 23c (yellow), 23d (blue) and 23e (white) to compare the aromatic substitutions bearing a double or a triple bond. (For interpretation of the references to color in

this ﬁgure legend, the reader is referred to the web version of this article.)

if the inhibition mode was purely competitive (Fig. S2). The two

ﬁtting models giving the lowest reduced Chi²value were compet-

itive (Chi²¼ 5.81) and mixed type (Chi²¼ 6.66). However, the K_i⁰

value determined in the mixed type model was unrealistic

(540 ꢂ 1430 mM). The competitive mode was therefore conﬁrmed

for 24e.

extract a clear picture from this analysis like crucial residues as the

size of compound is variable. Nevertheless, it seemed that (for this

family of compounds) a few more interactions are favorable in

respect to the activity (Asn250 and Lys 292). In contrast, two resi-

dues appeared to have an opposite effect (based on the appearance

frequency in inactives derivatives), like Lys215 and Ser251.

The analysis of intermolecular interactions between cN-II resi-

dues and the derivatives was carried out by measuring all the

hydrogen bonds and van der Waals contacts (Table 3). From these

data, several key residues were always found like Asn 52, Asp54,

Phe157, His209 and Tyr210. However, it remained complicated to

3. Conclusion

We previously shown that derivative 4 can interfere with cN-II,

the cytosine containing analogue being equipotent in terms of

Fig. 5. (A) Overlay of binding modes obtained for the different cytosine-based modiﬁed compounds 24a (pink), 24b (brown), 24c (yellow), 24d (cyan) and 24e (green). (B)

Comparison of the best cytosine-based compounds 4 (dark blue), 8 (orange), 24d (cyan) and 24e (green). (For interpretation of the references to color in this ﬁgure legend, the

reader is referred to the web version of this article.)

26

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

Fig. 6. Discerning between the two alternative binding modes observed for compound 24e. Snapshots of the molecular dynamics simulations (simulated annealing) of the two

docking poses (depicted in blue or red) are shown at different times, 0 ps (A), 250 ps (B) and 680 ps (C). Secondary structure elements of cN-II are colored according to the starting

conformation of ligand (blue or red and depicted in stick model). Distances are indicated in angströms. (D) Root-mean square deviation of ligand atoms for each conformation (blue

and red lines), temperature is indicated as a green dash line. (E) Motion of 24e during simulation monitored by the distance between its terminal carbon atom and Pro 293 (as

shown in panels AeC). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

inhibition to the hypoxanthine and adenine counterparts [15].

Therefore, this derivative was used as starting point for an SAR

study. Additional assays and present results obtained for com-

pounds 1e22, while modest, suggested that only minor modiﬁca-

In conclusion, we report an in-depth SAR study of b-hydrox-

yphosphonate nucleoside analogues as cN-II inhibitors. cN-II is also

named high-K_mnucleotidase owing to the high substrate concen-

tration required foractivity (in the mM range), therefore the K_ivalues

of about 1 mM obtained for derivatives 8 and 24e are relevant in this

particular context. We have already validated the concept of using

cN-II inhibitors to sensitize cancer cells to cytotoxic nucleoside an-

alogues with compounds identiﬁed through virtual screening

(exhibiting a K_iof 1.25 mM), and being structurally different from

substrate analogues [28]. In addition, the medical relevance for the

development of such inhibitors has been conﬁrmed by recent ob-

servations [12,13], thus comforting our hypothesis on the interest of

cN-II inhibitors. As these molecules are too polar (clog P are shown in

Table 2) and ionized at physiological pH to cross effectively biological

membrane, their biological evaluation will need the development of

corresponding prodrugs to temporarily masked phosphonate group

[29], which constitutes the next step of this research project.

tion of the sugar counterpart is accepted and conﬁrmed the

b-

hydroxyphosphonate scaffold as a potential lead structure.

Modiﬁcation of the nucleobase is tolerated but no real

improvement in terms of inhibition efﬁciency could be observed

with such compounds (K_ivalues ranging from 1.14 to 8.85 mM).

Nevertheless, the size of the modiﬁcation is clearly important to

keep the binding inside the cavity without overlapping to the

effector site. Compound 24d (K_ivalue of 2.01 mM), including a

single phenyl group as substituent and compound 24e (phenyl-

ethenyl group, K_ivalue of 1.14 mM) were equally potent to deriv-

ative 4 (without any base modiﬁcation) and found to be the most

promising compounds for future drug developments. The reduc-

tion of the ﬂexibility by introducing a double or triple bond be-

tween the nucleobase and the substituent was not really efﬁcient

except for compound 24e for which the inhibition constant was

around 1 mM with a constrained orientation near an aromatic area

(privileged orientation according to the stronger interactions). In

addition, the volume of the cavity is not extensible and may only

accommodate small size substituent as several derivatives were

shown to extend outside of the cavity. Even so the enzyme is able to

undergo small domain motions during the nucleotidase reaction

(molecular dynamics simulations, data not shown) and leading to

little variations of the volume of the cavity, the size of substituent

group is rather limited. Overall, the SAR data indicate that there are

two exploitable regions near the nucleobase scaffold within the

protein active site. The ﬁrst is hydrophobic and the second is more

polar including protein residues like Thr155 and Asn158. Therefore,

the size and the nature of the residues forming the cavity are of

interest and render possible to consider novel modiﬁcations of the

nucleobase, considering as new osidic scaffold the best compound

8 for instance (K_ivalue of 0.98 mM).

4. Experimental section

4.1. Molecular docking of the b-hydroxyphosphonates analogues

Molecular docking was carried out using Gold 5.1 program

(Genetic Optimization for Ligand Docking, CCDC Software limited)

installed on an IBM blade center. First of all, the different com-

pounds were modeled using VegaZZ molecular modeling software

[30] and the atomic charges were assigned using the Gasteigere

Marsili empirical atomic partial charges [31,32]. The potential en-

ergy for all compounds was minimized in two steps (500 of

steepest descent followed by 5000 steps of conjugate gradient with

ꢀ

a gradient tolerance of 0.01 kcal/mol A). The crystal structure of

human cN-II (2XCW) solved in presence of IMP [33] was used for

docking and the other available structures with various ligands

bound to the protein (pdb codes: 2XJC, 2XJB, 2XCV and 2XCW) were

used for calibrating the docking procedure. During this step of

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

27

Table 3

Summary of the cN-II residues found to interact with the derivatives. The ionic interactions always presents between the phosphonate oxygens and magnesium ion are not

ꢀ

included in the table for clarity. The cutoff values for hydrogen bonds and van der Waals contacts were 3.5 A and 4.5 A, respectively. Residues in green reﬂect the most

encountered interactions with active compounds and in red the most unfavorable interactions found with inactive compounds.

structure selection and comparison, we noted that the Mg^2þion

was perfectly coordinated with aspartate residues, water molecules

and oxygen from IMP or GMP (in regard to the previous structure

2JC9 for instance). Hydrogen atoms were added using Gold prior to

ﬂexible docking of the chemical compounds. Then, 50 genetic al-

at least 10 A and the system was neutralized by adding Na^þor Cl^ꢀ

ions. Temperature (300 K) and pressure (1 atm) were kept con-

stants during the simulation using Langevin dynamics and Nosé-

ꢀ

Hoover Langevin piston [37,38]. A 12 A cutoff was used for the van

der Waals interactions with a switching function starting from 10 A.

The PME (Particle Mesh Ewald) algorithm [39] was used to calcu-

late long-range electrostatics with a grid spacing of 1 A. The po-

ꢀ

gorithms (GA) were performed with a radius of 15 A around the

ꢀ

magnesium ion used as target atom for docking. The water mole-

cules present in the crystal structures were retained and allowed to

spin around their oxygen atom. For each docked molecule, two

independent docking runs (50 GA, each) were performed using

Goldscore as scoring function. The different docking poses were

analyzed by the clustering method (complete linkage) from the

RMSD matrix of ranking solutions. The structural analysis and

visualization of docking poses was carried out using the Pymol

software [34]. The determination of the intermolecular interactions

between cN-II and the derivatives was achieved using LigPlotþ [35]

tential energy of the entire system was minimized for 50,000 steps

(time step of 1 fs) with the conjugate gradient method and the

CHARMM all-atom parameter set 27 [40] followed by a gradual

heating from 0 K to 300 K in 30 ps (increment of 10 K) under

constant temperature and pressure. The system was further

equilibrated for 100,000 steps before simulated annealing (from

300 K to 400 K) using a two steps “heatingecooling” procedure

(increment or decrement of 10 K by reinitializing the atom veloc-

ities with the Langevin temperature and piston). The cooling step

was achieved two times slower than the heating step with an

equilibration of 50 ps between both and a time step of 2 fs in

combination with the shake algorithm for water molecules. The

simulation trajectories were analyzed using VMD [41].

ꢀ

program with an upper cutoff value of 3.5 and 4.5 A for hydrogen

bonds and van der Waals contacts, respectively.

4.2. Molecular dynamics simulations of the derivative 24e

Simulations were carried out on an IBM cluster in explicit water

(TIP3P model) using the NAMD 2.9 software package [36] with

periodic boundary conditions. The two docking poses of derivative

24e obtained by docking were immersed in a cubic box ﬁlled with

water molecules with a distance between protein and box edges of

4.3. Evaluation of the inhibition of the nucleotidase activity by

hydroxyphosphonate analogues

b-

The nucleotidase activity was determined in the presence or in

the absence of -hydroxyphosphonate analogues using the N30

b

D

28

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

recombinant puriﬁed cN-II. Brieﬂy, the pET24b plasmid encoding

for cN-II with a 30 aminoacids deletion in N-terminal and a His tag

in C-terminal was used to transform E. coli BL21 cells. After growth

and induction, cN-II was puriﬁed using an ÄKTA-puriﬁer fast

protein liquid chromatography equipment (GE Healthcare) con-

nected to an His-Trap FF 5 mL column followed by a HiLoad 16/60

Superdex 75 gel ﬁltration column according to the procedure

described previously [15]. Two different enzyme activity assays

were used, either using the Green Malachite Phosphate Assay Kit

(Gentaur) in order to quantify the inorganic phosphate release

upon hydrolysis of IMP by cN-II, or the measurement of the ki-

netics rate constants of cN-II at different IMP concentrations. For

analysis or for few compounds by HR-MS and HPLC analysis (at

254 nm, purity proved to be ꢃ 95%).

Starting nucleotides 1, 41 and 42 were obtained according pre-

viously published procedure [17].

4.4.1. General procedure for diethylphosphonate removal

(compounds 6, 8, 10, 12, 14, 16, 20, 22, 23aee, 24aee)

The protected phosphonate (1 eq.) was dissolved in anhydrous

DMF (20 mL/mmol) and trimethylsilyl bromide (10e15 eq.) was

added dropwise at 0 ^ꢁC. The reaction mixture was stirred at room

temperature until completion of the reaction was indicated by TLC

(7% Isopropanol: 2% water: 1% ammonia). Then, the reaction was

stopped by adding triethylammonium bicarbonate (TEAB 1M, pH 7)

and concentrated to dryness under high vacuum. Column chro-

matography of the crude materials on reverse phase (gradient:

water to acetonitrile 50%) gave the expected phosphonic acid (as

triethylammoium salt), which was passed through a Dowex Na^þ

ion exchange column, the desired fractions were collected and

lyophilized leading to the title compound as sodium salt.

the ﬁrst assay, cN-II was added to a ﬁnal concentration of 0.1

80 L of buffer containing 50 mM imidazole pH 6.5, 500 mM NaCl,

10 mM MgCl₂and 1 mM DTT on ice and the reaction was started

by addition of the substrate (50 M of IMP with an equimolar

concentration of Mg^2þ) and incubated at 37 ^ꢁC for 5 min. The

reaction was stopped by adding 20 L of Green Malachite Reagent

mM in

m

and free phosphate was quantiﬁed by measuring the absorbance

above 630 nm using a Tecan plate reader (Sunrise) and compari-

son with a 0e50

mM phosphate calibration curve. A second ki-

4.4.1.1. 1-[6-Deoxy-6-phosphono-

b

-D-altrofuranosyl]uracil

(diacid

netics assay was required for conﬁrming the inhibition observed

with the Green Malachite Phosphate assay kit and also to evaluate

the inhibition of compounds that react with the Green colorant.

salt) (6). Treatment of compound 5 (350 mg, 0.89 mmol) following

general procedure gave rise to derivative 6 contaminated by trie-

thylammonium salts. HPLC puriﬁcation using a Hypercarb analyt-

ical column only gave rise to a small quantity of the expected

Therefore, the enzyme (0.1

mM) and substrate (IMP, varying from

200 M to 3 mM) freshly prepared in the same buffer as above

m

product (9 mg, 26%). ¹H NMR (400 MHz, D₂O)

d

¼ 7.75 (d, J ¼ 8.13

were mixed in a thermostatically controlled beaker with magnetic

stirring. The reaction was stopped at different times by mixing

with 10% perchloric acid. Quantiﬁcation of inosine and IMP pre-

sent in the reaction mixtures quenched at different times was

performed by HPLC (Waters) using a Partisphere 5-SAX column

(Whatman) and 15 mM ammonium phosphate buffer pH 5.5 as

mobile phase. The raw data were ﬁtted with an equation for

competitive inhibition using GraFit 7 (Erithacus software) in order

to determine the K_ivalues.

Hz, 1H, H-6), 6.05 (d, J ¼ 4.68 Hz, 1H, H-1⁰), 5.75 (d, J ¼ 8.11 Hz, 1H,

H-5), 4.25e4.15 (m, 2H, H-2⁰, H-3⁰), 4.15e4.05 (m, 1H, H-5⁰), 3.7 (t,

J ¼ 4.55 Hz, 1H, H-4⁰), 1.9e1.6 (m, 2H, H-6⁰, H-6⁰⁰). ¹³C NMR

(100 MHz, D₂O)

d

¼ 166.3 (C-4), 15.4 (C-2), 143.3 (C-6), 101.0 (C-5),

85.8e85.6 (C-4⁰, d, J ¼ 8 Hz), 85.1 (C-1⁰), 75.8 (C-2⁰), 74.6 (C-3⁰), 67.0

(C-5⁰), 32.2e30.9 (C-6⁰, d, J ¼ 130 Hz). ³¹P NMR (121 MHz, D₂O)

d

¼ 20.6. MS FAB > 0 (NBA) m/z 339 (M-2Na þ 3H)^þ. FAB<0 (NBA)

m/z 383 (MꢀH)^-, 359 (M ꢀ Na)^ꢀ, 337 (M ꢀ 2NaeH)e UV

l_max¼ 263 nm (ε_max¼ 9500) (EtOH 95). Calculated for C₁₀H₁₃N₂

Na₂O₉P, 2H₂O: C, 28.72; H, 4.4; N, 6.7. Found: C, 28.65; H, 4.80; N,

6.64.

4.4. Chemical synthesis

Tetrahydrofuran (THF) was distilled from sodium/benzophe-

none immediately prior to use, DMF and methanol from CaH₂,

dichloromethane from P₂O₅, pyridine and Et₃N from KOH. Solids

were dried over P₂O₅under reduced pressure at rt. Moisture sen-

sitive reactions were performed under argon atmosphere using

oven-dried glassware. ¹H NMR spectra were recorded at 300 MHz

and ¹³C NMR spectra at 75 MHz with proton decoupling at 25 ^ꢁC

4.4.1.2. 1-[6-Deoxy-6-phosphono-b-D-altrofuranosyl]cytosine (diso-

dium salt) (8). Treatment of compound 7 (174 mg, 0.442 mmol)

following general procedure gave rise to the expected derivative 8

(90 mg, 53%). Rf (iPrOH/H₂O/NH₄OH, 7/2/1, v/v) 0.09. ¹H NMR

(400 MHz, D₂O)

d

¼ 7.75 (d, J ¼ 7.56 Hz,1H, H-6), 6.10 (d, J ¼ 4.32 Hz,

1H, H-1⁰), 5.95 (d, J ¼ 7.55 Hz, 1H, H-5), 4.3e4.2 (m, 2H, H-2⁰, H-3⁰),

4.2e4.1 (m, 1H, H-5⁰), 3.85 (t, J ¼ 4.54 Hz, 1H, H-4⁰), 1.85e1.2 (m, 2H,

using a Bruker 300 Advance. Chemical shifts are given in

d

values

H-6⁰, H-6⁰⁰). ¹³C NMR (100 MHz, D₂O)

d

¼ 168.1 (C-4), 157.2 (C-2),

referenced to the residual solvent peak (CHCl₃at 7.26 and 77 ppm

or DMSO-d₅at 2.49 and 39.5 ppm) relative to TMS. COSY experi-

ments were performed in order to conﬁrm proton assignments.

Coupling constants, J, are reported in Hertz (Hz). 2D ¹He³C heter-

onuclear COSY spectra were recorded for the attribution of ¹³C

signals. ³¹P NMR spectra were recorded at ambient temperature at

121 MHz with proton decoupling. Chemical shifts are reported

relative to external H₃PO₄. FAB mass spectra were recorded in the

positive-ion or negative-ion mode on a JEOL JMS DX 300 using

thioglycerol/glycerol (1:1, v/v, G-T) or NBA (nitrobenzylalcohol) as

matrix. ESI Mass and High Resolution Mass spectra were recorded

in the positive or negative-ion mode on a Micromass Q-TOF. UV

spectra were recorded on an Uvikon 931 (Kontron). Elemental an-

alyses were carried out by the Service de Microanalyses du CNRS,

Division de Vernaison (France). TLC was performed on pre-coated

aluminum sheets of silica gel 60 F₂₅₄(Merck, Art. 9385), visuali-

zation of products being accomplished by UV absorbance followed

by charring with 5% ethanolic sulfuric acid and then heating. The

purity of all tested compounds was determined by elemental

143.0 (C-6), 95.2 (C-5), 86.0e85.9 (C-4⁰, d, J ¼ 9 Hz), 85.9 (C-1⁰), 75.6

(C-2⁰), 75.2 (C-3⁰), 67.0 (C-5⁰, d, J ¼ 3 Hz), 32.3e31.0 (C-6⁰, d,

J ¼ 131 Hz). ³¹P NMR (81 MHz, D₂O)

d

¼ 21.9 MS FAB > 0 (GT) m/z

382 (M þ 3H)^þ, 338 (M ꢀ 2Na þ 3H)^þ. FAB < 0 (GT) m/z 358

(M ꢀ Na)^ꢀ, 336 (M ꢀ 2Na)^ꢀ. UV l_max¼ 273 nm (ε_max¼ 14,100)

(EtOH 95). Calculated for C₁₀H₁₄N₃O₈PNa₂, 1.5H₂O: C, 29.42; H,

4.60; N, 10.29. Found: C, 29.51; H, 4.90; N, 9.93.

4.4.1.3. 1-[6-Deoxy-6-phosphono-

b-

D-glucofuranosyl]uracil

(diso-

dium salt) (10). Treatment of compound 9 (290 mg, 0.736 mmol)

following general procedure gave rise to the expected derivative

(200 mg, 71%). Rf (iPrOH/H₂O/NH₄OH, 7/2/1, v/v) 0.11. ¹H NMR

(400 MHz, D₂O)

d

¼ 7.87 (d, J ¼ 8.16 Hz,1H, H-6), 5.75 (d, J ¼ 8.13 Hz,

1H, H-5), 5.7 (d, J ¼ 0.74 Hz, 1H, H-1⁰), 4.35e4.25 (m, 1H, H-5⁰), 4.2

(dd, J ¼ 1.03e3.06 Hz, 1H, H-3⁰), 4.15 (s, 1H, H-2⁰), 4.1 (dd, J ¼ 3.04e

7.72 Hz, 1H, H-4⁰), 2.1e1.7 (m, 2H, H-6⁰, H-6⁰⁰). ¹³C NMR (100 MHz,

D₂O)

d

¼ 166.5 (C-4), 151.5 (C-2), 142.4 (C-6), 100.9 (C-5), 91.7 (C-1⁰),

85.9e85.8 (C-4⁰, d, J ¼ 14 Hz), 80.4 (C-2⁰), 74.1 (C-3⁰), 65.3e65.2 (C-

5⁰, d, J ¼ 4 Hz), 33.4e32.1 (C-6⁰, d, J ¼ 131 Hz). ³¹P NMR (81 MHz,

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

29

D₂O)

d

¼ 20.9. MS FAB > 0 (GT) m/z 383 (M þ 3H)^þ, 361

3⁰), 31.5, 30.2 (C-6⁰, d, J ¼ 130 Hz). ³¹P NMR (121 MHz, D₂O)

d

¼ 21.7.

(M ꢀ 2Na þ 2H)^þ. FAB < 0 (GT) m/z 359 (M ꢀ Na)^ꢀ, 337

(M ꢀ 2Na þ H)^ꢀ. UV l_max¼ 263 nm (ε_max¼ 10,200) (EtOH 95).

Analysis Calculated for C₁₀H₁₃N₂O₉PNa₂, 3.5H₂O: C, 26.98; H, 4.53;

N, 6.29. Found: C, 26.91; H, 4.10; N, 5.91.

MS FAB > 0 (NBA) m/z 341 (M ꢀ 2Na þ H)^þ. FAB < 0 (NBA) m/z 339

(M ꢀ 2Na þ H)^ꢀ. UV l_max¼ 262 nm (ε_max¼ 10,100) (EtOH 95).

Calculated for C₁₀H₁₅N₂O₉PNa₂, 0.7H₂O: C, 30.41; H, 4.43; N, 7.09.

Found: C, 30.25; H, 4.79; N, 6.87.

4.4.1.4. 1-[6-Deoxy-6-phosphono-

b

-D

-glucofuranosyl]cytosine (mon-

4.4.1.8. 1-(5S)-[6-deoxy-6-phosphono-2,3-seco-b-D-ribohexofur-

osodium salt) (12). Treatment of compound 11 (350 mg,

0.89 mmol) following general procedure gave rise to the expected

derivative (90 mg, 27%). Rf (iPrOH/H₂O/NH₄OH, 7/2/1, v/v) 0.16. ¹H

anosyl]cytosine (disodium salt) (22). Treatment of compound 21

(520 mg, 1.31 mmol) following general procedure gave rise to the

derivative 22 as hygroscopic lyophilisate (120 mg, 24%). Rf (iPrOH/

NMR (400 MHz, D₂O)

d

¼ 7.60 (d, J ¼ 8.1 Hz, 1H, H-6), 5.68 (d,

NH₄OH/H₂O, 7/2/1, v/v/v) 0.13. ¹H NMR (400 MHz, D₂O)

d

¼ 7.49 (d,

J ¼ 8.0 Hz, 1H, H-5), 5.42 (s, 1H, H-1⁰), 4.0e3.8 (m, 4H, H-5⁰, H-3⁰, H-

J ¼ 7.5 Hz, 1H, H-6), 5.80 (d, J ¼ 7.5 Hz, 1H, H-5), 5.70 (t, J ¼ 5.37 Hz,

1H, H-1⁰), 3.58 (m, 1H, H-5⁰), 3.50 (m, 4H, H-3⁰, H-3⁰⁰, H-2⁰, H-2⁰⁰),

3.20 (m, 1H, H-4⁰), 1.2e1.6 (m, 2H, H-6⁰, H-6⁰⁰). ¹³C NMR (100 MHz,

2⁰, H-4⁰), 2.0e1.5 (m, 2H, H-6⁰, H-6⁰⁰). ¹³C NMR (100 MHz, D₂O)

d

¼ 165.6 (C-4), 156.5 (C-2), 142.4 (C-6), 94.9 (C-5), 92.5 (C-1⁰),

86.0e85.8 (C-4⁰, d, J ¼ 14 Hz), 80.5 (C-2⁰), 74.1 (C-3⁰), 65.2e65.1 (C-

D₂O)

d

¼ 165.8 (C-4), 157.8 (C-2), 142.4 (C-6), 96.4 (C-5), 84.1 (C-1⁰),

5⁰, d, J ¼ 4.7 Hz), 33.7e31.9 (C-6⁰, d, J ¼ 132 Hz). ³¹P NMR (81 MHz,

82.2e82.0 (C-4⁰, d, J ¼ 14 Hz), 67.0 (C-5⁰, d, J ¼ 7 Hz), 62.0, 59.4 (C-2⁰

D₂O)

d

¼ 21.7. MS FAB > 0 (GT) m/z 360 (M þ H)^þ, 338

and C-3⁰), 31.7, 30.4 (C-6⁰, d, J ¼ 140 Hz). ³¹P NMR (121 MHz, D₂O)

(M ꢀ Na þ H)^þ. FAB<0 (GT) m/z 336 (M ꢀ Na)^ꢀ. HR-MS Calculated

for C₁₀H₁₇N₃O₈P: 338.0753; Found: 338.0759. UV l_max¼ 2671 nm

(ε_max¼ 10,700) (H₂O). Analysis Calculated for C₁₀H₁₅N₃O₈PNa: C,

26.98; H, 4.53; N, 6.29. Found: C, 19.16; H, 2.95; N, 6.62.

d

¼ 20.5. MS FAB > 0 (NBA) m/z 679 (2M ꢀ 2Na þ H)^þ, 340

(M ꢀ 2Na þ H)^þ. FAB<0 (NBA) m/z 339 (M ꢀ 2Na þ H)^ꢀ. UV

l_max¼ 272 nm (ε_max¼ 9500) (EtOH 95) Calculated for C₁₄H₂₆N₃O₈P,

0.55H₂O: C, 41.49; H, 6.74; N, 10.37. Found: C, 41.49; H, 6.71; N,

10.30.

4.4.1.5. 1-[6-Deoxy-6-phosphono-b-D-mannofuranosyl]uracil (diso-

dium salt) (14). Treatment of compound 13 (200 mg, 0.507 mmol)

following general procedure gave rise to the expected derivative

(92 mg, 51%). Rf (iPrOH/H₂O/NH₄OH, 7/2/1, v/v) 0.12. ¹H NMR

4.4.1.9. 1-[6-Deoxy-6-phosphono-b-D-allofuranosyl]-5-hexyn-1-yl-

uracil (disodium salt) (23a). Treatment of compound 47a (80 mg,

0.18 mmol) following general procedure gave rise to the expected

derivative (73 mg, 88%). Rf (iPrOH/NH₄OH/H₂O, 7/2/1, v/v/v) 0.10.

(400 MHz, D₂O)

d

¼ 7.96 (d, J ¼ 8.4 Hz, 1H, H-6), 6.13 (d, J ¼ 6.9 Hz,

1H, H-1⁰), 5.80 (d, J ¼ 8.1 Hz, 1H, H-5), 4.65e4.58 (m, 1H, H-2⁰),

¹H NMR (300 MHz, DMSO-d₆)

d

¼ 8.38 (s, 1H, H-6), 5.85 (d,

4.38e4.31 (m, 2H, H-3⁰, H-5⁰), 3.90e3.82 (m, H-4⁰), 2.1e1.7 (m, 2H,

J ¼ 5.70 Hz, 1H H-1⁰), 4.07 (m, 3H, H-2⁰, H-3⁰, H-5⁰), 3.89 (m, 1H, H-

H-6⁰, H-6⁰⁰). ¹³C NMR (100 MHz, D₂O)

d

¼ 166.4 (C-4), 151.9 (C-2),

4⁰), 2.42 (t, J ¼ 6.75 Hz, 2H, CH₂), 1.56 (m, 6H, CH₂, H-6⁰, H-6⁰⁰), 0.90

143.8 (C-6), 100.8 (C-5), 84.4 (C-1⁰), 82.8e82.6 (C-4⁰, d, J ¼ 14 Hz),

(t, 3H, CH₃). ¹³C NMR (75 MHz, DMSO-d₆)

d

¼ 162.1 (C-4), 150.2 (C-

71.7 (C-2⁰), 69.0 (C-3⁰), 65.8e65.7 (C-5⁰, d, J ¼ 4 Hz), 33.2e31.5 (C-6⁰,

2), 143.3 (C-6), 99.6 and 93.6 (C^C), 89.2 (d, J ¼ 14.42 Hz, C-4⁰), 87.6

(C-1⁰), 75.0 (C-2⁰), 73.1 (C-3⁰), 68.7 (C-5⁰), 67.2 (C-5), 30.6 (C-6⁰, CH₂).

21.7 (CH₂), 18.8 (CH₂), 13.8 (CH₃). ³¹P NMR (81 MHz, DMSO-d₆)

d, J ¼ 130 Hz). ³¹P NMR (81 MHz, D₂O)

¼ 21.2. MS FAB > 0 (GT) m/z

d

361 (M ꢀ Na þ 2H)^þ, 339 (M ꢀ 2Na þ 3H)^þ. FAB < 0 (GT) m/z 359

(M

ꢀ

Na)^ꢀ, 337 (M

ꢀ

2Na

þ

H)^ꢀ. HR-MS Calculated for

d

¼ 18.6. MS ESI-QT of >0 m/z 837 (2M þ H)^þ, 419 (M þ H)^þ. HR-MS

C

₁₀H₁₆N₂O₉P: 339.0593; Found: 339.0586. UV l_max¼ 263 nm

Calculated for C₁₆H₂₄N₂O₉P: 419.1219; Found: 419.1208. UV (EtOH

(ε_max¼ 10,600) (H₂O). Analysis Calculated for C₁₀H₁₃N₂O₉PNa₂,

3.5H₂O: C, 26.98; H, 4.53; N, 6.29. Found: C,19.75; H, 3.10; N, 4.35; P,

4.92.

95) l_max¼ 293 nm (ε_max¼ 9200), l_max¼ 238 nm (ε_max¼ 9300).

4.4.1.10. 1-[6-Deoxy-6-phosphono-b-D-allofuranosyl]-5-(3,3-dimethyl-

1-butyn-1-yl)-uracil (disodium salt) (23b). Treatment of compound

47b (114 mg, 0.26 mmol) following general procedure gave rise to the

expected derivative (112 mg, 95%). Rf (iPrOH/NH₄OH/H₂O, 7/2/1, v/v/

4.4.1.6. 1-[6-Deoxy-6-phosphono-b-D-mannofuranosyl]cytosine

(disodium salt) (16). Treatment of crude compound 15 (46 mg,

0.09 mmol) following general procedure gave rise to the expected

derivative (14 mg, 37%). Rf (iPrOH/H₂O/NH₄OH, 7/2/1, v/v/v) 0.12. ¹H

v) 0.07. ¹H NMR (300 MHz, D₂O)

d

¼ 8.04 (s, 1H, H-6), 5.75 (d,

J ¼ 5.43 Hz, 1H, H-1⁰), 4.18 (m, 1H, H-3⁰), 4.09 (m, 2H, H-2⁰, H-5⁰), 3.99

NMR (400 MHz, D₂O)

d

¼ 8.01 (d, J ¼ 7.57 Hz, 1H, H-6), 6.22 (d,

(m, 1H, H-4⁰), 1.66 (m, 2H, H-6⁰, H-6⁰⁰), 1.11 (s, 9H, C(CH₃)₃). ¹³C NMR

J ¼ 6.74 Hz, 1H, H-1⁰), 6.06 (d, J ¼ 7.55 Hz, 1H, H-5), 4.67 (m, 1H, H-

(75 MHz, D₂O)

d

¼ 164.7 (C-4), 151.0 (C-2), 143.6 (C-6), 104.5 and

2⁰), 4.44 (m, 2H, H-3⁰, H-5⁰), 3.92 (m, H-4⁰), 2.01e1.71 (m, 2H, H-6⁰,

100.1 (C^C), 88.1 (C-1⁰), 87.3 (d, J ¼ 13.13 Hz, C-4⁰), 73.9 (C-2⁰), 69.4

H-6⁰⁰). ¹³C NMR (100 MHz, D₂O)

d

¼ 166.4 (C-4), 158.1 (C-2), 143.7

(C-5⁰), 68.1 (C-5), 7.2 (C-3⁰), 31.8 (d, J ¼ 127.5 Hz, C-6⁰), 29.9 (C(CH₃)₃).

(C-6), 95.3 (C-5), 85.0 (C-1⁰), 83.0 (C-4⁰, d, J ¼ 14 Hz), 71.9 (C-2⁰), 69.6

³¹P NMR (81 MHz, D₂O)

d

¼ 19.0. MS ESI-QT of >0 m/z 419 (M þ H)^þ,

(C-3⁰), 66.4 (C-5⁰, d, J ¼ 4 Hz), 33.3 (C-6⁰, d, J ¼ 130 Hz). ³¹P NMR

441 (M þ Na)^þ. MS ESI-QT of <0 m/z 417 (MꢀH)^-. HR-MS Calculated

(81 MHz, D₂O)

d

¼

19.4. MS ESI-QT of >0 m/z 338.2

for C₁₆H₂₄N₂O₉P: 419.1219; Found: 419.1228. UV (EtOH 95)

(M ꢀ 2Na þ 3H)^þ, 360.0 (M ꢀ Na þ 2H)^þ. MS ESI-QT of <0 m/z 336.3

(M ꢀ 2Na þ H)^ꢀ. HR-MS Calculated for C₁₀H₁₆N₃O₈PNa: 360.0573;

Found: 360.0559. UV l_max¼ 270 nm (ε_max¼ 7900) (H₂O).

l_max¼ 292 nm (ε_max¼ 6600), l_max¼ 228 nm (ε_max¼ 6700).

4.4.1.11. 1-[6-Deoxy-6-phosphono- -allofuranosyl]-5-phenyle-

b-D

thynyl-uracil (disodium salt) (23c). Treatment of compound 47c

(175 mg, 0.35 mmol) following general procedure gave rise to the

expected derivative (133 mg, 77%). Rf (iPrOH/NH₄OH/H₂O, 7/2/1, v/v/

4.4.1.7. 1-(5S)-[6-Deoxy-6-phosphono-2,3-seco-b-D-ribohexofur-

anosyl]uracil (disodium salt) (20). Treatment of compound 19

(300 mg, 0.75 mmol) following general procedure gave rise to the

derivative 20 as hygroscopic lyophilisate (138 mg, 54%). Rf (EtOAc/

v) 0.18.¹H NMR (400 MHz, D₂O)

d

¼ 8.29 (s,1H, H-6), 7.53 (m, 2H, He

Ar), 7.39 (m, 3H, HeAr), 5.90 (d, J ¼ 5.26 Hz, 1H, H-1⁰), 4.37e4.25 (m,

MeOH, 5/5, v/v) 0.12.¹H NMR (400 MHz, D₂O)

d

¼ 7.80 (d, J ¼ 8.05 Hz,

3H, H-2⁰, H-3⁰, H-5⁰), 4.14 (m, 1H, H-4⁰), 1.90 (m, 2H, H-6⁰, H-6⁰⁰). ¹³C

1H, H-6), 5.95 (t, J ¼ 5.63 Hz, 1H, H-1⁰), 5.87 (d, J ¼ 8.06 Hz, 1H, H-5),

3.95 (m, 1H, H-5⁰), 3.85e3.65 (m, 4H, H-3⁰, H-3⁰⁰, H-2⁰, H-2⁰⁰), 3.55 (m,

1H, H-4⁰), 1.8e1.5 (m, 2H, H-6⁰, H-6⁰⁰). ¹³C NMR (100 MHz, D₂O)

NMR (100 MHz, D₂O)

d

¼ 164.2 (C-4), 150.7 (C-2), 144.1 (C-6), 131.5,

129.1, 128.7, 121.8 (CeAr), 100.0 and 94.0 (C^C), 88.6 (C-1⁰), 87.4 (d,

J ¼ 13.48 Hz, C-4⁰), 79.9 (C-5), 74.2 (C-2⁰), 68.4 (C-3⁰), 67.2 (d,

J ¼ 2.69 Hz, C-5⁰), 32.7 (d, J ¼ 132.0 Hz, C-6⁰). ³¹P NMR (81 MHz, D₂O)

d

¼ 166.3 (C-4),152.3 (C-2),142.4 (C-6),102.4 (C-5), 83.9 (C-1⁰), 82.5e

82.3 (C-4⁰, d, J ¼ 13 Hz), 67.2 (C-5⁰, d, J ¼ 4 Hz), 61.7, 59.6 (C-2⁰and C-

d

¼ 20.7. MS ESI-QT of >0 m/z 439.1 (M ꢀ 2Na þ 3H)^þ. MS ESI-QT of

30

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

<0 m/z 437.2 (M ꢀ 2Na þ H)^ꢀ. HR-MS Calculated for C₁₈H₁₈N₂O₉P:

437.0750; Found: 437.0770. UV (H₂O) l_max¼ 263 nm (ε_max¼ 15,195),

l_max¼ 277 nm (ε_max¼ 14,182), l_max¼ 305 nm (ε_max¼ 18,041).

(Cq). ³¹P NMR (81 MHz, D₂O)

d

¼ 20.6. MS ESI-QT of >0 m/z 440.0

(M þ 2H e Na)^þ, 418.0 (M þ 3H e 2Na)^þMS ESI-QT of <0 m/z 416.2

(M þ H e 2Na)^ꢀ. HR-MS Calculated for C₁₆H₂₃N₃O₈Na₂P: 462.1018;

Found: 462.0995. UV (H₂O) l_max¼ 208 nm (ε_max¼ 20,574),

l_max¼ 235 nm (ε_max¼ 14,768), l_max¼ 297 nm (ε_max¼ 7454).

4.4.1.12. 1-[6-Deoxy-6-phosphono-b-D-allofuranosyl]-5-phenyl-ura-

cil (disodium salt) (23d). Treatment of compound 49d (97 mg,

0.21 mmol) following general procedure gave rise to the expected

derivative (84 mg, 88%). Rf (iPrOH/NH₄OH/H₂O, 7/2/1, v/v/v) 0.31.

4.4.1.16. 1-[6-Deoxy-6-phosphono-b-D-allofuranosyl]-5-phen-

ylethynyl-cytosine (disodium salt) (24c). Treatment of compound 48c

(162 mg, 0.33 mmol) following general procedure gave rise to the

expected derivative (126 mg, 80%). Rf (iPrOH/NH₄OH/H₂O, 9/9/2, v/v/

¹H NMR (400 MHz, D₂O)

d

¼ 8.11 (s, 1H, H-6), 7.46 (m, 5H, HeAr),

6.00 (d, J ¼ 4.26 Hz,1H, H-1⁰), 4.36 (m, 2H, H-2⁰, H-3⁰), 4.25e4.17 (m,

2H, H-4⁰, H-5⁰), 1.87 (m, 2H, H-6⁰, H-6⁰⁰). ¹³C NMR (100 MHz, D₂O)

v) 0.39. ¹H NMR (400 MHz, D₂O)

d

¼ 8.25 (s,1H, H-6), 7.55e7.35 (2m,

d

¼ 164.6 (C-2),151.4 (C-4),138.9 (C-6), 131.8, 128.6,128.4,128.3 (Ce

5H, HeAr), 5.85 (d, J ¼ 4.78 Hz,1H, H-1⁰), 4.35e4.05 (2m, 4H, H-2⁰, H-

Ar), 115.8 (C-5), 88.4 (C-1⁰), 87.1 (d, J ¼ 13.09 Hz, C-4⁰), 73.9 (C-2⁰),

68.3 (C-3⁰), 67.0 (d, J ¼ 2.30 Hz, C-5⁰), 32.3 (d, J ¼ 130.7 Hz, C-6⁰). ³¹P

3⁰, H-5⁰, H-4⁰), 2.05e1.80 (m, 2H, H-6⁰, H-6⁰⁰). ¹³C NMR (100 MHz,

D₂O)

d

¼ 164.4 (C-4), 155.9 (C-2), 144.5 (C-6), 131.3, 129.0,128.6,121.5

NMR (81 MHz, D₂O)

d

¼ 20.4. MS ESI-QT of >0 m/z 459.0 (M þ H)^þ,

(CeAr), 95.4 and 92.7 (C^C), 89.5 (C-1⁰), 86.6 (d, J ¼ 13.63 Hz, C-4⁰),

437.1 (M ꢀ Na þ 2H)^þ, 415.1 (M ꢀ 2Na þ 3H)^þ. MS ESI-Qt of <0 m/z

79.1 (C-2⁰), 74.4 (C-3⁰), 68.2 (C-5), 67.0 (C-5⁰), 32.3 (d, J ¼ 131.0 Hz, C-

413.2 (M ꢀ 2Na þ H)^ꢀ. HR-MS Calculated for C₁₆H₁₈N₂Na₂O₉P:

6⁰). ³¹P NMR (81 MHz, D₂O)

d

¼ 20.4. MS ESI-QT of >0 m/z 482.0

459.0545; Found: 459.0532. UV (H₂O) l_max

(ε_max¼ 12,191), l_max¼ 279 nm (ε_max¼ 10,092).

¼

235 nm

(M þ H)^þ, 460.0 (M ꢀ Na þ 2H)^þ, 438.0 (M ꢀ 2Na þ 3H)^þ. MS ESI-Qt

of<0 m/z 436.1 (M

ꢀ

2Na

þ

H)^þ. HR-MS Calculated for

C

₁₈H₁₉N₃O₈Na₂P: 482.0705; Found: 482.0694. UV l_max¼ 210 nm

4.4.1.13. 1-[6-Deoxy-6-phosphono-b-D-allofuranosyl]-5-(2-(E)-phe-

(ε_max¼ 15,161), l_max¼ 283 nm (ε_max¼ 8125) (H₂O).

nylvinyl)-uracil (disodium salt) (23e). Treatment of compound 49e

(71 mg, 0.14 mmol) following general procedure gave rise to the

expected derivative (39 mg, 57%). Rf (iPrOH/NH₄OH/H₂O, 7/2/1, v/v/

4.4.1.17. 1-[6-Deoxy-6-phosphono- -allofuranosyl]-5-phenyl-cyto-

b-D

sine (disodium salt) (24d). Treatment of compound 50d (109 mg,

0.23 mmol) following general procedure gave rise to the expected

derivative (85 mg, 80%). Rf (iPrOH/NH₄OH/H₂O, 7/2/1, v/v/v) 0.15.

v) 0.27. ¹H NMR (400 MHz, D₂O)

5H, HeAr), 7.23 (d, J ¼ 16.51 Hz, HC]C), 6.85 (d, J ¼ 16.54 Hz, 1H,

HC]C), 5.93 (d, J ¼ 5.03 Hz, 1H, H-1⁰), 4.38e4.30 (m, 3H, H-2⁰, H-3⁰,

H-5⁰), 4.17 (m, 1H, H-4⁰), 1.90 (m, 2H, H-6⁰, H-6⁰⁰). ¹³C NMR

d

¼ 8.14 (s, 1H, H-6), 7.50e7.29 (m,

¹H NMR (400 MHz, D₂O)

d

¼ 7.96 (s, 1H, H-6), 7.52e7.41 (m, 5H, He

Ar), 5.98 (d, J ¼ 4.31 Hz, 1H, H-1⁰), 4.35e4.21 (m, 3H, H-2⁰, H-3⁰, H-

(100 MHz, D₂O)

d

¼ 164.1 (C-2), 150.8 (C-4), 137.4 (C-6), 136.8,129.5,

5⁰), 4.16 (m, 1H, H-4⁰), 1.87 (m, 2H, H-6⁰, H-6⁰⁰). ¹³C NMR (100 MHz,

128.8, 127.9, 126.3 (CeAr, C]C), 119.0 (C]C), 112.7 (C-5), 88.3 (C-

1⁰), 87.0 (d, J ¼ 13.5 Hz, C-4⁰), 74.0 (C-2⁰), 68.2 (C-3⁰), 67.1 (C-5⁰), 32.3

D₂O)

d

¼ 162.9 (C-4), 155.6 (C-2), 138.7 (C-6), 130.5, 127.7, 127.5,

127.2 (CeAr), 109.0 (C-5), 87.7 (C-1⁰), 85.1 (d, J ¼ 13.3 Hz, C-4⁰), 72.5

(d, J ¼ 129.2 Hz, C-6⁰). ³¹P NMR (81 MHz, D₂O)

d

¼ 20.0. MS ESI-QT

(C-2⁰), 66.6 (C-3⁰), 65.4 (d, J ¼ 2.4 Hz, C-5⁰), 30.7 (d, J ¼ 130.5 Hz, C-

of >0 m/z 463.1 (M ꢀ Na þ 2H)^þ, 441.1 (M ꢀ 2Na þ 3H)^þ. MS ESI-Qt

6⁰). ³¹P NMR (81 MHz, D₂O)

d

¼ 20.3. MS ESI-QT of >0 m/z 458.2

of <0 m/z 439.2 (M

ꢀ

2Na

þ

H)^ꢀ. HR-MS Calculated for

(M þ H)^þ, 436.2 (M ꢀ Na þ 2H)^þ, 414.2 (M ꢀ 2Na þ 3H)^þ. MS ESI-Qt

C

₁₈H₂₀N₂Na₂O₉P: 485.0702; Found: 485.0710. UV l_max¼ 271 nm

of<0 m/z 412.1 (M

ꢀ

2Na

þ

H)^ꢀ. HR-MS Calculated for

(ε_max¼ 8281) (H₂O).

C₁₆H₂₀N₃NaO₈P: 436.0886; Found: 436.0866. UV (H₂O)

l_max¼ 232 nm (ε_max¼ 7489), 282 nm (ε_max¼ 4110).

4.4.1.14. 1-[6-Deoxy-6-phosphono-b-D-allofuranosyl]-5-hexyn-1-yl-

cytosine (disodium salt) (24a). Treatment of compound 48a

(135 mg, 0.29 mmol) following general procedure gave rise to the

expected derivative (30 mg, 23%). Rf (iPrOH/NH₄OH/H₂O, 7/2/1, v/v/

4.4.1.18. 1-[6-Deoxy-6-phosphono- -allofuranosyl]-5-(2-(E)-phe-

b-D

nylvinyl)-cytosine (disodium salt) (24e). Treatment of compound

50e (95 mg, 0.19 mmol) following general procedure gave rise to

the expected derivative (22 mg, 24%). Rf (iPrOH/NH₄OH/H₂O, 7/2/1,

v) 0.35. ¹H NMR (400 MHz, D₂O)

d

¼ 8.14 (s, 1H, H-6), 5.93 (d,

J ¼ 5.08 Hz, 1H, H-1⁰), 4.33e4.23 (m, 3H, H-2⁰, H-3⁰, H-5⁰), 4.13 (m,

1H, H-4⁰), 2.44 (t, J ¼ 7.07 Hz, 2H, CH₂C^C), 1.87 (m, 2H, H-6⁰, H-6⁰⁰),

1.55e1.40 (m, 4H, CH₂), 0.90 (t, J ¼ 7.27 Hz, CH₃). ¹³C NMR (100 MHz,

v/v/v) 0.13 ¹H NMR (400 MHz, D₂O)

d

¼ 8.31 (s, 1H, H-6), 7.55e7.25

(m, 6H, HeAr, HC]C), 6.84 (d, J ¼ 4.1 Hz, 1H, HC]C), 5.92 (d,

J ¼ 3.8 Hz, 1H, H-1⁰), 4.48e4.11 (m, 4H, H-2⁰, H-3⁰, H-4⁰, H-5⁰), 1.90e

D₂O)

d

¼ 165.1 (C-2), 156.3 (C-4), 143.8 (C-6), 98.3 and 93.5 (C^C),

1.62 (m, 2H, H-6⁰, H-6⁰⁰). ¹³C NMR (100 MHz, D₂O)

d

¼ 165.6 (C-4),

89.1 (C-1⁰), 86.9 (d, J ¼ 13.78 Hz, C-4⁰), 74.2 (C-2⁰), 70.0 (C-3⁰), 68.2

(C-5), 67.0 (C-5⁰), 32.2 (d, J ¼ 130.05 Hz, C-6⁰), 29.8 (CH₂C^C), 214

(CH₂CH₂eC^C), 18.3 (CH₃CH₂), 12.8 (CH₃). ³¹P NMR (81 MHz, D₂O)

158.3 (C-2), 139.4 (C-6), 137.9, 132.6, 130.3, 129.6, 127.9 (CeAr, HC]

C), 119.4 (HC]C), 108.8 (C-5), 90.6 (C-1⁰), 88.0 (d, J ¼ 12.7 Hz, C-4⁰),

76.0 (C-2⁰), 69.2 (C-3⁰), 68.85 (C-5⁰), 33.7 (d, J ¼ 125.2 Hz, C-6⁰). ³¹P

d

¼ 20.2. MS ESI-QT of >0 m/z 440.1 (M ꢀ Na þ 2H)^þ, 418.1

NMR (81 MHz, D₂O)

d

¼ 19.2. MS ESI-QT of >0 m/z 440.2

(M ꢀ 2Na þ 3H)^þ. MS ESI-QT of<0 m/z 416.2 (M ꢀ 2Na þ H)^ꢀ. HR-

(M ꢀ 2Na þ 3H)^þ. MS ESI-Qt of <0 m/z 438.2 (M ꢀ 2Na þ H)^ꢀ. HR-

MS Calculated for C₁₈H₂₃N₃NaO₈P: 440.1223; Found: 440.1234. UV

(H₂O) l_max¼ 278 nm (ε_max¼ 9351).

MS Calculated for C₁₆H₂₅N₃O₈P: 418.1379; Found: 418.1386. UV

(H₂O) l_max

(ε_max¼ 15,324), l_max¼ 296 nm (ε_max¼ 7593).

¼

209 nm (ε_max

¼

20,758), l_max

¼

235 nm

4.4.2. 1-(5S)-[6-deoxy-6-diethylphosphono-2,3-seco-b-D-

4.4.1.15. 1-[6-Deoxy-6-phosphono- -allofuranosyl]-5-(3,3-dimethyl-

b

-

D

ribohexofuranosyl]uracil (19)

1-butyn-1-yl)-cytosine (disodium salt) (24b). Treatment of compound

48b (145 mg, 0.31 mmol) following general procedure gave rise to

the expected derivative (103 mg, 73%). Rf (iPrOH/NH₄OH/H₂O, 7/2/1,

Compound 1 (2 g, 5.07 mmol) was dissolved in a mixture of

dioxane (53 mL) and water (10 mL) and a solution of NaIO₄(1.21 g,

5.57 mmol, 1.1 eq.) in water (10 mL) was added. The reaction

mixture was stirred for 1.5 h at rt, diluted with dioxane, and then

ﬁltered. The solid residue was washed with dioxane, and then so-

dium borohydride (0.192 g, 5.07 mmol, 1 eq.) was added to the

ﬁltrate. The reaction mixture was stirred for 15 min, concentrated

and freeze-dried. Column chromatography of the crude materials

on reverse phase (gradient: water to acetonitrile) gave the expected

v/v/v) 0.38. ¹H NMR (400 MHz, D₂O)

d

¼ 8.15 (s, 1H, H-6), 5.92 (d,

J ¼ 5.09 Hz, 1H, H-1⁰), 4.33e4.13 (m, 4H, H-2⁰, H-3⁰, H-4⁰, H-5⁰), 1.90

(m, 2H, H-6⁰, H-6⁰⁰), 1.29 (s, 9H, CH₃). ¹³C NMR (100 MHz, D₂O)

d

¼ 164.8 (C-2), 156.2 (C-4), 144.0 (C-6), 106.0 and 93.2 (C^C), 89.2

(C-1⁰), 86.8 (d, J ¼ 13.76 Hz, C-4⁰), 74.2 (C-2⁰), 68.8 (C-3⁰), 68.2 (C-5),

66.9 (d, J ¼ 2.69 Hz, C-5⁰), 32.2 (d, J ¼ 131.1 Hz, C-6⁰), 29.8 (CH₃), 27.7

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

31

product 19 as a white solid (2.0 g, 99%). Rf (EtOAc/MeOH, 5/5, v/v)

0.65. ¹H NMR (400 MHz, DMSO-d₆)

375 (M ꢀ H)^ꢀ. UV l_max¼ 248 nm (ε_max¼ 7100), 224 nm

(ε_max¼ 8700), l_min¼ 235 nm (ε_min¼ 6500), (EtOH 95). Calculated

for C₁₄H₂₃N₂O₈P, 0.3H₂O: C, 44.69; H, 5.62; N, 7.44. Found: C, 44.45;

H, 6.10; N, 7.13.

d

¼ 11.25 (s, 1H exchangeable,

NH), 7.78 (d, J ¼ 8.06 Hz, 1H, H-6), 5.91 (t, J ¼ 5.81 Hz, 1H, H-1⁰), 5.88

(d, J ¼ 8.04 Hz, 1H, H-5), 4.10e3.90 (m, 5H, H-5⁰, OCH₂CH₃), 3.85e

3.65 (m, 4H, H-3⁰, H-3⁰⁰, H-2⁰, H-2⁰⁰), 3.57 (m, 1H, H-4⁰), 2.15e1.80

(m, 2H, H-6⁰, H-6⁰⁰), 1.22e1.18 (m, 6H, OCH₂CH₃). ¹³C NMR

4.4.5. 1-[6-Deoxy-6-diethylphosphono-b-D-altrofuranosyl]uracil

(100 MHz, DMSO-d₆)

d

¼ 166.1 (C-4), 152.3 (C-2), 142.3 (C-6), 102.6

(5)

(C-5), 84.1 (C-1⁰), 82.2e82.0 (C-4⁰, d, J ¼ 13 Hz), 65.6e65.5 (C-5⁰, d,

J ¼ 7 Hz), 63.3 (m, OCH₂CH₃), 59.3 (C-2⁰), 57.4 (C-3⁰), 29.0e27.5 (C-

6⁰, d, J ¼ 140 Hz), 15.6e15.5 (OCH₂CH₃, d, J ¼ 6 Hz). ³¹P (121 MHz,

The intermediate 29 (1 g, 2.65 mmol) was dissolved in a

mixture of methanol and water (27 mL, 1/1, v/v). A solution of

NaOH 1 N was added (26.5 mL) and the reaction mixture was

stirred for 1 h, neutralized with AcOH, concentrated, diluted in

water and freeze-dried. Column chromatography of the crude

materials on reverse phase (water/acetonitrile, 9/1 then 5/5, v/v)

gave the expected product as a white powder (1 g, 96%). Rf (EtOAc/

DMSO-d₆)

d

¼ 32.1. MS FAB>0 (GT) m/z 793 (2M þ H)^þ, 397

(M þ H)^þ. FAB<0 (GT) m/z 791 (2M ꢀ H)^ꢀ, 395 (M ꢀ H)^ꢀ. UV

l_max¼ 263 nm (ε_max¼ 11,800) (EtOH 95).

4.4.3. 1-(5S)-[6-Deoxy-6-diethylphosphono-2,3-seco-

ribohexofuranosyl]cytosine (21)

b

-

D

-

MeOH, 7/3, v/v) 0.47. ¹H NMR (400 MHz, D₂O)

d

¼ 7.7 (d,

J ¼ 8.12 Hz, 1H, H-6), 6.07 (d, J ¼ 4.57 Hz, 1H, H-1⁰), 5.67 (d,

J ¼ 8.11 Hz, 1H, H-5), 4.28 (dd, J ¼ 3.42e4.53 Hz, 1H, H-2⁰), 4.22

(dd, J ¼ 3.5e4.53 Hz, 1H, H-3⁰), 4.17 (m, 1H, H-5⁰), 4.1e4.0 (m, 4H,

OCH₂CH₃), 3.75 (dd, J ¼ 4.53e5.99 Hz, 1H, H-4⁰), 2.3e2.05 (m, 2H,

H-6⁰, H-6⁰⁰), 1.21 (m, 6H, OCH₂CH₃). ¹³C NMR (100 MHz, D₂O)

Compound 19 (0.30 g, 0.76 mmol) was suspended in anhydrous

acetonitrile (7.8 mL), N-methyl pyrrolidine (0.76 mL, 7.27 mmol)

and trimethylsilyl chloride (0.29 mL, 2.27 mmol) were added to the

reaction mixture. After 1 h of stirring, triﬂuoroacetic anhydride

(0.32 mL, 2.27 mmol) was added at 0 ^ꢁC, the reaction mixture was

stirred for 1 h more at 0 ^ꢁC and followed by the addition of 4-

nitrophenol (0.315 g, 2.27 mmol). Three hours later, the volatiles

were evaporated under reduced pressure, the crude was diluted in

dioxane (20 mL) and NH₄OH 28% (7.57 mL) was added. The reaction

mixture was stirred overnight at 50 ^ꢁC, neutralized with a solution

of acetic acid 10% aqueous, freeze-dried and then puriﬁed twice on

reverse phase column chromatography (gradient: water to aceto-

nitrile) to give the cytosine containing acycloderivative 21 as a

yellow solid (105 mg, 35%). Rf (EtOAc/MeOH, 5/5, v/v) 0.34. ¹H NMR

d

¼ 166.4 (C-4), 151.5 (C-2), 143.0 (C-6), 101.0 (C-5), 85.5 (C-1⁰),

85.5e85.3 (C-4⁰, d, J ¼ 15 Hz), 75.6 (C-2⁰), 75.3 (C-3⁰), 65.6e65.5

(C-5⁰, d, J ¼ 5 Hz), 63.4 (m, OCH₂CH₃), 29.5e28.1 (C-6⁰, d,

J ¼ 140 Hz), 15.6e15.5 (OCH₂CH₃, d, J ¼ 6 Hz). ³¹P NMR (121 MHz,

D₂O)

d

¼ 32.0. MS FAB > 0 (NBA) m/z 789 (2M þ H)^þ, 395

(M þ H)^þ. FAB < 0 (NBA) m/z 393 (M ꢀ H)^ꢀ. UV l_max¼ 263 nm

(ε_max¼ 10,000) (EtOH 95). Calculated for C₁₄H₂₃N₂O₉P, 0.5H₂O: C,

41.69; H, 6.00; N, 6.95. Found: C, 41.42; H, 5.76; N, 6.73.

4.4.6. 1-[6-Deoxy-6-diethylphosphono-b-D-altrofuranosyl]cytosine

(400 MHz, D₂O)

d

¼ 7.70 (d, J ¼ 7.5 Hz, 1H, H-6), 6.05 (d, J ¼ 7.49 Hz,

(7)

1H, H-5), 5.95 (d, J ¼ 5.61 Hz, 1H, H-1⁰), 4.15e3.95 (m, 4H,

OCH₂CH₃), 3.95e3.88 (m, 1H, H-5⁰), 3.85e3.65 (m, 4H, H-3⁰, H-3⁰⁰,

H-2⁰, H-2⁰⁰), 3.45 (m, 1H, H-4⁰), 2.15e1.85 (m, 2H, H-6⁰, H-6⁰⁰), 1.22e

The derivative 5 (300 mg, 0.761 mmol) was suspended in

anhydrous acetonitrile (7.6 mL). N-methyl pyrrolidine (0.76 mL,

7.30 mmol) and trimethylsilyl chloride (0.29 mL, 2.28 mmol) were

added to the reaction mixture. After 1 h of stirring, TFA anhydride

(0.32 mL, 2.28 mmol) was added at 0 ^ꢁC, the reaction mixture was

stirred for 1 h more at 0 ^ꢁC and followed by the addition of 4-

nitrophenol (0.316 g, 2.28 mmol). Three hours later, the mixture

was concentrated under reduced pressure, the crude was diluted in

dioxane (23 mL) and NH₄OH 28% (7.61 mL) was added. The reaction

mixture was stirred overnight, neutralized with a solution of acetic

acid 10% aqueous, freeze-dried and then puriﬁed twice on reverse

phase column chromatography (gradient:water to acetonitrile) to

give the desired product 7 (130 mg, 43%). Rf (CH₂Cl₂/MeOH, 8/2, v/

1.15 (m, 6H, OCH₂CH₃). ¹³C NMR (100 MHz, D₂O)

d

¼ 166.0 (C-4),

158.1 (C-2), 142.1 (C-6), 96.6 (C-5), 84.4 (C-1⁰), 82.0e81.9 (C-4⁰, d,

J ¼ 14 Hz), 65.7e65.6 (C-5⁰, d, J ¼ 7 Hz), 63.3 (m, OCH₂CH₃), 61.8 (C-

2⁰), 59.2 (C-3⁰), 29.1e27.7 (C-6⁰, d, J ¼ 140 Hz), 15.6e15.5 (OCH₂CH₃,

d, J ¼ 6 Hz). ³¹P NMR (121 MHz, D₂O)

¼ 29.4. MS FAB > 0 (NBA) m/

d

z 791 (2M þ H)^þ, 396 (M þ H)^þ. FAB < 0 (NBA) m/z 789 (2M ꢀ H)^ꢀ,

394 (M ꢀ H)^ꢀ. UV l_max¼ 272 nm (ε_max¼ 10,200) (EtOH 95).

Calculated for C₁₄H₂₆N₃O₈P, 0.55H₂O: C, 41.49; H, 6.74; N, 10.37.

Found: C, 41.49; H, 6.71; N, 10.30.

4.4.4. 1-[2,2⁰-Anhydro-6-deoxy-6-diethylphosphono-

altrofuranosyl]uracil (29)

b-

D

-

v) 0.09. ¹H NMR (400 MHz, D₂O)

d

¼ 7.65 (d, J ¼ 7.56 Hz, 1H, H-6),

6.1 (d, J ¼ 4.23 Hz, 1H, H-1⁰), 5.9 (d, J ¼ 7.55 Hz, 1H, H-5), 4.29 (dd,

J ¼ 2.83e4.19 Hz, 1H, H-2⁰), 4.21 (dd, J ¼ 2.9e3.93 Hz, 1H, H-3⁰),

4.20e4.15 (m, 1H, H-5⁰), 4.15e4.05 (m, 4H, OCH₂CH₃), 3.75 (dd,

J ¼ 4e6.31 Hz,1H, H-4⁰), 2.4e2.0 (m, 2H, H-6⁰, H-6⁰⁰), 1.3e1.2 (m, 6H,

Diphenyl-carbonate (0.956 g, 4.46 mmol) was added to a solu-

tion of the nucleotide 1 (1.6 g, 4.06 mmol) in anhydrous DMF

(20 mL) under argon. The reaction mixture was heated at 90 ^ꢁC, the

addition of Na₂CO₃(16 mg, 10% weight) was performed and stirring

was pursued at 110 ^ꢁC during 1.5 h. The solution was concentrated

under high vacuum. Column chromatography of the crude mate-

rials on silica gel (dichloromethane/methanol, 9/1 then 8/2, v/v)

gave the anhydro-derivative as a light yellow powder (1.06 g, 70%).

Rf (EtOAc/MeOH, 5/5, v/v) 0.44. ¹H NMR (400 MHz, DMSO-d₆)

OCH₂CH₃). ¹³C NMR (100 MHz, D₂O)

d

¼ 166.1 (C-4), 157.3 (C-2),

142.7 (C-6), 95.2 (C-5), 86.3 (C-1⁰), 85.8e85.6 (C-4⁰, d, J ¼ 17 Hz),

75.6 (C-2⁰), 74.5 (C-3⁰), 65.7 (C-5⁰, d, J ¼ 5 Hz), 63.5e63.4 (OCH₂CH₃),

29.6e28.2 (C-6⁰, d, J ¼ 140 Hz),15.6e15.9 (OCH₂CH₃, d, J ¼ 5 Hz). ³¹P

NMR (81 MHz, D₂O)

d

¼ 33.2. MS FAB > 0 (NBA) m/z 787 (2M ꢀ H)^þ,

394 (M ꢀ H)^þ, 112 (base-H)^þ, FAB<0 (NBA) m/z 785 (2M ꢀ H)^ꢀ, 392

(M ꢀ H)^ꢀ. UV l_max¼ 273 nm (ε_max¼ 10,300) (EtOH 95). Calculated

for C₁₄H₂₄N₃O₈P,1.3H₂O: C, 40.35; H, 6.43; N,10.08. Found: C, 40.09;

H, 6.24; N, 9.86.

d

¼ 7.91 (d, J ¼ 7.47 Hz, 1H, H-6), 6.50 (d, J ¼ 5.78 Hz, 1H, H-1⁰), 6.15

(d, J ¼ 7.45 Hz,1H, H-5), 5.42 (d, J ¼ 5.72 Hz,1H, H-2⁰), 4.71 (s,1H, H-

3⁰), 4.09 (d, J ¼ 8.11 Hz, 1H, H-4⁰), 4.05e3.9 (m, 4H, OCH₂CH₃), 3.55e

3.41 (m, 1H, H-5⁰), 2.11e1.86 (m, 2H, H-6⁰, H-6⁰⁰), 1.30 (m, 6H,

OCH₂CH₃). ¹³C NMR (100 MHz, DMSO-d₆)

d

¼ 175.3 (C-4), 160.8 (C-

4.4.7. 3-O-Benzyl-1,2-5,6-O-diisopropylidene-a-D-glucofuranose

(30) [42,43]

2), 138.3 (C-6), 109.2 (C-5), 91.6e91.5 (C-4⁰, d, J ¼ 17 Hz), 91.2 (C-1⁰),

89.3 (C-2⁰), 74.1 (C-3⁰), 65.5e65.45 (C-5⁰, d, J ¼ 5 Hz), 63.4, 63.37 (2d,

J ¼ 6.0 Hz, OCH₂CH₃), 29.6e28.2 (C-6⁰, d, J ¼ 141 Hz), 15.6e15.5

Diacetonide-D-glucose (5 g, 19.2 mmol) was added to a sus-

pension of KOH (4.3 g, 76.8 mmol) in anhydrous DMSO (32 mL).

After 30 min stirring, BnBr (4.6 mL, 38.4 mmol) was added. The

reaction mixture was stirred for 14 h at room temperature, diluted

(OCH₂CH₃, d, J ¼ 5 Hz). ³¹P NMR (121 MHz, DMSO-d₆)

¼ 30.8. MS

d

FAB > 0 (NBA) m/z 753 (2M þ H)^þ, 377 (M þ H)^þ. FAB<0 (NBA) m/z

32

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

with water (20 mL), extracted three times with diethyl ether. The

organic layers were dried over Na₂SO₄, concentrated under reduced

pressure. The oil obtained was puriﬁed on silica gel chromatog-

raphy column (hexane/ethyl acetate, 9/1 then 8/2, v/v) to give the

corresponding protected compound as yellow oil (6.4 g, 95%). Rf

(Hexane/EtOAc, 8/2, v/v) 0.28. ¹H NMR (400 MHz, DMSO-d₆)

4.4.10. 3-O-Benzyl-6-deoxy-6-diethylphosphono-1,2-O-

isopropylidene- -glucofuranose (34) [45]

a-D

A solution of iodo derivative 33 (0.80 g, 2 mmol) in triethyl-

phosphite (4 mL) was heated at 110 ^ꢁC under argon for two days.

The solution was evaporated under high vacuum at 70 ^ꢁC. Column

chromatography of the crude mixture on silica gel (ethyl acetate)

gave the expected phosphonate derivative as colorless oil (0.692 g,

83%). Rf (CH₂Cl₂/EtOAc, 8/2, v/v) 0.10. ¹H NMR (400 MHz, DMSO-d₆)

d

¼ 7.40e7.25 (m, 5H, HeAr), 5.85 (d, J ¼ 3.76 Hz, 1H, H-1), 4.72 (d,

J ¼ 3.8 Hz, 1H, H-2), 4.69 (d, J ¼ 11.9 Hz, 1H, H-7), 4.54 (d,

J ¼ 11.86 Hz, 1H, H-7⁰), 4.28 (dd, J ¼ 6.14e6.78 Hz, 1H, H-5), 4.1 (dd,

J ¼ 3.14e6.78 Hz, 1H, H-4), 3.98 (dd, J ¼ 6.14e8.35 Hz, 1H, H-6), 3.93

(d, J ¼ 3.14 Hz, 1H, H-3), 3.8 (dd, J ¼ 6.38e8.35 Hz, 1H, H-6⁰), 1.4e1.1

d

¼ 7.35e7.25 (m, 5H, HeAr), 5.8 (d, J ¼ 3.75 Hz, 1H, H-1), 5.07 (d,

J ¼ 7.11 Hz, 1H exchangeable, OH-5), 4.7 (d, J ¼ 3.76 Hz, 1H, H-2),

4.67 (d, J ¼ 11.5 Hz, 1H, H-7), 4.58 (d, J ¼ 11.5 Hz, 1H, H-7⁰), 4.15e

4.05 (m, 1H, H-3), 4.0e3.85 (m, 6H, H-4, H-5, OCH₂CH₃), 2.05e1.9

(m, 2H, H-6, H-6⁰), 1.4e1.25 (2s, 6H, C(CH₃)₂), 1.24e1.19 (m, 6H,

(4s, 12H, C(CH₃)₂). ¹³C NMR (100 MHz, DMSO-d₆)

d

¼ 138.3, 128.7,

128.1, 128.0 (CeAr), 111.3 (C(CH₃)₂), 108.5 (C(CH₃)₂), 105.2 (C-1),

82.0 (C-2), 81.6 (C-3), 80.9 (C-4), 72.7 (C-5), 71.5 (C-7), 66.6 (C-6),

27.1, 27.0, 26.5, 25.7 (C(CH₃)₂).

OCH₂CH₃). ¹³C NMR (100 MHz, DMSO-d₆)

d

¼ 138.6, 128.6, 128.1,

128.0 (CeAr), 111.2 (C(CH₃)₂), 104.9 (C-1), 83.4e83.3 (C-4, d,

J ¼ 15 Hz), 82.0 (C-2), 81.0 (C-3), 71.6 (C-7), 63.2 (C-5, d, J ¼ 6 Hz),

61.4e61.1 (OCH₂CH₃, m), 31.7e30.3 (C-6, d, J ¼ 138.9 Hz), 27.1, 26.6

(C(CH₃)₂), 16.8e16.7 (OCH₂CH₃, m). ³¹P NMR (81 MHz, DMSO-d₆)

4.4.8. 3-O-Benzyl-1,2-O-isopropylidene-

a

-

D

-glucofuranose (31)

-glucose

A solution of 3-O-benzyl-1,2-5,6-O-diisopropylidene-

D

d

¼ 27.2. MS FAB > 0 (NBA) m/z 861 (2M þ H)^þ, 431 (M þ H)^þ, 91

(26.9 g, 76.8 mmol) in aqueous acetic acid (9/1, v/v, 385 mL) was

warmed for 1 h at 60 ^ꢁC. The solution was then concentrated and

co-evaporated three times with toluene. Column chromatography

of crude materials on silica gel (dichloromethane/ethyl acetate, 8/2

to 0/10, v/v) gave the titled compound as colorless oil (22.35 g, 93%).

Rf (CH₂Cl₂/EtOAc, 8/2, v/v) 0.12. ¹H NMR (400 MHz, DMSO-d₆)

(Bn)^þ. FAB < 0 (NBA) m/z 859 (2M ꢀ H)^ꢀ, 429 (M ꢀ H)^ꢀ.

4.4.11. 5-O-Benzoyl-3-O-benzyl-6-deoxy-6-diethylphosphono-1,2-

O-isopropylidene-a-D-gluco-furanose (35)

Benzoyl chloride (0.88 mL, 7.54 mmol) was added at room

temperature to a solution of intermediate 34 (2.5 g, 5.8 mmol) in

anhydrous pyridine (15 mL) under argon. The mixture was stirred

overnight, then iced-water (100 mL) was added and the resulting

aqueous phase was extracted with ethyl acetate. The organic layers

were successively washed with HCl 1 N, saturated NaHCO₃, and

water, then dried over Na₂SO₄and concentrated. Column chro-

matography of the crude mixture on silica gel (dichloromethane/

ethyl acetate, 7/3, v/v) gave the expected phosphonate derivative as

a yellow oil (2.9 g, 93%). Rf (CH₂Cl₂/EtOAc, 7/3, v/v) 0.22. ¹H NMR

d

¼ 7.40e7.25 (m, 5H, HeAr), 5.8 (d, J ¼ 3.7 Hz, 1H, H-1), 4.75 (d,

J ¼ 6.08 Hz, 1H exchangeable, OH-5), 4.7 (d, J ¼ 11.6 Hz, 1H, H-7),

4.67 (d, J ¼ 3.7 Hz, 1H, H-2), 4.6 (d, J ¼ 11.4 Hz, 1H, H-7⁰), 4.45 (d,

J ¼ 5.58 Hz, 1H exchangeable, OH-6), 3.95 (m, 2H, H-3, H-4), 3.75

(m,1H, H-5), 3.6 (m, H-6), 3.4 (m, 1H, H-3),1.4e1.2 (2s, 6H, C(CH₃)₂).

¹³C NMR (100 MHz, DMSO-d₆)

d

¼ 138.2, 128.2, 127.5, 127.4 (CeAr),

110.5 (C(CH₃)₂), 104.6 (C-1), 81.4 (C-2), 81.1 (C-3), 79.7 (C-4), 71.2

(C-7), 68.1 (C-5), 63.7 (C-6), 26.5, 26.1 (C(CH₃)₂). MS FAB > 0 (NBA)

m/z 621 (2M þ H)^þ, 311 (M þ H)^þ. FAB < 0 (NBA) m/z 619

(2M ꢀ H)^ꢀ, 309 (M ꢀ H)^ꢀ.

(400 MHz, DMSO-d₆)

d

¼ 8.00e7.90 (m, 2H, HeAr, Bz), 7.70e7.60

(m, 1HeAr, Bz), 7.50e7.40 (m, 2HeAr, Bz), 7.2e7.1 (m, 5H, HeAr,

Bn), 5.9 (d, J ¼ 3.73 Hz, 1H, H-1), 5.52 (m, J ¼ 3.9e19.9 Hz, 1H, H-5),

4.8 (d, J ¼ 3.73 Hz, 1H, H-2), 4.65 (d, J ¼ 11.4 Hz, 1H, H-7), 4.51 (dd,

J ¼ 3.22e7.17 Hz, 1H, H-4), 4.35 (d, J ¼ 11.4 Hz, 1H, H-7⁰), 4.0 (d,

J ¼ 3.27 Hz, 1H, H-3), 3.95e3.80 (m, 4H, OCH₂CH₃), 2.45e2.23 (m,

2H, H-6, H-6⁰), 1.4e1.27 (2s, 6H, C(CH₃)₂), 1.1e1.0 (2t, J ¼ 7.05 Hz, 6H,

4.4.9. 3-O-Benzyl-6-deoxy-6-iodo-1,2-O-isopropylidene-a-D-

glucofuranose (33) [44]

3-O-benzyl-1,2-O-isopropylidene-D-glucose (1 g, 3.2 mmol) was

dissolved in anhydrous pyridine (32 mL). N,N-dimethylaminopyr-

idine (0.393 g, 3.2 mmol) was added at 0 ^ꢁC, and then the solution

was stirred for 15 min before adding the tosylchloride (1.22 g,

6.4 mmol). The solution was allowed to room temperature and

stirred overnight. The reaction mixture was cooled to 0 ^ꢁC; water

and ethyl acetate were added. After decantation and extraction

with ethyl acetate, the combined organic layers were dried over

Na₂SO₄, concentrated under reduced pressure and co-evaporated

with toluene. The oil obtained (containing tosyl intermediate 32)

was partially dissolved into acetone and the insoluble salts were

removed by ﬁltration. The ﬁltrate was concentrated and the residue

dissolved in anhydrous acetone (10 mL) under argon, sodium iodide

(623 mg, 4.16 mmol) was added and the mixture was reﬂuxed

overnight and then concentrated. Column chromatography of the

crude materials on silica gel (cyclohexane/ethyl acetate, 8/2, v/v)

gave the iodo derivative as a yellow oil (0.93 g, 70%). Rf (Cyclo-

hexane/EtOAc, 8/2, v/v) 0.29. ¹H NMR (400 MHz, DMSO-d₆)

OCH₂CH₃). ¹³C NMR (100 MHz, DMSO-d₆)

d

¼ 164.9 (C]O), 137.8e

125.8 (CeAr), 111.6 (C(CH₃)₂), 105.4 (C-1), 81.5 (C-2), 80.9 (C-3),

80.1e80.0 (C-4, d, J ¼ 10 Hz), 71.4 (C-7), 66.9e66.8 (C-5, d, J ¼ 6 Hz),

61.6e61.5 (OCH₂CH₃, m), 27.8e26.4 (C-6, d, J ¼ 140 Hz), 27.1, 26.6

(C(CH₃)₂), 16.5e16.4 (OCH₂CH₃, m). ³¹P NMR (81 MHz, DMSO-d₆)

d

¼ 26.9. MS FAB>0 (NBA) m/z 1069 (2M þ H)^þ, 535 (M þ H)^þ, 91

(Bn)^þ.

4.4.12. 5-O-Benzoyl-3-O-benzyl-6-deoxy-1,2-O-diacetyl-6-

diethylphosphono-a,b-D-glucofuranose (36)

A solution of the fully protected sugar phosphonate 35 (8.0 g,

14.9 mmol) in glacial acetic acid (56.7 mL), acetic anhydride

(20.3 mL) and concentrated sulfuric acid (7.25 mL added at 0 ^ꢁC)

was stirred overnight at room temperature. The reaction was

diluted in dichloromethane, washed with aqueous saturated

NaHCO₃solution. The organic layer was washed with brine, dried

over Na₂SO₄and concentrated under reduced pressure. Silica gel

column chromatography of the crude materials (toluene/acetone,

7/3, v/v) gave the acetylated derivative as a yellow oil (8.0 g, 90%). Rf

(Toluene/Acetone, 7/3, v/v) 0.44. ¹H NMR(400 MHz, DMSO-d₆)

d

¼ 7.40e7.25 (m, 5H, HeAr), 5.81 (d, J ¼ 3.7 Hz, 1H, H-1), 5.4 (l, 1H

exchangeable, OH-5), 4.7 (d, J ¼ 3.9 Hz, 1H, H-2), 4.68 (d, J ¼ 11.9 Hz,

1H, H-7), 4.59 (d, J ¼ 11.6 Hz, 1H, H-7⁰), 3.97 (d, J ¼ 2.8 Hz, 1H, H-3),

3.89 (dd, J ¼ 2.8 and 8.66 Hz, 1H, H-4), 3.5e3.3 (m, 3H, H-5, H-6, H-

6⁰), 1.4e1.25 (2s, 6H, C(CH₃)₂). ¹³C NMR (100 MHz, DMSO-d₆)

d

¼ 8.00e7.90 (m, 2H, HeAr, Bz), 7.70e7.60 (m, 1HeAr, Bz), 7.55e

d

¼ 138.0, 128.2e127.4 (CeAr), 111.0 (C(CH₃)₂), 104.5 (C-1), 82.5 (C-

7.45 (m, 2HeAr, Bz), 7.35e7.2 (m, 5H, HeAr, Bn), 6.35 (d, J ¼ 4.61 Hz,

1H, H-1 anomer min), 6.05 (s, 1H, H-1 anomer maj), 5.7e5.5 (m, 1H,

H-5), 5.25 (m, 1H, H-2), 4.8e4.7 (m, 1H, H-4), 4.7e4.6 (m, 1H, H-7),

4.6e4.4 (m, 2H, H-3 anomer min, H-7⁰), 4.3 (d, J ¼ 5.34 Hz, 1H, H-3

4), 81.5 (C-2), 80.5 (C-3), 71.2 (C-7), 65.4 (C-5), 26.7, 26.1 (C(CH₃)₂),

16.7 (C-6). MS FAB > 0 (NBA) m/z 421 (M þ H)^þ, 405 (M ꢀ OH þ H)^þ,

91 (Bn)^þ.

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

33

anomer maj), 3.9e3.8 (m, 4H, OCH₂CH₃), 2.45e2.2 (m, 1H, H-6, H-

6⁰), 2.2e1.9 (m, 6H, C(CH₃)₂), 1.1e0.95 (m, 6H, OCH₂CH₃). ¹³C NMR

l_max¼ 262 nm (ε_max¼ 11,400) (EtOH 95). Analysis Calculated for

₁₄H₂₃N₂O₉P, 0.9H₂O: C, 40.96; H, 6.09; N, 6.82. Found: C, 40.93; H,

C

(100 MHz, DMSO-d₆)

d

¼ 169.5e164.4 (C]O), 137.4e125.3 (CeAr),

6.23; N, 6.80.

98.6 (C-1 anomer maj), 93.0 (C-1 anomer min), 83.1e83.0 (C-4

anomer maj, d, J ¼ 11 Hz), 79.5e79.3 (C-3 anomer maj, d, J ¼ 16 Hz),

78.8e78.7 (C-3 anomer min, d, J ¼ 12 Hz and C-4 anomer min), 78.0

(C-2 anomer maj), 76.0 (C-2 anomer min), 71.7e71.3 (C-7), 61.1e

61.0 (OCH₂CH₃), 20.6e20.3 (CH₃, Ac, m), 16.0e15.9 (OCH₂CH₃, m).

4.4.15. 1-[2-O-Acetyl-5-O-benzoyl-3-O-benzyl-6-deoxy-6-

diethylphosphono-b-D-glucofuranosyl] N-4-benzoyl-cytosine (38)

The dried protected nucleobase (2.97 g, 13.84 mmol) was dis-

solved in anhydrous acetonitrile (35 mL) and N,O-bis(trimethylsilyl)

acetamide (6.76 mL, 27.6 mmol) was added under argon. The so-

lution was reﬂuxed for 2 h, and then the reaction mixture was

allowed to cool to room temperature. A solution of phosphonate

intermediate 36 (4 g, 6.92 mmol) in anhydrous acetonitrile (35 mL)

was added, followed by tin (IV) chloride (3.24 mL, 27.68 mmol). The

resulting mixture was stirred overnight at room temperature,

poured into a cold saturated NaHCO₃solution and ﬁltered over

celite. The celite cake was washed with ethyl acetate. The organic

layer was washed with saturated NaHCO₃solution, dried over

Na₂SO₄and concentrated under reduced pressure. Silica gel column

chromatography of the crude materials (dichloromethane/meth-

anol, 97/3, v/v) gave the expected product (3.25 g, 65%). Rf (CH₂Cl₂/

³¹P NMR (121 MHz, DMSO-d₆)

d

¼ 27.0. MS FAB > 0 (NBA) m/z 1157

(2M þ H)^þ, 579 (M þ H)^þ, 105 (Bz)^þ, 91 (Bn)^þ. FAB < 0 (NBA) m/z

535 (M ꢀ Ac)^ꢀ.

4.4.13. 1-[2-O-Acetyl-5-O-benzoyl-3-O-benzyl-6-deoxy-6-

diethylphosphono-b-D-glucofuranosyl] uracil (37)

The dried uracil (1.55 g,13.84 mmol) was dissolved in anhydrous

acetonitrile (36 mL) and N,O-bis(trimethylsilyl)acetamide (6.8 mL,

27.68 mmol) was added under argon. The solution was reﬂuxed 2 h

and then the reaction mixture was allowed to cool to room tem-

perature. A solution of sugar-phosphonate 36 (4 g, 6.92 mmol) in

anhydrous acetonitrile (36 mL) was added, followed by tin (IV)

chloride (3.24 mL, 27.68 mmol). The resulting mixture was stirred

overnight at room temperature, poured into a cold saturated

NaHCO₃solution and ﬁltered over celite. The celite cake was

washed with ethyl acetate. The organic layer was washed with

saturated NaHCO₃solution, dried over Na₂SO₄and concentrated

under reduced pressure. Silica gel column chromatography of the

crude materials (dichloromethane/methanol, 97/3, v/v) gave the

expected product (3.2 g, 73%). Rf (CH₂Cl₂/MeOH, 9/1, v/v) 0.56. ¹H

MeOH, 95/5, v/v) 0.48. ¹H NMR (400 MHz, DMSO-d₆)

d

¼ 11.2 (s, 1H,

exchangeable, NH), 8.0 (d, J ¼ 7.49 Hz, 1H, H-6), 8.0e7.4 (m, 10H,

Bz), 7.2e7.1 (m, 5H, Bn), 7.05 (d, J ¼ 7.27 Hz, 1H, H-5⁰), 6.0 (s, 1H, H-

1⁰), 5.85e5.75 (m, 1H, H-5), 5.40 (s, 1H, H-2⁰), 4.7 (dd, J ¼ 3.4e

7.35 Hz, 1H, H-4⁰), 4.6, 4.4 (2d, J ¼ 11.2 Hz, 2H, H-7⁰, H-7⁰’), 4.2 (d,

J ¼ 3.61 Hz,1H, H-3⁰), 4.0e3.8 (m, 4H, OCH₂CH₃), 2.6e2.3 (m, 2H, H-

6⁰, H-6⁰⁰), 2.2 (s, 3H, Ac), 1.2e1.0 (2t, 6H, OCH₂CH₃). ¹³C NMR

(75 MHz, DMSO-d₆)

d

¼ 169.3 (C]O, Ac), 164.3 (C]O, Bz), 163.2 (C-

NMR (400 MHz, DMSO-d₆)

d

¼ 11.3 (s, 1H, exchangeable, NH), 7.90e

4), 154.3 (C-2), 144.8 (C-6), 136.7e127.8 (CeAr), 95.9 (C-5), 89.7 (C-

1⁰), 82.4e82.3 (C-4⁰, d, J ¼ 10 Hz), 78.8 (C-2⁰), 78.19 (C-3⁰), 71.0 (CH₂,

Bn), 65.6 (C-5⁰, d, J ¼ 6 Hz), 61.3e61.2 (m, OCH₂CH₃, C-6⁰), 20.7 (Ac),

7.80 (m, 2H, HeAr, Bz), 7.70e7.60 (m, 1H, HeAr, Bz), 7.5 (d,

J ¼ 8.13 Hz, 1H, H-6), 7.50e7.40 (m, 2H, HeAr, Bz), 7.3e7.2 (m, 5H,

HeAr, Bn), 6.00 (d, J ¼ 1.5 Hz,1H, H-1⁰), 5.8e5.6 (m, 1H, H-5⁰), 5.3 (d,

J ¼ 1.4 Hz, 1H, H-2⁰), 5.2 (m, 1H, H-5), 4.7 (d, J ¼ 10.99 Hz, 1H, H-7⁰),

4.5 (dd, J ¼ 3.66e6.61 Hz, 1H, H-4⁰), 4.4 (d, J ¼ 10.97 Hz, 1H, H-7⁰⁰),

4.2 (d, J ¼ 3.7 Hz, 1H, H-3⁰), 3.95e3.8 (m, 4H, OCH₂CH₃), 2.5e2.25

(m, 2H, H-6⁰, H-6⁰⁰), 2.1 (s, 3H, Ac), 1.1e0.9 (2t, 6H, OCH₂CH₃). ¹³C

16.0e15.8 (OCH₂CH₃). ³¹P NMR (121 MHz, DMSO-d₆)

d

¼ 28.9. MS

FAB > 0 (NBA) m/z 734 (M þ H)^þ. FAB < 0 (NBA) m/z 732 (M ꢀ H)^ꢀ.

4.4.16. 1-[6-Deoxy-6-diethylphosphono-b-D-glucofuranosyl]

cytosine (11)

NMR (75 MHz, DMSO-d₆)

d

¼ 169.9 (C]O, Ac), 164.8 (C]O, Bz),

The nucleotide compound 38 (300 mg, 0.517 mmol) was dis-

solved in anhydrous dichloromethane (44 mL) under argon. The

reaction mixture was cooled to ꢀ78 ^ꢁC and BCl₃1 M in heptane

(18.74 mL, 18.74 mmol) was added. The solution was stirred over-

night, heated slowly to room temperature. Then methanol (80 mL)

and NH₄OH (28 mL) were added at 0 ^ꢁC and the reaction mixture

was concentrated under reduced pressure. Silica gel column chro-

matography of the crude materials (dichloromethane/methanol, 9/

1 then 0/1, v/v) gave the desired compound (153 mg, 75%). Rf

163.4 (C-4), 150.6 (C-2), 140.8 (C-6), 137.3e128.4 (CeAr), 102.2 (C-

5), 88.4 (C-1⁰), 81.8 (C-4⁰), 79.7 (C-3⁰), 79.0 (C-2⁰), 71.5 (C-7⁰), 66 (C-

5⁰), 61.7e61.6 (OCH₂CH₃, m), 21.2 (CH₃, Ac), 16.52, 16.46, 16.40.16.34

(OCH₂CH₃). ³¹P NMR (81 MHz, DMSO-d₆)

d

¼ 27.5. MS FAB>0 (NBA)

m/z 631 (M þ H)^þ, 105 (Bz)^þ, 91 (Bn)^þ. FAB<0 (NBA) m/z 629

(M ꢀ H)^ꢀ.

4.4.14. 1-[6-Deoxy-6-diethylphosphono-b-D-glucofuranosyl]uracil

(9)

(CH₂Cl₂/MeOH, 9/1, v/v) 0.16. ¹H NMR (400 MHz, D₂O)

d

¼ 7.7 (d,

The Pd/C (520 mg, 100% weight) was added to a solution of

nucleotide compound 37 (520 mg, 0.825 mmol) in methanol

(36 mL) at 0 ^ꢁC under argon. Ammonium formate (261 mg,

4.2 mmol) was added at room temperature. The solution was

reﬂuxed for 4.5 h, ﬁltered over celite. The celite cake was washed

with methanol and the ﬁltrate was concentrated under reduced

pressure. Silica gel column chromatography of the crude materials

(dichloromethane/methanol, 95/5, v/v) and then reverse phase

chromatography gave the expected product (154 mg, 50%). Rf

J ¼ 7.59 Hz, 1H, H-6), 5.9 (d, J ¼ 7.58 Hz, 1H, H-5), 5.7 (s, 1H, H-1⁰),

4.4e4.3 (m, 1H, H-5⁰), 4.2 (s, 1H, H-2⁰), 4.15e4.05 (m, 6H, H-3⁰, H-4⁰,

OCH₂CH₃), 2.5e2.15 (m, 2H, H-6⁰, H-6⁰⁰), 1.3e1.2 (m, 6H, OCH₂CH₃).

¹³C NMR (100 MHz, D₂O)

d

¼ 166.3 (C-4), 157.4 (C-2), 142.0 (C-6),

95.1 (C-5), 92.8 (C-1⁰), 85.5e85.3 (C-4⁰, d, J ¼ 15 Hz), 80.6e74.0 (C-

2⁰, C-3⁰), 63.6e63.3 (C-5⁰, OCH₂CH₃, m), 30.5e29.1 (C-6⁰, d,

J ¼ 140 Hz), 15.6e15.5 (OCH₂CH₃, d, J ¼ 6 Hz). ³¹P NMR (121 MHz,

D₂O)

d

¼ 30.8. MS ESI-QT of >0 m/z 394.2 (M þ H)^þ. HR-MS

Calculated for C₁₄H₂₅N₃O₈P: 394.1379; Found: 394.1401.

(CH₂Cl₂/MeOH, 9/1, v/v) 0.16. ¹H NMR (400 MHz, D₂O)

d

¼ 7.75 (d,

J ¼ 8.15 Hz, 1H, H-6), 5.75 (d, J ¼ 8.15 Hz, 1H, H-5), 5.70 (d,

J ¼ 0.78 Hz, 1H, H-1⁰), 4.4e4.3 (m, 1H, H-5⁰), 4.2 (m, 2H, H-2⁰, H-3⁰),

4.15e4.05 (m, 5H, H-4⁰, OCH₂CH₃), 2.4e2.1 (m, 2H, H-6⁰, H-6⁰⁰), 1.3e

4.4.17. 1-[5-O-Benzoyl-3-O-benzyl-6-deoxy-6-diethylphosphono-

b-

D-glucofuranosyl]uracil (39)

The uracil derivative 37 (1.3 g, 2.06 mmol) was dissolved in a

1.1 (m, 6H, OCH₂CH₃). ¹³C NMR (75 MHz, D₂O)

d

¼ 166.5 (C-4), 151.5

mixture of pyridine and glacial acetic acid (22 mL, 4/1, v/v), then

hydrated hydrazine (0.14 mL, 28.06 mmol) was added and the re-

action mixture was heated 5 h at 100 ^ꢁC. The solution was allowed

to room temperature, diluted with acetone, dichloromethane and

water. The resulting organic layer was washed with a saturated

NaHCO₃solution, brine, then dried over Na₂SO₄and concentrated

(C-2), 142.2 (C-6), 101.1 (C-5), 91.9 (C-1⁰), 85.4e85.3 (C-4⁰, d,

J ¼ 14 Hz), 80.4 (C-2⁰), 73.8 (C-3⁰), 63.5e63.3 (C-5⁰, OCH₂CH_3,m),

30.5e29.1 (C-6⁰, d, J ¼ 139 Hz), 15.6e15.5 (OCH₂CH_3,m). ³¹P NMR

(121 MHz, D₂O)

d

¼ 29.2. MS FAB > 0 (NBA) m/z 789 (2M þ H)^þ, 395

(M þ H)^þ. FAB < 0 (NBA) m/z 787 (2M ꢀ H)^ꢀ, 393 (M ꢀ H)^ꢀ. UV

34

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

under reduced pressure. Silica gel column chromatography of the

crude materials (dichloromethane/methanol, 9/1, v/v) gave the

expected product (0.820 g, 70%). Rf (CH₂Cl₂/MeOH, 9/1, v/v) 0.42. ¹H

H-6⁰, H-6⁰⁰), 1.3e1.2 (m, 6H, OCH₂CH₃). ¹³C NMR (100 MHz, D₂O)

d

¼ 166.4 (C-4), 151.8 (C-2), 143.6 (C-6), 100.9 (C-5), 84.5 (C-1⁰),

82.3e82.2 (C-4⁰, d, J ¼ 15 Hz), 71.6 (C-2⁰), 68.8 (C-3⁰), 63.9e63.8 (C-

5⁰, d, J ¼ 6 Hz), 63.3 (m, OCH₂CH₃), 30.0e28.6 (C-6⁰, d, J ¼ 140 Hz),

NMR (400 MHz, DMSO-d₆)

d

¼ 11.35 (s, 1H exchangeable, NH), 7.9e

7.4 (m, 5HeAr, Bz), 7.6 (d, J ¼ 8.13 Hz, 1H, H-6), 7.2 (sl, 5HeAr, Bn),

6.1 (l, 1H exchangeable, OH), 5.8 (d, J ¼ 1.07 Hz,1H, H-1⁰), 5.7 (m,1H,

H-5⁰), 5.2 (d, J ¼ 8.13 Hz, 1H, H-5), 4.6 (m, 2H, H-4⁰, H-7⁰), 4.4 (m, 2H,

H-2⁰, H-7⁰’), 4.0 (d, 1H, J ¼ 3.7 Hz, H-3⁰), 3.95e3.8 (m, 4H, OCH₂CH₃),

2.6e2.3 (m, 2H, H-6⁰, H-6⁰⁰), 1.1e0.9 (m, 6H, OCH₂CH₃). ¹³C NMR

15.6e15.5 (OCH₂CH₃, d, J ¼ 6 Hz). ³¹P NMR (121 MHz, D₂O)

¼ 29.4.

d

MS FAB > 0 (NBA) m/z 395 (M þ H)^þ. FAB<0 (NBA) m/z 393

(M ꢀ H)^ꢀ. UV l_max¼ 262 nm (ε_max¼ 16,900) (EtOH 95). Analysis

Calculated for C₁₄H₂₃N₂O₉P, 0.8H₂O: C, 41.14; H, 6.07; N, 6.85.

Found: C, 41.09; H, 6.44; N, 6.65.

(75 MHz, DMSO-d₆)

d

¼ 164.3 (C]O), 163.0 (C-4), 150.4 (C-2), 140.7

(C-6), 140.7e127.8 (CeAr), 101.0 (C-5), 90.8 (C-3⁰), 81.8 (C-1⁰), 81.4e

81.3 (C-4⁰, d, J ¼ 11 Hz), 76.6 (C-2⁰), 70.8 (C-7), 66.0e65.9 (C-5⁰, d,

J ¼ 6.0 Hz), 61.2e61.1 (m, OCH₂CH₃), 27.3e25.9 (C-6⁰, d, J ¼ 140 Hz),

4.4.20. 1-[6-Deoxy-6-diethylphosphono-b-D-mannofuranosyl]

cytosine (15)

The nucleotide compound 13 (282 mg, 0.566 mmol) was sus-

pended in anhydrous acetonitrile (5.7 mL). N-methyl pyrrolidine

(0.56 mL, 5.436 mmol) and trimethylsilyl chloride (0.22 mL,

1.70 mmol) were added to the reaction mixture. After 1 h stirring,

TFA anhydride (0.24 mL, 1.70 mmol) was added at 0 ^ꢁC, the reaction

mixture was stirred for 1 h more at 0 ^ꢁC and followed by the

addition of 4-nitrophenol (236 mg, 1.70 mmol). After 3 h, the re-

action mixture was concentrated under reduced pressure, the

crude was diluted in dioxane (17 mL) and NH₄OH 28% (5.7 mL) was

added. The reaction mixture was stirred overnight, neutralized

with an aqueous solution of acetic acid (10%, v/v) and freeze-dried.

Due to the small amount of compound obtained (46 mg, 16%), the

crude was directly engaged in the next step without further puri-

ﬁcation. Rf (CH₂Cl₂/MeOH, 9.1, v/v) 0.12. ³¹P NMR (81 MHz, D₂O)

16.0e15.9 (m, OCH₂CH₃). ³¹P NMR (81 MHz, DMSO-d₆)

d

¼ 27.7. MS

FAB > 0 (NBA) m/z 1177 (2M þ H)^þ, 589 (M þ H)^þ. FAB < 0 (NBA) m/

z 1175 (2M ꢀ H)^ꢀ, 587 (M ꢀ H)^ꢀ.

4.4.18. 1-[5-O-Benzoyl-3-O-benzyl-6-deoxy-6-diethylphosphono-

b-

D-mannofuranosyl]uracil (40)

A

solution of compound 39 (1.13 g, 1.92 mmol) in dry

dichloromethane (20 mL), at 0 ^ꢁC, was treated by Dess-Martin

periodinane (1.63 g, 3.84 mmol, 2 eq.). The reaction mixture was

stirred for 2 h at room temperature, diluted with ethyl acetate, and

quenched by adding a saturated solution of NaHCO₃containing

Na₂SO₃. After decantation, the organic layer was dried over Na₂SO₄,

concentrated under reduced pressure and coevaporated with

anhydrous toluene to afford the keto-intermediate. This last was

dissolved in absolute ethanol (20 mL) under argon. A solution of

sodium borohydride (0.227 g, 6.14 mmol) in absolute ethanol

(20 mL) was added dropwise at 0 ^ꢁC. The mixture was stirred for 1 h

at room temperature, and neutralized with HCl 1 N. After adding

dichloromethane and water, the resulting organic layer was

washed with water, dried over Na₂SO₄and concentrated under

reduced pressure. Silica gel column chromatography of the crude

materials (dichloromethane/methanol, 97/3, v/v) gave the expected

product (0.510 g, 45%). Rf (CH₂Cl₂/MeOH, 9/1, v/v) 0.41. ¹H NMR

d

¼ 27.5.

4.4.21. 1-[3-O-Benzoyl-6-deoxy-2,5-O-diacetyl-6-

diethylphosphono- -allofuranosyl]-5-iodo-uracil (43)

b-D

The nucleotide 41 (1 g, 1.72 mmol) was dissolved in anhydrous

dichloromethane (20 mL), iodine chloride (0.51 g, 3.18 mmol) was

added, the mixture was reﬂuxed for 15 h. The organic layer was

washed with aqueous NaHSO₃(2% w/v), dried over MgSO₄and

concentrated. The crude product was puriﬁed on silica gel (DCM/

EtOAc, 1/2, v/v) yielding derivative 43 (1.00 g, 82%) as a pale yellow

solid. Rf (CH₂Cl₂/MeOH, 9/1, v/v) 0.55. ¹H NMR (400 MHz, CDCl₃)

(400 MHz, DMSO-d₆)

d

¼ 11.15 (s,1H exchangeable, NH), 7.9e7.3 (m,

5HeAr, Bz), 7.7 (d, J ¼ 8.12 Hz,1H, H-6), 7.3e7.1 (m, 5HeAr, Bn), 5.95

(d, J ¼ 6.69 Hz, 1H, H-1⁰), 5.75 (d, J ¼ 5.56 Hz, 1H exchangeable, OH),

5.6e5.5 (m, 1H, H-5⁰), 5.1 (dd, J ¼ 1.98e8.1 Hz, 1H, H-5), 4.7 (d,

J ¼ 10.69 Hz, 1H, H-7⁰), 4.5 (m, 1H, H-2⁰), 4.3 (d, J ¼ 10.69 Hz, 1H, H-

7⁰’), 4.15 (m, 1H, H-4⁰), 4.1 (m, 1H, H-3⁰), 3.80e3.65 (m, 4H,

OCH₂CH₃), 2.4e2.1 (m, 2H, H-6⁰, H-6⁰⁰), 1.0e0.8 (m, 6H, OCH₂CH₃).

d

¼ 8.93 (bs, 1H, NH), 8.08 (m, 2H, HeAr), 7.70 (s, 1H, H-6), 7.62 (m,

1H, HeAr), 7.48 (m, 2H, HeAr), 6.08 (d, J ¼ 6.43 Hz, 1H, H-1⁰), 5.78

(dd, J ¼ 4.00, 6.34 Hz, 1H, H-3⁰), 5.49 (m, 1H, H-5⁰), 5.40 (t,

J ¼ 6.40 Hz, 1H, H-2⁰), 4.60 (t, J ¼ 3.62 Hz, 1H, H-4⁰), 4.12 (m, 4H,

OCH₂CH₃), 2.31 (m, 2H, H-6⁰, H-6⁰⁰), 2.22, 1.99 (2s, 6H, Ac), 1.30 (td,

J ¼ 7.06 and 1.82 Hz, 6H, OCH₂CH₃). ¹³C NMR (100 MHz, CDCl₃)

¹³C NMR (75 MHz, DMSO-d₆)

d

¼ 164.9 (C]O), 163.5 (C-4), 151.3 (C-

d

¼ 169.7, 169.6 (C]O, Ac),165.0 (C]O, Bz), 159.3 (C-4), 149.6 (C-2),

2), 144.0 (C-6), 138.3e128.2 (CeAr), 100.4 (C-5), 83.4 (C-1⁰), 77.0,

144.1 (C-6), 133.8, 129.8 (Cq, CeAr), 128.7 (CeAr), 87.4 (C-1⁰), 83.0

(d, J ¼ 7.64 Hz, C-4⁰), 72.6 (C-2⁰), 69.7 (C-5), 69.6 (C-3⁰), 68.2 (C-5⁰),

62.2 (dd, J ¼ 6.14 and 15.03 Hz, OCH₂CH₃), 28.1 (d, J ¼ 140.81 Hz, C-

6⁰), 21.3, 20.3 (CH₃, Ac), 16.3 (d, J ¼ 5.54 Hz, OCH₂CH₃). ³¹P NMR

73.6, 71.183.4 (C-4⁰, C-2⁰, C-3⁰), 61.6e61.5 (d, J ¼ 6 Hz, C-7), 16.5e

16.4 (m, OCH₂CH₃). ³¹P NMR (81 MHz, DMSO-d₆)

d

¼ 27.6. MS

FAB > 0 (NBA) m/z 589 (M þ H)^þ. FAB < 0 (NBA) m/z 587 (M ꢀ H)^ꢀ.

(81 MHz, CDCl₃)

d

¼ 24.7. MS ESI-QT of >0 m/z 709.0 (M þ H)^þ. HR-

4.4.19. 1-[6-Deoxy-6-diethylphosphono-

b-

D-mannofuranosyl]uracil

MS Calculated for C₂₅H₃₁N₂O₁₂PI: 709.0659; Found: 709.0659.

(13)

The nucleotide compound 40 (0.675 g, 1.14 mmol) was dissolved

in anhydrous dichloromethane (50 mL) under argon. The reaction

mixture was cooled to ꢀ78 ^ꢁC and BCl₃1 M in heptane (22.8 mL,

22.8 mmol) was added. The solution was stirred overnight, heated

slowly to room temperature. Then methanol (100 mL) and NH₄Cl

(27 mL) were added at 0 ^ꢁC and the middle was concentrated under

reduced pressure. Two silica gel column chromatographies of the

crude materials (dichloromethane/methanol, 8/2 then 9/1, v/v) and

reverse phase chromatography gave the expected product (0.290 g,

65%). Rf (CH₂Cl₂/MeOH, 9.1, v/v) 0.22. ¹H NMR (400 MHz, D₂O)

4.4.22. 1-[3-O-Benzoyl-6-deoxy-2,5-O-diacetyl-6-

diethylphosphono-b-D-allofuranosyl]-N-4-benz-oyl-5-iodo-cytosine

(44)

Iodine (0.75 g, 2.92 mmol) was added to a suspension of 42

(3.34 g, 4.87 mmol) in a mixture of CH₃COOH and CCl₄(20 mL, 1/1,

v/v). After heating to 40 ^ꢁC, iodic acid (0.77 g, 4.38 mmol) was

added and the reaction mixture was stirred at 40 ^ꢁC overnight. The

suspension was concentrated in vacuo and the residue taken up in

dichloromethane. The organic layer was washed with saturated

aqueous NaHCO₃and aqueous NaHSO₃(2%, w/v), dried with MgSO₄

and concentrated. Column chromatography of the crude product

(dichloromethane/ethyl acetate, 1/2, v/v) yielded compound 44

(2.39 g, 61%) as a pale yellow solid. Rf (CH₂Cl₂/MeOH, 9/1, v/v) 0.64.

d

¼ 7.85 (d, J ¼ 8.16 Hz, 1H, H-6), 6.10 (d, J ¼ 6.87 Hz, 1H, H-1⁰), 5.75

(d, J ¼ 8.15 Hz, 1H, H-5), 4.6 (dd, J ¼ 4.78e6.86 Hz, 1H, H-2⁰), 4.4e

4.35 (m, 1H, H-5⁰), 4.3 (dd, J ¼ 3.14e4.78 Hz, 1H, H-3⁰), 4.15e4.0 (m,

4H, OCH₂CH₃), 3.8 (dd, J ¼ 3.09e8.32 Hz,1H, H-4⁰), 2.4e2.05 (m, 2H,

¹H NMR (400 MHz, CDCl₃)

d

¼ 13.14 (bs, 1H, NH), 8.38 and 8.09 (2m,

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

35

4H, HeAr), 7.81 (s, 1H, H-6), 7.49 (m, 6H, HeAr), 6.13 (d, J ¼ 6.57 Hz,

1H, H-1⁰), 5.81 (dd, J ¼ 3.75 and 6.25 Hz, 1H, H-3⁰), 5.51 (m, 1H, H-

5⁰), 5.41 (t, J ¼ 6.41 Hz, 1H, H-2⁰), 4.63 (t, J ¼ 3.45 Hz, 1H, H-4⁰), 4.12

(m, 4H, OCH₂CH₃), 2.34 (m, 2H, H-6⁰, H-6⁰⁰), 2.23 and 2.00 (2s, 6H,

CH₃, Ac), 1.30 (t, J ¼ 7.06 Hz, 6H, OCH₂CH₃). ¹³C NMR (100 MHz,

4.4.25.1. 1-[6-Deoxy-6-diethylphosphono-

hexyn-1-yl-uracil (47a). According to general procedure, the title

compound (39 mg, 35%) was obtained, as yellow solid, from 45

b

-

D

-allofuranosyl]-5-

(120 mg, 0.24 mmol) and n-hexyne (223

mL, 1.94 mmol). Rf (EtOAc/

MeOH, 8/2, v/v) 0.38. ¹H NMR (300 MHz, DMSO-d₆)

d

¼ 11.58 (bs,

CDCl₃)

d

¼ 180.0 (C ¼ 0, Bz), 169.7, 169.8 (C]O, Ac),165.0 (C]O, Bz),

1H, NH), 8.03 (s, 1H, H-6), 5.79 (d, J ¼ 4.20 Hz, 1H, H-1⁰), 5.58 (d,

J ¼ 5.75 Hz, 1H, OH), 5.41 (d, J ¼ 6.01 Hz, 1H, OH), 5.09 (d,

J ¼ 4.77 Hz, 1H, OH), 4.03e3.95 (m, 7H, OCH₂CH₃, H-2⁰, H-3⁰, H-5⁰),

3.79 (dd, J ¼ 3.25 Hz,1H, H-4⁰), 2.37 (t, J ¼ 6.73 Hz, 2H, CH₂), 2.01 (m,

2H, H-6⁰, H-6⁰⁰), 1.45 (m, 4H, CH₂), 1.22 (td, J ¼ 7.06 and 1.96 Hz,

156.1 (C-4), 147.4 (C-2), 144.8 (C-6), 136.4, 133.9, 133.1, 130.4, 129.8,

128.7 (Cq, CeAr),128.3 (CeAr), 87.5 (C-1⁰), 83.4 (d, J ¼ 7.60 Hz, C-4⁰),

72.7 (C-2⁰), 70.9 (C-5), 69.6 (C-3⁰), 68.3 (C-5⁰), 62.4 (dd, J ¼ 15.38 and

6.22 Hz, OCH₂CH₃), 28.2 (d, J ¼ 140.8 Hz, C-6⁰), 21.3 and 20.3 (CH₃,

Ac), 16.3 (d, J ¼ 5.87 Hz, OCH₂CH₃). ³¹P NMR (81 MHz, CDCl₃)

OCH₂CH₃), 0.85 (t, 3H, CH₃). ¹³C NMR (75 MHz, DMSO-d₆)

d

¼ 161.1

d

¼ 24.7. MS ESI-QT of >0 m/z 812.0 (M þ H)^þ. HR-MS Calculated for

(C-4), 149.8 (C-2), 142.8 (C-6), 95.4 and 93.4 (C^C), 87.2 (C-1⁰), 87.1

(d, J ¼ 13.98 Hz, C-4⁰), 73.1 (C-2⁰), 72.6 (C-3⁰), 68.3 (C-5⁰), 65.4 (C-5),

61.9 (dd, J ¼ 23.73 and 5.95 Hz, OCH₂CH₃), 30.2 (CH₂), 28.4 (d,

J ¼ 137.67 Hz, C-6⁰), 21.3 and 18.5 (CH₂), 16.2 (OCH₂CH₃), 13.4 (CH₃).

C

₃₂H₃₆N₃O₁₂PI: 812.1081; Found: 812.1066.

³¹P NMR (81 MHz, DMSO-d₆)

d

¼ 31.8. MS ESI-QT of >0 m/z 447

4.4.23. 1-[6-Deoxy-6-diethylphosphono-b-D-allofuranosyl]-5-iodo-

uracil (45)

(M þ H)^þ. HR-MS Calculated for C₁₈H₂₈N₂O₉P: 447.1532; Found:

The nucleotide 43 (1.4 g, 2.40 mmol), was dissolved in meth-

anolic ammonia (48 mL) and stirred at room temperature until

completion of the reaction was indicated by TLC. Puriﬁcation by

column chromatography (dichloromethane/methanol, 9/1, v/v)

gave the expected compound 45 (0.79 g, 84%) as a white powder. Rf

(CH₂Cl₂/MeOH, 9/1, v/v) 0.38. ¹H NMR (400 MHz, DMSO-d₆)

447.1535.

4.4.25.2. 1-[6-Deoxy-6-diethylphosphono-b-D-allofuranosyl]-5-(3,3-

dimethyl-1-butyn-1-yl)-uracil (47b). According to general proce-

dure, the title compound (114 mg, 42%) was obtained, as a yellow

solid, from 45 (300 mg, 0.6 mmol) and 3,3-dimethyl-1-butyne

¼ 8.30 (s, 1H, H-6), 5.74 (d, J ¼ 5.81 Hz, 1H, H-1⁰), 5.61 (d,

(448

(300 MHz, DMSO-d₆)

m

L, 3.6 mmol). Rf (EtOAc/MeOH, 8/2, v/v) 0.31. ¹H NMR

d

J ¼ 5.32 Hz, 1H, OH), 5.41 (d, J ¼ 5.30 Hz, 1H, OH), 5.07 (d,

J ¼ 4.65 Hz, 1H, OH), 4.01 (m, 7H, OCH₂CH₃, H-2⁰, H-3⁰, H-5⁰), 3.84

(m, 1H, H-4⁰), 1.99 (m, 2H, H-6⁰, H-6⁰⁰), 1.24 (t, J ¼ 7.05 Hz, 6H,

d

¼ 11.60 (bs, 1H, NH), 8.00 (s, 1H, H-6), 5.75

(d, J ¼ 6.24 Hz, 1H, H-1⁰), 5.58 (d, J ¼ 5.69 Hz, 1H, OH), 5.39 (d,

J ¼ 5.59 Hz, 1H, OH), 5.09 (d, J ¼ 4.83 Hz, 1H, OH), 4.05e3.97 (m, 7H,

OCH₂CH₃, H-2⁰, H-3⁰, H-5⁰), 3.80 (m, 1H, H-4⁰), 2.02 (m, 2H, H-6⁰, H-

6⁰⁰), 1.25 (s, 9H, C(CH₃)₃), 1.22 (td, J ¼ 7.06 and 1.96 Hz, OCH₂CH₃).

OCH₂CH₃). ¹³C NMR (100 MHz, DMSO-d₆)

d

¼ 160.4 (C-4), 150.4 (C-

2), 145.0 (C-6), 87.4 (C-1⁰), 86.9 (d, J ¼ 13.13 Hz, C-4⁰), 73.4 (C-2⁰),

69.7 (C-5), 68.1 (C-3⁰), 65.4 (d, J ¼ 2.38 Hz, C-5⁰), 61.1 (dd, J ¼ 15.12

and 6.19 Hz, OCH₂CH₃), 30.3 (d, J ¼ 139.06 Hz, C-6⁰), 16.2 (d,

¹³C NMR (75 MHz, DMSO-d₆)

d

¼ 162.7 (C-4), 151.0 (C-2), 144.1 (C-

6), 102.3 and 100.5 (C^C), 88.5 (C-1⁰), 88.2 (d, J ¼ 13.89 Hz, C-4⁰),

74.3 (C-2⁰), 72.3 (C-3⁰), 69.4 (C-5⁰), 66.6 (C-5), 61.0 (dd, J ¼ 23.73 and

5.95 Hz, OCH₂CH₃), 31.9 (C(CH₃)₃), 29.7 (d, J ¼ 137.60 Hz, C-6⁰), 16.2

J ¼ 5.99 Hz, OCH₂CH₃). ³¹P NMR (81 MHz, DMSO-d₆)

d

¼ 28.9. MS

ESI-QT of >0 m/z 520.9 (M

þ

H)^þ. HR-MS Calculated for

(OCH₂CH₃). ³¹P NMR (81 MHz, DMSO-d₆)

d

¼ 31.8. MS ESI-QT of

C

₁₄H₂₃N₂O₉PI: 521.0186; Found: 521.0196.

>0 m/z 447 (M þ H)^þ. HR-MS Calculated for C₁₈H₂₈N₂O₉P:

447.1532; Found: 447.1535.

4.4.24. 1-[6-Deoxy-diethylphosphono-b-D-allofuranosyl]-5-iodo-

cytosine (46)

4.4.25.3. 1-[6-Deoxy-6-diethylphosphono-b-D-allofuranosyl]-5-

phenylethynyl-uracil (47c). According to general procedure, the ti-

tle compound (184 mg, 94%) was obtained, as a yellow solid, from

Derivative 46 (1.48 g, 2.85 mmol, 97%), was obtained by treat-

ment of 44 (2.39 g, 2.95 mmol) as described for 45. Rf (CH₂Cl₂/

MeOH, 9/1, v/v) 0.44. ¹H NMR (400 MHz, DMSO-d₆)

d

¼ 8.23 (s, 1H,

45 (205 mg, 0.39 mmol) and phenylacetylene (260

mL, 2.34 mmol).

H-6), 7.88 (bs, 1H, NH), 6.68 (bs, 1H, NH), 5.71 (d, J ¼ 5.14 Hz, 1H, H-

1⁰), 5.62 (d, J ¼ 5.10 Hz,1H, OH), 5.35 (d, J ¼ 5.61 Hz,1H, OH), 5.02 (d,

J ¼ 5.16 Hz, 1H, OH), 4.00 (m, 7H, OCH₂CH₃, H-2⁰, H-3⁰, H-5⁰), 3.82

(m, 1H, H-4⁰), 1.97 (m, 2H, H-6⁰, H-6⁰⁰), 1.23 (t, J ¼ 7.4 Hz, 6H,

Rf (CH₂Cl₂/MeOH, 9/1, v/v) 0.20. ¹H NMR (400 MHz, DMSO-d₆)

d

¼ 11.74 (bs, 1H, NH), 8.31 (s, 1H, H-6), 7.50e7.40 (m, 5H, HeAr),

5.80 (d, J ¼ 5.62 Hz, 1H, H-1⁰), 5.67 (d, J ¼ 5.46 Hz, 1H, OH), 5.44 (d,

J ¼ 5.48 Hz, 1H, OH), 5.09 (d, J ¼ 5.07 Hz, 1H, OH), 4.04e3.97 (m, 7H,

OCH₂CH₃, H-2⁰, H-3⁰, H-5⁰), 3.86 (m, 1H, H-4⁰), 2.14e1.86 (m, 2H, H-

6⁰, H-6⁰⁰), 1.23 (td, J ¼ 7.06 and 1.96 Hz, OCH₂CH₃). ¹³C NMR

OCH₂CH₃). ¹³C NMR (100 MHz, DMSO-d₆)

d

¼ 163.6 (C-4), 154.1 (C-

2), 147.8 (C-6), 88.9 (C-1⁰), 86.3 (d, J ¼ 13.34 Hz, C-4⁰), 73.6 (C-2⁰),

68.0 (C-3⁰), 65.3 (d, J ¼ 2.56 Hz, C-5⁰), 61.0 (dd, J ¼ 6.23 and 19.75 Hz,

OCH₂CH₃), 56.76 (C-5), 29.9 (d, J ¼ 137.55 Hz, C-6⁰), 16.2 (d,

(100 MHz, DMSO-d₆)

d

¼ 161.2 (C-4), 149.7 (C-2), 143.9 (C-6), 131.1,

128.6, 122.3 (CeAr), 98.4 and 91.8 (C^C), 87.5 (C-1⁰), 86.9 (d,

J ¼ 13.32 Hz, C-4⁰), 82.2 (C-5), 73.5 (C-2⁰), 68.1 (C-3⁰), 65.3 (C-5⁰),

61.0 (dd, J ¼ 23.73 and 5.95 Hz, OCH₂CH₃), 30.1 (d, J ¼ 137.50 Hz, C-

J ¼ 5.97 Hz, OCH₂CH₃). ³¹P NMR (81 MHz, DMSO-d₆)

d

¼ 29.0. MS

ESI-QT of >0 m/z 520.0 (M

þ

H)^þ. HR-MS Calculated for

C

₁₄H₂₄N₃O₈PI: 520.0346; Found: 520.0319.

6⁰), 16.2 (OCH₂CH₃). ³¹P NMR (81 MHz, DMSO-d₆)

d

¼ 29.0. MS ESI-

QT of >0 m/z 495.3 (M þ H)^þ. HR-MS Calculated for C₂₂H₂₈N₂O₉P:

4.4.25. General procedure for Sonogashira coupling reaction

(compounds 47aec and 48aec)

495.1532; Found: 495.1540.

The C5-iodonucleotide (1 eq.) was dissolved in anhydrous DMF

(30 mL/mmol), Pd(PPh₃)₄(0.2 eq.) and CuI (0.4 eq.) were added and

then the required alcyne derivative (6 eq.) and Et₃N (4 eq.) were

added dropwise under Ar atmosphere. The reaction mixture was

stirred at room temperature in the dark, until completion of the

reaction was indicated by TLC. Then, the reaction was concentrated

to dryness under high vacuum. Column chromatography of the

crude mixture on silica gel (dichloromethane/methanol, 9/1, v/v)

and eventually reverse phase (gradient: water to methanol, 30%)

gave the expected derivative.

4.4.25.4. 1-[6-Deoxy-diethylphosphono-b-D-allofuranosyl]-5-hexyn-

1-yl-cytosine (48a). According to general procedure, the title

compound (135 mg, 68%) was obtained, as a white solid from 46

(217 mg, 0.42 mmol) and n-hexyne (286

m

L, 2.52 mmol). Rf (CH₂Cl₂/

MeOH, 9/1, v/v) 0.32. ¹H NMR (400 MHz, DMSO-d₆)

d

¼ 8.03 (s, 1H,

H-6), 7.73 (bs, 1H, NH), 6.77 (bs, 1H, NH), 5.77 (d, J ¼ 5.47 Hz, 1H H-

1⁰), 5.54 (d, J ¼ 5.20 Hz,1H, OH), 5.31 (d, J ¼ 5.39 Hz,1H, OH), 5.01 (d,

J ¼ 4.92 Hz, 1H, OH), 4.01 (m, 7H, OCH₂CH₃, H-2⁰, H-3⁰, H-5⁰), 3.80

(m, 1H, H-4⁰), 2.40 (t, J ¼ 6.98 Hz, 2H, CH₂eC]C), 1.96 (m, 2H, H-6⁰,

H-6⁰⁰), 1.48 (m, 4H, CH₂), 1.23 (t, J ¼ 7.05 Hz, 6H, OCH₂CH₃), 0.89 (t,

36

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

J ¼ 7.20 Hz, CH₃). ¹³C NMR (100 MHz, DMSO-d₆)

d

¼ 164.3 (C-4),

OH), 5.43 (d, J ¼ 5.41 Hz, 1H, OH), 5.09 (d, J ¼ 4.78 Hz, 1H, OH), 4.13

(m, 2H, H-2⁰, H-3⁰), 3.99 (m, 5H, OCH₂CH₃, H-5⁰), 3.87 (m, 1H, H-4⁰),

2.00 (m, 2H, H-6⁰, H-6⁰⁰), 1.21 (td, J ¼ 7.05 and 2.96 Hz, 6H,

153.8 (C-2), 144.2 (C-6), 95.7 and 90.7 (C^C), 88.6 (C-1⁰), 86.6 (d,

J ¼ 14.32 Hz, C-4⁰), 73.6 (C-2⁰), 71.7 (C-3⁰), 68.1 (C-5), 65.4 (d,

J ¼ 2.97 Hz, C-5⁰), 61.1 (dd, J ¼ 17.63, 6.07 Hz, OCH₂CH₃), 30.2 (d,

J ¼ 137.79 Hz, C-6⁰), 30.1 (CH₂C^C), 21.5, 18.7 (CH₂), 16.2 (d,

J ¼ 5.91 Hz, OCH₂CH₃), 13.4 (CH₃). ³¹P NMR (81 MHz, DMSO-d₆)

OCH₂CH₃). ¹³C NMR (100 MHz, DMSO-d₆)

d

¼ 162.0 (C-4), 150.2 (C-

2), 138.0 (C-6), 132.9 (Cq), 128.0, 127.2 (CeAr), 113.7 (C-5), 87.5 (C-

1⁰), 86.8 (d, J ¼ 12.85 Hz, C-4⁰), 73.4 (C-2⁰), 68.3 (C-3⁰), 65.5 (d,

J ¼ 2.28 Hz, C-5⁰), 61.1 (dd, J ¼ 23.60 and 6.05 Hz, OCH₂CH₃), 30.1 (d,

J ¼ 137.39 Hz, C-6⁰), 16.2 (d, J ¼ 5.87 Hz, OCH₂CH₃). ³¹P NMR

d

¼ 29.1. MS ESI-QT of >0 m/z 474.0 (M þ H)^þ. HR-MS Calculated for

C

₂₀H₃₃N₃O₈P: 474.2005; Found: 474.1999.

(81 MHz, DMSO-d₆)

d

¼ 28.9. MS ESI-QT of >0 m/z 471.2474

4.4.25.5. 1-[6-Deoxy-diethylphosphono-

b

-

D

-allofuranosyl]-5-(3,3-

(M þ H)^þ. HR-MS Calculated for C₂₀H₂₈N₂O₉P: 471.1532; Found:

dimethyl-1-butyn-1-yl)-cytosine (48b). According to general pro-

cedure, the title compound (145 mg, 80%) was obtained, as a white

solid, from 46 (200 mg, 0.39 mmol) and 3,3-dimethyl-1-butyne

471.1518.

4.4.26.2. 1-[6-Deoxy-6-diethylphosphono-b-D-allofuranosyl]-5-(2-

(283

(400 MHz, DMSO-d₆)

m

L, 2.34 mmol). Rf (CH₂Cl₂/MeOH, 9/1, v/v) 0.17. ¹H NMR

(E)-phenylvinyl)-uracil (49e). According to general procedure, the

title compound (71 mg, 25%) was obtained, as a white solid, from 45

(301 mg, 0.58 mmol) and 2-(E)-phenylvinyl-boronic acid (111 mg,

0.75 mmol). Rf (CH₂Cl₂/MeOH, 9/1, v/v) 0.30. ¹H NMR (400 MHz,

d

¼ 8.02 (s, 1H, H-6), 7.75 (bs, 1H, NH), 6.49

(bs, 1H, NH), 5.76 (d, J ¼ 5.52 Hz, 1H, H-1⁰), 5.53 (d, J ¼ 5.29 Hz, 1H,

OH), 5.29 (d, J ¼ 5.75 Hz, 1H, OH), 4.98 (d, J ¼ 5.16 Hz, 1H, OH), 3.99

(m, 7H, OCH₂CH₃, H-2⁰, H-3⁰, H-5⁰), 3.81 (m, 1H, H-4⁰), 2.05 (m, 2H,

H-6⁰, H-6⁰⁰), 1.27 (s, 9H, CH₃), 1.23 (t, J ¼ 7.05 Hz, 6H, OCH₂CH₃). ¹³C

DMSO-d₆)

d

¼ 11.41 (bs, 1H, NH), 8.10 (s, 1H, H-6), 7.49e7.23 (m, 6H,

HeAr, HC]C), 6.94 (d, J ¼ 16.47 Hz, 1H, HC]C), 5.84 (d, J ¼ 5.61 Hz,

1H, H-1⁰), 5.73 (bs, 1H, OH), 5.45 (bs, 1H, OH), 5.13 (bs, 1H, OH), 4.02

(m, 7H, OCH₂CH₃, H-2⁰, H-3⁰, H-5⁰), 3.85 (m, 1H, H-4⁰), 2.02 (m, 2H,

H-6⁰, H-6⁰⁰), 1.24 (q, J ¼ 6.80 Hz, OCH₂CH₃). ¹³C NMR (100 MHz,

NMR (100 MHz, DMSO-d₆)

d

¼ 164.0 (C-4), 153.8 (C-2), 144.4 (C-6),

103.1 and 90.5 (C^C), 88.6 (C-1⁰), 86.4 (d, J ¼ 13.82 Hz, C-4⁰), 73.5

(C-2⁰), 70.2 (C-3⁰), 68.1 (C-5), 65.4 (d, J ¼ 2.73 Hz, C-5⁰), 61.1 (dd,

J ¼ 26.42 and 5.99 Hz, OCH₂CH₃), 30.5 (CH₃), 30.0 (d, J ¼ 137.97 Hz,

C-6⁰), 27.8 (Cq), 16.2 (d, J ¼ 6.05 Hz, OCH₂CH₃). ³¹P NMR (81 MHz,

DMSO-d₆)

d

¼ 162.1 (C-4), 149.8 (C-2), 138.3 (C-6), 137.5 (Cq), 128.6,

127.6, 127.3, 125.9 (CeAr, C]C), 121.2 (C]C), 110.9 (C-5), 87.4 (C-1⁰),

87.0 (d, J ¼ 14.42 Hz, C-4⁰), 73.5 (C-2⁰), 68.4 (C-3⁰), 65.5 (C-5⁰), 61.1

(dd, J ¼ 31.48 and 6.0 Hz, OCH₂CH₃), 30.1 (d, J ¼ 138.0 Hz, C-6⁰), 13.2

DMSO-d₆)

d

¼ 29.2. MS ESI-QT of >0 m/z 474.2 (M þ H)^þ. HR-MS

Calculated for C₂₀H₃₃N₃O₈P: 474.2005; Found: 474.2017.

(d, J ¼ 5.77 Hz, OCH₂CH₃). ³¹P NMR (81 MHz, DMSO-d₆)

¼ 29.3. MS

d

ESI-QT of >0 m/z 497.2 (M

þ

H)^þ. HR-MS Calculated for

4.4.25.6. 1-[6-Deoxy-diethylphosphono-b-D-allofuranosyl]-5-

phenylethynyl-cytosine (48c). According to general procedure, the

title compound (165 mg, 80%) was obtained, as a white solid, from

C₂₂H₃₀N₂O₉P: 497.1689; Found: 497.1682.

46 (217 mg, 0.42 mmol) and phenylacetylene (275

mL, 2.52 mmol).

Rf (CH₂Cl₂/MeOH, 9/1, v/v) 0.17. ¹H NMR (400 MHz, DMSO-d₆)

4.4.26.3. 1-[6-Deoxy-diethylphosphono-b-D-allofuranosyl]-5-phenyl-

cytosine (50d). According to general procedure, the title compound

(203 mg, 59%) was obtained, as a white lyophilisate, from 46

(203 mg, 0.39 mmol) and phenyl-boronic acid (62 mg, 0.51 mmol).

d

¼ 8.30 (s, 1H, H-6), 7.83 (bs, 1H, NH), 7.59 (m, 2H, HeAr), 7.39 (m,

3H, HeAr), 7.09 (bs, 1H, NH), 5.80 (d, J ¼ 5.23 Hz, 1H, H-1⁰), 5.64 (d,

J ¼ 5.27 Hz, 1H, OH), 5.36 (d, J ¼ 5.70 Hz, 1H, OH), 5.01 (d,

J ¼ 5.37 Hz, 1H, OH), 3.99 (m, 7H, OCH₂CH₃, H-2⁰, H-3⁰, H-5⁰), 3.85

(m, 1H, H-4⁰), 1.99 (m, 2H, H-6⁰, H-6⁰⁰), 1.23 (td, J ¼ 7.04 and 2.24 Hz,

Rf (CH₂Cl₂/MeOH, 9/1, v/v) 0.17. ¹H NMR (400 MHz, D₂O)

d

¼ 7.75 (s,

1H, H-6), 7.49e7.41 (m, 5H, HeAr), 5.94 (d, J ¼ 4.10 Hz, 1H, H-1⁰),

4.34e4.17 (m, 3H, H-2⁰, H-3⁰, H-5⁰), 4.10 (m, 4H, OCH₂CH₃), 3.98 (t,

J ¼ 4.41 Hz, 1H, H-4⁰), 2.18 (m, 2H, H-6⁰, H-6⁰⁰), 1.26 (t, J ¼ 7.09 Hz,

6H, OCH₂CH₃). ¹³C NMR (100 MHz, DMSO-d₆)

d

¼ 163.7 (C-4), 153.7

(C-2), 145.6 (C-6), 131.2, 128.4, 122.5 (CeAr), 93.7 and 89.8 (C^C),

88.7 (C-1⁰), 86.4 (d, J ¼ 13.92 Hz, C-4⁰), 81.4 (C-2⁰), 73.9 (C-3⁰), 68.0

(C-5), 65.3 (C-5⁰), 61.1 (dd, J ¼ 26.11 and 6.14 Hz, OCH₂CH₃), 30.0 (d,

J ¼ 137.41 Hz, C-6⁰), 16.2 (d, J ¼ 5.98 Hz, OCH₂CH₃). ³¹P NMR

OCH₂CH₃). ¹³C NMR (100 MHz, D₂O)

d

¼ 162.4 (C-4), 154.9 (C-2),

138.0 (C-6), 130.0, 127.2, 127.0, 126.7 (CeAr), 108.5 (C-5), 87.9 (C-1⁰),

83.9 (d, J ¼ 17.87 Hz, C-4⁰), 71.6 (C-2⁰), 66.8 (C-3⁰), 63.6 (d,

J ¼ 4.58 Hz, C-5⁰), 61.3 (d, J ¼ 7.16 Hz, OCH₂CH₃), 27.0 (d,

J ¼ 140.95 Hz, C-6⁰), 13.4 (d, J ¼ 5.74 Hz, OCH₂CH₃). ³¹P NMR

(81 MHz, DMSO-d₆)

d

¼ 29.1. MS ESI-QT of >0 m/z 494.2 (M þ H)^þ.

HR-MS Calculated for C₂₂H₂₉N₃O₈P: 494.1692; Found: 494.1708.

(81 MHz, D₂O)

d

¼ 31.68. MS ESI-QT of >0 m/z 470.0 (M þ H)^þ. HR-

MS Calculated for C₂₀H₂₉N₃O₈P: 470.1692; Found: 470.1705.

4.4.26. General procedure for Suzuki coupling reaction (compounds

49d, 49e and 50d, 50e)

A round bottom ﬂask containing C5-iodonucleotide (1 eq.),

boronic acid derivative (1.3 eq.) and KOH (2 eq.) was purged with

Ar. Upon addition of TPPS (0.025 eq.), PdNa₂Cl₄(0.01 eq.) and

degazed H₂O/DMF (1/1, v/v, 5 mL/mmol), the reaction mixture was

stirred at 100 ^ꢁC until completion of the reaction was indicated by

HPLC. Then, the reaction mixture was concentrated to dryness

under high vacuum. Column chromatography of the crude mate-

rials on silica gel (gradient: dichloromethane/methanol, 9/1, v/v)

and eventually by reverse phase chromatography (water to meth-

anol 50%) gave the expected derivative.

4.4.26.4. 1-[6-Deoxy-diethylphosphono-b-D-allofuranosyl]-5-(2-(E)-

phenylvinyl)cytosine (50e). According to general procedure, the ti-

tle compound (86 mg, 45%) was obtained, as a white solid, from 46

(202 mg, 0.39 mmol) and 2-(E)-phenylvinylboronic acid (75 mg,

0.51 mmol). Rf (CH₂Cl₂/MeOH, 7/2/1, v/v/v) 0.57. ¹H NMR (400 MHz,

DMSO-d₆)

d

¼ 8.28 (s, 1H, H-6), 7.58e7.21 (m, 7H, HeAr, NH₂), 7.09

(d, J ¼ 16.11 Hz, 1H, HC¼), 6.89 (d, J ¼ 16.04 Hz, 1H, HC¼), 5.81 (d,

J ¼ 4.56 Hz, 1H, H-1⁰), 5.70 (d, J ¼ 5.29 Hz, 1H, OH), 5.38 (d,

J ¼ 5.22 Hz, 1H, OH), 5.01 (d, J ¼ 5.33 Hz, 1H, OH), 4.13e3.99 (m, 7H,

OCH₂CH₃, H-2⁰, H-3⁰, H-5⁰), 3.87 (m, 1H, H-4⁰), 2.18e1.89 (m, 2H, H-

6⁰, H-6⁰⁰), 1.22 (m, OCH₂CH₃). ¹³C NMR (100 MHz, DMSO-d₆)

4.4.26.1. 1-[6-Deoxy-6-diethylphosphono-

b

-

D

-allofuranosyl]-5-

d

¼ 164.0 (C-4), 155.0 (C-2), 138.4 (C-6), 137.7, 128.9, 127.8, 127.7,

phenyl-uracil (49d). According to general procedure, the title

compound (97 mg, 35%) was obtained, as a white solid, from 45

(305 mg, 0.59 mmol) and phenyl-boronic acid (93 mg, 0.77 mmol).

Rf (CH₂Cl₂/MeOH, 9/1, v/v) 0.35. ¹H NMR (400 MHz, DMSO-d₆)

126.9 (CeAr, C]C), 119.9 (C]C), 104.7 (C-5), 89.9 (C-1⁰), 86.5 (d,

J ¼ 13.89 Hz, C-4⁰), 74.4 (C-2⁰), 68.6 (C-3⁰), 65.9 (C-5⁰), 61.6 (dd,

J ¼ 29.58 and 6.18 Hz, OCH₂CH₃), 30.5 (d, J ¼ 137.36 Hz, C-6⁰), 16.7

(d, J ¼ 5.95 Hz, OCH₂CH₃). ³¹P NMR (81 MHz, DMSO-d₆)

¼ 29.2. MS

d

¼ 11.56 (bs, 1H, NH), 8.07 (s, 1H, H-6), 7.53 (m, 2H, HeAr), 7.37 (m,

ESI-QT of>0 m/z 496.1 (M

þ

H)^þ. HR-MS Calculated for

3H, HeAr), 5.87 (d, J ¼ 5.19 Hz, 1H, H-1⁰), 5.54 (d, J ¼ 5.51 Hz, 1H,

C₂₂H₃₁N₃O₈P: 496.1849; Found: 496.1848.

M. Meurillon et al. / European Journal of Medicinal Chemistry 77 (2014) 18e37

37

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Acknowledgments

This work was supported by Institutional funds from the Institut

National du Cancer (INCa, project n^ꢁ2010-200 “Nucleotarg”) and

Agence Nationale de la Recherche (ANR Programme Blanc 2011-

SIMI7, projet cN-II Focus). M.M. and A.H. are grateful for their

doctoral fellowships to the Centre National de la Recherche Scien-

tiﬁque & Région Languedoc-Roussillon and the Ministère National

de l’Enseignement, de la Recherche et des Technologies, respec-

tively. We kindly thank M.-C. Bergogne for manuscript editing.

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studies of novel 5-substituted alkynylpyrimidine nucleosides as potent in-

hibitors of mycobacteria, Journal of Medicinal Chemistry 48 (2005) 7012e

7017.

Appendix A. Supplementary data

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substituted 2 ’-Deoxyuridine using Suzuki-Miyaura cross-coupling in water,

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of 5-iodo to 5-alkynyl and derived 5-substituted uracil bases and nucleosides,

Journal of Organic Chemistry 48 (1983) 1854e1862.

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Article Doi

Structure-activity relationships of β-hydroxyphosphonate nucleoside analogues as cytosolic 5′-nucleotidase II potential inhibitors: Synthesis, in vitro evaluation and molecular modeling studies

DOI: 10.1016/j.ejmech.2014.02.055

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