Acyclic nucleoside bisphosphonates: Synthesis and properties of chiral 2-amino-4,6-bis[(phosphonomethoxy)alkoxy]pyrimidines

European Journal of Medicinal Chemistry 44 (2009) 2408–2424

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European Journal of Medicinal Chemistry

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Original article

Acyclic nucleoside bisphosphonates: Synthesis and properties of chiral

2-amino-4,6-bis[(phosphonomethoxy)alkoxy]pyrimidines

*

ˇ

´

ꢀ

´

ˇ´

´

Petra Dolakova , Martin Dracınsky, Milena Masojıdkova, Veronika Solınova,

ꢀ

´

ˇ ˇ

´

Vaclav Kasicka, Antonın Holy

´

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i. Flemingovo nam. 2, CZ-166 10 Prague 6, Czech Republic

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 28 May 2008

Received in revised form 15 September 2008

Accepted 18 September 2008

Available online 2 October 2008

2-Amino-4,6-bis[(phosphonomethoxy)alkoxy]pyrimidines bearing two equal or different achiral or

chiral phosphonoalkoxy chains have been prepared either by aromatic nucleophilic substitution of

2-amino-4,6-dichloropyrimidine or by alkylation of 4,6-dihydroxy-2-(methylsulfanyl)pyrimidine with

appropriate phosphonate-bearing building block. Alkylation of 4,6-dihydroxy-2-(methylsulfanyl)py-

rimidine proved to be the method of choice for efﬁcient preparation of variety of bisphosphonates. The

enantiomeric purity of selected compounds was determined by capillary electrophoresis. Antiviral

activity of bisphosphonates is discussed.

Keywords:

Acyclic nucleoside phosphonates

Pyrimidine

Bisphosphonates

Alkylation

1. Introduction

(3a–d) and 2-amino-4,6-dihydroxypyrimidine signiﬁcantly inhibited

replication of retroviruses and herpes viruses in cell culture.

Acyclic nucleoside phosphonates (ANPs) [1] represent a key class

of nucleotide analogs with a broad spectrum of antiviral and cyto-

static activity. Among ANPs, particularly 9-[2-(phosphonomethox-

y)ethyl]adenine (PMEA, adefovir, 1a, Fig. 1) is active against DNA and

retroviruses [2]; its prodrug, adefovir dipivoxil [3], was approved for

hepatitis B therapy (Hepsera) [4]. 9-(R)-[2-(Phosphonomethoxy)-

propyl]adenine (PMPA, tenofovir, 1c) is a promising anti-HIV drug;

its prodrug Viread was approved for treatment of AIDS [5]. A third

type of antiviral compounds is represented by 9-(S)-[3-hydroxy-2-

(phosphonomethoxy)propyl]cytosine (HPMPC, cidofovir, Vistide, 2)

which possesses general anti-DNA-viral activity [6]. Cidofovir was

approved for treatment of cytomegalovirus retinitis in AIDS patients.

The 2,6-diaminopurine derivatives (1b and 1d) and their guanine

counterparts are potent antivirals and exhibit powerful antitumor

activity [7].

Compounds 3 can be considered as analogs of 2,6-diaminopurine

with an open imidazole ring of the purine moiety. This structural

relation is strongly supported by the ﬁnding that the corresponding

analog of PMPDAP, i.e. 2,4-diamino-6-[2-(phosphonomethoxy)-

propoxy]pyrimidine (3d), has the same selective antiretroviral

activity as (R)-PMPDAP (1d). This activity is also limited only to the

(R)-enantiomer, while the (S)-enantiomer is similarly devoid

of antiviral activity as is (S)-PMPDAP. Further SAR studies

demonstrated that 5-substituted derivatives of 2,4-diamino-6-[2-

(phosphonomethoxy)ethoxy]pyrimidine (3a) markedly inhibited

retrovirus replication in cell culture [9]. The 5-methyl derivative 3b

was exquisitely inhibitory to human immunodeﬁciency virus and

Moloney murine sarcoma virus-induced cytopathicity in cell culture

but also cytostatic to CEM cell cultures. The 5-halogen-substituted

derivatives (3c) showed pronounced antiretroviral activity, compa-

rable to that of the reference drugs adefovir and tenofovir, but were

devoid of any measurable toxicity.

The isomeric compounds 6a and 7a (Fig. 2) bearing two phos-

phonoalkoxy chains [8a] may be considered as a second group of

‘‘open-ring’’ ANPs and potential antivirals. Direct alkylation of

2-amino-4,6-dihydroxypyrimidine (5) with 2-(diisopropoxyph-

osphorylmethoxy)ethylchloride gave a mixture of disubstituted

regioisomers 6a and 7a approximately in the same ratio. While 2-

amino-4-[2-(phosphonomethoxy)ethoxy]-1-[2-(phosphonometh-

oxy)ethyl]pyrimidin-6(1H)-one (7a) was not antivirally active,

2-amino-4,6-bis[2-(phosphonomethoxy)ethoxy]pyrimidine (6a)

We have recently described a new type of antiviral acyclic

nucleoside phosphonates originating from 2-substituted 4-amino-

6-hydroxypyrimidines [8]. Alkylation of 6-hydroxypyrimidines by

phosphonate-bearing building block afforded a mixture of O⁶- and

N¹-regioisomer. While none of the isomeric 1-[2-(phosphono-

methoxy)ethyl]pyrimidin-6-one derivatives

4 was antivirally

active, compounds derived from 2,4-diamino-6-hydroxypyrimidine

* Corresponding author. Tel.: þ420 220 183 262; fax: þ420 220 183 560.

´

E-mail address: dolakova@uochb.cas.cz (P. Dolakova).

doi:10.1016/j.ejmech.2008.09.031

´

P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

2409

pyrimidine derivatives bearing two equal or different chiral phos-

phonoalkoxy chains as potentially biologically active compounds

from a large family of phosphonate antivirals.

2. Results and discussion

2.1. Stepwise synthesis of bisphosphonates from 2-amino-4,6-

dichloropyrimidine

Synthesis of bisphosphonates bearing two 3-hydroxy-2-(phos-

phonomethoxy)propoxy (HPMPO) chains at positions 4 and 6 of the

pyrimidine moiety started from 2-amino-4,6-dichloropyrimidine

(10) (Scheme 1). While alkylation of 2-amino-4,6-dihydroxypy-

rimidine (5) afforded a mixture of O- and N-alkylated products 6a

and 7a [8a], nucleophilic aromatic substitution of 10 with appro-

priate alkylating agent can give only O-regioisomer. Reaction of 10

with 2 equiv of (S)-1,2-isopropylideneglycerol (11) was performed

in THF in the presence of NaH as a base; subsequent deprotection

by diluted hydrochloric acid gave 2,3-dihydroxypropoxy derivative

12 in 64% yield. Primary hydroxyl groups were protected by treat-

ment with 4,4⁰-dimethoxytrityl chloride and alkylation of

secondary hydroxy groups by diisopropoxyphosphorylmethyl

bromide followed by deprotection with acetic acid afforded bis-

HPMPO derivative 13a. Diisopropyl esters were cleaved under

standard conditions (bromotrimethylsilane in acetonitrile, fol-

lowed by hydrolysis) to afford free phosphonic acid 13b. The

enantiomer 14b was prepared by the same procedure as compound

13b from pyrimidine 10 and (R)-1,2-isopropylideneglycerol (16).

The diastereoisomer 18b was prepared by reaction of pyrimidine 10

with 1 equiv of 11 and 1 equiv of NaH in THF; the monoalkylated

product 15 was treated with 16 under the same conditions to afford

dihydroxypropoxy derivative 17. Further procedure was identical

with that described for compound 13b. Compounds 13b, 14b and

18b were puriﬁed by preparative HPLC; triethylammonium salts of

phosphonic acids were converted to the free phosphonic acids on

a column of Dowex 50 ꢀ 8 in H^þform.

Fig. 1. Acyclic nucleoside phosphonates.

was reported to show antiretroviral activity [8a]. Another study

concerning bisphosphonate derivatives 8 and 9 [10] was also per-

formed in our laboratory.

In this paper, we describe efﬁcient synthesis of series of

bisphosphonates derived from 4,6-(dihydroxy)pyrimidine. We

were interested in regioselective synthesis of O-alkylated

Fig. 2. Acyclic nucleoside bisphosphonates.

´

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P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

Scheme 1. Synthesis of bis-HPMPO derivatives.

Synthesis of bisphosphonates bearing two 2-(phosphono-

6b and 7b in approximately 1:1 ratio in a very low yield. Alkylation

of 5 in DMSO slightly increased the yield. Alkylation of commer-

cially available 4,6-dihydroxy-2-(methylsulfanyl)pyrimidine (19) in

DMSO gave O-alkylated regioisomer 21 as a major product in 54%

yield; regioisomer 22 was formed in 12% yield (Scheme 2). Alkyl-

ation of 19 in DMF did not proceed at all because of poor solubility

of disodium salt of pyrimidine 19 in DMF; this might be also

a reason of a low yield in the reaction described in literature [8a].

Thus, the alkylation of pyrimidine 19 was the method of choice for

the synthesis of bisphosphonates. This method afforded compound

methoxy)propoxy (PMPO) chains or two different phosphonoalkoxy

chains by this method failed. Reaction of 10 with phosphonoalkox-

yalkanol gives only product of monosubstitution. 2-Amino-4-chloro-

6-[2-(diisopropoxyphosphorylmethoxy)ethoxy]pyrimidine is further

unreactive towards nucleophilic aromatic substitution. The ether

bond at position 6 of the pyrimidine moiety is relatively labile and

under harsh reaction conditions (basic, acidic or high temperature)

decomposes. For details see Supporting information.

2.2. Synthesis of bisphosphonates from 4,6-dihydroxy-2-

(methylsulfanyl)pyrimidine

Table 1

Substitution pattern of compounds 20, 23 and 24

Compd. 20

R¹, R²

Y

Compd. 23, 24

R¹

Since synthesis of bisphosphonates bearing two different chains

from 10 failed, we focused on alkylation of dihydroxypyrimidines 5

and 19 with phosphonate 20a [11] (Table 1). Previously described

[8a] alkylation of 2-amino-4,6-dihydroxypyrimidine (5) in the

presence of NaH as a base in DMF afforded a mixture of compounds

a

b

c

d

e

H

Cl

23a

23b

23c

24a

24b

H

(S)-CH₃

(R)-CH₃

(S)-CH₂OH

(R)-CH₂OH

OTs

(S)-CH₃

(R)-CH₃

(S)-CH₃

(R)-CH₃

´

P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

2411

Scheme 2. Alkylation of 4,6-(dihydroxy)pyrimidines.

6a in 18% overall yield compared to the previously reported 3.5%

yield; and moreover, formation of N-alkylated regioisomers was

suppressed. The reaction sequence further allows introduction of

modiﬁcations at positions 2, 4 and 6 of the 4,6-(dihydroxy)pyr-

imidine moiety in satisfactory yields.

Alkylation of pyrimidine 19 with 1 equiv of appropriate phos-

phonate 20 (Table 1) in DMSO afforded a mixture of mono- and

dialkylated products 23 and 24 (Scheme 3, Table 1), respectively.

Monoalkylated product 23 was subsequently alkylated by 20 in

DMF to afford bis derivative bearing two different substituents

25a–i (Table 2). 2-Methylsulfanyl group of compounds 24 and 25

(Table 2) was oxidized by m-CPBA in dichloromethane [12] to give

2-methylsulfonyl derivatives 26, which were further converted to

2-amino congeners using liquid ammonia in THF at r.t. [13]. Final

deprotection of diisopropyl esters by bromotrimethylsilane affor-

ded free phosphonic acids 6a and 27a–k.

Compound 26j upon treatment with primary or secondary amine

[12b] and subsequent deprotection with bromotrimethylsilane

afforded N²-substituted bisphosphonates 28a–f (Table 3). 2-Meth-

ylsulfonyl derivative 26j was hydrolyzed by sodium hydroxide in

a mixture of water and THF [13] to give 2-hydroxy derivative 29a;

treatment of 26j with sodium methylate in methanol [14] gave 2-

methoxy derivative 29b. Treatment of 29a and 29b with bromo-

trimethylsilane led to the decomposition of pyrimidine derivatives.

2-Methylsulfanyl derivative 21 was reduced with Raney-Nickel [11]

to afford 4,6-bis[2-(phosphonomethoxy)ethoxy]pyrimidine, which

was subsequently deprotected by bromotrimethylsilane to give free

phosphonic acid 30.

phosphonates 20a–c (Table 1) gave unequivocally S-alkylated

products 32a–c (Table 4). Sulfur derivatives are better nucleophiles

than their oxygen and nitrogen analogs, so the alkylation of sulfur

at positions 4 and 6 took place smoothly even at room temperature.

Alkylation of pyrimidine 31 with 1 equiv of alkylating agent 20a

or 20b gave monoalkylated products 34a and 34b together with

dialkylated product. Further alkylation of 34a and 34b afforded

pyrimidine with two different substituents 35a–c. Diisopropyl

esters of bisphosphonates 32a–c and 35a–c were cleaved by stan-

dard procedure with bromotrimethylsilane.

2.4. Alkoxyalkyl esters of bisphosphonates

For further biological activity screening lipid esters of

compound 6a and its 5-bromo and 5-methyl congener (Scheme 5)

were prepared by the method described by J.R. Beadle and K.Y.

Hostetler [17].

Pyrimidine 10 in neat ethyleneglycol in the presence of t-BuOK

gave hydroxyethoxy derivative 37 in 71% yield (Scheme 5).

Pyrimidine 37 in THF was treated with NaH, heated to 50 ^ꢁC and

then hexadecyloxyethyl toluenesulfonyloxymethylphosphonate

(38) was added. Monoalkylated derivative 39a was isolated

together with bis derivative 40a. Alkylation in DMF or in mixture of

triethylamine and THF (1:1) gave lower yields; reaction in tri-

ethylamine [17] as solvent did not proceed at all. Bromination of 37

with elemental bromine in DMF/CCl₄[9a] gave smoothly the

5-bromo derivative 41. 5-Substituted derivatives 41 and 42 [Petr

Jansa, unpublished results] were similarly converted to esters 39b

and 39c and dialkylated products 40b and 40c by above described

alkylation. Monoalkylated compounds 39a–c were fully characte-

rized and submitted for biological activity screening, however

dialkylated products 40a–c were nearly insoluble in any solvent;

therefore their NMR spectra could not be measured. Thus

compounds 40a–c were characterized only by mass spectroscopy

2.3. Bisphosphonates derived from 2-amino-4,6-

disulfanylpyrimidne

Pyrimidine [15] 10 was converted to the disulfanyl analog 31 by

reaction with thiourea (Scheme 4) [16]. Alkylation of 31 with

´

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P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

Scheme 3. Synthesis of bisphosphonates by alkylation of 4,6-(dihydroxy)-2-(methylsulfanyl)pyrimidine.

and elemental analysis and were not tested for biological activity.

Our attempts to convert compound 6a to cycloSal, cycloAmb [18] or

POM [3] esters failed due to instability of ether bonds at positions 4

and 6 under reaction conditions.

2.5. Capillary electrophoresis

Enantiomeric purity of compounds 27a and 27b was analyzed

by capillary electrophoresis. Baseline separation of enantiomers

27a and 27b, with resolution 1.67, was achieved in chiral back-

ground electrolyte (BGE) composed of 50 mM borax, adjusted by

NaOH to pH 10.0, with chiral selector b-cyclodextrin (20 mg/mL)

Table 2

Substitution pattern of compounds 25–27

(Fig. 3A). Compound 27a was found enantiomerically pure as

Compd. 25, 26

R¹

R²

Product

R¹

R²

25a, 26a

25b, 26b

25c, 26c

25d, 26d

25e, 26e

25f, 26f

25g, 26g

25h, 26h

25i, 26i

26j

H

(S)-CH₃

(R)-CH₃

27a

27b

27c

27d

27e

27f

27g

27h

27i

H

(S)-CH₃

(R)-CH₃

(S)-CH₂OH

(R)-CH₂OH

(R)-CH₃

(S)-CH₂OH

(R)-CH₂OH

(S)-CH₂OH

(R)-CH₂OH

H

(S)-CH₂OH

(R)-CH₂OH

(R)-CH₃

(S)-CH₂OH

(R)-CH₂OH

(S)-CH₂OH

(R)-CH₂OH

H

Table 3

Substituents at position 2 of compound 28

(S)-CH₃

(R)-CH₃

H

(S)-CH₃

(R)-CH₃

H

Compd. 28

R³

R⁴

a

b

c

d

e

f

H

–

Cyclopropyl

Cyclopentyl

Methyl

Benzyl

4-Methoxybenzyl

Morpholino

6a

27j

27k

26k

26l

(S)-CH₃

(R)-CH₃

(S)-CH₃

(R)-CH₃

(S)-CH₃

(R)-CH₃

(S)-CH₃

(R)-CH₃

´

P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

2413

Scheme 4. Alkylation of 2-amino-4,6-(disulfanyl)pyrimidine.

demonstrated by single peak of CE analysis of this compound in

chiral BGE (Fig. 3B) whereas a very small admixture of enantiomer

27a was found in the CE analysis of compound 27b (Fig. 3C).

It was conﬁrmed that optically active phosphonates are stable

and do not tend to racemize. No racemization did occur during the

whole multistep synthesis from chiral precursors.

3. Conclusion

In conclusion, in the SAR studies of ‘‘open-ring‘‘ ANPs a series of

bisphosphonates derived from 2-amino-4,6-(dihydroxy)pyr-

imidine were prepared. Bisphosphonates bearing two identical or

different achiral or chiral phosphonoalkoxy chains were prepared

either by nucleophilic aromatic substitution of 2-amino-4,6-

dichloropyrimidine (10) or by alkylation of 4,6-(dihydroxy)-2-

(methylsulfanyl)pyrimidine (19). The second method proved to be

the universal method for regioselective preparation of O-alkylated

pyrimidines at positions 4 and 6; furthermore 2-methylsulfanyl

function is a versatile leaving group for introduction of various

substituents at position

2 of the pyrimidine moiety. Disul-

fanylpyrimidine 31 was alkylated in the same manner to give

exclusively S-alkylated product. Alkoxyalkyl esters of selected

bisphosphonates were prepared to improve their bioavailability;

however introduction of two lipid esters to bisphosphonates

dramatically decreased their solubility. Enantiomeric purity of

compounds 27a and 27b was successfully determined by capillary

electrophoresis.

The whole series of bisphosphonates (6a, 13b, 14b, 18b, 27a–k,

28a–f, 30, 33a–c, 36a–c and 39a–c) was investigated for their

Table 4

Substitution pattern of compounds 32–36

Compd. 32–34

R¹

Compd. 35, 36

R¹

R²

a

b

c

H

a

b

c

H

(S)-CH₃

(R)-CH₃

(S)-CH₃

(R)-CH₃

(S)-CH₃

Scheme 5. Synthesis of alkoxyalkyl esters of bisphosphonates.

´ ´

P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

2414

Fig. 3. A: CE separation of enantiomers 27a (S), 0.1 mM and 27b (R), 0.2 mM in chiral BGE (background electrolyte) composed of 50 mM borax, adjusted by NaOH to pH 10.0, with

chiral selector

ﬂow marker.

b-cyclodextrin (20 mg/mL). B: CE analysis of enantiomer 27a in the above chiral BGE. C: CE analysis of enantiomer 27b in the above chiral BGE. eof ¼ electroosmotic

inhibitory activity against several DNA and retroviruses. None of

the prepared bisphosphonates showed any appreciable antiviral

activity. Finally, antiretroviral activity of resynthesized parent

compound 6a was not conﬁrmed. The previously reported activity

[8a] might probably had been caused by undetectable admixture of

several orders more active monoalkylated 2-amino-4-hydroxy-6-

[2-(phosphonomethoxy)ethoxy]pyrimidine. Compounds are devoid

of any measurable toxicity to cell cultures.

spectrometer using electrospray ionization (ESI). Preparative HPLC

puriﬁcation was performed on a column packed with 10 m C18

reversed phase (Luna), 250 ꢀ 21 mm; in ca. 300 mg portions of

mixtures using linear gradient 0.1 M triethylammonium hydrogen

carbonate (TEAB) in water and in 50% MeOH (linear gradient of

TEAB in 50% MeOH, 0–100%).

All new compounds were fully characterized by mass spec-

trometry, elemental analysis or high resolution mass spectrometry

(for intermediates) and NMR spectroscopy (including complete

assignment of all NMR signals using a combination of H,H-COSY,

H,C-HSQC, and H,C-HMBC methods). Diastereoisomers gave iden-

tical NMR spectra. To prove that we have two different diaste-

reoisomers we prepared mixed samples of two diastereoisomers;

two sets of signals were found for OCH₂-1⁰protons in ¹H NMR

spectra.

m

4. Experimental section

4.1. Materials and general procedures

Solvents were dried by standard procedures. Tetrahydrofuran

(THF) was freshly distilled from sodium/benzophenone under

argon. NMR spectra were recorded with Bruker Avance 500

(500 MHz for ¹H and 125.8 MHz for ¹³C) and Bruker Avance 400 (¹H

at 400, ¹³C at 100.6 MHz) spectrometers in CDCl₃, DMSO-d₆, or D₂O.

4.1.1. General procedure 1 (GP1) – deprotection of diisopropyl esters

of bisphosphonates

Chemical shifts (in ppm,

d

scale) were referenced to TMS (for ¹H

Bisphosphonate (1 mmol) in acetonitrile (20 mL) was treated

with bromotrimethylsilane (1.5 mL) at r.t. overnight. Volatiles were

removed under reduced pressure, the residue was codistilled with

water (3 ꢀ 50 mL) and 0.1 M TEAB (2 ꢀ 50 mL). Crude products

were puriﬁed by preparative HPLC using linear gradient 0.1 M

triethylammonium hydrogen carbonate in water and in 50% MeOH

(linear gradient of TEAB in 50% MeOH, 0–100%) and triethyl-

ammonium salts of phosphonates were converted to free phos-

phonic acids by application onto a column of Dowex 50 ꢀ 8 in H^þ

form and elution with water.

NMR spectra in CDCl₃) and/or to the solvent signal (CDCl₃

d

¼ 7.26 ppm for ¹H NMR and

d

¼ 77.0 ppm for ¹³C NMR; DMSO-d₆

for ¹H NMR

d

¼ 2.5 ppm and for ¹³

C

d

¼ 39.7). Chemical shifts in D₂O

were referenced to 1,4-dioxane for ¹H NMR

NMR

Point B-545 apparatus and are uncorrected. TLC was performed on

plates of Kieselgel 60 F254 (Merck). Mass spectra were measured

on a ZAB-EQ (VG Analytical) spectrometer using FAB (ionization by

Xe, accelerating voltage 8 kV, glycerol matrix) or on an LCQ classic

d

¼ 3.75 and for ¹³C

d

¼ 67.19. Melting points were determined on a Bu¨chi Melting

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P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

2415

4.1.2. General procedure 2 (GP2) – dialkylation of 4,6-dihydroxy-2-

(methylsulfanyl)pyrimidine (19) in DMSO

60% in parafﬁn oil, 61 mmol) in THF (80 mL) at r.t. After stirring for

1 h, pyrimidine 10 (5 g, 30.5 mmol) was added in one portion and

the reaction mixture was reﬂuxed for 6 h. After cooling to r.t.,

solvent was removed under reduced pressure and the residue was

dissolved in hot CHCl₃and ﬁltered through Celite. Chromatography

on silica gel (CHCl₃/MeOH 0–2%) afforded intermediate 2-amino-

4,6-(S,S)-bis(2,2-dimethyl-4-methoxy-1,3-dioxolane)pyrimidine

Pyrimidine 19 (0.16 g, 1 mmol) in DMSO (5 mL) was treated with

NaH (0.084 g, 60% in parafﬁn oil, 2.1 mmol) and heated at 80 ^ꢁC for

30 min; appropriate phosphonates 20a–e (2.1 mmol) were added

and the resulting mixture was heated at 120 ^ꢁC for 8–16 h. The

mixture was cooled to r.t., DMSO was evaporated in vacuo at 60 ^ꢁC.

The residue was codistilled with DMF and EtOH, taken to CHCl₃

(100 mL) and washed with water (3 ꢀ 50 mL). Organic fraction was

dried over MgSO₄and evaporated in vacuo. The residue was

separated by ﬂash chromatography (CHCl₃/MeOH).

(10.1 g, 93%) as a yellow oil. ¹H NMR (DMSO-d₆):

d

¼ 6.60 (br s, 2H,

NH₂), 5.36 (s, 1H, H-5), 4.33 (m, 2H, H-2⁰), 4.22 (dd, J(1⁰a,2⁰) ¼ 4.6,

J_gem¼ 11.1, 2H, H-1⁰a), 4.16 (dd, J(1⁰b,2⁰) ¼ 6.3, J_gem¼ 11.1, 2H, H-1⁰b),

4.04 (dd, J(3⁰a,2⁰) ¼ 6.4, J_gem¼ 8.4, 2H, H-3⁰a), 3.68 (dd,

J(3⁰b,2⁰) ¼ 6.2, J_gem¼ 8.4, 2H, H-3⁰b), 1.33 and 1.28 (2 ꢀ s, 2 ꢀ 6H,

4.1.3. General procedure 3 (GP3) – monoalkylation of 4,6-

dihydroxy-2-(methylsulfanyl)-pyrimidine (19) in DMSO

CH₃) ppm. ¹³C NMR (DMSO-d₆):

d

¼ 171.19 (2C, C-4 and C-6), 162.73

(C-2), 108.96 (2C, CHMe₂), 78.56 (C-5), 73.59 (2C, C-2⁰), 66.37 and

65.93 (2 ꢀ 2C, C-1⁰, C-3⁰), 26.81 and 25.52 (2 ꢀ 2C, CH₃) ppm. MS

(FAB): m/z (%) ¼ 356.1 (85) [MH]^þ. Anal. C₁₆H₂₅N₃O₆(C, H, N).

Solution of the intermediate (9 g, 25.3 mmol) in methanol/

water mixture (1:4, 100 mL) was acidiﬁed with hydrochloric acid to

pH 2 and stirred for 4 h at r.t. Reaction mixture was applied onto

a column of Dowex 50 ꢀ 8, washed with water until neutral reac-

tion of eluate and eluted with 2.5% ammonia. UV absorbing eluate

was collected and evaporated. Crystallization from ethanol/ether

mixture afforded 12 as a white solid (4.74 g, 66%); m.p. 110 ^ꢁC. ¹H

Pyrimidine 19 (0.16 g, 1 mmol) and NaH (0.04 g, 60% in parafﬁn

oil, 1 mmol) in DMSO (5 mL) were heated at 60 ^ꢁC for 30 min,

phosphonates 20a–e (1 mmol) in DMSO (1 mL) were added drop-

wise and the reaction mixture was heated at 120 ^ꢁC for 8 h, cooled

to r.t. and evaporated in vacuo at 60 ^ꢁC. The residue was codistilled

with DMF and EtOH, diluted with CHCl₃, washed with 3 portions of

water and dried over MgSO₄. Products were separated by ﬂash

chromatography (CHCl₃/MeOH).

4.1.4. General procedure 4 (GP4) – 2nd alkylation of monoalkylated

product 23

NMR (DMSO-d₆):

d

¼ 6.50 (br s, 2H, NH₂), 5.32 (s, 1H, H-5), 4.90 and

4.63 (2 ꢀ br s, 2 ꢀ 2H, OH), 4.17 (dd, J(1⁰a,2⁰) ¼ 4.3, J_gem¼ 10.9, 2H,

H-1⁰a), 4.06 (dd, J(1⁰b,2⁰) ¼ 6.6, J_gem¼ 10.9, 2H, H-1⁰b), 3.72 (m, 2H,

H-2⁰), 3.38 (d, J(3⁰,2⁰) ¼ 4.0, 4H, H-3⁰) ppm. ¹³C NMR (DMSO-d₆):

Pyrimidine 23 (1 mmol) and NaH (0.044 g, 60% in parafﬁn oil,

1.1 mmol) in DMF (5 mL) were heated at 40 ^ꢁC for 30 min, appro-

priate phosphonates 20a–e (1.1 mmol) were added and the mixture

was stirred at 100 ^ꢁC for 8–12 h, evaporated in vacuo, and codis-

tilled with EtOH.

d

¼ 171.62 (2C, C-4, C-6), 162.82 (C-2), 78.58 (C-5), 69.89 (2C, C-2⁰),

67.73 (2C, C-1⁰), 62.97 (C-3⁰) ppm. MS (FAB): m/z (%) ¼ 276.1 (100)

[MH]^þ. Anal. C₁₀H₁₇N₃O₆.½H₂O (C, H, N).

4.1.5. General procedure 5 (GP5) – oxidation of 2-methylsufanyl

group to 2-methylsulfonyl group by m-CPBA

4.3. 2-Amino-4,6-(2R,2⁰R)-bis[2-(diisopropoxyphosphorylmethoxy)-

3-hydroxypropoxy]pyrimidine (13a)

2-Methylsulfanyl derivative (1 mmol) in CH₂Cl₂(6 mL) was

treated with m-chloroperoxybenzoic acid (3 mmol) at r.t. for 3–

12 h. The reaction mixture was diluted with CH₂Cl₂, washed with

saturated aqueous Na₂S₂O₅, saturated NaHCO₃and water. Organic

fraction was dried over MgSO₄and puriﬁed by ﬂash chromatog-

raphy (CHCl₃/MeOH).

Compound 12 (3 g, 11.7 mmol) in pyridine (400 mL) was treated

with DMTrCl (11.95 g, 35 mmol) and the mixture was stirred for 3 h.

The reaction was quenched by addition of EtOH and the solvent was

removed in vacuo. The residue was partitioned between CHCl₃and

saturated aqueous NaHCO₃, and the separated organic layer was

washed with brine, dried (MgSO₄) and concentrated in vacuo. The

residue in DMF (150 mL) was treated with NaH (1 g, 60% suspen-

sion in mineral oil, 25 mmol) at 0 ^ꢁC and stirred for 30 min; diiso-

propoxyphosphorylmethyl bromide (6.25 g, 25 mmol) was added

and the mixture was stirred at r.t. overnight; the solvent was

removed in vacuo and the residue was dissolved in 80% acetic acid

(100 mL). After stirring at r.t. for 1 h, acetic acid was evaporated and

the residue was codistilled with water. Flash chromatography in

CHCl₃/MeOH (0–3%) afforded colorless oil (2.6 g, 39%). ¹H NMR

4.1.6. General procedure 6 (GP6) – ammonolysis of 2-

methylsulfonyl group to amino group

To a cooled (ꢂ78 ^ꢁC) and stirred solution of compound 26

(1 mmol) in dry THF (30 mL), in a pressure tube, 20–30 mL of liquid

ammonia was added. The pressure tube was sealed and allowed to

warm to r.t. and the reaction mixture was stirred for 5–12 h. The

reaction mixture was concentrated in vacuo and the crude product

in CHCl₃was applied onto a pad of silica gel and washed with 10%

MeOH in CHCl₃(150 mL).

(DMSO-d₆):

d

¼ 6.57 (br s, 2H, NH₂), 5.29 (s, 1H, H-5), 4.78 (t,

J(OH,3⁰) ¼ 5.4, 2H, OH), 4.58 (m, 4H, CHipr.), 4.32 (dd, J(1⁰a,2⁰) ¼ 3.6,

J_gem¼ 11.5, 2H, H-1⁰a), 4.19 (dd, J(1⁰b,2⁰) ¼ 6.1, J_gem¼ 11.5, 2H, H-

1⁰b), 3.90 (dd, J(P,CH) ¼ 8.7, J_gem¼ 13.8, 2H) and 3.86 (dd,

J(P,CH) ¼ 8.9, J_gem¼ 13.8, 2H, PCH₂), 3.69 (m, 2H, H-2⁰), 3.51 (t,

J(3⁰,2⁰) ¼ J(3⁰,OH) ¼ 5.4, 4H, H-3⁰), 1.235 (d, 6H), 1.23 (d, 6H), 1.22 (d,

6H) and 1.21 (d, J(CH₃,CH) ¼ 6.2, 6H, CH₃) ppm. ¹³C NMR (DMSO-

4.1.7. General procedure 7 (GP7) – alkylation of hydroxyethoxy

derivatives 37, 41 and 42 with phosphonate 38

2-Hydroxyethoxy derivative (0.25 mmol), NaH (30 mg, 60% in

parafﬁn oil, 0.75 mmol) and 4-dimethylaminopyridine (6 mg) in

THF were heated at 50 ^ꢁC for 30 min and phosphonate 38 (292 mg,

0.525 mmol) was added in one portion. The resulting mixture was

heated at 70 ^ꢁC for 16 h and evaporated in vacuo. The residue in

CHCl₃(50 mL) was washed with brine (2 ꢀ 50 mL) and water

(1 ꢀ50 mL) and evaporated under reduced pressure. Flash chro-

matography (EtOAc:EtOH:acetone:H₂O, 6:1:1:0.5 and EtOAc:EtO-

H:acetone:H₂O, 4:1:1:1) gave compounds 39 and 40.

d₆):

d

¼ 171.34 (2C, C-4, C-6), 162.77 (C-2), 80.53 (d, 2C, J(P,C) ¼ 11.7,

C-2⁰), 78.46 (C-5), 70.37 (d, 2C) and 70.32 (d, 2C, J(P,C) ¼ 6.3, CHipr.),

65.25 (2C, C-1⁰), 63.99 (d, 2C, J(P,C) ¼ 164.6, PC), 60.08 (2C, C-3⁰),

23.99 (d, 4C, J(P,C) ¼ 3.9) and 23.82 (d, 4C, J(P,C) ¼ 4.4, CH₃) ppm. MS

(FAB): m/z (%) ¼ 632.6 (56) [MH]^þ. Anal. C₂₄H₄₇N₃O₁₂P₂(C, H, N, P).

4.4. 2-Amino-4,6-(2R,2⁰R)-bis[2-(phosphonomethoxy)-3-

hydroxypropoxy]pyrimidine (13b)

4.2. 2-Amino-4,6-(2R,2⁰R)-bis(1,2-dihydroxypropoxy)pyrimidine (12)

(S)-1,2-Isopropylideneglycerol (11) (7.53 mL, 61 mmol) in THF

(15 mL) was added dropwise to a stirred suspension of NaH (2.5 g,

Compound 13a (2 g, 3.17 mmol) was deprotected by GP1 to give

13b (1.05 g, 71%) as colorless foam. ¹H NMR (D₂O):

d

¼ 4.56 (dd,

´ ´

P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

2416

J(1⁰a,2⁰) ¼ 3.8, J_gem¼ 11.2, 2H, H-1⁰a), 4.43 (dd, J(1⁰b,2⁰) ¼ 5.6,

J_gem¼ 11.2, 2H, H-1⁰b), 3.94 (m, 2H, H-2⁰), 3.89 (dd, 2H) and 3.84

(dd, J(P,CH) ¼ 9.3, J_gem¼ 13.3, 2H, PCH₂), 3.82 (dd, J(3⁰a,2⁰) ¼ 4.3,

J_gem¼ 12.2, 2H, H-3⁰a), 3.75 (dd, J(3⁰b,2⁰) ¼ 5.7, J_gem¼ 12.2, 2H, H-

evaporated in vacuo. Flash chromatography in chloroform/methanol

(0–2%) gave protected intermediate [2-amino-4,6-(2R,2⁰S)-bis(2,2-

dimethyl-4-methoxy-1,3-dioxolane)pyrimidine, 2.95 g, 72%] as

a yellow oil. ¹H NMR (DMSO-d₆) and ¹³C NMR (DMSO-d₆) identical

with 2-amino-4,6-(2S,2⁰S)-bis(2,2-dimethyl-4-methoxy-1,3-dioxo-

lane)pyrimidine. MS (FAB): m/z (%) ¼ 356.0 (100) [MH]^þ. Anal.

C₁₆H₂₅N₃O₆(C, H, N).

3⁰b) ppm. ¹³C NMR (D₂O):

d

¼ 169.14 (2C, C-4, C-6), 155.56 (C-2),

79.62 (C-5), 79.57 (d, J(P,C) ¼ 11.2, 2C, C-2⁰), 68.16 (2C, C-1⁰), 65.74

(d, J(P,C) ¼ 158.7, 2C, PC), 60.11 (2C, C-3⁰) ppm. MS (FAB): m/z

25

(%) ¼ 464 (49) [MH]^þ.

[

a]

¼ ꢂ1.0 (c 0.502, H₂O). Anal.

The intermediate was deprotected by the same procedure as

was described for compound 12. White solid, yield 69%; m.p. 116 ^ꢁC.

D

C₁₂H₂₃N₃O₁₂P₂.H₂O (C, H, N, P).

¹H NMR (DMSO-d₆):

d

¼ 6.51 (br s, 2H, NH₂), 5.32 (s, 1H, H-5), 4.90

4.5. 2-Amino-4,6-(2S,2⁰S)-bis[2-(diisopropoxyphosphorylmethoxy)-

3-hydroxypropoxy]pyrimidine (14a)

and 4.65 (2 ꢀ br s, 2 ꢀ 2H, OH), 4.17 (dd, J(1⁰a,2⁰) ¼ 4.3, J_gem¼ 10.9,

2H, H-1⁰a), 4.06 (dd, J(1⁰b,2⁰) ¼ 6.6, J_gem¼ 10.9, 2H, H-1⁰b), 3.72 (m,

2H, H-2⁰⁰), 3.38 (d, J(3⁰,2⁰) ¼ 4.0, 4H, H-3⁰) ppm. ¹³C NMR (DMSO-d₆):

Prepared from 10 and 16 by the same procedure as compound 13a.

2-Amino-4,6-(2R,2⁰R)-bis(2,2-dimethyl-4-methoxy-1,3-dioxola-

ne)pyrimidine, yellow oil, yield 9.6 g (89%). ¹H NMR (DMSO-d₆)

d

¼ 171.62 (2C, C-4, C-6), 162.82 (C-2), 78.58 (C-5), 69.89 (2C, C-2⁰),

67.73 (2C, C-1⁰), 62.97 (C-3⁰) ppm. MS (FAB): m/z (%) ¼ 276.1 (100)

[MH]^þ. Anal. C₁₀H₁₇N₃O₆.½H₂O (C, H, N).

13

and C NMR (DMSO-d₆) identical with (2S,2⁰S) enantiomer. MS

(FAB): m/z (%) ¼ 356.0 (54) [MH]^þ. Anal. C₁₆H₂₅N₃O₆(C, H, N).

2-Amino-4,6-(2S,2⁰S)-bis(1,2-dihydroxypropoxy)pyrimidine,

white solid, yield 5 g (70%); m.p. 99 ^ꢁC. ¹H NMR (DMSO-d₆) and ¹³C

NMR (DMSO-d₆) identical with (2R,2⁰R) enantiomer. MS (FAB):

m/z (%) ¼ 276.0 (80) [MH]^þ. Anal. C₁₀H₁₇N₃O₆.½H₂O (C, H, N).

2-Amino-4,6-(2S,2⁰S)-bis[2-(diisopropoxyphosphorylmethoxy)-

3-hydroxypropoxy]pyrimidine (14a), colorless oil, yield 2.2 g (33%).

¹H NMR (DMSO-d₆) and ¹³C NMR (DMSO-d₆) identical with 13a.

MS (FAB): m/z (%) ¼ 632.6 (100) [MH]^þ. Anal. C₂₄H₄₇N₃O₁₂P₂(C, H,

N, P).

4.9. 2-Amino-4,6-(2S,2⁰R)-bis[2-(diisopropoxyphosphorylmethoxy)-

3-hydroxypropoxy]pyrimidine (18a)

Prepared from compound 17 by the same procedure as was

described for 13a, colorless oil, yield 2.7 g (41%). ¹H NMR (DMSO-d₆)

and ¹³C NMR (DMSO-d₆) identical with 13a. MS (FAB):

m/z (%) ¼ 632.1 (100) [MH]^þ. Anal. C₂₄H₄₇N₃O₁₂P₂(C, H, N, P).

4.10. 2-Amino-4,6-(2S,2⁰R)-bis[2-(phosphonomethoxy)-3-

hydroxypropoxy]pyrimidine (18b)

4.6. 2-Amino-4,6-(2S,2⁰S)-bis[2-(phosphonomethoxy)-3-

hydroxypropoxy]pyrimidine (14b)

Prepared by the same procedure as compound 13b, colorless

foam, yield 63%. ¹H NMR (D₂O) and ¹³C NMR (D₂O) identical with

25

13b. MS (FAB): m/z (%) ¼ 464 (35) [MH]^þ. [

a]

¼ þ0.02 (c 0.383,

Prepared from 14a by the same procedure as compound 13b.

D

Colorless foam, yield 0.95 g (64%). ¹H NMR (D₂O) and ¹³C NMR

H₂O). Anal. C₁₂H₂₃N₃O₁₂P₂.H₂O (C, H, N, P).

(D₂O) identical with 13b. MS (FAB): m/z (%) ¼ 464 (15) [MH]^þ.

25

[

a]

¼ þ1.9 (c 0.267, H₂O). Anal. C₁₂H₂₃N₃O₁₂P₂.H₂O (C, H, N, P).

D

4.11. 4,6-Bis[2-(diisopropoxyphosphorylmethoxy)ethoxy]-2-

(methylsulfanyl)pyrimidine (21) and 4-[2-(diisopropoxy-

phosphorylmethoxy)ethoxy]-1-[2-(diisopropoxyphosphoryl-

methoxy)ethyl]-2-(methylsulfanyl)pyrimidine-6(1H)-one (22)

4.7. 2-Amino-4-chloro-6-(2S)-(2,2-dimethyl-4-methoxy-1,3-

dioxolane)pyrimidine (15)

Compound 11 (12.34 mL, 100 mmol) was added dropwise to the

suspension of NaH (4 g, 60% suspension in mineral oil, 100 mmol)

in THF (130 mL); the mixture was stirred for 1 h and pyrimidine 10

(16.4 g, 100 mmol) was added in one portion. Reaction mixture was

heated at reﬂux for 6 h, cooled to r.t. and evaporated in vacuo. The

residue in chloroform was washed with brine; the organic extract

was dried over magnesium sulfate and evaporated. Chromatog-

raphy in chloroform/methanol (0–3%) afforded 23.1 g (89%) of

compound 15 as a white solid, m.p. 130 ^ꢁC. ¹H NMR (DMSO-d₆):

GP2, pyrimidine 19 (3 g, 19 mmol), phosphonate 20a (10.6 g,

40 mmol), yield 6.15 g (54%) of 21, colorless syrup. ¹H NMR (DMSO-

d₆):

d

¼ 5.88 (s, 1H, H-5), 4.58 (dh, J(CH,P) ¼ 7.7, J(CH,CH₃) ¼ 6.3, 4H,

CHipr.), 4.43 (m, 4H, H-1⁰), 3.82 (m, 4H, H-2⁰), 3.79 (d, J(C,H,P) ¼ 8.3,

4H, PCH₂), 2.48 (s, 3H, SCH₃), 1.23 (m, 24H, CH₃ipr.) ppm. ¹³C NMR

(DMSO-d₆):

d

¼ 170.61 (C-2), 170.26 (2C, C-4,6), 85.55 (C-5), 70.57

(d, J(2⁰,P) ¼ 11.9, C-2⁰), 70.36 (d, J(CH,P) ¼ 6.2, CHipr.), 65.67 (C-1⁰),

64.95 (d, J(C,P) ¼ 164.6, PCH₂), 24.00 (d, J(CH₃,P) ¼ 3.7) and 23.85 (d,

J(CH₃,P) ¼ 4.6, CH₃ipr.), 13.66 (SCH₃) ppm. MS (ESI): m/z (%) ¼ 625.1

(100) [MNa]^þ, 603 (15) [MH]^þ. HR MS (FAB) calcd. for

C₂₃H₄₅N₂O₁₀P₂S [MH]^þ603.2192, found 603.2185. Anal.

C₂₃H₄₅N₂O₁₀P₂S (C, H, N, P, S).

d

¼ 7.10 (br s, 2H, NH₂), 6.11 (s, 1H, H-5), 4.35 (m, 1H, H-2⁰), 4.28 (dd,

J(1⁰a,2⁰) ¼ 4.4, J_gem¼ 11.0, 1H, H-1⁰a), 4.21 (dd, J(1⁰b,2⁰) ¼ 6.3,

J_gem¼ 11.0, 1H, H-1⁰b), 4.04 (dd, J(3⁰a,2⁰) ¼ 6.5, J_gem¼ 8.6, 1H, H-3⁰a),

3.70 (dd, J(3⁰b,2⁰) ¼ 6.0, J_gem¼ 8.6, 1H, H-3⁰b), 1.32 and 1.27 (2 ꢀ s,

2 ꢀ 3H, CH₃) ppm. ¹³C NMR (DMSO-d₆):

d

¼ 170.48 (C-6), 162.95

Further elution of column gave 22, yield 1.35 g (12%), colorless

(C-2), 160.21 (C-4), 109.05 (CHMe₂), 94.48 (C-5), 73.35 (C-2⁰), 66.85

and 65.82 (C-1⁰, C-3⁰), 26.79 and 25.51 (CH₃) ppm. MS (ESI): m/z

(%) ¼ 282.43 (100) [MNa]^þ, 260.0 (37) [MH]^þ. Anal. C₁₀H₁₄ClN₃O₃

(C, H, Cl, N).

syrup. ¹H NMR (DMSO-d₆):

d

¼ 5.40 (s, 1H, H-5), 4.58 (m, 4H,

CHipr.), 4.31 (m, 2H, H-1⁰⁰), 4.10 (t, J(1⁰,2⁰) ¼ 5.4, 2H, H-1⁰), 3.82 (m,

2H, H-2⁰⁰), 3.79 (d, 2H) and 3.76 (d, J(P,CH) ¼ 8.3, 2H, PCH₂), 3.75

(t, J(2⁰,1⁰) ¼ 5.4, 2H, H-2⁰), 2.54 (s, 3H, SCH₃), 1.24 (d, 6H), 1.23 (d,

6H), 1.22 (d, 6H) and 1.20 (d, J(CH₃,CH) ¼ 6.2, 6H, CH₃) ppm. ¹³C

4.8. 2-Amino-4,6-(2R,2⁰S)-bis(1,2-dihydroxypropoxy)pyrimidine (17)

NMR (DMSO-d₆):

d

¼ 167.34 (C-2), 162.75 (C-4), 159.48 (C-6),

85.65 (C-5), 70.76 (d, J(C,P) ¼ 11.9, C-2⁰), 70.44 (d, J(C,P) ¼ 11.89, C-

Compound 16 (1.43 mL, 11.6 mmol) was added dropwise to the

suspension of NaH (0.46 g, 60% suspension in mineral oil,11.6 mmol)

in THF (10 mL); the mixture was stirred for 1 h and compound 15

(3 g, 11.6 mmol) in THF (5 mL) was added. Reaction mixture was

heated at reﬂux for 6 h, ﬁltered through Celite while hot, Celite was

washed with chloroform and combined organic extracts were

2⁰⁰), 70.04 (m, CHipr.), 69.53 (d, J(C,P) ¼ 6.61, CHipr.), 65.31 (C-1⁰),

64.80 (d, J(C,P) ¼ 164.41, PCH₂), 64.75 (d, J(C,P) ¼ 163.72, PCH₂⁰⁰),

0

43.69 (C-1⁰⁰), 23.61 (m, CH₃ipr.), 14.43 (SCH₃) ppm. MS (ESI): m/z

(%) ¼ 625.1 (100) [MNa]^þ. HR MS (FAB) calcd. for C₂₃H₄₅N₂O₁₀P₂S

[MH]^þ603.2192, found 603.2193. Anal. C₂₃H₄₅N₂O₁₀P₂S (C, H, N,

P, S).

´

P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

2417

4.12. 6-[2-(Diisopropoxyphosphorylmethoxy)ethoxy]-4-hydroxy-2-

(methylsulfanyl)pyrimidine (23a)

washed with 10% MeOH in CHCl₃(150 mL). The ﬁltrate was evap-

orated in vacuo. The residue was without further puriﬁcation

oxidized by m-CPBA by GP5 to give 26a (0.52 g, 38%), colorless oil.

GP3, pyrimidine 19 (3 g, 19 mmol), phosphonate 20a (4.8 g,

18.1 mmol), yield 2.67 g (37%) of 23a, colorless syrup. ¹H NMR

¹H NMR (DMSO-d₆):

d

¼ 6.52 (s, 1H, H-5), 4.58 (m, 4H, CHipr.), 4.52

(m, 2H, H-1⁰), 4.43 (dd, J_gem¼ 11.4, J(1⁰⁰a,2⁰⁰) ¼ 3.5, 1H, H-1⁰⁰a), 4.34

(dd, J_gem¼ 11.4, J(1⁰⁰b,2⁰⁰) ¼ 6.0, 1H, H-1⁰⁰b), 3.92 (m, 2H, H-2⁰⁰), 3.86

(m, 2H, H-2⁰), 3.81 (m, 4H, 2 ꢀ PCH₂), 3.38 (s, 3H, SO₂CH₃), 1.19–1.24

(m, 24H, CH₃ipr.), 1.19 (d, J(CH₃, 2⁰⁰) ¼ 6.5, 3H, H-3⁰⁰) ppm. ¹³C NMR

(DMSO-d₆):

d

¼ 12.32 (br s, 1H, OH), 5.39 (br s, 1H, H-5), 4.59 (dh,

J(CH,CH₃) ¼ 6.2, J(CH,P) ¼ 7.8, 2H, CHipr.), 4.33 (m, 2H,

H-1⁰), 3.80 (m, 2H, H-2⁰), 3.79 (d, J(CH₂,P) ¼ 8.3, 2H, CH₂P), 2.47 (s,

3H, SCH₃), 1.24 (d, J(CH₃,CH) ¼ 6.0, 6H) and (1.22 d, J(CH₃,CH) ¼ 6.0,

(DMSO-d₆):

d

¼ 171.51 and 171.45 (C-4, C-6), 164.26 (C-2), 92.66 (C-

6H, CH₃ipr.) ppm. ¹³C NMR (DMSO-d₆):

d

¼ 169.19 (C-4), 86.06 (C-

5), 75.05 (d, J(2⁰⁰,P) ¼ 12.5, C-2⁰⁰), 70.12–70.40 (m, 6C, CHipr., C-2⁰

and C-1⁰⁰), 66.95 (C-1⁰), 64.95 (d, J(C,P) ¼ 164.5, C-3⁰), 63.03 (d,

J(C,P) ¼ 165.3, C-3⁰⁰), 38.94 (CH₃SO₂), 23.82–24.01 (m, 8C, CH₃ipr.),

16.21 (C-3⁰⁰) ppm. MS (FAB): m/z (%) ¼ 671 (23) [MNa]^þ, 649 (32)

[MH]^þ. HR MS (FAB) calcd. for C₂₄H₄₇N₂O₁₂P₂S [MH]^þ649.2325,

found 649.2328.

5), 70.66 (d, J(2,P) ¼ 11.9, C-2⁰), 70.43 (d, J(C,O,P) ¼ 6.3, CHipr.),

65.83 (C-1⁰), 64.97 (d, J(C,P) ¼ 164.5, CH₂P), 24.03 (d, J(CH₃,P) ¼ 3.6)

and 23.89 (d, J(CH₃,P) ¼ 4.4, CH₃ipr.),13.12 (SCH₃) ppm. MS (ESI): m/

z (%) ¼ 403 (100) [MNa]^þ. HR MS (FAB) calcd. for C₁₄H₂₆N₂O₆PS

[MH]^þ381.1249, found 381.1265. Compound 21 (4.18 g, 36%) was

isolated as a side product.

4.16. 4-[2-(Diisopropoxyphosphorylmethoxy)ethoxy]-6-(2R)-[2-

(diisopropoxyphosphorylmethoxy)propoxy]-2-

(methylsulfonyl)pyrimidine (26b)

4.13. 6-(2S)-[2-(Diisopropoxyphosphorylmethoxy)propoxy]-4-

hydroxy-2-(methylsulfanyl) pyrimidine (23b) and 4,6-(2S,2⁰S)-

bis[2-(diisopropoxyphosphorylmethoxy)propoxy]-2-

(methylsulfanyl)pyrimidine (24a)

Prepared by the same procedure as described for compound 26a

from 23a (800 mg, 2.1 mmol) and phosphonate 20c. Colorless oil,

460 mg (34%). ¹H NMR spectra identical with compound 26a. MS

(ESI): m/z (%) ¼ 671.1 (100) [MNa]^þ. HR MS (FAB) calcd. for

C₂₄H₄₇N₂O₁₂P₂S [MH]^þ649.2325, found 649.2318.

GP3, from pyrimidine 19 (2 g, 12.6 mmol) and phosphonate 20b

(5.68 g, 13.9 mmol). Yield 1.25 g (25%) of 23b, colorless oil. ¹H NMR

(CDCl₃):

d

¼ 12.3 (br s, 1H, OH), 5.40 (br s, 1H, H-5), 4.58 (m, 2H,

CHipr.), 4.23 (dd, J_gem¼ 11.3, J(1⁰a,2⁰) ¼ 3.8, 1H, H-1⁰a), 4.16 (dd,

J_gem¼ 11.3, J(1⁰b,2⁰) ¼ 6.1, 1H, H-1⁰b), 3.87 (m, 2H, H-2⁰), 3.80 (m, 2H,

OCH₂P), 2.47 (s, 3H, SCH₃), 1.22 (m, 12H, CH₃ipr.), 1.15 (d,

4.17. 4-[2-(Diisopropoxyphosphorylmethoxy)ethoxy]-6-(2S)-[2-

(diisopropoxyphosphorylmethoxy)-3-hydroxypropoxy]-2-

(methylsulfonyl)pyrimidine (26c)

J(CH₃,2⁰) ¼ 6.4, 3H, H-3⁰) ppm. ¹³C NMR (CDCl₃):

d

¼ 170.52 (C-2),

169.15 (C-4), 162.50 (C-6), 86.08 (C-5), 75.24 (d, J(2⁰,P) ¼ 12.8, C-2⁰),

70.32 (m, 2C, CHipr.), 69.20 (C-1⁰), 63.01 (d, J(OCH₂,P) ¼ 165.2,

OCH₂P), 23.92 (m, 4C, CH₃ipr.), 16.28 (C-3⁰), 13.10 (SCH₃) ppm. MS

(FAB): m/z (%) ¼ 395 (100) [MH]^þ. HR MS (FAB) calcd. for

C₁₅H₂₈N₂O₆PS [MH]^þ395.1405, found 395.1418.

Prepared by the same procedure (GP4 and GP5) as compound

26a from 23a (800 mg, 2.1 mmol) and phosphonate 20d (1.07 g,

2.52 mmol). Colorless oil, 870 mg (62%). ¹H NMR (CDCl₃):

d

¼ 6.21

(s, 1H, H-5), 4.59 (m, 2H, H-1⁰), 4.51 (m, 2H, H-1⁰⁰), 4.05 (dd,

J_gem¼ 14.2, J(H,C,P) ¼ 7.3, 1H, PCH₂⁰⁰a), 3.95 (m, 2H, H-2⁰), 3.88 (m,

2H, H-2⁰⁰), 3.83 (dd, J_gem¼ 14.2, J(H,C,P) ¼ 8.4, 1H, PCH₂⁰⁰b), 3.81 (d,

Dialkylated derivative 24a was isolated as a second product.

Yield 1.75 g (22%), colorless oil.¹H NMR (CDCl₃):

d

¼ 5.69 (s,1H, H-5),

0

4.74 (m, 4H, CHipr.), 4.307 (d, J(1⁰,2⁰) ¼ 5.17, 4H, H-1⁰), 3.89 (m, 2H, H-

2⁰), 3.82 (d, J(CH₂,P) ¼ 9.11, 4H, OCH₂P), 2.49 (s, 3H, SCH₃), 1.31 (m,

24H, CH₃ipr.),1.23 (d, J(3⁰,2⁰) ¼ 6.39, 6H, H-3⁰) ppm.¹³C NMR (CDCl₃):

J(H,C,P) ¼ 8.2, 2H, PCH₂), 3.80 (m, 1H, H-3⁰⁰a), 3.70 (dd, J_gem¼ 12.3,

J(3⁰⁰b,2⁰⁰) ¼ 5.9, 1H, H-3⁰⁰b), 3.30 (s, 3H, CH₃SO₂), 1.34 (m, 24H,

CH₃ipr.) ppm. ¹³C NMR (CDCl₃):

d

¼ 171.35 (C-6), 171.21 (C-4), 164.18

d

¼ 170.92 (C-2), 170.17 (C-4,6), 86.12 (C-5), 75.85 (d, J(2⁰,P) ¼ 11.98,

(C-2), 93.69 (C-5), 81.49 (d, J(2⁰⁰,P) ¼ 7.4, C-2⁰⁰), 71.78 (d,

J(CH,P) ¼ 6.6), 71.40 (d, J(CH,P) ¼ 6.9) and 71.15 (d, J(CH,P) ¼ 6.5,

CHipr.), 70.73 (d, J(2⁰,P) ¼ 10.5, C-2⁰), 66.86 (2C, C-1⁰,1⁰⁰), 66.06 (d,

C-2⁰), 71.05 (d, J(C,P) ¼ 6.68) and 70.96 (d, J(C,P) ¼ 6.66, CHipr.),

69.35 (C-1⁰), 64.04 (d, J(C,P) ¼ 168.56, PCH₂), 24.06 (d, J(C,P) ¼ 3.69)

and 23.92 (d, J(C,P) ¼ 4.64, CH₃ipr.), 16.55 (C-3⁰), 14.00 (SCH₃) ppm.

MS (ESI): m/z (%) ¼ 653 (100) [MNa]^þ, 631 (100) [MH]^þ. HR MS (FAB)

calcd. for C₂₅H₄₉N₂O₁₀P₂S [MH]^þ631.2583, found 631.2579.

0

J(C,P) ¼ 167.7, PCH₂), 65.35 (d, J(C,P) ¼ 168.8, PCH₂⁰⁰), 61.62 (C-3⁰⁰),

38.88 (CH₃SO₂), 23.97 (m, CH₃ipr.) ppm. MS (ESI): m/z (%) ¼ 687

(100) [MNa]^þ, 665 (44) [MH]^þ. HR MS (FAB) calcd. for

C₂₄H₄₇N₂O₁₃P₂S [MH]^þ645.2274, found 645.2268.

4.14. 6-(2R)-[2-(Diisopropoxyphosphorylmethoxy)propoxy]-4-

hydroxy-2-(methylsulfanyl) pyrimidine (23c) and 4,6-(2R,2⁰R)-

bis[2-(diisopropoxyphosphorylmethoxy)propoxy]-2-

(methylsulfanyl)pyrimidine (24b)

4.18. 4-[2-(Diisopropoxyphosphorylmethoxy)ethoxy]-6-(2R)-[2-

(diisopropoxyphosphorylmethoxy)-3-hydroxypropoxy]-2-

(methylsulfonyl)pyrimidine (26d)

GP3, from pyrimidine 19 (4 g, 25.2 mmol) and phosphonate 20c

(10.3 g, 25.2 mmol). Yield 2.5 g (25%) of compound 23c, colorless oil.

¹H NMR (CDCl₃) and ¹³C NMR (CDCl₃) identical with compound 23b.

MS (FAB): m/z (%) ¼ 395 (100) [MH]^þ. HR MS (FAB) calcd. for

C₁₅H₂₈N₂O₆PS [MH]^þ395.1405, found 395.1413. Compound 24b

isolated as a colorless oil, yield 1.2 g (7.5%).¹H NMR (CDCl₃) identical

with compound 24a. MS (FAB): m/z (%) ¼ 631 (100) [MH]^þ. HR MS

(FAB) calcd. for C₂₅H₄₉N₂O₁₀P₂S [MH]^þ631.2583, found 631.2599.

Prepared by the same procedure (GP4 and GP5) as compound

26a from 23a (800 mg, 2.1 mmol) and phosphonate 20e (1.07 g,

2.52 mmol). Colorless oil, 700 mg (50%). ¹H NMR (CDCl₃) spectra

identical with compound 26c. MS (ESI): m/z (%) ¼ 687 (25) [MNa]^þ,

665 (12) [MH]^þ. HR MS (FAB) calcd. for C₂₄H₄₇N₂O₁₃P₂S [MH]^þ

645.2274, found 645.2288.

4.19. 4,6-(2R,2⁰S)-Bis[2-(diisopropoxyphosphorylmethoxy)propoxy]-

2-(methylsulfonyl)pyrimidine (26e)

4.15. 4-[2-(Diisopropoxyphosphorylmethoxy)ethoxy]-6-(2S)-[2-

(diisopropoxyphosphorylmethoxy)propoxy]-2-(methylsulfonyl)-

pyrimidine (26a)

Prepared by the same procedure (GP4 and GP5) as compound 26a

from 23b (500 mg, 1.27 mmol) and phosphonate 20c (0.57 g,

1.4 mmol). Colorless oil, 740 mg (88%). ¹H NMR (DMSO-d₆):

d

¼ 6.35

GP4, 23a (800 mg, 2.1 mmol), 20b (0.94 g, 2.3 mmol). The crude

product was taken to CHCl₃, ﬁltered through a pad of silica gel and

(s, 1H, H-5), 4.58 (m, 4H, CHipr.), 4.43 (dd, J(1⁰a,2⁰) ¼ 3.4, J_gem¼ 11.4,

2H, H-1⁰a), 4.34(dd, J(1⁰b,2⁰) ¼ 5.9, J_gem¼ 11.4, 2H, H-1⁰b), 3.92(m, 2H,

´ ´

P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

2418

H-2⁰), 3.82 (m, 4H, OCH₂P), 2.38 (s, 3H, SO₂CH₃), 1.18–1.24 (m, 30H,

CH₃ipr., H-3⁰) ppm.¹³C NMR (DMSO-d₆):

2), 92.66(C-5), 75.05 (d, J(2⁰,P) ¼ 12.7, C-2⁰), 70.30(m, CHipr), 70.13(C-

1⁰), 63.04 (d, J(C,P) ¼ 165.5, PCH₂), 38.95 (CH₃SO₂), 23.92 (m, CH₃ipr.),

16.22 (C-3⁰) ppm. MS (ESI): m/z (%) ¼ 685 (100) [MNa]^þ. HR MS (FAB)

calcd. for C₂₅H₄₉N₂O₁₂P₂S [MH]^þ663.2476, found 663.2469.

m/z (%) ¼ 679 (18) [MH]^þ, 701 (100) [MNa]^þ. HR MS (FAB) calcd. for

d

¼ 171.50 (C-4,6),164.25 (C-

C₂₅H₄₉N₂O₁₃P₂S [MH]^þ679.2430, found 679.2432.

4.24. 4,6-Bis[2-(diisopropoxyphosphorylmethoxy)ethoxy]-2-

(methylsulfonyl)pyrimidine (26j)

GP4, 21 (5.8 g, 9.62 mmol). Colorless oil, yield 4.2 g (69%). ¹H

4.20. 4-(2S)-[2-(Diisopropoxyphosphorylmethoxy)-3-hydroxypro-

poxy]-6-(2S)-[2-(diisopropoxyphosphorylmethoxy)propoxy]-2-

(methylsulfonyl)pyrimidine (26f)

NMR (DMSO-d₆):

d

¼ 6.20 (s, 1H, H-5), 4.58 (dh, J(CH,P) ¼ 7.6,

J(CH,CH₃) ¼ 6.4, 4H, CHipr.), 4.58 (m, 4H, H-1⁰), 3.94 (m, 4H, H-2⁰),

3.80 (d, J(C,H,P) ¼ 8.17, 4H, PCH₂), 3.30 (s, 3H, SO₂CH₃), 1.33 (d, 12H)

and 1.32 (d, 12H, J(CH₃,CH) ¼ 6.16, CH₃ipr.) ppm. MS (FAB):

m/z (%) ¼ 635 (100) [MH]^þ. HR MS (FAB) calcd. for C₂₃H₄₅N₂O₁₂P₂S

[MH]^þ635.2168, found 635.2152.

GP4, GP5, 23b (1.25 g, 3.17 mmol), 20d (1.75 g, 4.12 mmol).

Colorless oil, 394 mg (18%). ¹H NMR (DMSO-d₆):

d

¼ 6.21 (s, 1H,

H-5), 4.75 (m, 4H, CHipr.), 4.52 (dd, J_gem¼ 11.4, J(1⁰⁰a,2⁰⁰) ¼ 5.9, 1H,

H-1⁰⁰a), 4.49 (dd, J_gem¼ 11.4, J(1⁰⁰b,2⁰⁰) ¼ 4.8, 1H, H-1⁰⁰b), 4.44 (dd,

J_gem¼ 11.3, J(1⁰a,2⁰) ¼ 3.8, 1H, H-1⁰a), 4.40 (dd, J_gem¼ 11.3,

J(1⁰b,2⁰) ¼ 6.2, 1H, H-1⁰b), 4.06 (dd, J_gem¼ 14.2, J(C,H,P) ¼ 7.3, 1H,

PCH₂₀⁰⁰a), 3.95 (m, 2H, H-2⁰), 3.87 (m, 2H, H-2⁰⁰), 3.83 (m, 4H, PCH₂⁰⁰b,

PCH₂, H-3⁰⁰), 3.70 (m, 2H, H-3b⁰⁰), 3.30 (s, 3H, SO₂CH₃),1.34 (m, 24H,

CH₃ipr.), 1.28 (d, J(3⁰,2⁰) ¼ 6.4, 3H, H-3⁰) ppm. ¹³C NMR (DMSO-d₆):

4.25. 4,6-(2S,2⁰S)-Bis[2-(diisopropoxyphosphorylmethoxy)propoxy]-

2-(methylsulfonyl)pyrimidine (26k)

GP5, 24a (840 mg, 1.33 mmol). Yield 800 mg (72%), colorless oil.

¹H NMR (DMSO-d₆) identical with spectra of 26e. MS (ESI):

m/z (%) ¼ 685 (100) [MNa]^þ. HR MS (FAB) calcd. for C₂₅H₄₉N₂O₁₂P₂S

[MH]^þ663.2476, found 663.2475.

d

¼ 171.39 (C-4), 171.20 (C-6), 164.17 (C-2), 93.65 (C-5), 81.53 (d,

J(2⁰⁰,P) ¼ 7.5, C-2⁰⁰), 75.54 (d, J(2⁰,P) ¼ 11.9, C-2⁰), 71.81 (d,

J(C,O,P) ¼ 6.7), 71.42 (d, J(C,O,P) ¼ 6.9) and 71.08 (d, 2C,

J(C,O,P) ¼ 6.7, CHipr.), 70.54 (C-1⁰), 66.83 (C-1⁰⁰), 65.35 (d,

4.26. 4,6-(2R,2⁰R)-Bis[2-(diisopropoxyphosphorylmethoxy)propoxy]-

2-(methylsulfonyl)pyrimidine (26l)

J(C,P) ¼ 168.8, PCH₂⁰⁰), 64.00 (d, J(C,P) ¼ 169.2, PCH₂), 61.62 (C-3⁰⁰),

0

38.89 (CH₃SO₂), 24.00 (m, CH₃ipr.), 16.34 (C-3⁰) ppm. MS (ESI): m/z

(%) ¼ 679 (10) [MH]^þ, 701 (100) [MNa]^þ. HR MS (FAB) calcd. for

C₂₅H₄₉N₂O₁₃P₂S [MH]^þ679.2430, found 679.2429.

GP5, 24b (1.2 g, 1.9 mmol). Yield 1.1 g (87%), colorless oil. ¹H

NMR (DMSO-d₆) identical with spectra of 26e. MS (ESI):

m/z (%) ¼ 685 (100) [MNa]^þ. HR MS (FAB) calcd. for C₂₅H₄₉N₂O₁₂P₂S

[MH]^þ663.2476, found 663.2484.

4.21. 4-(2R)-[2-(Diisopropoxyphosphorylmethoxy)-3-hydroxypro-

poxy]-6-(2S)-[2-(diisopropoxyphosphorylmethoxy)propoxy]-2-

(methylsulfonyl)pyrimidine (26g)

4.27. Preparation of compounds 27 – general method

2-Methylsulfonyl derivatives 26 were converted to 2-amino

derivatives by GP6 and diisopropyl esters of 2-amino pyrimidines

were without further puriﬁcation deprotected to free phosphonic

acids by GP1.

GP4, GP5, 23b (1.25 g, 3.17 mmol), 20e (1.75 g, 4.12 mmol).

Colorless oil, 360 mg (16%). ¹H NMR (DMSO-d₆) and ¹³C NMR

(DMSO-d₆) identical with compound 26f. MS (ESI): m/z (%) ¼ 679

(6) [MH]^þ, 701 (100) [MNa]^þ. HR MS (FAB) calcd. for

C₂₅H₄₉N₂O₁₃P₂S [MH]^þ679.2430, found 679.2438.

4.27.1. 2-Amino-4-[2-(phosphonomethoxy)ethoxy]-6-(2S)-[2-

(phosphonomethoxy)propoxy] pyrimidine (27a)

4.22. 4-(2S)-[2-(Diisopropoxyphosphorylmethoxy)-3-hydroxypro-

poxy]-6-(2R)-[2-(diisopropoxyphosphorylmethoxy)propoxy]-2-

(methylsulfonyl)pyrimidine (26h)

26a (500 mg, 0.77 mmol), freeze dried, white hydroscopic foam,

yield 210 mg (61%). ¹H NMR (DMSO-d₆):

d

¼ 5.92 (s, 1H, H-5), 4.52

(m, 5H, H-1⁰), 4.45 (dd, J_gem¼ 11.2, J(1⁰⁰a,2⁰) ¼ 2.9, 1H, H-1⁰⁰a), 4.30

(dd, J_gem¼ 11.3, J(1⁰⁰b,2⁰⁰) ¼ 6.3, 1H, H-1⁰⁰b), 4.05 (dt,

J(2⁰⁰,3⁰⁰) ¼ J(2⁰⁰,1⁰⁰b) ¼ 6.4, J(2⁰⁰,1⁰⁰a) ¼ 3.0, 2H, H-2⁰⁰), 3.98 (m, 2H, H-

2⁰), 3.74–3.84 (m, 4H, PCH₂), 1.27 (d, J(3⁰⁰,2⁰⁰) ¼ 6.5, 3H, H-3⁰⁰) ppm.

GP4, GP5, 23c (1.25 g, 3.17 mmol), 20d (1.75 g, 4.12 mmol).

Colorless oil, 960 mg (42%). ¹H NMR (DMSO-d₆):

d

¼ 6.21 (s, 1H, H-

5), 4.75 (m, 4H, CHipr.), 4.51 (m, 2H, H-1⁰⁰), 4.54 (dd, J_gem¼ 11.4,

J(1⁰a,2⁰) ¼ 3.9, 1H, H-1⁰a), 4.41 (dd, J_gem¼ 11.3, J(1⁰b,2⁰) ¼ 6.1, 1H, H-

1⁰b), 4.05 (dd, J_gem¼ 14.1, J(C,H,P) ¼ 7.4, 1H, PCH₀₂⁰⁰a), 3.95 (m, 2H, H-

2⁰), 3.78–3.89 (m, 5H, H-2⁰⁰, PCH₂⁰⁰b, PCH₂, H-3⁰⁰), 3.70 (dd,

J_gem¼ 12.4, J(3⁰⁰b,2⁰⁰) ¼ 5.9, 1H, H-3⁰⁰b), 3.30 (s, 3H, SO₂CH₃), 1.33 (m,

24H, CH₃ipr.), 1.28 (d, J(3⁰,2⁰) ¼ 6.4, 3H, H-3⁰) ppm. ¹³C NMR (DMSO-

¹³C NMR (DMSO-d₆):

d

¼ 169.98 (C-4), 169.91 (C-6), 156.14 (C-2),

79.67 (C-5), 76.06 (d, J(2⁰⁰,P) ¼ 11.0, C-2⁰⁰), 72.42 (C-1), 70.72 (d,

J(2⁰,P) ¼ 10.3, C-2⁰), 67.19 (d, J(P,CH₂) ¼ 157.2, PCH₂), 65.05 (d,

J(P,CH₂) ¼ 159.1, PCH₂), 15.72 (C-3⁰⁰) ppm. MS (ESI): m/z (%) ¼ 418

25

(100) [MH]^þ. [

(C, H, N, P).

a]

¼ þ12.1 (c 0.248, H₂O). Anal. C₁₁H₂₁N₃O₁₀P₂.H₂O

D

d₆):

d

¼ 171.35 (C-4), 171.17 (C-6), 164.14 (C-2), 93.60 (C-5), 81.43

(d, J(2⁰⁰,P) ¼ 7.7, C-2⁰⁰), 75.51 (d, J(2⁰,P) ¼ 11.8, C-2⁰), 71.75 (d,

J(C,O,P) ¼ 6.7), 71.37 (d, J(C,O,P) ¼ 6.9), 71.06 (d, J(C,O,P) ¼ 6.7) and

71.04 (d, J(C,O,P) ¼ 6.5, CHipr.), 70.52 (C-1⁰), 66.84 (C-1⁰⁰), 65.28 (d,

4.27.2. 2-Amino-4-[2-(phosphonomethoxy)ethoxy]-6-(2R)-[2-

(phosphonomethoxy)propoxy] pyrimidine (27b)

26b (400 mg, 0.62 mmol), freeze dried, white hydroscopic foam,

J(C,P) ¼ 168.8, PCH₂⁰⁰), 63.95 (d, J(C,P) ¼ 169.1, PCH₂), 61.53 (C-3⁰⁰),

yield 190 mg (70%). NMR spectrum identical with compound 27a.

0

25

38.89 (CH₃SO₂), 23.96 (m, CH₃ipr.), 16.30 (C-3⁰) ppm. MS (ESI):

m/z (%) ¼ 679 (35) [MH]^þ, 701 (15) [MNa]^þ. HR MS (FAB) calcd. for

C₂₅H₄₉N₂O₁₃P₂S [MH]^þ679.2430, found 679.2441.

MS (ESI): m/z (%) ¼ 418 (100) [MH]^þ. [

a]

¼ ꢂ10.3 (c 0.347, H₂O).

D

Anal. C₁₁H₂₁N₃O₁₀P₂.H₂O (C, H, N, P).

4.27.3. 2-Amino-4-[2-(phosphonomethoxy)ethoxy]-6-(2S)-[2-

(phosphonomethoxy)-3-hydroxypropoxy]pyrimidine (27c)

26c (870 mg, 1.3 mmol), freeze dried, white hydroscopic foam,

4.23. 4-(2R)-[2-(Diisopropoxyphosphorylmethoxy)-3-hydroxypro-

poxy]-6-(2R)-[2-(diisopropoxyphosphorylmethoxy)propoxy]-2-

(methylsulfonyl)pyrimidine (26i)

yield 320 mg (54%). ¹H NMR (DMSO-d₆):

d

¼ 5.36 (s, 1H, H-5), 4.29

(m, 2H, H-1⁰), 4.25 (dd, J_gem¼ 11.3, J(1⁰⁰a,2⁰⁰) ¼ 4.3, 1H, H-1⁰⁰a), 4.20

(dd, J_gem¼ 11.3, J(1⁰⁰b,2⁰⁰) ¼ 5.7, 1H, H-1⁰⁰b), 3.75 (m, 2H, H-2⁰), 3.71

(dd, J_gem¼ 13.6, J(H–C–P) ¼ 8.7, 1H, OCH₂P⁰⁰a), 3.67 (dd, J_gem¼ 13.6,

GP3, GP4, 23c (1.25 g, 3.17 mmol), 20e (1.75 g, 4.12 mmol).

Colorless oil,1.05 g (46%). NMR spectra identical with 26h. MS (ESI):

´

P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

2419

25

J(H–C–P) ¼ 8.9, 1H, OCH₂P⁰⁰b), 3.67 (m, 2H, H-2⁰⁰), 3.58 (d, J(H–C–

27g. MS (ESI): m/z (%) ¼ 448.2 (100) [MH]^þ. [

a

]

¼ ꢂ4.4 (c 0.182,

D

P) ¼ 8.7, 2H, OCH₂P⁰), 3.51 (m, 2H, H-3⁰⁰) ppm. ¹³C NMR (DMSO-d₆):

H₂O). Anal. C₁₂H₂₃N₃O₁₁P₂.H₂O (C, H, N, P).

d

¼ 171.46 (C-6), 171.36 (C-4), 162.82 (C-2), 80.41 (d, J(2⁰⁰,P) ¼ 9.9, C-

2⁰⁰), 78.64 (C-5), 70.76 (d, J(2⁰,P) ¼ 11.4, C-2⁰), 66.97 (d, J(C,P) ¼ 160.4,

4.27.9. 2-Amino-4-(2R)-[2-(phosphonomethoxy)-3-hydroxypro-

poxy]-6-(2R)-[2-(phosphonomethoxy)propoxy]pyrimidine (27i)

From 26i (1 g, 1.62 mmol), freeze dried, white hydroscopic foam,

OCH₂P), 65.93 (d, J(C,P) ¼ 160.4, OCH₂P⁰⁰), 65.39 (C-1⁰⁰), 64.93 (C-1⁰),

25

60.33 (C-3⁰⁰) ppm. MS (ESI): m/z (%) ¼ 434 (100) [MH]^þ. [

(c 0.625, H₂O). Anal. C₁₁H₂₁N₃O₁₁P₂.H₂O (C, H, N, P).

a

]

¼ þ5.8

D

yield 616 mg (81%). NMR spectra identical with compound 39f. MS

25

(ESI): m/z (%) ¼ 448.0 (100) [MH]^þ. [

a

]

¼ ꢂ5.8 (c 0.312, H₂O). Anal.

D

4.27.4. 2-Amino-4-[2-(phosphonomethoxy)ethoxy]-6-(2R)-[2-

(phosphonomethoxy)-3-hydroxypropoxy]pyrimidine (27d)

26d (700 mg, 1.05 mmol), freeze dried, white hydroscopic foam,

C₁₂H₂₃N₃O₁₁P₂.H₂O (C, H, N, P).

4.27.10. 2-Amino-4,6-(2S,2⁰S)-bis[2-

yield 215 mg (45%). NMR spectrum identical with compound 27c.

(phosphonomethoxy)propoxy]pyrimidine (27j)

25

MS (ESI): m/z (%) ¼ 434 (100) [MH]^þ. [

a

]

D

¼ ꢂ6.7 (c 0.341, H₂O).

26k (350 mg, 0.58 mmol), yield 135 mg (52%), colorless foam.

Anal. C₁₁H₂₁N₃O₁₁P₂.H₂O (C, H, N, P).

¹H NMR (DMSO-d₆):

d

¼ 3.57 (s, 1H, H-5), 4.17 (dd, J_gem¼ 11.1,

J(1⁰a,2⁰) ¼ 5.8, 2H, H-1⁰a), 4.11 (dd, J_gem¼ 11.1, J(1⁰b,2⁰) ¼ 4.4, 2H,

H-1⁰b), 3.81 (m, 2H, H-2⁰), 3.60 (d, J(CH₂,P) ¼ 9.3, 4H, PCH₂), 1.14

(d, J(H-3⁰,H-2⁰⁰) ¼ 6.3, 6H, H-3⁰) ppm. ¹³C NMR (DMSO-d₆):

4.27.5. 2-Amino-4,6-(2R,2⁰S)-bis[2-(phosphonomethoxy)-

propoxy]pyrimidine (27e)

26e (840 mg, 1.27 mmol), freeze dried, white hydroscopic foam,

d

¼ 171.41 (C-4, C-6), 162.80 (C-2), 78.54 (C-5), 75.02 (d, J(C-

yield280 mg (49%).¹H NMR (DMSO-d₆):

d

¼ 3.58(s,1H, H-5), 4.18 (dd,

2⁰,P) ¼ 11.6, C-2⁰⁰), 68.59 (C-1⁰), 65.02 (d, J(CH₂,P) ¼ 161.7, PCH₂),

J_gem¼ 11.1, J(1⁰a,2⁰⁰) ¼ 5.8, 2H, H-1⁰a), 4.12 (dd, J_gem¼ 11.1,

J(1⁰b,2⁰) ¼ 4.4, 2H, H-1⁰⁰b), 3.82 (m, 2H, H-2⁰), 3.60 (d, J(CH₂,P) ¼ 9.3,

4H, PCH₂),1.14 (d, J(H-3⁰,H-2⁰) ¼ 6.4, 6H, H-3⁰) ppm. ¹³C NMR (DMSO-

16.82 (C-3⁰) ppm. MS (ESI): m/z (%) ¼ 432.1 (100) [MH]^þ; 454.1

(26) [MNa]^þ. [

a

]

¼ þ20.9 (c 0.291, H₂O). Anal. C₁₂H₂₃N₃O₁₀P₂.

25

D

H₂O (C, H, N, P).

d₆):

d

¼ 171.40 (C-4, C-6), 162.77 (C-2), 78.54 (C-5), 74.96 (d, J(C-

2⁰,P) ¼ 11.4, C-2⁰), 68.58 (C-1⁰), 65.07 (d, J(CH₂,P) ¼ 161.6, PCH₂),16.80

4.27.11. 2-Amino-4,6-(2R,2⁰R)-bis[2-(phosphonomethoxy)-

propoxy]pyrimidine (27k)

(C-3⁰⁰) ppm. MS (ESI): m/z (%) ¼ 432.1 (100) [MH]^þ, 454.1 (37) [MNa]^þ.

25

[a]

¼ þ0.9 (c 0.521, H₂O). Anal. C₁₂H₂₃N₃O₁₀P₂.H₂O (C, H, N, P).

From 26l (1.2 g, 1.8 mmol), white hydroscopic foam, yield

D

450 mg (55%). NMR spectra identical with compound 27j. MS (ESI):

25

4.27.6. 2-Amino-4-(2S)-[2-(phosphonomethoxy)-3-hydroxypropoxy]-

6-(2S)-[2-(phosphonomethoxy)propoxy]pyrimidine (27f)

m/z (%) ¼ 432.0 (100) [MH]^þ; 454.1 (14) [MNa]^þ. [

(c 0.254, H₂O). Anal. C₁₂H₂₃N₃O₁₀P₂.H₂O (C, H, N, P).

a

]

¼ ꢂ20.5

D

26f (394 mg, 0.64 mmol), freeze dried, white hydroscopic

foam, yield 200 mg (67%). ¹H NMR (DMSO-d₆):

d

¼ 5.36 (s, 1H, H-

4.28. 2-Substituted bisphosphonates

5), 4.26 (dd, J_gem¼ 11.3, J(1⁰⁰a,2⁰⁰) ¼ 4.3, 1H, H-1⁰⁰a), 4.20 (dd,

J_gem¼ 11.3, J(1⁰⁰b,2⁰⁰) ¼ 5.8, 1H, H-1⁰⁰b), 4.16 (dd, J_gem¼ 11.1,

J(1⁰a,2⁰) ¼ 5.8, 1H, H-1⁰a), 4.12 (dd, J_gem¼ 11.1, J(1⁰b,2⁰) ¼ 4.4, 1H, H-

1⁰b), 3.81 (m, 1H, H-2⁰), 3.71 (dd, J_gem¼ 13.6, J(C–H–P) ¼ 8.7, 1H,

OCH₂,P⁰⁰a), 3.67 (dd, J_gem¼ 13.6, J(C–H–P) ¼ 8.9, 1H, OCH₂,P⁰⁰b),

3.67 (m, 1H, H-2⁰⁰), 3.60 (d, 2H, J(C–H–P) ¼ 9.4, OCH₂,P⁰), 3.51 (m,

2H, H-3⁰⁰), 1.13 (d, J(3⁰,2⁰) ¼ 6.4, 3H, H-3⁰) ppm. ¹³C NMR (DMSO-

4.28.1. 2-Cyclopropylamino-4,6-bis[2-(phosphonomethoxy)-

ethoxy]pyrimidine (28a)

Compound 26j (1 g, 1.57 mmol) in dry THF (45 mL) was treated

with cyclopropylamine (5.5 mL) and the mixture was reﬂuxed in

a sealed ﬂask for 2 h. Volatiles were removed in vacuo and the

residue was puriﬁed by ﬂash chromatography (EtOAc/EtOH 0–10%)

to give 330 mg (34%) of 2-cyclopropylamino-4,6-bis[2-(diisopro-

poxyphosphorylmethoxy)ethoxy]pyrimidine as a thick oil. ¹H NMR

d₆):

d

¼ 171.48, 171.42 (C-4, C-6), 162.83 (C-2), 80.40 (d,

J(2⁰⁰,P) ¼ 9.7, C-2⁰⁰), 78.59 (C-5), 75.08 (d, J(2⁰,P) ¼ 11.7, C-2⁰), 68.63

(C-1⁰), 66.00 (d, J(P,C) ¼ 161.0, PCH₂⁰⁰), 65.45 (C-1⁰⁰), 65.01 (d,

(DMSO-d₆):

d

¼ 7.25 (br d, J(NH,CH) ¼ 3.6, 1H, NH), 5.32 (s, 1H, H-5),

J(P,C) ¼ 161.8, PCH₂), 60.35 (C-3⁰⁰), 16.83 (C-3⁰) ppm. MS (ESI): m/z

4.58 (m, 4H, P–OCH), 4.34 (m, 4H, C-1⁰), 3.79 (m, 4H, C-2⁰), 3.78 (d,

J(OCH₂,P) ¼ 8.3, 4H, PCH₂), 2.67 (m, 1H, CH cycloprop.), 1.23 (d, 12H)

and 1.22 (d, J(CH₃,CH) ¼ 6.3, 12H, CH₃), 0.62 (m, 2H) and 0.45 (m,

0

25

(%) ¼ 448.1 (100) [MH]^þ. [

a

]

¼ þ12.2 (c 0.181, H₂O). Analysis

D

C₁₂H₂₃N₃O₁₁P₂.H₂O (C, H, N, P).

2H, CH₂cycloprop.) ppm. ¹³C NMR (DMSO-d₆):

d

¼ 170.99 2C (C-4,

4.27.7. 2-Amino-4-(2R)-[2-(phosphonomethoxy)-3-hydroxypro-

poxy]-6-(2S)-[2-(phosphonomethoxy)propoxy]pyrimidine (27g)

From 26g (360 mg, 0.58 mmol), freeze dried, white hydroscopic

C-6), 162.51 (C-2), 78.66 (C-5), 70.80 (C-2⁰), 70.34 (d, 4C,

J(CH,P) ¼ 6.4, P–OCH), 64.96 (d, J(C-3⁰,P) ¼ 164.5, P–OCH₂), 64.48

(C-1⁰), 23.87 (CH cycloprop.), 23.92 (CH₃), 6.34 (CH₂cycloprop.)

ppm. MS (FAB): m/z (%) ¼ 612 (100) [MH]^þ. HR MS (FAB) calcd. for

C₂₅H₄₈N₃O₁₀P₂[MH]^þ612.2814, found 612.2813.

foam, yield 200 mg (73%). ¹H NMR (DMSO-d₆):

d

¼ 5.36 (s, 1H, H-5),

4.25 (dd, J_gem¼ 11.3, J(1⁰⁰a,2⁰⁰) ¼ 4.3, 1H, H-1⁰⁰a), 4.20 (dd, J_gem¼ 11.3,

J(1⁰⁰b,2⁰⁰) ¼ 5.8, 1H, H-1⁰⁰b), 4.17 (dd, J_gem¼ 11.1, J(1⁰a,2⁰) ¼ 5.8, 1H, H-

1⁰a), 4.11 (dd, J_gem¼ 11.1, J(1⁰b,2⁰) ¼ 4.4,1H, H-1⁰b), 3.81 (m,1H, H-2⁰),

3.71 (dd, J_gem¼ 13.6, J(C–H–P) ¼ 8.7, 1H, OCH₂,P⁰⁰a), 3.67 (dd,

J_gem¼ 13.6, J(C–H–P) ¼ 8.9,1H, OCH₂,P⁰⁰b), 3.67 (m,1H, H-2⁰⁰), 3.60 (d,

J(C–H–P) ¼ 9.4, 2H, OCH₂,P⁰), 3.51 (m, 2H, H-3⁰⁰),1.13 (d, J(3⁰,2⁰) ¼ 6.4,

Diisopropyl esters were deprotected by GP1 to give 28a (140 mg,

62%) as a white foam. ¹H NMR (D₂O):

d

¼ 4.53 (m, 4H, H-1⁰⁰), 3.98

(m, 4H, H-2⁰), 3.76 (d, J(PCH₂) ¼ 9.2, 4H, PCH₂), 2.72 (m, 1H, CH

cycloprop.), 0.93 and 0.71 (2 ꢀ m, 2 ꢀ 2H, CH₂cycloprop.) ppm. ¹³

C

NMR (D₂O):

d

¼ 171.05 (C-4, C-6),159.38 (C-2), 78.79 (C-5), 70.85 (d,

3H, H-3⁰) ppm.¹³C NMR (DMSO-d₆):

d

¼ 171.48 and 171.42 (C-4, C-6),

J(C-2⁰,P) ¼ 10.4, C-2⁰), 68.23 (C-1⁰), 67.60 (d, J(CH₂,P) ¼ 156.4, PCH₂),

23.41 (CH cycloprop.), 7.19 (CH₂cycloprop.) ppm. MS (ESI): m/z

(%) ¼ 444.1 (100) [MH]^þ, 466.0 (26) [MNa]^þ. Anal. C₁₃H₂₃N₃O₁₀P₂.

H₂O (C, H, N, P).

162.83 (C-2), 80.40 (d, J(2⁰⁰,P) ¼ 9.7, C-2⁰⁰), 78.59 (C-5), 75.08 (d,

J(2⁰,P) ¼ 11.7, C-2⁰), 68.63 (C-1⁰), 66.00 (d, J(P,C) ¼ 161.0, PCH₂⁰⁰), 65.45

0

(C-1⁰⁰), 65.01 (d, J(P,C) ¼ 161.8, PCH₂), 60.35 (C-3⁰⁰),16.83 (C-3⁰) ppm.

MS (ESI): m/z (%) ¼ 448.1 (100) [MH]^þ, 470.1 (35) [MNa]^þ.

25

[a]

¼ þ8.6 (c 0.561, H₂O). Anal. C₁₂H₂₃N₃O₁₁P₂.H₂O (C, H, N, P).

4.28.2. 2-Cyclopentylamino-4,6-bis[2-(phosphonomethoxy)-

ethoxy]pyrimidine (28b)

D

4.27.8. 2-Amino-4-(2S)-[2-(phosphonomethoxy)-3-hydroxypro-

poxy]-6-(2R)-[2-(phosphonomethoxy)propoxy]pyrimidine (27h)

From 26h (960 mg, 1.55 mmol), freeze dried, white hydroscopic

foam, yield 605 mg (83%). NMR spectra identical with compound

Compound 26j (1 g, 1.57 mmol) in dry THF (30 mL) was treated

with cyclopentylamine (1.6 mL) and the mixture was reﬂuxed in

a sealed ﬂask for 3 h. THF was removed under reduced pressure and

the residue was puriﬁed by ﬂash chromatography (CHCl₃/MeOH

´ ´

P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

2420

0–2%)

to

give

2-cyclopentylamino-4,6-bis[2-(diisopropox-

The intermediate was treated with bromotrimethylsilane (GP1)

yphosphorylmethoxy)ethoxy]pyrimidine as an oil, yield 540 mg

to give 28d, white hydroscopic foam, yield 120 mg (42%). ¹H NMR

(54%). ¹H NMR (CDCl₃):

d

¼ 5.37 (s, 1H, H-5), 4.88 (d, J(NH-1⁰) ¼ 7.0,

(DMSO-d₆):

d

¼ 7.66 (br t, J(NH, H-1⁰⁰) ¼ 6.0, 1H, NH), 7.16–7.34 (m,

1H, NH), 4.76 (dh, J(CH,CH₃) ¼ 6.2, J(CH,P) ¼ 7.7, 4H, CHipr.), 4.39

(m, 4H, H-1⁰), 4.18 (m, 1H, H-1⁰⁰), 3.89 (m, 4H, H-2⁰), 3.82 (d,

J(P,CH₂) ¼ 8.2, 4H, PCH₂), 2.01 (m, 2H, H-2⁰⁰a), 1.71 and 1.61 (2 ꢀ m,

2 ꢀ 2H, H-3⁰⁰), 1.45 (m, 2H, H-2⁰⁰b), 1.33 (m, 24H, CH₃ipr.) ppm. ¹³C

5H, arom.), 5.37 (s, 1H, H-5), 4.42 (d, J(H-1⁰⁰, NH) ¼ 5.6, 2H, H-1⁰⁰),

4.27 (m, 4H, H-1⁰), 3.71 (m, 4H, H-2⁰), 3.53 (m, 4H, PCH₂) ppm. ¹³C

NMR (DMSO-d₆):

d

¼ 171.15 (C-4, C-6), 161.52 (C-2), 140.75, 128.35,

127.46, 126.70 (arom.), 78.68 (C-5), 70.60 (d, J(C-2⁰,P) ¼ 10.9, C-2⁰),

67.18 (d, J(CH₂,P) ¼ 160.6, PCH₂), 64.97 (C-1⁰), 44.41 (C-1⁰⁰) ppm. MS

(FAB): m/z (%) ¼ 494 (100) [MH]^þ, 516 (35) [MNa]^þ. Anal.

C₁₇H₂₅N₃O₁₀P₂.H₂O (C, H, N, P).

NMR (CDCl₃):

d

¼ 171.20 (C-4, C-6), 161.10 (C-2), 79.48 (C-5), 71.33

(d, J(2⁰-P) ¼ 11.0, C-2⁰⁰), 71.08 (d, J(CH–P) ¼ 6.6, CHipr.), 65.97 (d,

J(3⁰-P) ¼ 167.1, PCH₂), 64.76 (C-1⁰), 52.91 (C-1⁰⁰), 33.29 (C-2⁰⁰), 24.07

(d, J(CH₃-P) ¼ 3.7) and 23.93 (d, J(CH₃-P) ¼ 4.6, CH₃ipr.), 23.72 (C-

3⁰⁰) ppm. MS (FAB): m/z (%) ¼ 640.5 (60) [MH]^þ. HR MS (FAB) calcd.

for C₂₇H₅₂N₃O₁₀P₂[MH]^þ640.3128, found 640.3140.

4.28.5. 2-(4-Methoxybenzyl)amino-4,6-bis[2-

(phosphonomethoxy)ethoxy]pyrimidine (28e)

Diisopropyl esters were cleaved by GP1 to afford 28b (220 mg,

2-Methylsulfonyl derivative 26j (1 g, 1.57 mmol) in dry THF

(30 mL) was treated with 4-methoxybenzylamine (0.61 mL,

4.1 mmol) and the reaction mixture was reﬂuxed in a sealed tube for

2 h. The solvent was removed under reduced pressure and the

residue was puriﬁed by ﬂash chromatography to give 2-(4-

methoxybenzyl)amino-4,6-bis[2-(diisopropoxyphosphorylmethox-

y)ethoxy]pyrimidine (550 mg, 50%) as colorless oil. ¹H NMR (CDCl₃):

53%) as a white hydroscopic foam. ¹H NMR (DMSO-d₆):

d

¼ 5.34 (s,

1H, H-5), 4.31 (m, 4H, H-1⁰), 4.09 (m, 1H, H-1⁰⁰), 3.76 (m, 4H, H-2⁰),

3.58 (d, J(CH₂P) ¼ 8.7, 4H, PCH₂), 1.88 (m, 2H, H-2⁰⁰a), 1.65 (m, 2H, H-

3⁰⁰a), 1.49 (m, 4H, H-2⁰⁰b and H-3⁰⁰b) ppm. ¹³C NMR (DMSO-d₆):

d

¼ 171.04 (C-4, C-6), 161.23 (C-2), 78.17 (C-5), 70.73 (d, J(2,P) ¼ 11.2,

C-2⁰), 66.95 (d, J(3,P) ¼ 160.3, PCH₂), 64.83 (C-1⁰), 52.60 (C-1⁰⁰),

32.44 (C-2⁰⁰), 23.71 (C-3⁰⁰) ppm. MS (FAB): m/z (%) ¼ 472.1 (100)

[MH]^þ. Anal. C₁₅H₂₇N₃O₁₀P₂.H₂O (C, H, N, P).

d

¼ 7.25 (m, 2H, Ph-2), 6.86 (m, 2H, Ph-3), 5.40 (s, 1H, H-5), 5.20 (t,

J(NH,CH₂) ¼ 5.9, 1H, NH), 4.75 (dh, J(CH,CH₃) ¼ 6.2, J(CH,P) ¼ 7.7, 4H,

CHipr.), 4.49 (d, J(CH₂,NH) ¼ 5.9, 2H, PhCH₂NH), 4.33 (m, 4H, H-1),

3.86 (m, 4H, H-2), 3.81 (d, J(CH₂,P) ¼ 8.3, 4H, PCH₂), 3.80 (s, 3H,

4.28.3. 2-Methylamino-4,6-bis[2-(phosphonomethoxy)-

ethoxy]pyrimidine (28c)

OCH₃), 1.33 (m, 24H, CH₃ipr.) ppm. ¹³C NMR (CDCl₃):

d

¼ 171.29 (C-4,

2-Methylsulfonyl derivative 26j (1 g, 1.57 mmol) in EtOH

(33.75 mL) was treated with methylamine (8 M solution in EtOH,

11.25 mL) and the reaction mixture was heated at 50 ^ꢁC in a sealed

tube for 6 h. Volatiles were removed under reduced pressure and

the residue was puriﬁed by ﬂash chromatography (EtOAc/EtOH 0–

5%) to give colorless oil of 2-methylamino-4,6-bis[2-(diisopropox-

yphosphorylmethoxy)ethoxy]pyrimidine, yield 440 mg (48%). ¹H

C-6), 161.20 (C-2), 158.73 (Ph-4), 131.41 (Ph-1), 128.74 (Ph-2), 113.85

(Ph-3), 80.01 (C-5), 71.29 (d, J(C-2⁰,P) ¼ 11.1, C-2⁰), 71.08 (d,

J(CH,P) ¼ 6.6, CHipr.), 65.96 (d, J(CH₂,P) ¼ 167.1, PCH₂), 64.86 (C-1⁰),

55.24 (OCH₃), 44.90 (PhCH₂NH), 24.06 (d, J(CH₃,CH) ¼ 3.7) and 23.93

(d, J(CH₃,CH) ¼ 4.6, CH₃ipr.) ppm. MS (FAB): m/z (%) ¼ 692.2 (15)

[MH]^þ. HR MS (FAB) calcd. for C₃₀H₅₂N₃O₁₁P₂[MH]^þ692.3077,

found 692.3074.

NMR (DMSO-d₆):

d

¼ 6.97 (br q, J(NH,CH₃) ¼ 4.7, 1H, NH), 5.28 (s,

Diisopropyl esters were deprotected by GP1. The ﬁnal product

was applied onto a column of Dowex 50 ꢀ 8 in Na^þform and eluted

with water to give 28e (160 mg, 23%) as a tetrasodium salt;

1H, H-5), 4.59 (dh, J(CH,CH₃) ¼ 6.2, J(CH,P) ¼ 7.8, 4H, CHipr.), 4.33

(m, 4H, H-1⁰), 3.78 (m, 8H, H-2⁰, H-3⁰), 2.75 (d, J(CH₃,NH) ¼ 4.7, 3H,

CH₃NH) ppm. ¹³C NMR (DMSO-d₆):

d

¼ 170.82 (C-2), 161.74 (C-4, C-

white hydroscopic foam. ¹H NMR (DMSO-d₆):

d

¼ 7.60 (br t,

6), 77.83 (C-5), 70.36 (m, CHipr.), 64.97 (d, J(CH₂,P) ¼ 164.2, CH₂P),

64.49 (C-1⁰), 27.86 (CH₃NH), 23.93 m (CH₃ipr.) ppm. MS (FAB):

m/z (%) ¼ 586.2 (75) [MH]^þ. HR MS (FAB) calcd. for C₂₃H₄₆N₃O₁₀P₂

[MH]^þ586.2658, found 586.2659.

J(NH,CH₂) ¼ 6.4, NH), 7.23 (m, 2H, H-2⁰⁰), 6.85 (m, 2H, H-3⁰⁰), 5.36 (d,

1H, H-5), 4.33 (d, J(CH₂,NH) ¼ 6.0, 2H, CH₂N), 4.25 (m, 4H, H-1⁰),

3.70 (s, 3H, OCH₃), 3.70 (m, 4H, H-2⁰), 3.50 (d, 4H, J(H,P) ¼ 8.6,

PCH₂). ¹³C NMR (DMSO-d₆):

d

¼ 171.14 (C-4,6), 161.46 (C-2), 158.22

Diisopropyl esters were deprotected by GP1 to give 28c (210 mg,

(C-4⁰⁰), 132.68 (C-1⁰⁰), 128.83 (C-2⁰⁰), 113.76 (C-3⁰⁰), 78.60 (C-5), 70.54

(d, J(2⁰,P) ¼ 10.6, C-2⁰), 67.57 (d, J(C,P) ¼ 159.5, PCH₂), 64.97 (C-1⁰),

55.21 (OCH₃), 43.80 (CH₂N). MS (ESI): m/z (%) ¼ 524.1 (100) [MH]^þ,

546.1 (69) [MNa]^þ. Anal. C₁₈H₂₃N₃Na₄O₁₁P₂(C, H, N, P).

67%) as a white hydroscopic foam. ¹H NMR (D₂O):

d

¼ 4.54 (m, 4H, H-

1⁰), 3.98 (m, 4H, H-2⁰), 3.77 (d, J(CH₂,P) ¼ 9.2, 4H, PCH₂), 3.00 (s, 3H,

CH₃) ppm. ¹³C NMR (D₂O):

d

¼ 173.02 (C-4, C-6), 162.95 (C-2), 70.72

d (C-2⁰), 69.21 (C-1⁰⁰), 67.20 (d, PCH₂), 28.21 (CH₃) ppm. MS (FAB):

m/z (%) ¼ 418 (100) [MH]^þ. Anal. C₁₁H₂₁N₃O₁₀P₂.H₂O (C, H, N, P).

4.28.6. 2-Morpholino-4,6-bis[2-(phosphonomethoxy)-

ethoxy]pyrimidine (28f)

4.28.4. 2-Benzylamino-4,6-bis[2-(phosphonomethoxy)-

ethoxy]pyrimidine (28d)

Compound 26j (1 g, 1.57 mmol) in dry THF (30 mL) was treated

with morpholine (1.4 mL) and the mixture was reﬂuxed in a sealed

ﬂask for 2 h and the solvent was removed under reduced pressure.

Flash chromatography (CHCl₃/MeOH 0–1%) gave thick oil of

2-morpholino-4,6-bis[2-(diisopropoxyphosphorylmethoxy)ethox-

Pyrimidine 26j (1 g, 1.57 mmol) in dry THF was treated with

benzylamine (6 mL) and the reaction mixture was heated at 50 ^ꢁC

in a sealed tube for 4 h. The solvent was removed under reduced

pressure and the residue was puriﬁed by ﬂash chromatography

(EtOAc/EtOH 0–5%) to afford 2-benzylamino-4,6-bis[2-(diisopro-

poxyphosphorylmethoxy)ethoxy]pyrimidine (390 mg, 37%) as

y]pyrimidine (720 mg, 71%). ¹H NMR (CDCl₃):

d

¼ 5.39 (s, 1H, C-5),

4.76 (dh, J(CH,CH₃) ¼ 6.2, J(CH,P) ¼ 7.7, 4H, CHipr.), 4.41 (m, 4H, H-

1⁰), 3.90 (m, 4H, H-2⁰), 3.82 (d, J(CH₂,P) ¼ 8.2, 4H, PCH₂), 3.72 (s, 8H,

H-2⁰⁰, H-3⁰⁰), 1.34 (d, J(CH₃,CH) ¼ 6.3, 12H) and 1.33 (d,

a colorless oil. ¹H NMR (DMSO-d₆):

d

¼ 7.63 (br t, J(NH,H-1⁰⁰) ¼ 6.4,

1H, NH), 7.15–7.34 (m, 5H, arom.), 5.29 (s, 1H, H-5), 4.58 (m, 4H,

CHipr.), 4.41 (d, J(H-1⁰⁰,NH) ¼ 6.3, 2H, H-1⁰⁰), 4.29 (m, 4H, H-1⁰), 3.75

(m, 8H, H-2⁰, PCH₂), 1.23 (d, J(CH₃,CH) ¼ 6.2, 12H) and 1.21 (d,

J(CH₃,CH) ¼ 6.3, 12H, CH₃ipr.) ppm. ¹³C NMR (CDCl₃):

¼ 171.13 (C-

d

4, C-6), 160.62 (C-2), 79.50 (C-5), 71.27 (d, J(2⁰,P) ¼ 10.8, C-2⁰), 71.07

(d, J(CH,P) ¼ 6.61, CHipr.), 66.74 (C-3⁰⁰), 66.02 (d, J(CH,P) ¼ 167.3,

PCH₂), 64.80 (C-1⁰), 44.24 (C-2⁰⁰), 24.07 (d, J(CH₃,P) ¼ 3.5) and 23.94

(d, J(CH₃,P) ¼ 4.5, CH₃ipr.) ppm. MS (FAB): m/z (%) ¼ 642.5 (22)

[MH]^þ. HR MS (FAB) calcd. for C₂₆H₅₀N₃O₁₁P₂[MH]^þ642.2921,

found 642.2911.

J(CH₃,CH) ¼ 6.2, 12H, CH₃ipr.) ppm. ¹³C NMR (DMSO-d₆):

¼ 170.94

d

(C-4, C-6), 161.46 (C-2), 140.71, 128.28, 127.42, 126.67 (arom.), 78.59

(C-5), 70.74 (C-2⁰), 70.35 (d, J(CH,P) ¼ 6.3, 4C, CHipr.), 64.95 (d,

J(CH₂,P) ¼ 164.3, 2C, PCH₂), 64.58 (2C, C-1⁰), 44.39 (C-1⁰⁰), 24.00 (d,

J(CH₃,P) ¼ 3.6, 4C) and 23.84 (d, J(CH₃,P) ¼ 4.3, 4C, CH₃ipr.) ppm. MS

(FAB): m/z (%) ¼ 662 (25) [MH]^þ. HR MS (FAB) calcd. for

C₂₉H₅₀N₃O₁₀P₂[MH]^þ662.2971, found 662.2981.

Deprotection of diisopropyl esters by GP1 gave 28f (360 mg,

65%) as a white hydroscopic foam. ¹H NMR (DMSO-d₆):

d

¼ 5.45

(s, 1H, H-5), 4.33 (m, 4H, H-1⁰), 3.77 (m, 4H, H-2⁰), 3.64 (m, 8H,

´

P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

2421

CH₂–morpholine), 3.57 (d, J(P,CH) ¼ 8.7, 4H, PCH₂) ppm. ¹³C NMR

MS (ESI): m/z (%) ¼ 389 (76) [MH]^þ. Anal. C₁₀H₁₈N₂O₁₀P₂.H₂O (C, H,

(DMSO-d₆):

d

¼ 171.13 (2C, C-4, C-6),160.51 (C-2), 78.84 (C-5), 70.62

N, P).

(d, J(C-2,P) ¼ 11.2, C-2⁰), 66.93 (d, J(C-3,P) ¼ 160.4, PCH₂), 66.13 (C-

3⁰⁰), 65.10 (C-1⁰), 44.13 (C-2⁰⁰) ppm. MS (ESI): m/z (%) ¼ 474 (86)

[MH]^þ. Anal. C₁₄H₂₅N₃O₁₁P₂.H₂O (C, H, N, P).

4.29. 4,6-Disulfanylpyrimidine derivatives

4.29.1. 2-Amino-4,6-disulfanylpyrimidine (31)

4.28.7. 4,6-Bis[2-(diisopropoxyphosphorylmethoxy)ethoxy]-2-

hydroxypyrimidine (29a)

Thiourea (9.5 g, 90 mmol) was added to the solution of

dichloropyrimidine 10 (5 g, 30 mmol) in EtOH (250 mL) and the

reaction mixture was reﬂuxed for 2 h. Solvent was removed in

vacuo and the residue in aq. NaOH (0.5 M, 250 mL) was heated at

80 ^ꢁC for 16 h. The reaction mixture was cooled to r.t., acidiﬁed with

acetic acid to pH 4 and evaporated to half of its volume. The

precipitate was ﬁltered off, washed with water and dried to give

yellow solid (4.78 g, 98%), m.p. 259 ^ꢁC dec. ¹H NMR (DMSO-d₆):

Solution of NaOH (0.072 g) in water (10 mL) was added in one

portion to the 2-methylsulfonyl derivative 26j (1 g, 1.57 mmol) in

THF (5 mL) and the resulting mixture was heated at 60 ^ꢁC for 1 h.

Reaction mixture was cooled to r.t., neutralized with acetic acid and

volatiles were removed in vacuo. The residue was partitioned

between CHCl₃and water. Organic fraction was washed with water

(3 ꢀ 50 mL) and dried over MgSO₄. Flash chromatography (CHCl₃/

MeOH 0–5%) gave colorless oil, 450 mg (50%). ¹H NMR (CDCl₃):

d

¼ 11.2–11.8 (br s, 2H, SH), 7.17 (br s, 2H, NH₂), 6.24 (s, 1H, H-5)

ppm. ¹³C NMR (DMSO-d₆):

d

¼ 173.91 (2C, C-4, C-6), 149.10 (C-2),

d

¼ 5.29 (s, 1H, H-5), 4.76 (dh, J(CH,P) ¼ 7.7, J(CH,CH₃) ¼ 6.2, CHipr.),

116.36 (C-5) ppm. MS (EI): m/z (%) ¼ 159.2 (100) [M]^þ. Anal.

4.38 (m, 4H, H-1⁰⁰), 3.92 (m, 4H, H-2⁰), 3.81 (d, J(CH₂,P) ¼ 8.2, PCH₂),

1.34 and 1.33 (2 ꢀ d, J(CH₃,CH) ¼ 6.2, 2 ꢀ12H, CH₃ipr.) ppm. ¹³C

C₄H₅N₃S₂(C, H, N, S).

NMR (CDCl₃):

d

¼ 162.57 (C-4,6),158.72 (C-2), 75.35 (C-5), 71.18 (d,

4.29.2. 2-Amino-4,6-bis{[2-(diisopropoxyphosphorylmethoxy)-

ethyl]sulfanyl}pyrimidine (32a) and 2-amino-4,6-bis{[2-

(phosphonomethoxy)ethyl]sulfanyl}pyrimidine (33a)

Phosphonate 20a (6.9 g, 26.4 mmol) was added to a stirred

mixture of disulfanylpyrimidine 31 (2 g, 12.56 mmol) and NaH

(1.25 g, 60% in parafﬁn oil, 31 mmol) in DMF (50 mL) and the

resulting mixture was stirred at r.t. for 24 h and evaporated in

vacuo. The residue was adsorbed onto silica gel from methanol and

separated by ﬂash chromatography (CHCl₃/MeOH 0–5%) to give 32a

J(C,P) ¼ 6.6, CHipr.), 70.59 (d, J(C,P) ¼ 10.6, C-2⁰), 67.29 (C-1⁰⁰), 66.08

(d, J(C,P) ¼ 167.6, PCH₂), 24.05 (d, J(C,P) ¼ 4.0, CH₃ipr.), 23.95 (d,

J(C,P) ¼ 4.6, CH₃ipr.) ppm. MS (FAB): m/z (%) ¼ 573 (100) [MH]^þ. HR

MS (FAB) calcd. for C₂₂H₄₃N₂O₁₁P₂[MH]^þ573.2342, found

573.2347.

4.28.8. 2-Methoxy-4,6-bis[2-(diisopropoxyphosphorylmethoxy)-

ethoxy]pyrimidine (29b)

26j (2 g, 3.15 mmol) in MeOH (40 mL) was treated with MeONa

(1 M in MeOH, 3.5 mL) and the resulting mixture was heated at

80 ^ꢁC for 4 h. Reaction mixture was cooled to r.t. and solvent was

removed in vacuo. Flash chromatography afforded 29b (600 mg,

(5.6 g, 74%), pale yellow oil. ¹H NMR (DMSO-d₆):

d

¼ 6.74 (br s, 2H,

NH₂), 6.41 (s, 1H, H-5), 4.58 (m, 4H, CHipr.), 3.79 (d, J(P,CH) ¼ 8.3,

4H, PCH₂), 3.69 (t, J(1⁰,2⁰) ¼ 6.4, 4H, H-1⁰), 3.26 (t, J(2⁰⁰,1⁰) ¼ 6.4, 4H,

H-2⁰⁰), 1.24 (d, 12H) and 1.23 (d, J(CH₃,CH) ¼ 6.2, 12H, CH₃) ppm. ¹³C

32%) as an oil. ¹H NMR (CDCl₃):

d

¼ 5.71 (s, 1H, H-5), 4.76 (dh,

NMR (DMSO-d₆):

d

¼ 167.60 (2C, C-4, C-6), 161.79 (C-2), 103.07 (C-

J(CH,CH₃) ¼ 6.2, J(H,C,P) ¼ 7.7, 4H, CHipr), 4.48 (m, 4H, H-1⁰), 3.94 (s,

5), 71.18 (d, J(P,C) ¼ 12.2, 2C, C-2⁰), 70.35 (d, J(P,C) ¼ 6.3, 4C, CHipr.),

64.81 (d, J(P,C) ¼ 164.6, 2C, PCH₂), 27.60 (2C, C-1⁰), 24.03 (d,

J(P,C) ¼ 3.9, 4C) and 23.92 (d, J(P,C) ¼ 4.4, 4C, CH₃) ppm. MS (ESI):

m/z (%) ¼ 604.2 (100) [MH]^þ. Anal. C₂₂H₄₃N₃O₈P₂S₂(C, H, N, P, S).

Subsequent deprotection of 32a (2.6 g, 4.3 mmol) by GP1 gave

free phosphonic acid 33a (1.2 g, 65%) as a white foam. ¹H NMR

3H, OCH₃), 3.92 (m, 4H, H-2⁰⁰), 3.82 (d, J(H,C,P) ¼ 8.2, 4H, OCH₂P),

1.33 (m, 24H, CH₃ipr.) ppm. ¹³C NMR (CDCl₃):

d

¼ 171.99 (C-4, C-6),

164.52 (C-2), 84.20 (C-5), 71.08 (m, CHipr., H-2⁰), 65.97 (d,

J(C,P) ¼ 167.4, OCH₂P), 65.51 (C-1⁰), 54.65 (OCH₃), 24.05 (d,

J(C,C,O,P) ¼ 3.7) and 23.91 (d, J(C,C,O,P) ¼ 4.6, CH₃ipr.) ppm. MS

(FAB): m/z (%) ¼ 587 (62) [MH]^þ. HR MS (FAB) calcd. for

C₂₃H₄₅N₂O₁₁P₂[MH]^þ587.2498, found 587.2516.

(D₂O):

d

¼ 6.75 (s, 1H, H-5), 3.97 (t, J(2⁰,1⁰⁰) ¼ 6.4, 4H, H-2⁰), 3.80 (d,

J(P,CH) ¼ 8.7, 4H, PCH₂), 3.45 (t, J(1⁰,2⁰⁰) ¼ 6.4, 4H, H-1⁰) ppm. ¹³C

NMR (D₂O):

d

¼ 169.49 (2C, C-4, C-6), 161.20 (C-2), 104.15 (C-5),

4.28.9. 4,6-Bis[2-(phosphonomethoxy)ethoxy]pyrimidine (30)

Compound 21 (2 g, 3.22 mmol) in MeOH (200 mL) was treated

with suspension of Raney-Nickel (ca. 15 g) in MeOH (30 mL). The

reaction mixture was reﬂuxed for 6 h, ﬁltered while hot through

Celite and the precipitate was washed with MeOH (500 mL). The

ﬁltrate was evaporated in vacuo, and the residue was puriﬁed by

ﬂash chromatography (CHCl₃/MeOH 0–5%) to give 4,6-bis[2-(dii-

sopropoxyphosphorylmethoxy)ethoxy]pyrimidine as a thick oil

70.59 (d, J(P,C) ¼ 10.7, 2C, C-2⁰), 66.82 (d, J(P,C) ¼ 156.2, 2C, PCH₂),

28.38 (2C, C-1⁰) ppm. MS (ESI): m/z (%) ¼ 436 (35) [MH]^þ. Anal.

C₁₀H₁₉N₃O₈P₂S₂(C, H, N, P, S).

4.29.3. 2-Amino-4,6-(2S,2⁰S)-bis{[2-(diisopropoxyphosphorylmeth-

oxy)propyl]sulfanyl}pyrimidine (32b) and 2-amino-4,6-(2S,2⁰S)-

bis{[2-(phosphonomethoxy)propyl]sulfanyl}pyrimidine (33b)

Prepared by the same procedure as compounds 32a and 33a.

From pyrimidine 31 (200 mg, 1.25 mmol) and phosphonate 20b

(1.06 g, 2.6 mmol). Thick oil, 534 mg (68%). ¹H NMR (DMSO-d₆):

(1.5 g, 81%). ¹H NMR (DMSO-d₆):

d

¼ 8.38 (d, J(H-2,H-5) ¼ 0.9, 1H,

H-2), 6.05 (d, J(H-5,H-2) ¼ 0.9, 1H, H-5), 4.76 (dh, J(CH,CH₃) ¼ 6.2,

J(CH,P) ¼ 7.7, 4H, CHipr.), 4.50 (m, 4H, H-1⁰), 3.93 (m, 4H, H-2⁰), 3.83

(d, J(H,C,P) ¼ 8.2, 4H, POCH₂), 1.33 (m, 24H, CH₃ipr.) ppm. ¹³C NMR

d

¼ 6.69 (s, 2H, NH₂), 6.14 (s, 1H, H-5), 4.59 (m, 4H, CHipr.), 3.78 (d,

J(P,CH) ¼ 9.2, 4H, PCH₂), 3.74 (m, 2H, H-2⁰), 3.26 (dd, J(1⁰⁰a,2⁰) ¼ 5.5,

J_gem¼ 13.6, 2H, H-1⁰⁰a), 3.20 (dd, J(1⁰b,2⁰) ¼ 5.8, J_gem¼ 13.6, 2H, H-

1⁰b), 1.24 (d, 12H), 1.23 (d, 6H) and 1.16 (d, J(CH₃,CH) ¼ 6.2, 6H,

(DMSO-d₆):

d

¼ 170.51 (C-4, C-6), 157.21 (C-2), 91.15 (C-5), 71.12 (d,

J(2⁰,P) ¼ 10.8, C-2⁰), 71.09 (d, J(CH,P) ¼ 6.7, CHipr.), 65.99 (d,

J(C,P) ¼ 167.3, POCH₂), 65.52 (C-1⁰), 24.05 (d, J(CH₃,P) ¼ 3.7) and

23.93 (d, J(CH₃,P) ¼ 4.6, CH₃ipr.) ppm. MS (ESI): m/z (%) ¼ 579.2

(100) [MNa]^þ. Anal. C₂₂H₄₂N₂O₁₀P₂(C, H, N, P).

CH₃ipr.) ppm. ¹³C NMR (DMSO-d₆):

d

¼ 167.63 (2C, C-4,6), 161.64

(C-2), 103.30 (C-5), 76.19 (d, J(P,C) ¼ 12.7, 2C, C-2⁰), 70.30 (d,

J(P,C) ¼ 6.3, 4C, CHipr.), 62.84 (d, J(P,C) ¼ 165.0, 2C, PCH₂), 33.21

(2C, C-1⁰), 24.02 (d, J(P,C) ¼ 3.6, 4C) and 23.88 (d, J(P,C) ¼ 4.6, 4C,

CH₃ipr.), 18.73 (2C, C-3⁰) ppm. MS (ESI): m/z (%) ¼ 654.2 (100)

[MNa]^þ.

The intermediate was deprotected by bromotrimethylsilane

(GP1) to give 30 (350 mg, 48%), freeze dried, white hydroscopic

foam. ¹H NMR (DMSO-d₆):

d

¼ 8.44 (d, J(H-2,H-5) ¼ 0.9, 1H, H-2),

6.28 (d, J(H-5,H-2) ¼ 0.9, 1H, H-5), 4.39 (m, 4H, H-1⁰), 3.80 (m, 4H,

Phosphonic acid 33b, yield (280 mg, 73%), white foam. ¹H NMR

H-2⁰), 3.57 (d, J(H,C,P) ¼ 8.7, 4H, OCH₂P) ppm. ¹³C NMR (DMSO-d₆):

(D₂O):

d

¼ 6.81 (s, 1H, H-5), 3.94 (m, 2H, H-2⁰), 3.77 (dd, 2H) and

d

¼ 170.70 (C-4, C-6), 157.79 (C-2), 90.36 (C-5), 70.46 (d,

3.65 (dd, J(P,CH) ¼ 9.3, J_gem¼ 13.2, 2H, PCH₂), 3.38 (dd, 2H) and 3.35

J(2⁰,P) ¼ 11.0, C-2⁰), 67.10 (d, J(C,P) ¼ 160.2, PCH₂), 66.01 (C-1⁰) ppm.

(dd, J(1⁰,2⁰) ¼ 5.5, J_gem¼ 13.6, 2H, H-1⁰⁰), 1.29 (d, J(3⁰,2⁰⁰) ¼ 6.2, 6H,

´

2422

P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

25

H-3⁰) ppm. MS (ESI): m/z (%) ¼ 464.0 (100) [MH]^þ. [

a

]

¼ þ48.3

(400 mg, 82%) as an oil. ¹H NMR (DMSO-d₆):

d

¼ 6.70 (br s, 2H, NH₂),

D

(c 0.357, H₂O). Anal. C₁₂H₂₃N₃O₈P₂S₂.H₂O (C, H, N, P, S).

6.41 (s, 1H, H-5), 4.59 (m, 4H, CHipr.), 3.775 (d, 2H) and 3.77 (d,

J(P,CH) ¼ 8.4, 2H, PCH₂), 3.76 (m, 1H, H-2⁰⁰), 3.71 (t, J(2⁰,1⁰) ¼ 5.4, 2H,

H-2⁰), 3.28 (dd, J(1⁰⁰a,2⁰⁰) ¼ 6.1, J_gem¼ 13.6, 1H, H-1⁰⁰a), 3.26 (t,

J(1⁰,2⁰) ¼ 5.4, 2H, H-1⁰), 3.20 (dd, J(1⁰⁰b,2⁰⁰) ¼ 5.8, J_gem¼ 13.6, 1H, H-

1⁰⁰b),1.245 (d,12H) and 1.23 (d, J(CH₃,CH) ¼ 6.2,12H, CH₃ipr.),1.18 (d,

4.29.4. 2-Amino-4,6-(2R,2⁰R)-bis{[2-(diisopropoxyphosphoryl-

methoxy)propyl]sulfanyl}pyrimidine (32c) and 2-amino-4,6-

(2R,2⁰R)-bis{[2-(phosphonomethoxy)propyl]sulfanyl}pyrimidine

(33c)

Prepared by the same procedure as compounds 32a and 33a.

From pyrimidine 31 (500 mg, 3.1 mmol) and phosphonate 20c

(2.65 g, 6.5 mmol). Thick oil, 1.48 g (75%). NMR spectra identical

with compound 32b. MS (ESI): m/z (%) ¼ 654.0 (100) [MNa]^þ.

Phosphonic acid 33c, yield 702 mg (71%), white foam. NMR

J(CH₃,CH) ¼ 6.2, 3H, H-3⁰⁰) ppm. ¹³C NMR (DMSO-d₆):

¼ 167.71 and

d

167.47 (C-4,6),161.70 (C-2),103.83 (C-5), 75.70 (d, J(P,C) ¼ 11.7, C-2⁰⁰),

71.08 (d, 4C) and 70.64 (d, 4C, J(P,C) ¼ 6.3, CHipr.), 70.03 (d,

J(P,C) ¼ 10.7, C-2⁰), 64.91 (d) and 62,05 (d, J(P,C) ¼ 164.5, PCH₂), 32.10

(C-1⁰⁰), 27.60 (C-1⁰), 24.66 (d, 4C) and 24.18 (d, 4C, J(P,C) ¼ 3.9), 23.59

(d, 4C) and 23.32 (d, 4C, J(P,C) ¼ 4.4, CH₃), 17.20 (C-3⁰⁰) ppm. MS

(FAB): m/z (%) ¼ 618.2 (100) [MH]^þ. HR MS (FAB) calcd. for

C₂₃H₄₆N₃O₈P₂S₂[MH]^þ618.2203, found 618.2205.

spectra identical with compound 33b. MS (ESI): m/z (%) ¼ 464.0

25

(100) [MH]^þ. [

a

]

¼ ꢂ40.2 (c 0.589, H₂O). Anal. C₁₂H₂₃N₃O₈P₂S₂.

D

H₂O (C, H, N, P, S).

Diisopropylester 35a was deprotected by GP1 to give 36a

(100 mg, 53%) as a white foam. ¹H NMR (D₂O):

d

¼ 6.79 (s, 1H, H-5),

4.29.5. 2-Amino-6-{[2-(diisopropoxyphosphorylmethoxy)ethyl]-

sulfanyl}-4-sulfanylpyrimidine (34a)

3.94 (m, 1H, H-2⁰⁰), 3.87 (t, J(2⁰,1⁰) ¼ 6.1, 2H, H-2⁰), 3.74 (dd,

J(P,CH) ¼ 9.2, J_gem¼ 13.3,1H) and 3.70 (d, J(P,CH) ¼ 8.9, 2H) and 3.66

(dd, J(P,CH) ¼ 9.4, J_gem¼ 13.3, 1H, PCH₂), 3.38 (t, J(1⁰,2⁰) ¼ 6.1, 2H, H-

1⁰), 3.36 (dd, J(1⁰⁰a,2⁰⁰) ¼ 5.4, J_gem¼ 14.4, 1H, H-1⁰⁰a), 3.32 (dd,

Tothesolutionofpyrimidine 31 (3 g,18.84 mmol)andNaH(0.76 g,

60% in parafﬁn oil, 19 mmol) in DMF (70 mL) was added dropwise

phosphonate 20a (5 g,19 mmol). The resulting mixture was stirred at

r.t. for 3 days and evaporated in vacuo. The residue in CHCl₃(200 mL)

was washed with water (3 ꢀ 100 mL), dried over MgSO₄and evapo-

rated under reduced pressure. The residue was separated by ﬂash

chromatography (CHCl₃/MeOH 0–5%) to give 32a (3.18 g, 28%) and

J(1⁰⁰b,2⁰⁰) ¼ 6.1, J_gem¼ 14.4, 1H, H-1⁰⁰b), 1.29 (d, J(3⁰⁰,2⁰⁰) ¼ 6.3, 3H, H-

25

3⁰⁰). MS (ESI): m/z (%) ¼ 450 (100) [MH]^þ. [

a

]

¼ þ32.5 (c 0.142,

D

H₂O). Anal. C₁₁H₂₁N₃O₈P₂S₂.H₂O (C, H, N, P, S).

4.29.8. 2-Amino-4-{[2-(diisopropoxyphosphorylmethoxy)-

ethyl]sulfanyl}-6-(2R)-{[2-(diisopropoxyphosphorylmethoxy)-

propyl]sulfanyl}pyrimidine (35b) and 2-amino-

34a (2.92 g, 40%)asa paleyellowoil.¹H NMR (DMSO-d₆):

d

¼ 11.90 (br

s, 1H, SH), 7.00 (br s, 2H, NH₂), 6.33 (s, 1H, H-5), 4.59 (m, 2H, CHipr.),

3.78 (d, J(P,CH) ¼ 8.3, 2H, PCH₂), 3.71 and 3.22 (2 ꢀ t, J(1⁰,2⁰) ¼ 6.3,

2 ꢀ 2H, H-1⁰, H-2⁰⁰),1.24 (d, 6H) and 1.23 (d, J(CH₃,CH) ¼ 6.2, 6H, CH₃)

4-{[2-(phosphonomethoxy)ethyl]sulfanyl}-6-(2R)-

{[2-(phosphonomethoxy)propyl]sulfanyl}-pyrimidine (36b)

Prepared by the same procedure as compounds 35a and 36a

from pyrimidine 34a and phosphonate 20c.

ppm.¹³C NMR (DMSO-d₆):

d

¼ 177.97 (C-4),167.60 (C-2),154.04 (C-6),

109.86 (C-5), 70.24 (d, J(P,C) ¼ 12.2, C-2⁰⁰), 70.36 (d, 2C, J(P,C) ¼ 6.3,

CHipr.), 64.83 (d, J(P,C) ¼ 164.1, PCH₂), 28.33 (C-1⁰), 23.98 (d, 2C,

J(P,C) ¼ 3.9) and 23.91 (d, 2C, J(P,C) ¼ 4.4, CH₃) ppm. MS (FAB): m/z

(%) ¼ 382 (100) [MH]^þ. Anal. C₁₃H₂₄N₃O₄PS₂(C, H, N, P, S).

35b: thick oil, yield 390 mg (80%). NMR spectra identical with

compound 35a. MS (FAB): m/z (%) ¼ 618.0 (100) [MH]^þ. HR MS

(FAB) calcd. for C₂₃H₄₆N₃O₈P₂S₂[MH]^þ618.2203, found 618.2206.

36b: white foam, yield 95 mg (50%). NMR spectra identical with

4.29.6. 2-Amino-6-(2S)-{[2-(diisopropoxyphosphorylmethoxy)-

propyl]sulfanyl}-4-sulfanylpyrimidine (34b)

compound 36a. MS(ESI):m/z(%) ¼ 450(100)[MH]^þ;472(50)[MNa]^þ.

25

[a]

¼ ꢂ19.3 (c 0.216, H₂O). Anal. C₁₁H₂₁N₃O₈P₂S₂.H₂O (C, H, N, P, S).

D

To the solution of pyrimidine 31 (1 g, 6.3 mmol) and NaH

(0.252 g, 60% in parafﬁn oil, 6.3 mmol) in DMF (25 mL) was added

dropwise phosphonate 20b (2.57 g, 6.3 mmol) at 0 ^ꢁC. The

resulting mixture was stirred at 60 ^ꢁC for 8 h and evaporated in

vacuo. The residue was puriﬁed by ﬂash chromatography to give

32b (750 mg, 19%) and 34b (780 mg, 31%) as a thick oil. ¹H NMR

4.29.9. 2-Amino-4,6-(2R,2⁰S)-bis{[2-(diisopropoxyphosphoryl-

methoxy)propyl]sulfanyl}pyrimidine (35c) and 2-amino-4-(2R,2⁰S)-

bis{[2-(phosphonomethoxy)propyl]sulfanyl}pyrimidine (36c)

Prepared by the same procedure as compounds 35a and 36a

from pyrimidine 34b (400 mg, 1.01 mmol) and phosphonate 20c

(0.45 g, 1.1 mmol).

(DMSO-d₆):

d

¼ 11.88 (br s, 1H, SH), 7.00 (br s, 2H, NH₂), 6.33 (s,

1H, H-5), 4.59 (m, 2H, CHipr.), 3.79 (dd, 1H) and 3.75 (dd,

J_gem¼ 13.8, J(P,CH) ¼ 9.2, 1H, PCH₂), 3.74 (m, 1H, H-2⁰), 3.22 (dd,

J(1⁰⁰a,2⁰) ¼ 5.4, J_gem¼ 13.6, 1H, H-1⁰a), 3.17 (dd, J(1⁰b,2⁰) ¼ 5.8,

J_gem¼ 13.6, 1H, H-1⁰b), 1.24 (d, 6H), 1.235 (d, 6H) and 1.18 (d, 3H,

35c: thick oil, yield 440 mg (71%). NMR spectra identical with

compound 32b. MS (ESI): m/z (%) ¼ 654.0 (100) [MNa]^þ.

36c: white foam, yield 180 mg (61%). ¹H NMR (D₂O):

d

¼ 6.86 (s,

1H, H-5), 3.95 (m, 2H, H-2⁰), 3.79 (dd, J_gem¼ 13.2, J(CH₂,P) ¼ 9.3, 2H)

and 3.68 (dd, J_gem¼ 13.2, J(CH₂,P) ¼ 9.5, 2H, PCH₂), 3.43 (dd,

J_gem¼ 14.3, J(1,2) ¼ 4.4, 2H) and 3.33 (dd, J_gem¼ 14.3, J(1,2) ¼ 6.2,

J(CH₃,CH) ¼ 6.2, CH₃) ppm. ¹³C NMR (DMSO-d₆):

d

¼ 178.56 (C-4),

167.86 (C-2), 153.95 (C-6), 109.94 (C-5), 75.97 (d, J(P,C) ¼ 12.8, C-

2⁰), 70.37 and 70.34 (d, J(P,C) ¼ 6.3, CHipr.), 62.78 (d, J(P,C) ¼ 165.6,

PCH₂), 34.35 (d, J(P,C) ¼ 3.9, C-1⁰), 24.04 (d, 2C, J(P,C) ¼ 3.0) and

23.90 (2C, J(P,C) ¼ 4.6, CH₃), 18.17 (C-3⁰) ppm. MS (ESI): m/z

(%) ¼ 418 (100) [MNa]^þ. HR MS (FAB) calcd. for C₁₄H₂₇N₃O₄PS₂

[MH]^þ396.1180, found 396.1176.

2H, H-1⁰), 1.30 (d, J(CH₃,2⁰) ¼ 6.7, 6H, H-3⁰) ppm. MS (ESI): m/z

25

(%) ¼ 464.0 (100) [MH]^þ.

[

a

]

D

¼ þ0.3 (c 0.358, H₂O). Anal.

C₁₂H₂₃N₃O₈P₂S₂.H₂O (C, H, N, P, S).

4.30. Alkoxyalkyl esters of bisphosphonates

4.29.7. 2-Amino-4-{[2-(diisopropoxyphosphorylmethoxy)-

ethyl]sulfanyl}-6-(2S)-{[2-(diisopropoxyphosphorylmethoxy)-

propyl]sulfanyl}pyrimidine (35a) and 2-amino-4-{[2-(phosphono-

methoxy)ethyl]sulfanyl}-6-(2S)-{[2-(phosphonomethoxy)propyl]-

sulfanyl}pyrimidine (36a)

Monoderivative 34a (300 mg, 0.79 mmol), NaH (0.035 g, 60% in

parafﬁn oil, 0.87 mmol) and phosphonate 20b (0.35 g, 0.86 mmol) in

DMF (10 mL) were stirred at 60 ^ꢁC for 4 h and evaporated in vacuo.

The residue was treated with hot chloroform and ﬁltered, and the

ﬁltrate was evaporated in vacuo. Flash chromatography afforded 35a

4.30.1. 2-Amino-4,6-bis(2-hydroxyethoxy)pyrimidine (37)

A solution of t-BuOK (13.5 g, 120 mmol) in ethyleneglycol

(50 mL) was heated at 80 ^ꢁC for 30 min and dichloropyrimidine 10

(5 g, 30 mmol) was added. The resulting mixture was stirred at

100 ^ꢁC for 1 h, cooled to r.t. and neutralized by addition of Dowex

50 ꢀ 8; the resulting mixture was diluted with water (100 mL),

applied onto a column of Dowex 50 ꢀ 8 and washed with water (1 L).

Column was then eluted with 2.5% aq. ammonia, UV absorbing

fraction was collected and evaporated under reduced pressure. The

crude product was recrystallized from water to give a white solid

´

P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

2423

(4.65 g, 71%), m.p. 158 ^ꢁC. ¹H NMR (DMSO-d₆):

d

¼ 6.48 (br s, 2H,

(C-1⁰), 63.11 (br, C-4⁰), 59.77 (C-2⁰⁰), 31.57 (CH₃CH₂CH₂), 29.55,

29.31, 29.21, 28.98, 25.94 (alif.), 22.37 (CH₃CH₂), 14.24 (CH₃), 7.09

(5-CH₃) ppm. MS (ESI): m/z (%) ¼ 590.3 (100) [M ꢂ Na]^ꢂ. Anal.

C₂₈H₅₃N₃NaO₈P (C, H, N, P).

NH₂), 5.32 (s, 1H, H-5), 4.82 (t, J(OH,2⁰) ¼ 5.4, 2H, OH), 4.17 (t,

J(1⁰,2⁰) ¼ 5.1, 4H, H-1⁰), 3.63 (q, J(2⁰,1⁰) ¼ J(2⁰,OH) ¼ 5.2, 4H, H-2⁰)

ppm. ¹³C NMR (DMSO-d₆):

d

¼ 171.57 (C-4, C-6), 162.87 (C-2), 76.69

(C-5), 67.53 (C-1⁰), 59.61 (C-2⁰) ppm. MS (ESI): m/z (%) ¼ 216 (100)

[MH]^þ. Anal. C₈H₁₃N₃O₄(C, H, N).

4.30.8. Bis[2-(hexadecyloxy)ethyl] 2-amino-5-methyl-4,6-bis[2-

(phosphonomethoxy)ethoxy]pyrimidine, sodium salt (40c)

GP7, white solid (106 mg, 21%), m.p. 142 ^ꢁC. MS (ESI): m/z

(%) ¼ 952.5 (48) [M ꢂ (2 ꢀ Na) þ H]^ꢂ. Anal. C₄₇H₉₁N₃Na₂O₁₂P₂(C, H,

N, P).

4.30.2. Hexadecyloxyethyl toluenesulfonyloxymethylphosphonate,

sodium salt (38)

Prepared by previously described procedure [17a]; yield 47%. ¹H

NMR (CDCl₃):

d

¼ 7.77 (br d, J(CH,CH) ¼ 5.81, 2H, CHarom.), 7.30 (br

d, J(CH,CH) ¼ 6.0, 2H, CHarom.), 4.07 (br, 2H, PCH₂), 3.97 (br, 2H,

OCH₂), 3.45 (br, 2H, OCH₂), 3.34 (br, 2H, OCH₂), 2.38 (s, 3H,

CH₃arom.), 1.48 (br, 2H, OCH₂CH₂CH₂), 1.25 (br s, 26H, 13 ꢀ CH₂),

0.87 (t, J(CH₃,CH₂) ¼ 6.83, CH₃CH₂) ppm. MS (ESI): m/z (%) ¼ 579

(100) [MNa]^þ. Anal. C₂₆H₄₆NaO₇PS (C, H, N, P, S).

4.30.9. 2-Amino-5-bromo-4,6-bis(2-hydroxyethoxy)pyrimidine (41)

Pyrimidine 37 (1 g, 4.65 mmol) in DMF (15 mL) was treated with

bromine (0.7 M solution in CCl₄, 10 mL) and the mixture was stirred

at r.t. overnight. The mixture was evaporated in vacuo and codis-

tilled with EtOH (3 ꢀ 100 mL). The crude product was recrystallized

from EtOH to give pale yellow needles (530 mg, 39%), m.p. 178 ^ꢁC.

4.30.3. 2-(Hexadecyloxy)ethyl 2-amino-4-(2-hydroxyethoxy)-6-

[2-(phosphonomethoxy)ethoxy]pyrimidine, sodium salt (39a)

GP7, white solid (47 mg, 31%), m.p. 129 ^ꢁC. ¹H NMR (DMSO-d₆):

¹H NMR (DMSO-d₆):

d

¼ 6.71 (br s, 2H, NH₂), 4.26 (t, J(1⁰,2⁰) ¼ 5.3,

4H, H-1⁰); 3.67 (t, J(2⁰,1⁰) ¼ 5.3, 4H, H-2⁰) ppm. ¹³C NMR (DMSO-d₆):

d

¼ 166.42 (C-4, C-6), 160.88 (C-2), 73.60 (C-5), 68.46 (C-1⁰), 59.46

d

¼ 6.51 (br s, 2H, NH₂), 5.31 (s, 1H, H-5), 4.24 (m, 2H, H-1⁰), 4.15 (t,

(C-2⁰) ppm. MS (ESI): m/z (%) ¼ 294 (18) [MH^þ], 276 (100)

[M ꢂ H₂O þ H]^þ. Anal. C₈H₁₂BrN₃O₄(C, H, Br, N).

J(1⁰⁰,2⁰⁰) ¼ 5.2, 2H, H-1⁰⁰), 3.75 (m, 2H, H-4⁰), 3.68 (m, 2H, H-2⁰), 3.62

(m, 2H, H-2⁰⁰), 3.39 (m, 6H, H-3⁰, 5⁰, 6⁰), 1.45 (m, 2H, H-7⁰), 1.22 (m,

26H, alif.), 0.84 (t, J(CH₃,CH₂) ¼ 6.9, 3H, CH₃) ppm. ¹³C NMR (DMSO-

4.31. Capillary electrophoresis

d₆):

d

¼ 171.52 and 171.37 (C-4, C-6), 162.83 (C-2), 78.62 (C-5), 70.79

(d, J(5⁰,P) ¼ 5.6, C-5⁰), 70.46 (C-6⁰⁰), 69.99 (d, J(2⁰,P) ¼ 8.9, C-2⁰), 67.47

(C-1⁰⁰), 64.98 (C-1⁰), 62.88 (d, J(4⁰,P) ¼ 5.1, C-4⁰), 59.55 (C-2⁰⁰), 31.51

(CH₃CH₂CH₂), 29.52, 29.25, 29.15, 28.92 and 25.90 (alif.), 22.31

(CH₃CH₂), 14.18 (CH₃) ppm. MS (ESI): m/z (%) ¼ 598.4 (80) [M ꢂ H]^ꢂ.

Anal. C₂₇H₅₁N₃NaO₈P (C, H, N, P).

Analyses were performed in a commercial P/ACE MDQ capillary

electrophoresis (CE) apparatus (Beckman Coulter, Fullerton, CA, USA),

equipped with an internally non-coated fused silica capillary with

outer polyimide coating, total length 390 mm, effective length (from

injection end to the detector) 288 mm, I.D./O.D. 50/375 mm (Poly-

micro Technologies, Phoenix, AR, USA). The analytes were monitored

by UV–vis absorption spectrophotometric photodiode array detector

(190–600 nm) at twowavelengths, 206 and 254 nm, respectively. The

temperature of capillary liquid coolant was set at 20 ^ꢁC.

The samples were injected hydrodynamically, by pressure

13.8 mbar for 10 s. The analytes were dissolved in deionized water,

the concentration of enantiomers in their individual CZE analyses

was 0.1 mM, whereas in enantiomeric mixtures the concentration

of R-isomer was 0.2 mM and of S-isomer 0.1 mM, in order to

distinguish their migration order. Separation voltage was 15 kV.

The analyses were performed both in non-chiral and chiral

background electrolytes (BGEs) of the following composition:

4.30.4. Bis[2-(hexadecyloxy)ethyl] 2-amino-4,6-bis-

[2-(phosphonomethoxy)ethoxy]pyrimidine, sodium salt (40a)

GP7, white solid (59 mg, 24%), m.p. 184 ^ꢁC. MS (ESI): m/z (%) ¼

962.5 (88) [M ꢂ Na þ H]^þ. Anal. C₄₆H₈₉N₃Na₂O₁₂P₂(C, H, N, P).

4.30.5. 2-(Hexadecyloxy)ethyl 2-amino-5-bromo-4-(2-

hydroxyethoxy)-6-[2-(phosphonomethoxy)ethoxy]pyrimidine,

sodium salt (39b)

GP7, white solid (44 mg, 26%), m.p. 83 ^ꢁC. ¹H NMR (DMSO-d₆):

d

¼ 6.72 (br s, 2H, NH₂), 4.86 (br s, 1H, OH), 4.34 (m, 2H, H-1⁰), 4.25

(m, 2H, H-1⁰⁰), 3.61–3.80 (m, 6H, H-2⁰, 4⁰, 2⁰⁰), 3.38 (m, 6H, 2 ꢀ OCH₂,

PCH₂),1.44 (m, 2H, H-7⁰),1.22 (m, 26H, alif.), 0.84 (t, J(CH₃,CH₂) ¼ 6.8,

3H, CH₃) ppm.¹³C NMR (DMSO-d₆):

d

¼ 166.40 and 166.28 (C-4, C-6),

Non-chiral BGEs: 25–50 mM borax, adjusted by NaOH to pH

10.0–10.5.

Chiral BGEs: 25–50 mM borax, adjusted by NaOH to pH 10.0–

160.87 (C-2), 73.57 (C-5), 70.78 (d, J(5⁰,P) ¼ 5.7, C-5⁰), 70.48 (C-6⁰),

69.88 (C-2⁰), 68.41 (C-1⁰⁰), 66.20 (C-1⁰), 63.02 (C-4⁰), 59.42 (C-2⁰⁰),

31.52 (CH₃CH₂CH₂), 29.54, 29.22, 29.18, 28.94, 25.91 (alif.), 22.33

(CH₃CH₂),14.19 (CH₃) ppm. MS (ESI): m/z (%) ¼ 654 (100) [M ꢂ Na]^ꢂ.

Anal. C₂₇H₅₀BrN₃NaO₈P (C, H, N, P).

10.5 þ chiral selector

b-cyclodextrin (5–20 mg/mL).

4.32. Biological activity assays

4.30.6. Bis[2-(hexadecyloxy)ethyl] 2-amino-5-bromo-4,6-bis[2-

(phosphonomethoxy)ethoxy]pyrimidine, sodium salt (40b)

GP7, white solid (61 mg, 23%), m.p. 160 ^ꢁC dec. MS (ESI): m/z

(%) ¼ 1038 (26) [M ꢂ Na]^ꢂ, 1016 (63) [M-(2 ꢀ Na) þ H]^ꢂ. Anal.

C₄₆H₈₈BrN₃Na₂O₁₂P₂(C, H, N, P).

In vitro cytostatic activity tests (cell growth inhibition) were

performed with cultures of murine leukemia L1210 cells (ATCC CCL

219), human promyelocytic leukemia HL60 cells (ATCC CCL 240),

human cervix carcinoma HeLa S3 cells (ATCC CCL 2.2) and the

human T lymphoblastoid CCRF-CEM cell line (ATCC CCL 119) [19].

The methodology of the antiviral activity assays followed

previously described procedures [20,21].

4.30.7. 2-(Hexadecyloxy)ethyl 2-amino-4-(2-hydroxyethoxy)-5-

methyl-6-[2-(phosphonomethoxy)ethoxy]pyrimidine, sodium

salt (39c)

Analyses indicated by the symbols of the elements or functions

were within ꢃ0.4% of the theoretical values.

GP7, white solid (122 mg, 40%), m.p. 95 ^ꢁC. ¹H NMR (DMSO-d₆):

d

¼ 6.20 (br s, 2H, NH₂), 4.85 (br,1H, OH), 4.28 (m, 2H, H-1⁰), 4.19 (m,

2H, H-1⁰⁰), 3.62–3.81 (m, 6H, H-2⁰, 4⁰, 2⁰⁰), 3.39 (m, 6H, H-3⁰, 5⁰, 6⁰),

Acknowledgments

1.77 (s, 3H, 5-CH₃), 1.43 (m, 2H, H-7⁰), 1.22 (m, 26H, alif.), 0.84 (t,

J(CH₃,CH₂) ¼ 7.0, 3H, CH₃) ppm. ¹³C NMR (DMSO-d₆):

d

¼ 168.48

This study was performed as a part of research project

OZ40550506 of the Institute of Organic Chemistry and Biochem-

istry, v.v.i. This study was supported by Centre of new antivirals and

and 168.32 (C-4, C-6), 160.37 (C-2), 86.86 (C-5), 70.73 (d,

J(5⁰,P) ¼ 5.5, C-5⁰), 70.52 (C-6⁰), 70.41 (C-2⁰), 67.49 (C-1⁰⁰), 65.15

´ ´

P. Dolakova et al. / European Journal of Medicinal Chemistry 44 (2009) 2408–2424

2424

(b) J. Balzarini, C. Pannecouque, E. De Clercq, S. Aquaro, C.F. Perno, H. Egberink,

antineoplastics 1M0508 by the Ministry of Education, Youth and

Sports of the Czech Republic, by Gilead Sciences and IOCB Research

Centre and by the NIH grant 1UC1AI062540-01. The authors’ thanks

are due to Prof. E. De Clercq and his group in the Rega Institute,

Katholic University Leuven (Belgium) for the evaluation of antiviral

activity; and to Dr I. Votruba of the Institute of Organic Chemistry

and Biochemistry for the evaluation of cytostatic activity.

ꢀ

A. Holy, Antimicrob. Agents Chemother. 46 (2002) 2185–2193;

ꢀ

(c) C. Ying, A. Holy, D. Hockova´, Z. Havlas, E. De Clercq, J. Neyts, Antimicrob.

Agents Chemother. 49 (2005) 1177–1180;

ꢀ

(d) D. Hockova´, M. Masojı´dkova´, A. Holy, Collect. Czech. Chem. Commun. 70

(2005) 247–258.

ꢀ

[9] (a) D. Hockova, A. Holy, M. Masojıdkov

a´, G. Andrei, R. Snoeck, E. De Clercq,

´

J. Balzarini, J. Med. Chem. 46 (2003) 5064–5073;

ꢀ

´

(b) D. Hockova, A. Holy, M. Masojıdkova, G. Andrei, R. Snoeck, E. De Clercq,

J. Balzarini, Bioorg. Med. Chem. 12 (2004) 3197–3202;

(c) J. Balzarini, D. Schols, K. Van Laethem, E. De Clercq, D. Hockova´,

Appendix. Supplementary material

ꢀ

M. Masojı´dkova´, A. Holy, J. Antimicrob. Chemother. 59 (2007) 80–86.

ꢀ

[10] (a) S. Vrbovska´, A. Holy, R. Pohl, M. Masojı´dkova´, Collect. Czech. Chem.

Commun. 71 (2006) 543–566;

Supplementary data associated with this article can be found in

the online version, at doi:10.1016/j.ejmech.2008.09.031.

ꢀ

ˇ´

(b) S. Vrbkova´, M. Dracınsky, A. Holy, Collect. Czech. Chem. Commun. 72

(2007) 965–983;

ꢀ

ˇ´

(c) S. Vrbkova´, M. Dracınsky, A. Holy, Tetrahedron 63 (2007) 11391–11398;

ꢀ

ˇ´

(d) S. Vrbkova´, M. Dracınsky, A. Holy, Tetrahedron: Asymmetry 18 (2007)

2233–2247.

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Article Doi

Acyclic nucleoside bisphosphonates: Synthesis and properties of chiral 2-amino-4,6-bis[(phosphonomethoxy)alkoxy]pyrimidines

DOI: 10.1016/j.ejmech.2008.09.031

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