Bifunctional acyclic nucleoside phosphonates: 2. Symmetrical 2-{[bis(phosphono)methoxy]methyl}ethyl derivatives of purines and pyrimidines

Article abstract of DOI:10.1135/cccc20070965

Novel bisphosphonate alkylating agent, tetraisopropyl (2-[(mesyloxy)methyl] propane-1,3-diyl]bis(oxymethylene)bisphosphonate 19, was synthesized from diethyl 2,2-bis-(hydroxymethyl)malonate. Decarbethoxylation of the diethyl 2,2-dimethyl-1,3-dioxane-5,5-dicarboxylate was followed by chloromethylation of 2-[(benzyloxy)methyl]propane-1,3-diol and Arbuzov reaction with triisopropyl phosphite. Bisphosphonate building block 19 was used in the alkylation of various nucleobases (2-amino-6-chloropurine, adenine, 2-amino-6-(cyclopropyl) aminopurine, cytosine, uracil and 4-methoxy-5-methylpyrimidin-2(1H)-one). N ⁹-Substituted purines and N¹-substituted pyrimidines were converted to appropriate free bisphosphonic acids. No antiviral or cytostatic activity was detected.

Full text of DOI:10.1135/cccc20070965

Bifunctional Acyclic Nucleoside Phosphonates
965
BIFUNCTIONAL ACYCLIC NUCLEOSIDE PHOSPHONATES:
2. SYMMETRICAL 2-{[BIS(PHOSPHONO)METHOXY]METHYL}ETHYL
DERIVATIVES OF PURINES AND PYRIMIDINES
1,
2
3
Silvie VRBKOVÁ *, Martin DRAČÍNSKÝ and Antonín HOLÝ
Centre for New Antivirals and Antineoplastics, Gilead Sciences & IOCB Research Centre,
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
1
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; e-mail: vrbkova@uochb.cas.cz,
2
3
dracinsky@uochb.cas.cz, holy@uochb.cas.cz
Received March 12, 2007
Accepted April 4, 2007
Novel bisph osph on ate alkylatin g agen t, tetraisopropyl {2-[(m esyloxy)m eth yl]propan e-
1,3-d iyl}bis(o xym et h ylen e)bisp h o sp h o n at e 1 9 , was syn t h esized fro m d iet h yl 2,2-bis-
(h ydroxym eth yl)m alon ate. Decarbeth oxylation of th e dieth yl 2,2-dim eth yl-1,3-dioxan e-
5,5-dicarboxylate was followed by ch lorom eth ylation of 2-[(ben zyloxy)m eth yl]propan e-
1,3-diol an d Arbuzov reaction with triisopropyl ph osph ite. Bisph osph on ate buildin g block
19 was used in th e alkylation of various n ucleobases (2-am in o-6-ch loropurin e, aden in e,
2-am in o-6-(cyclopropyl)am in opurin e, cytosin e, uracil an d 4-m eth oxy-5-m eth ylpyrim idin -
2(1H)-on e). N9-Substituted purin es an d N1-substituted pyrim idin es were con verted to appro-
priate free bisph osph on ic acids. No an tiviral or cytostatic activity was detected.
Keyw ord s: Acyclic n ucleoside ph osph on ates; ANPs; Acyclic n ucleotide an alogues;
Ph osph on om eth yl eth ers; Adefovir; Bisph osph on ates; Nucleobases.
An alogues of n ucleic acids com pon en ts belon g to an tim etabolite group.
Th eir ability to in terfere with th e m etabolic path ways of n ucleic acids syn -
th esis de n ovo m ay cause an tiviral, an tin eoplastic or oth er biological activ-
ity. First gen eration of n ucleic acid an alogues aim ed at th eir close structural
sim ilarity to n atural m etabolites1–3, h owever, led to th eir lim ited stability in
th e organ ism due to th eir easy degradation in th e m etabolic path ways.
Th e secon d gen eration of an tim etabolites, acyclic n ucleosides, h as over-
com e th is lim itation by replacin g th e sugar m oiety with aliph atic ch ain
boun d to th e N9 n itrogen of purin es (e.g., acyclovir4,5, gan ciclovir6,7, AHPA 8
or (S)-DHPA 9). Th ese com poun ds are in vivo ph osph orylated to th eir 5′-n u-
cleotide. However, th e direct application of such n ucleotides is n ot possible
due to th e lability of th e ph osph om on oester bon d in blood plasm a an d
durin g th e tran sm em bran e tran sport. Th erefore, th ere is a n eed for n ew
isopolar an d isosteric ph osph ate an alogues resistan t to en zym atic reaction s.
On e of th e solution s was replacem en t of ph osph ate by en zym atically stable
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 7, pp. 965–983
© 2007 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20070965

966
Vrbková, Dračínský, Holý:
ph osph on ate group. Very in terestin g in th is respect are ph osph on om eth yl
eth ers10.
Structure–activity relation sh ip in vestigation s in th e series of acyclic n u-
cleotide an alogues bearin g a m odified ph osph oric acid residue in th e side
ch ain h ave revealed so far several biologically active acyclic n ucleoside
ph osph on ates (ANPs). Som e of th em are curren tly used in clin ical
practice11,12. Cidofovir or (S)-HPMPC 1 (Vistide®, an injectable form of Cidofovir)
(Ch art 1) is an an tiviral m edication for th e treatm en t of cytom egalovirus
(CMV) retin itis in patien ts with AIDS 13. Ten ofovir or (R)-PMPA 2, an d its
prodrug Viread®, belon gs to n ucleotide reverse tran scriptase in h ibitors
(NtRTIs) wh ich block an en zym e crucial to viral production in HIV-in fected
people14. Adefovir or PMEA 3, an d its prodrug Hepsera®, is an orally-
adm in istered NtRTI used for treatm en t of h epatitis B 15
.
CHART 1
First gen eration of acyclic n ucleoside ph osph on ates
Recen tly, atten tion was turn ed to th e syn th esis of a n ew type of ANPs
origin atin g from 2-substituted 4-am in o-6-h ydroxypyrim idin es16. In th ese
in vestigation s,
a sign ifican t poten tial activity of 6-[2-(ph osph on o-
m eth oxy)eth oxy]pyrim idin e derivatives, com poun ds derived from
2,4-diam in opyrim idin e (4a, 4c, 4d )17 an d 2-am in o-4-h ydroxypyrim idin e
18a,18b
(4b)17, an d th eir C5-substituted con gen ers 5
was discovered (Ch art 2).
CHART 2
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Bifunctional Acyclic Nucleoside Phosphonates
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Am on g th e products isolated in th ese studies bisph osph on ates 6 an d 7
were also iden tified (Sch em e 1)16. Despite th e fact th at th ese com poun ds
con stitute a n ew class of possible an tiviral agen ts, th ey h ave n ot yet re-
ceived m uch atten tion . Th e aim of th is work was to explore th e poten tial
biological activity of such substan ces, wh ich could be also con sidered as an a-
logues of n ucleotide bisph osph ate an tagon ists of th e P2Y1 receptor19
.
SCHEME 1
RESULTS AND DISCUSSION
In th e previous paper20 of th is series we h ave described th e syn th esis of
sym m etrical 1,3-bis(ph osph on om eth oxy)propan -2-yl com poun ds 8, de-
rived from th e 2-(ph osph on om eth oxy)eth yl (PME) ch ain , wh ere an addi-
tion al (ph osph on om eth oxy)m eth yl group is attach ed at th e C1 position of
th e eth yl ch ain of th e paren t PME com poun d 9 (Ch art 3).
In th is paper we discuss th e syn th esis of an oth er sym m etrical acyclic
n ucleoside bisph osph on ate (ANbP) con gen er 10 with th e bisph osph on ate
ch ain exten ded by on e carbon atom (Ch art 3).
CHART 3
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968
Vrbková, Dračínský, Holý:
Th e strategy ch osen for th e syn th esis of ANbP 10, was based on
alkylation of an appropriate h eterocyclic base with reagen t 19 wh ich con -
tain s a good leavin g group on an alkyl ch ain bearin g an esterified
bisph osph on ate groupin g lin ked th rough an eth er bridge. Th is approach in
m ost cases does n ot require an y protection of th e n ucleobase an d can be
also applied to sen sitive h eterocyclic system s bearin g reactive substituen ts
(e.g., 6-ch loropurin es). In som e cases, regioselectivity of th e alkylation is
lim ited an d th e separation of th e form ed isom ers is in evitable.
Preparation of Bisphosphonate Alkylating Agent for Nucleobases (Scheme 2)
As sh own in Sch em e 2, th e syn th esis of th e correspon din g bisph osph on ate
alkylatin g agen t 19 started from com m ercially available dieth yl 2,2-bis-
(h ydroxym eth yl)m alon ate 11, wh ich was tran sform ed to an aceton ide
OH
COOEt
EtOOC
COOEt
EtOOC
O
COOEt
O
(i)
(ii)
(iii)
OH OH
O
O
O
O
11
12
13
14
(iv)
OBn
OBn
OBn
O
P
O
P
RO
RO
OR
(v)
O
O
(vi)
OR
O
O
OH OH
R = iPr
16
15
17
(vii)
OH
O
OMs
O
O
P
O
P
O
(viii)
RO
RO
OR
OR
RO
RO
OR
OR
P
O
O
O
P
18
19
(i) 2,2-dimethoxypropane, H2SO4, acetone; (ii) H2O, NaCl, DMSO, 190-195 °C (65%);
(iii) LiAlH4, ether, 0 °C (99%); (iv) BnBr, NaH, THF; (v) Dowex 50 (H+ form), 80%
methanol, reflux (80%); (vi) (CH2O)n, HCl (g), CaCl2, DCM, 0 °C, then P(OiPr)3, 120 °C
(85%); (vii) H2/Pd/C, conc. HCl, MeOH (95%); (viii) MsCl, Et3N, DCM (98%).
SCHEME 2
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Bifunctional Acyclic Nucleoside Phosphonates
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12 21. Th e m on o ester 13 was obtain ed by decarbeth oxylation of 12 in th e
presen ce of NaCl an d DMSO accordin g to th e procedure described by
Krapch o et al.22–24 Ester 13 was reduced to prim ary alcoh ol 14 with LAH in
eth er an d ben zylated with ben zyl brom ide to afford ben zyloxy derivative
15 wh ich was used in th e subsequen t reaction with out furth er purification .
Acid h ydrolysis of th e isopropyliden e protectin g group of 15 gave
2-[(ben zyloxy)m eth yl]propan e-1,3-diol 16. Ch lorom eth ylation of 16 with
paraform aldeh yde an d gaseous HCl in dich lorom eth an e followed by th e
Arbuzov reaction with triisopropyl ph osph ite gave th e bisph osph on ate 17.
Th e target bisph osph on ate alkylatin g agen t 19 was th en prepared by
h ydrogen olysis of 17 followed by m esylation of 18 with m eth an esulfon yl
ch loride in an h ydrous CH2Cl2 in th e presen ce of Et3N.
Alkylation of Nucleobases in General
Th e bisph osph on ate 19 was used in alkylation s of various n ucleobases –
2-am in o-6-ch loropurin e 20, aden in e 21, 6-(cyclopropyl)am in opurin e 22,
cytosin e 31, uracil 33, 4-m eth oxy-5-m eth ylpyrim idin -2(1H)-on e 35. All re-
action s were perform ed at 100 °C in th e presen ce of Cs2CO3 in DMF an d
CaCl2 protectin g tube.
Alkylation of Purines (Scheme 3)
Alkylation of purin es is depicted in Sch em e 3. Th e reaction of 2-am in o-
6-ch loropurin e 20 easily proceeded at N9 an d also at N7 position s to give
regioisom ers 20a an d 20b. Th ese isom ers were easily separated by silica gel
colum n ch rom atograph y. Th e in term ediate 20a was furth er con verted to
oth er base-m odified bisph osph on ates: th us, acid h ydrolysis led to th e gua-
n in e derivative 23; th e 2,6-diam in opurin e derivative 24 was prepared by
am m on olysis with m eth an olic am m on ia, an d replacem en t of ch lorin e with
th e cyclopropylam in o group in th e reaction with cyclopropylam in e in
dioxan e gave 2-am in o-6-(cyclopropyl)am in o derivative 25.
Th e alkylation of aden in e 21 an d 6-(cyclopropyl)am in opurin e 22 gave
exclusively th e N9-isom ers 21a an d 22a. In con trast to th e an alogous
alkylation of 20, essen tially n o N7-regioisom er form ation was observed in
alkylation of com poun ds 21 an d 22. In all th e discussed cases, NMR an aly-
sis was used to iden tify th e position of substitution of th e purin e rin g. All
sign als of h ydrogen an d carbon atom s were assign ed usin g 2D-1H,13C HSQC
an d 2D-1H,13C HMBC experim en ts. In th e case of N9-isom ers, proton s on
carbon atom n ext to th e purin e n itrogen h ave stron g crosspeaks in HMBC
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970
Vrbková, Dračínský, Holý:
SCHEME 3
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Bifunctional Acyclic Nucleoside Phosphonates
971
spectra with carbon s C-4 an d C-8 of th e purin e rin g. In N7-isom ers th ese
proton s correlate with C-5 an d C-8 atom s.
Subsequen t deprotection of com poun ds 21a, 22a, 23 with brom o-
trim eth ylsilan e in aceton itrile followed by h ydrolysis yielded th e corre-
spon din g free bisph osph on ic acids 26–28, wh ich were ultim ately purified
by ion exch an ge ch rom atograph y. Th e beh avior of com poun ds 24 an d 25
differed from th e above-m en tion ed substan ces. Th e deprotected an d h ydro-
lyzed products were applied on to Dowex 50X8 (H+ form ) as well. However,
th ey adsorbed on th e station ary ph ase an d h ad to be eluted with dilute am -
m on ia solution . Th erefore, th e obtain ed am m on ia salts were fin ally tran s-
form ed in to sodium salts of bisph osph on ic acids 29 an d 30 by Dowex 50X8
(Na+ form ) ch rom atograph y.
Alkylation of Cytosine (Scheme 4)
Th e reaction of cytosin e 31 with bisph osph on ate 19 un der th e above con -
dition s afforded a m ixture of N1-regioisom er (31a; yield 49%) an d
O2-regioisom er (31b; yield 15%). On ly th e N1 derivative was furth er con ver-
ted to th e correspon din g free bisph osph on ic acid 32 as sh own in Scheme 4.
O2- and N1-isom ers in the pyrim idine series could be readily distinguished by
NMR spectroscopy. The carbon atom chain linked to the oxygen shows δ 64 ppm;
when bonded to nitrogen its chemical shift was δ 48 ppm (cf. Experim en tal).
Furth erm ore, th e proton s bon ded to th is carbon h ave crosspeaks in
2D-1H,13C HMBC spectra to carbon atom s C-2 an d C-6 (in th e case of
N1-isom ers) or on ly to C-2 (in th e case of O2-isom er).
SCHEME 4
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972
Alkylation of Uracil (Scheme 5)
Vrbková, Dračínský, Holý:
Sch em e 5 sh ows th e course of alkylation of uracil 33 with com poun d 19
un der stan dard con dition s. Of th e two products form ed in th e reaction ,
N1-alkylated 33a an d N1,N3-bisalkylated 33b, th e desired 33a was th e m ajor
product. It was isolated in about 54% yield wh ile th e bisalkylate was ob-
tain ed in 15% yield. Subsequen t deprotection of th e tetraester 33a with
brom otrim eth ylsilan e in aceton itrile followed by h ydrolysis yielded th e
correspon din g free bisph osph on ic acid 34 wh ich was isolated from th e
deion ized product by ion exch an ge ch rom atograph y. Th e structure of all
products was iden tified usin g NMR spectroscopy. In 1H NMR spectra of th e
bisalkylated 33b th e NH sign al is m issin g. In 13C NMR, th ere are two sign als
of carbon s bon ded to th e pyrim idin e rin g with δ 48 an d 40 ppm for N1 an d
N3, respectively. Full assign m en t of all carbon an d h ydrogen atom s was
don e usin g 2D h eterocorrelated experim en ts.
SCHEME 5
Alkylation of 4-Methoxy-5-methylpyrimidin-2(1H)-one (Scheme 6)
An alogously to cytosin e, also th e alkylation of 4-m eth oxy-5-m eth yl-
pyrim idin -2(1H)-on e 35, th e precursor of th ym in e, provided two products
(N1-alkylate 35a an d O2-alkylate 35b) in th e ratio alm ost 1:1. Th e procedure
is depicted in Sch em e 6. Com poun d 35a was furth er h ydrolyzed un der acid
con dition s to provide th ym in e derivative 36. It is worth m en tion in g th at
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Bifunctional Acyclic Nucleoside Phosphonates
973
attem pts to prepare th ym in e derivative of 35b failed. No reaction was ob-
served in th e reaction with 80% CH3COOH an d Dowex 50 (H+ form ). De-
com position of th e ph osph on ate was ach ieved un der m ore drastic
con dition s (h eatin g with 2 M HCl). Th e subsequen t deprotection of tetraester
36 with brom otrim eth ylsilan e in aceton itrile followed by h ydrolysis
yielded th e correspon din g free ph osph on ic acid 37, wh ich was isolated
from th e deion ized product by ion exch an ge ch rom atograph y. Both iso-
lated isom ers 35a an d 35b could again be distin guish ed by NMR spectro-
scopy. All sign als of h ydrogen an d carbon atom s were assign ed usin g 1D
an d 2D NMR experim en ts. Th e position of th e substituen t is clear from th e
ch em ical sh ift of th e carbon atom bon ded to th e pyrim idin e rin g (δ 65 ppm
for th e O2 derivative an d 48 ppm for th e N1 derivative) an d is con firm ed
also by 2D HMBC spectra.
SCHEME 6
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974
Vrbková, Dračínský, Holý:
CONCLUSIONS
A n ovel bisph osph on ate buildin g block, tetraisopropyl {2-[(m esyloxy)-
m eth yl]propan e-1,3-diyl}bis(oxym eth ylen e)bisph osph on ate, was syn th e-
sized usin g tran sform ation of dieth yl 2,2-bis(h ydroxym eth yl)m alon ate as
startin g com poun d. Th e obtain ed alkylatin g agen t was furth er used in
alkylation of various n ucleobases in th e presen ce of Cs2CO3. Wh ile th e
alkylation of 2-am in o-6-ch loropurin e afforded a m ixture of N9- an d N7-sub-
stituted purin e derivatives, N9-substituted n ucleobases were obtain ed in th e
reaction with aden in e an d 2-am in o-6-(cyclopropyl)am in opurin e. A m ixture
of N1- an d O2-regioisom ers were obtain ed in th e alkylation of cytosin e an d
4-m eth oxy-5-m eth ylpyrim idin -2(1H)-on e. Th e alkylation of uracil afforded
a m ixture of N1-m on o- an d N1,N3-bisalkylated products. Th e correspon din g
free bisph osph on ic acids were obtain ed after th e ester cleavage from
N9-substituted purin e an d N1-substituted pyrim idin e derivatives. Non e of
th e prepared com poun ds exh ibited an y an tiviral activity again st DNA an d
RNA viruses. Th e com poun ds sh owed poor, if an y, cytostatic activity. How-
ever, as an alogues of n ucleotide bisph osph ate an tagon ists of th e P2Y1 re-
ceptor, th ey m igh t possess th is activity. Th is altern ative is curren tly bein g
in vestigated.
EXPERIMENTAL
Un less oth erwise stated, solven ts were evaporated at 40 °C/2 kPa, an d com poun ds were
dried over P2O5 at 2 kPa. Meltin g poin ts were determ in ed on a Büch i m eltin g poin t appa-
ratus an d are un corrected. NMR spectra were m easured on FT NMR spectrom eter Varian
Un ity 500 (1H at 500 MHz an d 13C at 125.7 MHz). Ch em ical sh ifts are given in ppm
(δ-scale), couplin g con stan ts (J) in Hz. Mass spectra were m easured on a ZAB-EQ (Microm ass,
Man ch ester, U.K.) spectrom eter usin g FAB (ion ization by Xe, acceleratin g voltage 8 kV,
glycerol m atrix) or usin g EI (electron en ergy 70 eV). UV spectra (λ in n m ) were taken on
a Beckm an Coulter™, DU® 800 spectroph otom eter. Elem en tal an alyses were carried out on
a Perkin –Elm er CHN An alyser 2400, Series II Sys (Perkin –Elm er, Norwolk, CT, U.S.A.). Ch em i-
cals were purch ased from Sigm a–Aldrich (Prague, Czech Republic). Dim eth ylform am ide an d
aceton itrile were distilled from P2O5 an d stored over m olecular sieves (4Å). Aceton e was
dried over an h ydrous CuSO4. Dieth yl eth er was distilled from LiAlH4.
Dieth yl 2,2-Dim eth yl-1,3-dioxan e-5,5-dicarboxylate (12)
Syn th etic strategy an d an alysis correspon ds to ref.21
Eth yl 2,2-Dim eth yl-1,3-dioxan e-5-carboxylate (13)
Syn th etic strategy an d an alysis correspon ds to refs22–24
.
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Bifunctional Acyclic Nucleoside Phosphonates
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(2,2-Dim eth yl-1,3-dioxan -5-yl)m eth an ol (14)
LiAlH4 (4.6 g, 120.9 m m ol) an d dry dieth yl eth er (250 m l) were in troduced in to a well-dried
th ree-n eck vessel with con den ser. Th e reaction m ixture was cooled to 0 °C. Subsequen tly,
com poun d 13 (28.08 g, 149.2 m m ol) was added dropwise in 150 m l of dieth yl eth er in argon
atm osph ere durin g 30 m in . Th e reaction proceeded un der gen tle reflux. Th e m ixture was
stirred un der th ese con dition s for an oth er 3 h an d at room tem perature overn igh t. Th e resi-
due was cooled with ice bath an d 4.7 m l of water was slowly added. After th at, 4.7 m l of
15% NaOH an d 14.1 m l of water were added. Th e solution was stirred un til a wh ite precipi-
tate form ed. Th e m ixture was filtered th rough Celite. Th e solid was wash ed with 200 m l of
dieth yl eth er (twice). Th e filtrate was evaporated an d used with out furth er purification .
FAB MS: 147.1 (MH+) (70). 1H NMR (500 MHz, DMSO-d6): 4.54 (t, 1 H, JOH-CH2 = 5.2, OH);
3.82 (dd, 2 H, J2a-3 = 4.4, Jgem = 11.8, H-2a); 3.61 (dd, 2 H, J2b-3 = 7.2, Jgem = 11.8, H-2b);
3.49 (dd, 2 H, J4-3 = 6.7, J4-OH = 5.2, H-4); 1.69 (m, 1 H, H-3); 1.30 and 1.29 (2 × s, 6 H, 2 × CH3).
13C NMR (125.7 MHz, DMSO-d6): 97.32 (C-1); 61.05 (C-2); 59.81 (C-4); 36.68 (C-3); 24.97
an d 23.33 (2 × CH3).
5-[(Ben zyloxy)m eth yl]-2,2-dim eth yl-1,3-dioxan e (15)
Com poun d 14 (19 g, 130 m m ol) ) was added at 0 °C un der argon atm osph ere to a suspen -
sion of NaH (7.28 g of 60% suspen sion in m in eral oil prewash ed with n-h exan e; 182 m m ol)
in dry THF (350 m l). Th e reaction m ixture was th en cooled to –10 °C an d ben zyl brom ide
(28.9 g, 169 m m ol) in THF (200 m l) was added dropwise durin g 1 h . Th e m ixture was stirred
at –10 °C for 30 m in an d at room tem perature overn igh t, un der argon . Wh en th e reaction
was com plete (TLC), m eth an olic am m on ia (30 m l) was added. After stirrin g th e solution for
1 h , th e solven t was evaporated. Th e residue in ch loroform (500 m l) was wash ed with water
(500 m l). Th e organ ic layer was dried with an h ydrous MgSO4, filtered an d evaporated to
yield crude product 15, wh ich was used with out furth er purification .
2-[(Ben zyloxy)m eth yl]propan e-1,3-diol (16)
A crude m ixture of 15 (30.7 g, 130 m m ol) in 80% m eth an ol (300 m l) was refluxed with
Dowex 50X8 in H+ form (10 g) for 4 h . Th e resin was filtered off, th e solution n eutralized
with aqueous am m on ia an d th e solven t was evaporated. Th e crude product was purified by
silica gel colum n ch rom atograph y, usin g th e ch loroform –m eth an ol gradien t 0–6%, to yield
20.4 g (80%) of pure 16 as colorless oil. FAB MS: 197.2 (MH+) (80). 1H NMR (500 MHz,
CDCl3): 7.36–7.25 (m , 5 H, Ph ); 4.44 (s, 2 H, OCH2Ph ); 4.40 (t, 2 H, JOH-3 = 5.2, OH); 3.44 (t,
4 H, J3-OH ~ J3-2 = 5.3, H-3); 3.43 (d, 2 H, J1-2 = 6.1, H-1); 1.80 (h , 1 H, J2-3 ~ J2-1 = 6.0, H-2).
13C NMR (125.7 MHz, CDCl3): 139.03; 128.46 (2 C); 127.57 (2 C); 127.54; 72.39 (OCH2Ph );
68.79 (d, JC-P = 12.7, C-1); 59.73 (C-3); 44.59 (C-2).
Diisopropyl [2-(Ben zyloxym eth yl)propan e-1,3-diyl]bis(oxy)bis(m eth ylen e)bisph osph on ate
(17)
A m ixture of com poun d 16 (12.3 g, 62.7 m m ol), paraform aldeh yde (2.2 equiv.) an d CaCl2
(4 g) was saturated with gaseous HCl at 0 °C for 45 m in . Th e reaction m ixture was stirred at
0 °C for 2 h , th en allowed to reach room tem perature, evaporated an d dried. Th e residue
was used with out furth er purification in th e Arbuzov reaction with triisopropyl ph osph ite.
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976
Vrbková, Dračínský, Holý:
Th e crude m ixture was stirred at 100 °C, P(OiPr)3 (30 m l, 2.1 equiv.) was slowly added an d
stirred for 1 h , keepin g th e tem perature at 130 °C. Th e excess of ph osph ite an d isopropyl
ch loride was distilled off (oil bath 80 °C) an d th e residue was purified by colum n ch rom a-
tograph y on silica gel, usin g th e ch loroform –m eth an ol gradien t 0–3%, to yield 25 g (73%) of
pure 17 as yellowish oil. FAB MS: 553.3 (MH+) (80). 1H NMR (500 MHz, CDCl3): 7.36–7.25
(m , 5 H, Ph ); 4.74 (m , 4 H, CHiPr); 4.48 (s, 2 H, OCH2Ph ); 3.69 (d, 4 H, JCH-P = 8.6, OCH2P);
3.62 (d, 4 H, J1-2 = 6.0, H-1); 3.53 (d, 2 H, J3-2 = 6.0, H-3); 2.26 (m , 1 H, J2-3 ~ J2-1 = 6.0, H-2);
1.32 (m , 24 H, CH3). 13C NMR (125.7 MHz, CDCl3): 138.37; 128.32 (2 C); 127.52; 127.48
(2 C); 73.15 (OCH2Ph ); 71.75 (d, JC-P = 12.7, C-1); 71.18 (m , CHiPr); 66.79 (C-3); 66.04 (d,
JC-P = 168.0, OCH2P); 40.20 (C-2); 24.06 (m , CH3).
Diisopropyl [2-(Hydroxym eth yl)propan e-1,3-diyl]bis(oxy)bis(m eth ylen e)bisph osph on ate
(18)
Palladium on activated ch arcoal (10% Pd, 0.2 g) an d con cen trated HCl (0.1 m l) were added
to a solution of 17 (3.68 g, 6.66 m m ol) in m eth an ol (30 m l). Th e reaction m ixture was h ydro-
gen ated at atm osph eric pressure an d room tem perature overn igh t. Th e catalyst was filtered
off th rough a Celite pad, th e filtrate was n eutralized with Et3N an d evaporated. Th e crude
product was purified by colum n ch rom atograph y on silica gel, usin g th e ch loroform –m eth an ol
gradien t 0–6%, to yield 2.95 g (96%) of pure 18 as yellowish oil. FAB MS: 463.4 (MH+) (80).
1H NMR (500 MHz, CDCl3): 4.74 (dh , 4 H, JCH-P = 7.6, JCH-CH3 = 6.2, CHiPr); 3.68 (d, 2 H, J3-2
= 5.6, H-3); 3.66 (d, 4 H, JCH-P = 8.6, OCH2P); 3.64 (m , 4 H, H-1); 3.44 (bt, 1 H, JOH-H3 = 6.5,
OH); 2.13 (m , 1 H, H-2); 1.34 an d 1.33 (2 × d, 24 H, JCH3-CH = 6.2 an d 6.2, CH3). 13C NMR
(125.7 MHz, CDCl3): 72.02 (d, JC-P = 9.8, C-1); 71.11 (m , CHiPr); 65.91 (d, JC-P = 167.4,
OCH2P); 61.29 (C-3); 41.73 (C-2); 24.02 (m , CH3).
Diisopropyl [2-(Mesyloxym eth yl)propan e-1,3-diyl]bis(oxy)bis(m eth ylen e)bisph osph on ate
(19)
A m ixture of 18 (1.3 g, 2.8 m m ol) an d Et3N (1.5 equiv.) in dry dich lorom eth an e (30 m l) was
stirred at 0 °C with a CaCl2 protectin g tube. Mesyl ch loride (1.1 equiv.) was added. Th e
m ixture was stirred at 0 °C for 1 h an d th en kept overn igh t in refrigerator. Th e solution was
diluted with ice water (300 m l) an d th e layers separated. Th e organ ic layer was dried with
an h ydrous MgSO4, filtered an d evaporated in vacuo. Th e residue was purified by colum n
ch rom atograph y on silica gel, usin g th e ch loroform –m eth an ol gradien t 0–4%, to yield 1.5 g
(99%) of pure 19 as yellowish oil. For C19H42O11P2S (540.5) calculated: 42.22% C, 7.83% H,
11.46% P, 5.93% S; foun d: 42.25% C, 7.87% H, 11.56% P, 6.05% S. FAB MS: 541.3 (MH+)
(90). 1H NMR (500 MHz, CDCl3): 4.74 (dh , 4 H, JCH-P = 7.6, JCH-CH3 = 6.2, CHiPr); 4.32 (d,
2 H, J3-2 = 5.6, H-3); 3.71 (d, 4 H, JCH-P = 8.6, OCH2P); 3.64 (m , 4 H, H-1); 3.04 (s, 3 H,
Ms-CH3); 2.39 (h , 1 H, J2-3 ~ J2-1 = 6.0, H-2); 1.34 an d 1.33 (2 × d, 24 H, JCH3-CH = 6.2 an d
6.1, CH3). 13C NMR (125.7 MHz, CDCl3): 71.07 (d, JC-P = 6.6, CHiPr); 70.35 (d, JC-P = 11.8,
C-1); 67.59 (C-3); 66.14 (d, JC-P = 168.5, OCH2P); 39.59 (C-2); 37.00 (Ms-CH3); 24.04 (m ,
CH3).
Alkylation of Nucleobases with Com poun d 19. Gen eral Procedure
A m ixtu re of an appropriate n u cleobase (20, 21, 22, 31, 33, 35; 1 eq u iv.) an d Cs2CO3
(0.5 equiv.) in dry DMF was stirred at room tem perature for 1 h un der a CaCl2 protectin g
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 7, pp. 965–983

Bifunctional Acyclic Nucleoside Phosphonates
977
tube. Th e reaction m ixture was h eated at 60 °C an d bisph osph on ate 19 (1.0 equiv.) was
added. Th e m ixture was th en stirred at 100 °C for 24 h . Th e solven t was evaporated an d th e
residue was co-evaporated with toluen e. Th e residue dissolved in h ot ch loroform was filtered
th rough a Celite pad, evaporated an d purified on a silica gel colum n in ch loroform –m eth an ol.
2-Amino-6-chloro-9-(2-{[bis(diisopropyloxyphosphoryl)methoxy]methyl}ethyl)purine (20a).
Material: 2.8 mmol of 20, 1.4 mmol of Cs2CO3, 2.8 mmol of 19, 50 ml of DMF. Column chromato-
graph y (silica gel, 1–4% gradien t of MeOH in CHCl3) afforded th e product 20a as yellowish
oil (yield 77%). FAB MS: 615.1 (MH+) (90). 1H NMR (500 MHz, CDCl3): 7.90 (s, 1 H, H-8);
5.46 (bs, 2 H, NH2); 4.78 (m , 4 H, CHiPr); 4.26 (d, 2 H, J3′,2′ = 7.5, H-3′); 3.72 (m , 4 H,
OCH2P); 3.50 (dd, 2 H, Jgem = 9.1 an d J1′a,2′ = 6.0, H-1′a); 3.48 (dd, 2 H, Jgem = 9.0 an d J1′b,2′
=
5.6, H-1′b); 2.52 (m , 1 H, H-2′); 1.36, 1.35, 1.34 an d 1.33 (4 × d, 4 × 6 H, JCH3-CH = 6.2, 6.2,
6.4 an d 6.3, CH3). 13C NMR (125.7 MHz, CDCl3): 159.33 (C-2); 154.17 (C-4); 151.16 (C-6);
143.43 (C-8); 124.96 (C-5); 71.18 an d 71.16 (2 × d, JC-P = 6.6 an d 6.7, CHiPr); 70.94 (d, JC-P
12.9, C-1′); 66.11 (d, JC-P = 169.3, OCH2P); 41.46 (C-3′); 39.84 (C-2′); 24.08 (m , CH3).
=
2-Amino-6-chloro-7-(2-{[bis(diisopropyloxyphosphoryl)methoxy]methyl}ethyl)purine (20b ).
Material: 2.8 mmol of 20, 1.4 mmol of Cs2CO3, 2.8 mmol of 19, 50 ml of DMF. Column chromato-
graph y (silica gel, 1–6% gradien t of MeOH in CHCl3) afforded th e product 20b as yellowish
oil (yield 10%). FAB MS: 615.1 (MH+) (90). 1H NMR (500 MHz, CDCl3): 8.18 (s, 1 H, H-8);
5.17 (bs, 2 H, NH2); 4.76 (m , 4 H, CHiPr); 4.46 (d, 2 H, J3′,2′ = 7.5, H-3′); 3.69 (m , 4 H,
OCH2P); 3.63 (dd, 2 H, Jgem = 9.5 an d J1′a,2′ = 5.2, H-1′a); 3.49 (dd, 2 H, Jgem = 9.5 an d J1′b,2′
=
5.0, H-1′b); 2.51 (m , 1 H, H-2′); 1.36, 1.35, 1.34 an d 1.33 (4 × d, 4 × 6 H, JCH3-CH = 6.2, 6.2,
6.4 an d 6.3, CH3). 13C NMR (125.7 MHz, CDCl3): 164.38 (C-4); 159.33 (C-2); 150.03 (C-8);
143.29 (C-6); 116.17 (C-5); 71.15 (m , CHiPr); 70.81 (d, JC-P = 11.6, C-1′); 66.11 (d, JC-P
168.8, OCH2P); 45.06 (C-3′); 41.06 (C-2′); 24.05 (m , CH3).
=
9-(2-{[Bis(diisopropyloxyphosphoryl)methoxy]methyl}ethyl)adenine (21a). Material: 0.74 m m ol
of 21, 0.37 m m ol of Cs2CO3, 0.74 m m ol of 19, 20 m l of DMF. Colum n ch rom atograph y (silica
gel, 1–8% gradien t of MeOH in CHCl3) afforded th e product 21a as yellowish oil (yield
76%). FAB MS: 580.5 (MH+) (80). 1H NMR (500 MHz, CDCl3): 8.33 (s, 1 H, H-8); 8.00 (s, 1 H,
H-2); 5.82 (bs, 2 H, NH2); 4.76 (m , 4 H, CHiPr); 4.32 (d, 2 H, J3′,2′ = 6.7, H-3′); 3.71 (m , 4 H,
OCH2P); 3.58 (dd, 2 H, Jgem = 9.6 an d J1′a,2′ = 5.8, H-1′a); 3.52 (dd, 2 H, Jgem = 9.6 an d J1′b,2′
=
5.6, H-1′b); 2.60 (m , 1 H, H-2′); 1.34 (m , 24 H, CH3). 13C NMR (125.7 MHz, CDCl3): 155.37
(C-6); 152.91 (C-2); 150.33 (C-4); 141.87 (C-8); 119.53 (C-5); 71.10 (m , CHiPr an d C-1′);
66.08 (d, JC-P = 168.5, OCH2P); 41.95 (C-3′); 39.90 (C-2′); 24.07 (m , CH3).
6-(Cyclopropyl)amino-9-(2-{[bis(diisopropyloxyphosphoryl)methoxy]methyl}ethyl)purine (22a).
Material: 0.74 m m ol of 22, 0.37 m m ol of Cs2CO3, 0.74 m m ol of 19, 20 m l of DMF. Colum n
ch rom atograph y (silica gel, 1–5% gradien t of MeOH in CHCl3) afforded th e product 22a as
yellowish oil (yield 60%). FAB MS: 620.6 (MH+) (70). 1H NMR (500 MHz, CDCl3): 8.45 (s, 1 H,
H-2); 7.91 (s, 1 H, H-8); 5.99 (bs, 1 H, NH); 4.76 (m , 4 H, CHiPr); 4.31 (d, 2 H, J3′,2′ = 6.7,
H-3′); 3.70 (d, 4 H, JH-C-P = 8.6, OCH2P); 3.57 (dd, 2 H, Jgem = 9.5 an d J1′a,2′ = 5.8, H-1′a);
3.51 (dd, 2 H, Jgem = 9.5 an d J1′b,2′ = 5.7, H-1′b); 3.04 (m , 1 H, CHcycl); 2.59 (m , 1 H, H-2′);
1.35 (d, 12 H, JCH3,CH = 6.1, CH3); 1.33 (d, 12 H, JCH3,CH = 6.1, CH3); 0.93 (m , 2 H, CH2cycl);
0.67 (m , 2 H, CH2cycl). 13C NMR (125.7 MHz, CDCl3): 155.72 (C-6); 153.16 (C-2); 149.22
(C-4); 141.21 (C-8); 119.81 (C-5); 71.10 (m , C-1′ an d CHiPr); 66.07 (d, JC-P = 168.4, OCH2P);
41.87 (C-3′); 39.94 (C-2′); 24.07 (m , CH3).
1-(2-{[Bis(diisopropyloxyphosphoryl)methoxy]methyl}ethyl)cytosine (31a). Material: 0.87 m m ol
of 31, 0.43 m m ol of Cs2CO3, 0.87 m m ol of 19, 40 m l of DMF. Colum n ch rom atograph y (silica
gel, 1–15% gradien t of MeOH in CHCl3) afforded th e product 31a as yellowish oil (yield
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 7, pp. 965–983

978
Vrbková, Dračínský, Holý:
49%). FAB MS: 556.4 (MH+) (45). 1H NMR (500 MHz, CDCl3): 7.51 (d, 1 H, JH-6,H-5 = 7.2,
H-6); 5.75 (d, 1 H, JH-5,H-6 = 7.2, H-5); 4.74 (m , 4 H, CHiPr); 3.81 (d, 2 H, J3′,2′ = 6.8, H-3′);
3.68 (m , 4 H, OCH2P); 3.60 (dd, 2 H, Jgem = 9.6 an d J1′a,2′ = 5.3, H-1′a); 3.54 (dd, 2 H, Jgem
=
9.6 an d J1′b,2′ = 5.8, H-1′b); 2.52 (m , 1 H, H-2′); 1.33 (m , 24 H, CH3). 13C NMR (125.7 MHz,
CDCl3): 165.87 (C-4); 155.75 (C-2); 147.26 (C-6); 93.78 (C-5); 71.32 (d, J1′,P = 12.9, C-1′);
71.06 (m , CHiPr); 65.93 (d, JC-P = 169.0, OCH2P); 48.60 (C-3′); 38.63 (C-2′); 24.07 (m , CH3).
2-(2-{[Bis(diisopropyloxyphosphoryl)methoxy]methyl}ethyl)cytosine (31b). Material: 0.87 m m ol
of 31, 0.43 m m ol of Cs2CO3, 0.87 m m ol of 19, 20 m l of DMF. Colum n ch rom atograph y
(silica gel, 1–7% gradien t of MeOH in CHCl3) afforded th e product 31b as yellowish oil
(yield 15%). FAB MS: 556.5 (MH+) (45). 1H NMR (500 MHz, CDCl3): 7.96 (d, 1 H, JH-6,H-5
=
5.5, H-6); 6.11 (d, 1 H, JH-5,H-6 = 5.7, H-5); 5.64 (bs, 1 H, NH2); 4.74 (m , 4 H, CHiPr); 4.34 (d,
2 H, J3′,2′ = 6.8, H-3′); 3.76–3.68 (m , 8H, H-1′ an d OCH2P); 2.44 (m , 1 H, H-2′); 1.32 (m ,
24 H, CH3). 13C NMR (125.7 MHz, CDCl3): 164.83 an d 164.78 (C-4 an d C-2); 156.90 (C-6);
99.19 (C-5); 71.13 (d, J1′,P = 12.6, C-1′); 70.80 (d, JCH-P = 6.6, CHiPr); 65.79 (d, JC-P = 168.2,
OCH2P); 64.19 (C-3′); 39.18 (C-2′); 23.82 (d, JC-P = 3.7, CH3); 23.72 (d, JC-P = 4.5, CH3).
1-(2-{[Bis(diisopropyloxyphosphoryl)methoxy]methyl}ethyl)uracil (33a). Material: 0.87 m m ol
of 33, 0.43 m m ol of Cs2CO3, 0.87 m m ol of 19, 20 m l of DMF. Colum n ch rom atograph y
(silica gel, 1–5% gradien t of MeOH in CHCl3) afforded th e product as a m ixture of 33a an d
33b . Com poun d 33a was separated from 33b on th in layer ch rom atograph y in 10%
MeOH/CHCl3 as yellowish oil (54%). FAB MS: 557.5 (MH+) (90). 1H NMR (500 MHz, CDCl3):
8.72 (bs, 1 H, NH); 7.51 (d, 1 H, JH-6,H-5 = 7.9, H-6); 5.68 (dd, 1 H, JH-5,H-6 = 7.9, JH-5,3′ = 2.3,
H-5); 4.75 (m , 4 H, CHiPr); 3.82 (d, 2 H, J3′,2′ = 6.7, H-3′); 3.69 (m , 4 H, OCH2P); 3.61 (dd, 2 H,
Jgem = 9.6 an d J1′a,2′ = 5.6, H-1′a); 3.57 (dd, 2 H, Jgem = 9.6 an d J1′b,2′ = 5.5, H-1′b); 2.42 (m , 1 H,
H-2′); 1.34 (m , 24 H, CH3). 13C NMR (125.7 MHz, CDCl3): 163.55 (C-4); 150.88 (C-2);
146.17 (C-6); 101.68 (C-5); 71.26 (d, J1′,P = 12.2, C-1′); 71.10 (m , CHiPr); 66.04 (d, JC-P
169.1, OCH2P); 47.62 (C-3′); 38.98 (C-2′); 24.06 (m , CH3).
=
1,3-[Bis(2,2′-{[bis(diisopropyloxyphosphoryl)methoxy]methyl}ethyl)]uracil (33b ). Material:
0.87 m m ol of 33, 0.43 m m ol of Cs2CO3, 0.87 m m ol of 19, 20 m l of DMF. Colum n ch rom ato-
graph y (silica gel, 1–5% gradien t of MeOH in CHCl3) afforded th e product as a m ixture of
33a an d 33b. Com poun d 33b was separated from 33a on th in layer ch rom atograph y in 10%
MeOH/CHCl3 as yellowish oil (15%). FAB MS: 1001.9 (MH+) (85). 1H NMR (500 MHz,
CDCl3): 7.45 (d, 1 H, JH-6,H-5 = 7.9, H-6); 5.68 (d, 1 H, JH-5,H-6 = 7.9, H-5); 4.74 (m , 8 H,
CHiPr); 4.00 (d, 2 H, J3′′,2′′ = 6.9, H-3′′); 3.80 (d, 2 H, J3′,2′ = 6.8, H-3′); 3.75–3.53 (m , 16 H,
H-1′, H-1′′, OCH2P, OCH2P′); 2.39 (m , 2 H, H-2′ an d H-2′′); 1.35–1.31 (m , 48 H, CH3). 13C NMR
(125.7 MHz, CDCl3): 163.32 (C-4); 151.72 (C-2); 144.06 (C-6); 100.99 (C-5); 72.38 (d, J1′′,P
=
12.8, C-1′′); 71.29–70.85 (m , H-1′ an d CHiPr); 66.08 an d 66.04 (d, JC-P = 167.8 an d 168.9,
OCH2P an d OCH2P′); 48.58 (C-3′); 40.77 (C-3′′); 38.85 (C-2′); 38.51 (C-2′′); 24.15–24.00 (m ,
CH3).
1-(2-{[Bis(diisopropyloxyphosphoryl)methoxy]methyl}ethyl)-4-methoxythymine (35a). Mate-
rial: 1.85 m m ol of 35, 0.92 m m ol of Cs2CO3, 1.85 m m ol of 19, 25 m l of DMF. Colum n
ch rom atograph y (silica gel, 1–3% gradien t of MeOH in CHCl3) afforded th e product as a m ix-
ture of 35a an d 35b. Com poun d 35a was separated from 35b on th in layer ch rom atograph y
in 5% MeOH/CHCl3 as yellowish oil (45%). FAB MS: 585.5 (MH+) (70). 1H NMR (500 MHz,
CDCl3): 7.51 (q, 1 H, JH-6,CH3 = 1.1, H-6); 4.74 (m , 4 H, CHiPr); 3.97 (s, 3 H, OCH3); 3.87 (d,
J3′,2′ = 6.9, H-3′); 3.68 (m , 4 H, OCH2P); 3.60 (dd, 2 H, Jgem = 9.5 an d J1′a,2′ = 5.2, H-1′a); 3.52
(dd, 2 H, Jgem = 9.5 an d J1′b,2′ = 6.0, H-1′b); 2.53 (m , 1 H, H-2′); 1.96 (d, 3 H, JCH3,H-6 = 1.1,
Py-CH3); 1.35–1.31 (m , 24 H, CH3). 13C NMR (125.7 MHz, CDCl3): 170.79 (C-4); 156.88
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 7, pp. 965–983

Bifunctional Acyclic Nucleoside Phosphonates
979
(C-2); 145.64 (C-6); 104.29 (C-5); 71.24–70.96 (m , C-1′ an d CHiPr); 65.97 (d, JC-P = 169.1,
OCH2P); 54.44 (OCH3); 48.43 (C-3′); 38.61 (C-2′); 24.07 (m , CH3); 11.80 (Py-CH3).
2-(2-{[Bis(diisopropyloxyphosphoryl)methoxy]methyl}ethyl)-4-methoxythymine (35b). Material:
1.85 m m ol of 35, 0.92 m m ol of Cs2CO3, 1.85 m m ol of 19, 20 m l of DMF. Colu m n
ch rom atograph y (silica gel, 1–3% gradien t of MeOH in CHCl3) afforded th e product as a
m ixture of 35a an d 35b. Com poun d 35b was separated from 35a on th in layer ch rom ato-
graph y in 5% MeOH/CHCl3 as yellowish oil (38%). FAB MS: 585.6 (MH+) (70). 1H NMR
(500 MHz, CDCl3): 7.95 (s, 1 H, H-6); 4.73 (m , 4 H, CHiPr); 4.37 (d, 2 H, J3′,2′ = 6.2, H-3′);
3.98 (s, 3 H, OCH3); 3.74–3.70 (m , 8 H, H-1′ an d OCH2P); 2.49 (m , 1 H, H-2′); 2.05 (d, 3 H,
JCH3,H-6 = 0.7, Py-CH3); 1.31 (m , 24 H, CH3). 13C NMR (125.7 MHz, CDCl3): 169.61 (C-4);
163.60 (C-2); 157.00 (C-6); 111.10 (C-5); 71.40 (d, J1′,P = 12.7, C-1′); 70.97 (d, JCH,P = 6.3,
CHiPr); 66.10 (d, JC-P = 168.1, OCH2P); 65.14 (C-3′); 53.86 (OCH3); 39.42 (C-2′); 24.04 (m ,
CH3); 11.84 (Py-CH3).
9-(2-{[Bis(diisopropyloxyph osph oryl)m eth oxy]m eth yl}eth yl)guan in e (23)
A solution of 20a (370 m g, 0.6 m m ol) in 80% acetic acid (20 m l) was refluxed for 6 h . Th e
solution was n eutralized with Et3N an d th e volatiles were evaporated in vacuo. Colum n
ch rom atograph y (silica gel, 1–15% gradien t of MeOH in CHCl3) afforded com poun d 23 as a
wh ite am orph ous powder (yield 63%). FAB MS: 596.3 (MH+) (70). 1H NMR (500 MHz,
CDCl3): 11.43 (bs, 1 H, NH); 7.58 (s, 1 H, H-8); 7.02 (bs, 2 H, NH2); 4.80 (m , 4 H, CHiPr);
4.09 (d, 2 H, J3′,2′ = 7.6, H-3′); 3.69 (m , 4 H, OCH2P); 3.56 (dd, 2 H, Jgem = 9.1 an d J1′a,2′
=
6.0, H-1′a); 3.47 (dd, 2 H, Jgem = 9.0 an d J1′b,2′ = 4.4, H-1′b); 2.49 (m , 1 H, H-2′); 1.35 (m ,
24 H, CH3). 13C NMR (125.7 MHz, CDCl3): 157.89 (C-6); 154.46 (C-2); 150.82 (C-4); 138.23
(C-8); 117.60 (C-5); 71.61 (m , CHiPr an d C-1′); 66.10 (d, JC-P = 170.7, OCH2P); 42.01 (C-3′);
39.42 (C-2′); 24.05 (m , CH3).
2,6-Diam in o-9-(2-{[bis(diisopropyloxyph osph oryl)m eth oxy]m eth yl}eth yl)purin e (24)
A solution of 20a (570 m g, 0.93 m m ol) in m eth an olic am m on ia (50 m l) was h eated (100 °C)
in an autoclave for 13 h . Th e solven t was th en evaporated. Purification of th e residue by
colum n ch rom atograph y (silica gel, 1–12% gradien t of MeOH in CHCl3) afforded com poun d
24 as yellowish foam (yield 65%). FAB MS: 595.3 (MH+) (70). 1H NMR (500 MHz, CDCl3):
7.63 (s, 1 H, H-8); 5.52 (bs, 2 H, 6-NH2); 4.91 (bs, 2 H, 2-NH2); 4.77 (m , 4 H, CHiPr); 4.16 (d,
2 H, J3′,2′ = 6.5, H-3′); 3.76 an d 3.70 (2 × dd, 4 H, Jgem = 13.5 an d 13.5, JH-C-P = 8.9 an d 8.8,
OCH2P); 3.54 (dd, 2 H, Jgem = 9.5 an d J1′a,2′ = 6.1, H-1′a); 3.49 (dd, 2 H, Jgem = 9.6 an d J1′b,2′
=
5.4, H-1′b); 2.53 (m , 1 H, H-2′); 1.35 (m , 24 H, CH3). 13C NMR (125.7 MHz, CDCl3): 159.90
(C-2); 155.73 (C-6); 152.41 (C-4); 139.16 (C-8); 114.20 (C-5); 71.07 (m , CHiPr an d C-1′);
66.03 (d, JC-P = 168.8, OCH2P); 41.16 (C-3′); 39.77 (C-2′); 24.08 (m , CH3).
2-Am in o-6-(cyclopropyl)am in o-9-(2-{[bis(diisopropyloxyph osph oryl)m eth oxy]m eth yl}-
eth yl)purin e (25)
A solution of 20a (430 m g, 0.7 m m ol) an d cyclopropylam in e (9 equiv.) in dioxan e (20 m l)
was refluxed for 2 days. Th e solven t an d excess of am in e were evaporated to dryn ess. Purifi-
cation of com poun d by colum n ch rom atograph y (silica gel, 1–6% gradien t of MeOH in
CHCl3) afforded the product 25 as yellowish oil (yield 84%). FAB MS: 635.6 (MH+) (90). 1H NMR
(500 MHz, CDCl3): 7.57 (s, 1 H, H-8); 5.81 (bs, 1 H, NHcycl); 4.93 (bs, 2 H, NH2); 4.77 (m , 4 H,
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980
Vrbková, Dračínský, Holý:
CHiPr); 4.15 (d, 2 H, J3′,2′ = 6.6, H-3′); 3.75 an d 3.70 (2 × dd, 4 H, Jgem = 13.5 an d 13.5, JH-C-P
9.0 an d 8.8, OCH2P); 3.53 (dd, 2 H, Jgem = 9.6 an d J1′a,2′ = 6.1, H-1′a); 3.48 (dd, 2 H, Jgem
=
=
9.6 an d J1′b,2′ = 5.4, H-1′b); 2.99 (bs, 1 H, CHcycl); 2.52 (m , 1 H, H-2′); 1.34 (m , 24 H, CH3);
0.85 (m , 2 H, CH2cycl); 0.61 (m , 2 H, CH2cycl). 13C NMR (125.7 MHz, CDCl3): 160.07 (C-2);
156.17 (C-6); 151.36 (C-4); 138.38 (C-8); 114.38 (C-5); 71.07 (m , CHiPr an d C-1′); 66.01 (d,
JC-P = 168.9, OCH2P); 41.08 (C-3′); 39.80 (C-2′); 24.07 (m , CH3); 23.60 (CHcycl); 7.37
(CH2cycl).
1-(2-{[Bis(diisopropyloxyph osph oryl)m eth oxy]m eth yl}eth yl)th ym in e (36)
A solution of 35a (480 m g, 0.8 m m ol) in 80% acetic acid (15 m l) was refluxed for 24 h . Th e
solution was n eutralized with Et3N. Solven t an d excess of acetic acid were evaporated. Th e
purification of th e residue by colum n ch rom atograph y (silica gel, 1–3% gradien t of MeOH in
CHCl3) afforded th e product 36 as yellowish oil (yield 78%). FAB MS: 571.2 (MH+) (60).
1H NMR (500 MHz, CDCl3): 8.59 (s, 1 H, NH); 7.33 (d, 1 H, JH-6,CH3 = 1.2, H-6); 4.75 (m , 4 H,
CHiPr); 3.79 (d, 2 H, J3′,2′ = 6.8, H-3′); 3.69 (m , 4 H, OCH2P); 3.60 (dd, 2 H, Jgem = 9.5 an d
J1′a,2′ = 5.5, H-1′a); 3.51 (dd, 2 H, Jgem = 9.5 an d J1′b,2′ = 5.6, H-1′b); 1.95 (d, 3 H, JCH3,H-6
=
1.1, Py-CH3); 1.34 (m , 24 H, CH3). 13C NMR (125.7 MHz, CDCl3): 164.16 (C-4); 150.91
(C-2); 141.89 (C-6); 110.32 (C-5); 71.23 (d, J1′,P = 12.7, C-1′); 71.07 (d, JC-O-P = 7.0, CHiPr);
71.03 (d, JC-O-P = 6.9, CHiPr); 66.01 (d, JC-P = 169.2, OCH2P); 47.19 (C-3′); 39.07 (C-2′); 24.05
(m , CH3); 12.06 (Py-CH3).
Tran sform ation of Esters to Free Ph osph on ic Acids. Gen eral Procedure
Th e dried startin g esters (21a, 22a, 23, 24, 25, 31a, 33a an d 36), aceton itrile (15 m l) an d
BrSiMe3 (excess) were stirred at room tem perature overn igh t. After evaporation an d co-
distillation with aceton itrile, th e residue was treated with water an d aqueous am m on ia. Th e
m ixture was evaporated to dryn ess, th e residue dissolved in water was applied on to a colum n
of Dowex 50X8 in H+ form an d wash ed with water.
A) Elution with water an d evaporation in vacuo afforded free ph osph on ic acids 26–28,
32, 34, an d 36.
B) Elution with dilute am m on ia an d evaporation afforded am m on ium salts, wh ich were
applied on to Dowex 50 (Na+ form ). Elution with water an d evaporation gave ph osph on ic acids
29 an d 30 as tetrasodium salts.
9-(2-{[Bis(phosphono)methoxy]methyl}ethyl)adenine (26). Material: 0.50 m m ol of 21a, 3.0 m l
of TMSBr, 15 m l of CH3CN. Wh ite solid (yield 80%), m .p. 248.9 °C (dec.). For C11H19N5O8P2
(411.2) calculated: 32.13% C, 4.66% H, 17.03% N, 15.06% P; foun d: 32.07% C, 4.76% H,
17.15% N, 15.13% P. FAB MS: 412.1 (MH+) (45). 1H NMR (500 MHz, D2O + NaOD): 8.22 an d
8.21 (s, 2 H, H-8 an d H-2); 4.37 (d, 2 H, J3′,2′ = 6.7, H-3′); 3.58 (m , 8 H, H-1′ an d OCH2P);
2.56 (m , 1 H, H-2′). 13C NMR (125.7 MHz, D2O + NaOD): 156.10 (C-6); 152.93 (C-2); 149.79
(C-4); 144.04 (C-8); 119.03 (C-5); 71.50 (d, J1′,P = 11.6, C-1′); 67.95 (d, JC-P = 156.2, OCH2P);
43.15 (C-3′); 39.85 (C-2′). UV: (0.01 M HCl) λm ax = 258 nm (εm ax = 13768); (H2O) λm ax = 259 nm
(εm ax = 14352); (0.01 M NaOH) λm ax = 259 n m (εm ax = 13402).
6-(Cyclopropyl)a mino-9-(2-{[bis(phosphono)methoxy]methyl}ethyl)purine (2 7 ). Mat erial:
0.39 m m ol of 22a, 3.0 m l of TMSBr, 15 m l of CH3CN. Wh ite solid (yield 80%), m .p. 205.0 °C
(dec.). For C14H23N5O8P2 (451.3) calculated: 37.26% C, 5.14% H, 15.52% N, 13.73% P;
found: 37.37% C, 5.25% H, 15.68% N, 13.82% P. FAB MS: 452.3 (MH+) (60). 1H NMR (500 MHz,
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Bifunctional Acyclic Nucleoside Phosphonates
981
D2O): 8.49 (s, 1 H, H-2); 8.38 (s, 1 H, H-8); 4.49 (d, 2 H, J3′,2′ = 6.7, H-3′); 3.69–3.60 (m , 8 H,
H-1′ an d OCH2P); 2.91 (bs, 1 H, CHcycl); 2.62 (m , 1 H, H-2′); 1.11 (m , 2 H, CH2cycl); 0.90 (m ,
2 H, CH2cycl). 13C NMR (125.7 MHz, D2O): 150.61 (C-6); 148.35 (C-4); 146.08 (C-8); 144.71
(C-2); 119.08 (C-5); 71.72 (d, J1′,P = 11.8, C-1′); 67.24 (d, JC-P = 158.2, OCH2P); 44.04 (C-3′);
39.89 (C-2′); 23.42 (CHcycl); 7.33 (CH2cycl). UV: (0.01 M HCl) λm ax = 266 n m (εm ax = 18646);
(H2O) λm ax = 266 n m (εm ax = 18340); (0.01 M NaOH) λm ax = 268 n m (εm ax = 17518).
9-(2-{[Bis(phosphono)methoxy]methyl}ethyl)guanine (28). Material: 0.37 m m ol of 23, 3.0 m l
of TMSBr, 15 m l of CH3CN. Wh ite solid (yield 67%), m .p. 206.9 °C (dec.). For C11H19N5O9P2
(427.2) calculated: 30.92% C, 4.48% H, 16.39% N, 14.50% P; foun d: 30.85% C, 4.58% H,
16.50% N, 14.56% P. FAB MS: 428.3 (MH+) (60). 1H NMR (500 MHz, D2O): 8.98 (s, 1 H,
H-8); 4.44 (d, 2 H, J3′,2′ = 6.5, H-3′); 3.72–3.61 (m , 8 H, H-1′ an d OCH2P); 2.64 (m , 1 H, H-2′).
13C NMR (125.7 MHz, D2O): 156.02 an d 155.97 (C-6 an d C-2); 150.92 (C-4); 138.95 (C-8);
108.45 (C-5); 71.73 (d, J1′,P = 12.4, C-1′); 67.28 (d, JC-P = 158.1, OCH2P); 45.17 (C-3′); 39.12
(C-2′). UV: (0.01 M HCl) λm ax = 252 n m (εm ax = 10558); (H2O) λm ax = 251 n m (εm ax = 10890);
(0.01 M NaOH) λm ax = 266 n m (εm ax = 9320).
Tetrasodium salt of 2,6-diamino-9-(2-{[bis(phosphono)methoxy]methyl}ethyl)purine (29).
Material: 0.60 m m ol of 24, 3.5 m l of TMSBr, 15 m l of CH3CN. Wh ite solid (yield 88%),
m .p. 327.1 °C (dec.). FAB MS: 515.0 (MH+) (30). HR MS for C11H16N6Na4O8P2 calculated:
515.01546, foun d: 515.01739. 1H NMR (500 MHz, D2O): 7.91 (s, 1 H, H-8); 4.23 (d, 2 H, J3′,2′
=
6.8, H-3′); 3.95 (m , 8 H, H-1′ an d OCH2P); 2.51 (m , 1 H, H-2′). 13C NMR (125.7 MHz, D2O):
160.35 (C-6); 156.61 (C-2); 151.79 (C-4); 141.86 (C-8); 113.49 (C-5); 71.51 (d, J1′,P = 11.8,
C-1′); 68.56 (d, JC-P = 154.5, OCH2P); 42.62 (C-3′); 39.68 (C-2′). UV: (0.01 M HCl) λm ax
=
284 n m (εm ax = 9092); (H2O) λm ax = 278 n m (εm ax = 10120); (0.01 M NaOH) λm ax = 278 n m
(εm ax = 9442).
Tetrasodium salt of 2-amino-6-(cyclopropyl)amino-9-(2-{[bis(phosphono)methoxy]methyl}ethyl)-
purine (30). Material: 0.54 m m ol of 25, 3.5 m l of TMSBr, 15 m l of CH3CN. Yellow solid
(yield 73%), m .p. 338.8 °C (dec.). FAB MS: 555.0 (MH+) (30). HR MS for C14H20N6Na4O8P2
calculated: 555.0460, foun d: 555.0487. 1H NMR (500 MHz, D2O): 7.89 (s, 1 H, H-8); 4.24 (d,
2 H, J3′,2′ = 6.7, H-3′); 3.54 (m , 8 H, H-1′ an d OCH2P); 2.86 (bs, 1 H, CHcycl); 2.52 (m , 1 H,
H-2′); 0.88 (m , 2 H, CH2cycl); 0.67 (m , 2 H, CH2cycl). 13C NMR (125.7 MHz, D2O): 160.59
(C-2); 156.99 (C-6); 150.75 (C-4); 141.39 (C-8); 113.85 (C-5); 71.52 (d, J1′,P = 9.6, C-1′); 68.83
(d, JC-P = 153.7, OCH2P); 42.58 (C-3′); 39.69 (C-2′); 23.88 (CHcycl); 7.37 (CH2cycl). UV: (0.01
M
HCl) λm ax = 282 n m (εm ax = 13712); (H2O) λm ax = 282 n m (εm ax = 13736); (0.01 M NaOH)
λm ax = 290 n m (εm ax = 12008).
1-(2-{[Bis(phosphono)methoxy]methyl}ethyl)cytosine (32). Material: 0.36 m m ol of 31a,
3.0 m l of TMSBr, 15 m l of CH3CN. Wh ite solid (yield 57%), m .p. 208.5 °C (dec.). For
C
10H19N3O9P2 (387.2) calculated: 31.02% C, 4.95% H, 10.85% N, 16.00% P; foun d: 31.05% C,
4.85% H, 10.75% N, 16.15% P. FAB MS: 388.3 (MH+) (30). 1H NMR (500 MHz, D2O): 7.93 (d,
1 H, JH-6,H-5 = 7.7, H-6); 6.17 (d, 1 H, JH-5,H-6 = 7.7, H-5); 4.00 (d, 2 H, J3′,2′ = 6.9, H-3′); 3.65 (m ,
8 H, H-1′ an d OCH2P); 2.47 (m , 1 H, H-2′). 13C NMR (125.7 MHz, D2O): 160.19 (C-4);
151.21 (C-6); 149.99 (C-2); 94.92 (C-5); 71.91 (d, J1′,P = 12.1, C-1′); 67.27 (d, JC-P = 158.1,
OCH2P); 50.03 (C-3′); 38.50 (C-2′). UV: (0.01 M HCl) λm ax = 281 nm (εm ax = 11478); (H2O) λm ax
279 nm (εm ax = 10066); (0.01 M NaOH) λm ax = 272 n m (εm ax = 7846).
=
1-(2-{[Bis(phosphono)methoxy]methyl}ethyl)uracil (34). Material: 0.50 m m ol of 33a, 3.5 m l of
TMSBr, 15 m l of CH3CN. Hygroscopic yellow solid (yield 63%). For C10H18N2O10P2·0.5H2O
(397.2) calculated: 30.24% C, 4.82% H, 7.05% N, 15.60% P; foun d: 30.37% C, 4.95% H,
7.19% N, 15.73% P. FAB MS: 389.3 (MH+) (90). 1H NMR (500 MHz, D2O): 7.67 (d, 1 H,
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982
Vrbková, Dračínský, Holý:
JH-6,H-5 = 7.9, H-6); 5.83 (d, 1 H, JH-5,H-6 = 7.8, H-5); 3.91 (d, 2 H, J3′,2′ = 7.1, H-3′); 3.75 (d,
4 H, JH-C-P = 8.7, OCH2P); 3.67 (d, 4 H, J1′,2′ = 5.6, H-1′); 2.45 (m, 1 H, H-2′). 13C NMR (125.7 MHz,
D2O): 167.59 (C-4); 153.11 (C-2); 148.68 (C-6); 102.00 (C-5); 71.89 (d, J1′,P = 11.9, C-1′);
66.67 (d, JC-P = 159.5, OCH2P); 48.82 (C-3′); 38.70 (C-2′). UV: (0.01 M HCl) λm ax = 264 n m
(εm ax = 6682); (H2O) λm ax = 265 n m (εm ax = 8416); (0.01 M NaOH) λm ax = 264 n m (εm ax
=
8516).
1-(2-{[Bis(phosphono)methoxy]methyl}ethyl)thymine (37). Material: 0.66 m m ol of 36, 3.5 m l
of TMSBr, 15 m l of CH3CN. Hygroscopic wh ite solid (yield 78%). For C11H20N2O10P2·0.5H2O
(411.2) calculated: 32.13% C, 5.15% H, 6.81% N, 15.06% P; foun d: 32.27% C, 5.27% H,
6.89% N, 15.22% P. FAB MS: 403.3 (MH+) (45). 1H NMR (500 MHz, D2O): 7.52 (q, 1 H,
JH-6,CH3 = 1.2, H-6); 3.87 (d, 2 H, J3′,2′ = 7.2, H-3′); 3.75 (d, 4 H, JH-C-P = 8.7, OCH2P); 3.66 (d,
4 H, J1′,2′ = 5.6, H-1′); 2.44 (m, 1 H, H-2′); 1.89 (d, 3 H, JCH3,H-6 = 1.2, CH3). 13C NMR (125.7 MHz,
D2O): 167.82 (C-4); 153.18 (C-2); 144.46 (C-6); 111.31 (C-5); 71.91 (d, J1′,P = 12.1, C-1′);
66.63 (d, JC-P = 159.7, OCH2P); 48.55 (C-3′); 38.74 (C-2′); 12.00 (CH3). UV: (0.01 M HCl) λm ax
=
270 n m (εm ax = 8660); (H2O) λm ax = 270 n m (εm ax = 8846); (0.01 M NaOH) λm ax = 269 n m
(εm ax = 6760).
This work is a part of the research project Z4 055 0506. It was supported by the Centre for New
Antivirals and Antineoplastics (1M0508), by the Programme of Targeted Projects of Academy of
Sciences of the Czech Republic (1QS400550501) and by Gilead Sciences, Inc. (Foster City, CA,
U.S.A.). The Descartes Prize of the European Union HPAW-2002-10096 also supported this project.
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