A regioselective synthesis of alkyl 2-(guanin-9-YL)acetates as PNA building blocks from 7-(4-nitrobenzyl)guanine derivatives

Article abstract of DOI:10.1135/cccc20050085

Guanine derivatives substituted at N⁷ with 4-R-benzyl groups (R = H, MeO, NO₂) have been evaluated in the regioselective N ⁹-alkylation of guanine. Given the capricious removal of (substituted) benzyl groups from guanine d

Full text of DOI:10.1135/cccc20050085

Alkyl 2-(Guanin-9-yl)acetates
85
A REGIOSELECTIVE SYNTHESIS OF ALKYL 2-(GUANIN-9-YL)ACETATES
AS PNA BUILDING BLOCKS FROM 7-(4-NITROBENZYL)GUANINE
DERIVATIVES
b
2a
3a,
Györgyi FERENC1a, Péter FORGÓ , Zoltán KELE and Lajos KOVÁCS
*
a
Department of Medicinal Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary;
e-mail: gyorgyi@ovrisc.mdche.u-szeged.hu, kele@ovrisc.mdche.u-szeged.hu,
1
2
3
kovacs@ovrisc.mdche.u-szeged.hu
b
Department of Organic Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary;
e-mail: pforgo@chem.u-szeged.hu
Received July 29, 2004
Accepted October 8, 2004
Guan in e derivatives substituted at N7 with 4-R-ben zyl groups (R = H, MeO, NO2) h ave been
evaluated in th e regioselective N9-alkylation of guan in e. Given th e capricious rem oval of
(substituted) ben zyl groups from guan in e derivatives an d pen t-4-en oylation of guan in ium
h ydroch loride, an im proved altern ative approach h as been elaborated con sistin g in th e
pen t-4-en oylation an d per-O-acetylation of guan osin e (8), 4-n itroben zylation at N7 followed
by N-glycoside h ydrolysis (10), N9-alkylation (13) an d deprotection with sodium dith ion ite
to afford th e peptide n ucleic acid buildin g block tert-butyl [N2-(pen t-4-en oyl)guan in -9-yl]-
acetate (15) in 36% overall yield. Th is avoids N7 regioisom er form ation , solubility problem s
an d an y ch rom atograph ic purification . A rem arkable in fluen ce of th e O- an d/or N2-acyl
groups on th e stability of N-glycosidic bon d an d reactivity of 2-am in o group was observed.
Th e structure of a pyrim idin e by-product 12 arisin g from th e im idazole rin g-open in g of
guan in ium salt 4d in alkalin e m edium h as been elucidated by 2D NMR.
Keyw ord s: Guan in e; Regioselective alkylation ; Guan osin e; Nucleoside an alogues; Nucleo-
sides; Purin es; Peptide n ucleic acids.
Th e regioselective alkylation of guan in e at N9 is an im portan t syn th etic
route to ph arm aceutically im portan t acyclic n ucleoside an alogues (e.g.
acyclovir, gan ciclovir, HPMPG) an d m on om eric buildin g blocks of oligo-
n ucleotide an alogues (e.g. peptide n ucleic acids, PNA); h owever th ese reac-
tion s are rarely regiospecific, leadin g to m ixtures of 9- an d 7-alkylated prod-
ucts wh ich can be very difficult to separate1.
Th e regioselectivity an d yield in th e syn th esis of 7- or 9-substituted
guan osin e an alogues can be h igh in th e alkylation of persilylated guan in e
derivatives e.g. O6-(N,N-diph en ylcarbam oyl)-N2-isobutyrylguan in e with
β-O-activated alkylation reagen ts1,2 e.g. peracylated sugars3. 9-Alkoxyalkyl-
Collect. Czech. Chem. Commun. (Vol. 70) (2005)
doi:10.1135/cccc20050085

86
Ferenc, Forgó, Kele, Kovács:
ated products can be obtain ed in h igh yields even if an N9/N7 isom eric m ix-
ture is form ed wh ile th e N7 to N9 rearran gem en t takes place upon h eat-
in g4–10 or even at room tem perature in DMF 11
.
Th e regioselectivity of th e alkylation with n on -β-O-activated, sm all-sized
alkyl h alides (e.g. alkyl h aloacetates in th e syn th esis of PNA m on om ers)
un der basic con dition s (e.g. K2CO3) is still in adequate. Con strain in g gua-
n in e in to its lactim form e.g. in th e case of 2-am in o-6-ch loropurin e12–17
,
2-am in o-6-(arylsulfan yl)purin es18 or O6-(N,N-diph en ylcarbam oyl) deriva-
tives19 im proves th e N9/N7 isom er ratio, but it is n ot sufficien t requirem en t
sin ce th e h igh est yield of N9-isom er was aroun d 75% an d ch rom atograph ic
purification could n ot be avoided in every case, furth erm ore 2-am in o-
6-ch loropurin e is m utagen ic an d expen sive, 2-am in o-6-(arylsulfan yl)pur-
in es require a stron g acidic treatm en t, th e N,N-diph en ylcarbam oyl group
can be labile20,21 an d th is im poses lim itation s on its applicability. Th ere is a
th ird approach , h owever, affordin g exclusively 9-alkylated derivatives.
Izawa et al. reported an N9-regioselective substitution startin g from guan o-
sin e22,23. In th eir approach guan osin e was protected on N7 with a (substi-
tuted) ben zyl group; th en , after acid h ydrolysis of N-glycosidic bon d an d
O
O
N
NH
N
NH
HOH2C
N
N
NH2
O
N
N
NHR2
R3
HO OH
a
e
R1
N
O
N
R1
N
O
NH
NHR2
NH
N
N
N
NH2
R3
X
β-D-Rf
X
b
d
R1
N
O
R1
N
O
N
· HCl
NH
c
NH
NHR2
N
N
N
NH2
SCHEME 1
Th e reaction sequen ce followed by Izawa et al.22,23 Reagen ts an d con dition s: a R1X, b HCl,
c R2X, d R3X, e rem oval of R1. β-D-Rf = β-D-ribofuran osyl, R1 = substituted ben zyl, R2 = acyl,
R3 = alkyl
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Alkyl 2-(Guanin-9-yl)acetates
87
acylation of th e am in o group, 7-substituted ben zyl-N2-acylguan in es were
alkylated selectively at N9, th en deprotected at N7 in a good overall yield
(ca. 80%, Sch em e 1). In th is case th e (substituted) ben zyl is n ot on ly a pro-
tective group but it activates th e purin e rin g in alkylation 22,23
.
RESULTS AND DISCUSSION
Th e problem of th e regioselectivity of th e alkylation of guan in e derivatives
was presen t in th e reaction of tert-butyl brom oacetate with N2-isobutyryl-
guan in e an d O6-(N,N-diph en ylcarbam oyl)-N2-isobutyrylguan in e as th e first
step in th e syn th esis of Fm oc/acyl-protected PNA m on om ers24. In our in i-
tial study th e Izawa m eth od h as been adopted for th e regioselective N9-
alkylation of guan in e. Th us, 7-ben zylguan in e h ydroch loride25 (2a), avail-
able from guan osin e (1) in a on e-pot procedure, was acylated with iso-
butyric an h ydride an d th e resultin g com poun d 3a was allowed to react
with tert-butyl brom oacetate (Sch em e 2). Th e guan in ium salt 4a was sub-
jected to h ydrogen olysis to rem ove th e ben zyl group givin g rise to ester 5a.
All th e steps afforded crystallin e com poun ds, th ere was n o n eed for ch ro-
m atograph ic purification an d th e overall yield of th e four-step process,
startin g from guan osin e, was 37%. However, th e last step often proved to
be capricious an d irreproducible. Th e n otorious beh avior of ben zylated
purin es un der h ydrogen olysis is well docum en ted26 an d altern ative N-
deben zylation with th e potassium tert-butoxide–dim eth yl sufoxide–oxygen
system is n ot com patible with th e vuln erable, base-sen sitive groups27,28
Con sequen tly, we decided to aban don th e ben zyl group.
.
Th e 4-m eth oxyben zyl (PMB) group is kn own to be cleaved from eth ers or
am in es un der oxidative con dition s29 th us th e above reaction was repeated
(Sch em e 2) em ployin g th is group with th e on ly differen ce bein g th e appli-
cation of m eth yl brom oacetate as alkylatin g agen t (th e correspon din g
tert-butyl ester was n ot crystallin e). Th e in term ediates 2b–4b were again
n ice crystallin e com poun ds an d th e tran sform ation s were un even tful un til
th e last step. Th e rem oval of th e PMB group from 4b un der oxidative
(cerium am m on ium n itrate30, DDQ 31, potassium peroxodisulfate32),
catalytic33 or tran sfer h ydrogen olytic34 or acid (AlCl3-an isole35,36, CBr4-
m eth an ol37) con dition s h as n ot been successful. Th e PMB group in th e
guan in ium m oiety of com poun d 4b proved to be very resistan t to th e
above reagen ts.
Th e reductive deprotection of 4-n itroben zyl (PNB) group is a well-
establish ed m eth od, th erefore we h ave em barked upon th e syn th esis an d
use of 7-(4-n itroben zyl)guan in es. Th e reaction sequen ce carried out with
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

88
Ferenc, Forgó, Kele, Kovács:
ben zyl an d PMB groups h as been repeated with 4-n itroben zyl brom ide an d
stan dard tran sform ation s led to guan in ium salt 4c (Sch em e 2). Th e rem oval
of PNB group h as been accom plish ed with Zn /AcOH or In /AcOH an d ester
5a h as been obtain ed from guan osin e in 38–40% overall yield; h owever,
th e last step required ch rom atograph ic purification an d rem oval of zin c
acetate an d th e yellow by-product was cum bersom e.
In our experien ce th e rem oval of guan in e isobutyryl group was n ot quan -
titative in th e cleavage an d deprotection of PNA oligom ers38. Th erefore a
ch an ge in th e protective group was n eeded. Pen t-4-en oyl group seem s to be
a better ch oice sin ce it can be rem oved, in addition to th e con ven tion al
am m on ia, with iodin e as well39–41
.
O
N
N
N
NH
NH2
a
HOH2C
O
70-97%
1
HO OH
R1
N
R1
N
O
N
O
NH
b
NH
c
· HCl
NH2
71-93%
71-82%
N
N
N
NHIbu
R1
2a Bn
R1
3a Bn
2b PMB
2c PNB
3b PMB
3c PNB
R1
O
O
N
N
N
d
44-60%
via 4c
NH
NH
NHIbu
N
N
N
O
NHIbu
O
X
R2O
R2O
R2
R1
4a Bn
4b PMB Me
R2
t-Bu Br
Br
X
5a t-Bu
5b Me
4c PNB t-Bu Br
4d PNB t-Bu PF6
PMB = 4-methoxybenzyl
PNB = 4-nitrobenzyl
SCHEME 2
Reagents and conditions: a 1. R1Br, DMSO; 2. HCl, then MeOH. b Ibu2O, Et3N, DMF, 150 °C, 3 h.
c BrCH2COOR2, DMF, 60 °C, 24 h . d Zn , aq. AcOH, r.t., 18 h
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Alkyl 2-(Guanin-9-yl)acetates
89
7-(4-Nitroben zyl)guan in e h ydroch loride (2c) did n ot dissolve well durin g
th e pen t-4-en oylation , h en ce an oth er route was n eeded to protect th e
2-am in o group. Con siderin g th e h igh price of pen t-4-en oic an h ydride, th e
reactivity of th e am in o group an d th e solubility of com poun ds th e se-
quen ce N2-acylation , N7-protection , N-glycoside h ydrolysis, N9-alkylation
seem ed to be th e on ly good ch oice.
N2-(Pen t-4-en oyl)guan osin e (6) was obtain ed in a h igh yield by tem po-
rary protection of h ydroxy groups with th e electron -don atin g trim eth ylsilyl
group40,42. Th e 7,9-bis(4-n itroben zyl)guan in ium salt 11, detected in th e re-
action m ixture by TLC/MS, was th e product in subsequen t reaction with
4-n itroben zyl brom ide (Sch em e 3), 7-(4-n itroben zyl)-N2-(pen t-4-en oyl)gua-
n in e (10) could n ot even be detected. Th e form ation of 7,9-bis(aryl-
m eth yl)guan in es un der forced con dition s (elevated tem peratures an d pro-
lon ged reaction tim es) h as been reported43
.
En h an cin g th e stability of th e N-glycosidic bon d is im perative for th e
m on oalkylation of guan osin e derivatives by in troduction of electron -
with drawin g, e.g. acetyl groups to th e h ydroxy groups of th e sacch aride
m oiety. Pen t-4-en oylation of 2′,3′,5′-tri-O-acetylguan osin e was a slow an d
low-yieldin g process even at h igh er tem perature, th erefore 2′,3′,5′-tri-O-
O
O
PNB
N
N
NH
NHR2
NH
NHPnt
b
78%
via 9
N
R1
N
R1
N
N
Br
R1
R2
R1
1
6
8
β-D-Rf
β-D-Rf
H
7
β-D-Ribf
a
c
Pnt
Pnt
9 Ac3-β-D-Rf
Ac3-β-D-Rf
O
O
PNB
PNB
N
N
NH
NHPnt
b
NH
NHPnt
N
N
N
N
PNB Br
10
11
SCHEME 3
Reagen ts an d con dition s: a 1. TMSCl, pyridin e, r.t., 1 h ; 2. pen t-4-en oic an h ydride, pyridin e,
r.t., 16 h ; 3. water, 0–5 °C, 5 m in ; 4. aq. NH3, r.t., 30 m in . b 1–10 equiv. 4-n itroben zyl bro-
m ide, DMF, r.t., up to 6 days, see th e text. c Ac2O, pyridin e, DMF, r.t., 16 h . a + c 71%. β-D-Rf =
β-D-ribofuran osyl, Ac3-β-D-Rf = 2′,3′,5′-tri-O-acetyl-β-D-ribofuran osyl
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

90
Ferenc, Forgó, Kele, Kovács:
acetyl-N2-(pen t-4-en oyl)guan osin e (8) was syn th esized by acetylation of
N2-(pen t-4-en oyl)guan osin e (6). Th e form ation of guan in ium salt 9 was
slow but com poun d 11 form ed on ly in a n egligible am oun t. Th e reaction
was com plete after 2.5 days with an usual 4-equivalen t excess of th e re-
agen t. Th e excess of 4-n itroben zyl brom ide was scaven ged by pyridin e in
order to avoid th e form ation of th e dialkylated product 11 durin g evapora-
tion of th e solven t (DMF). 4-Nitroben zylpyridin ium brom ide an d com -
poun d 10 was separated with extraction after th erm olysis of th e N-
glycoside takin g place at 70 °C with out acid treatm en t.
Th e exten t of th e in fluen ce of th e O- an d/or N2-acyl groups on th e stabil-
ity of N-glycosidic bon d an d th e reactivity of N2 was n otable in th e above
reaction s. 7-(4-Nitroben zyl)guan osin ium ion was stable but it decom posed
spon tan eously in th e absen ce of acid after acylation on N2. Acetylation of
h ydroxy groups in com poun d 7 stabilized th e N-glycosidic bon d at room
tem perature but th erm olysis took place with out acid at 70 °C. Th e acetyl
groups in 2′,3′,5′-tri-O-acetylguan osin e with drew th e electron den sity of
th e purin e rin g an d, at th e sam e tim e, from th e 2-am in o group to such an
exten t th at th e acylation was n ot com plete even after a prolon ged tim e at
100 °C. On th e oth er h an d, trim eth ylsilyl group activated th e 2-am in o
group an d th e acylation took place sm ooth ly.
Alkylation of com poun d 10 affordin g guan in ium salt 13 (Sch em e 4), with
3 equivalen ts of tert-butyl brom oacetate at 70 °C was com plete overn igh t.
O2N
H2N
O
O
N
N
N
N
a
c
NH
NHPnt
b
NH
NHPnt
10
86%
N
N
O
O
Br
Br
t-BuO
t-BuO
13
14
O
N
N
NH
NHPnt
+
HN
CH2
N
O
16
t-BuO
15
SCHEME 4
Reagen ts an d con dition s: a 3 equiv. tert-butyl brom oacetate, DMF, 70 °C, 16 h ; b 4 equiv.
Na2S2O4, aq. aceton e, pH 7.0, r.t., 30 m in ; c 70 °C, 16 h ; b + c 76%
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Alkyl 2-(Guanin-9-yl)acetates
91
As m en tion ed earlier, th e purification of ester 5a after PNB rem oval with
Zn /acetic acid was too laborious, th erefore oth er reducin g agen ts h ave been
studied.
Sodium sulfide was successfully used for th e deprotection of 4-n itro-
ben zyl esters an d carbam ates44 an d our in itial studies were con ducted with
salt 4d . In th e sodium sulfide treatm en t, a n ew substan ce 16 (Sch em e 5)
em erged, th e polarity of wh ich was sim ilar to th e expected product 5a.
However, in its MS spectrum th e m olecular ion [M + H]+ was observed at
m/z 489 in stead of at th e expected value m/z 336. Im idazole rin g open in g is
kn own to occur in alkalin e solution as C-8 is electroph ilic in salts45 like 4d .
Th e exact position of form yl group (N4 or N5) h as n ot been ascertain ed
earlier46 or on ly un con vin cin gly proved47. Our 2D NMR m easurem en ts
(HSQC, HMBC) h ave un equivocally dem on strated (Fig. 1) th at th e form yl
group is located on N5 con firm in g th at th e en suin g rin g scission takes place
between C-8 an d N-9 atom s of guan in ium salt 4d . Com poun d 12 exists as a
7:1 m ixture of rotam ers at room tem perature in DMSO-d6 solution .
Reduction of th e n itro group in com poun d 13 was com plete with in
30 m in in th e presen ce of 4 equivalen ts of sodium dith ion ite even at pH 4,
n ot on ly at pH 8–9 wh ere deprotection of 4-n itroben zyloxycarbon yl group
was ach ieved44. To en h an ce th e rearran gem en t of 4-am in oben zyl group
leadin g to th e deprotection , th e reaction m ixture was h eated at 70 °C in a
ph osph ate buffer (pH 7) an d aceton e was em ployed to obtain a h om oge-
n ous solution (th e in term ediacy of 14 was verified by TLC/MS, Sch em e 4).
O
O
N
PNB
PNB
OH
N
N
NH
NHIbu
N
H
H
O
HO
N
N
N
NHIbu
H
O
O
PF6
4d
Ot-Bu
Ot-Bu
O
O
N
PNB
N
PNB
O
O
N
O
N
NH
NHIbu
H
(E)
H
H
73%
HN
HN
N
NHIbu
O
Ot-Bu
Ot-Bu
12 E/Z
SCHEME 5
Im idazole rin g-open in g of guan in ium salt 4d in alkalin e solution
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

92
Ferenc, Forgó, Kele, Kovács:
Based on th e observation th at electron -don atin g substituen ts on ph en yl
rin g an d/or on α-carbon atom prom ote th e rearran gem en t by stabilizin g
th e positive ch arge on ben zyl m eth ylen e group in th e rem oval of 4-n itro-
ben zyl carbam ates48, it was n ot suprisin g th at h eatin g of th e reaction m ix-
ture was required for th e deprotection as, in our case, a positively ch arged
substituen t was attach ed to th e ben zyl m eth ylen e group decreasin g th e sta-
bility of th e form ed cation .
Im in oquin om eth an e 16 was th e elusive by-product of th is deprotection
regim e. Th is com poun d h as n ever been isolated in pure form due to its
in stability an d ten den cy to polym erize48–50. We h ave been able to detect
FIG. 1
HMBC spectrum (detail) of com poun d 12. Crucial correlation s are h igh ligh ted by boxes. For
pyrim idin e n um berin g, see Sch em e 5, prim ed n um bers den ote atom s in th e ben zen e rin g.
Som e sign als are doubled in th e m ajor (*) an d m in or (+) rotam ers, e.g. th e form yl proton s at
7.99 (*) an d 8.38 ppm (+), respectively
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Alkyl 2-(Guanin-9-yl)acetates
93
in th e ESI m ass spectrum peaks of th is substan ce at m/z 106 an d m/z 138
correspon din g to ion s [M + H]+ an d [M + MeOH + H]+, respectively, but our
furth er attem pts to ch aracterize th is com poun d h ave n ot been rewardin g.
Th e presen ce of th is substan ce as a con tam in an t was obvious in both th e
yellow-colored aqueous an d th e organ ic ph ases. It was attem pted to scav-
en ge th e substan ce with sulfites49 (oxidized form of dith ion ite), h owever,
it was im possible to com pletely rem ove th is substan ce from th e product.
Th e sligh tly yellowish am orph ous product 15 was easily obtain ed by wash -
in g with eth yl acetate; it was com pletely pure accordin g to TLC an d NMR
an alyses.
Th e site of th e alkylation (N9 vs N7) in guan in e derivatives can be un equi-
vocally ascertain ed by 1H 18,51,52 13C 24,51,52 an d 15N NMR 53 or MS/MS 54
,
m eth ods. In th is study our 13C NMR m eth od24 was used. Th e structures of
FIG. 2
HMBC spectrum (detail) of com poun d 4a. Crucial correlation s are h igh ligh ted by boxes. For
purin e n um berin g, see Sch em e 1
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

94
Ferenc, Forgó, Kele, Kovács:
com poun ds 3, 10 an d 15 were corroborated by th e ch em ical sh ift differen ce
param eters a = δC-4 – δC-5, b = δC-8 – δC-5 an d c = δC-5 – δC-1′, in volvin g C-1′,
C-4, C-5 an d C-8 wh ich are th e m ost sen sitive to th e site of alkylation (data
n ot sh own ). It is n oteworth y th at th e guan in ium salts display an un usually
1
large h eteron uclear couplin g con stan t between H-8 an d C-8 (e.g. JH-8,C-8
=
226 (4a) an d 227 Hz (4c)), wh ich in dicates th e im idazolium substruc-
ture55–57. Th e structure of com poun d 4a h as been corroborated by HSQC
an d HMBC in vestigation s as well (Fig. 2).
In our study directed to 9-substituted guan in es, th e application of gua-
n in e derivatives substituted at N7 with differen t ben zyl groups (ben zyl,
4-m eth oxyben zyl, 4-n itroben zyl) h as been evaluated. Th e gen eral sequen ce
exem plified in Sch em es 1 an d 2 proved to be problem atic in term s of easy
an d reproducible rem oval of protectin g group at N7 an d acylation of com -
poun ds 2 with pen t-4-en oic an h ydride. Our im proved altern ative approach
described in th is paper com prises th e followin g steps: (i) pen t-4-en oylation
of guan osin e at N2 (6), (ii) per-O-acetylation (8), (iii) 4-n itroben zylation at
N7 (9), (iv) h ydrolysis of th e N-glycoside 9 (10), (v) N9-alkylation (13) an d
(vi) deprotection with sodium dith ion ite. Th is seem in gly len gth y procedure
can be com bin ed in to four distin ct, well-reproducible steps with stan dard
tran sform ation s affordin g th e PNA buildin g block tert-butyl [N2-(pen t-
O
O
PNB
N
N
R1
N
NH
NHR2
NH
NHPnt
c
b
86%
78%
N
N
N
R1
β-D-Rf
R2
H
10
a,
71%
1
8
Ac3-β-D-Rf
Pnt
O
O
N
PNB
N
d
N
NH
NHPnt
NH
NHPnt
76%
N
N
N
Br
13
O
O
t-BuO
t-BuO
15
SCHEME 6
Reagen ts an d con dition s: a 1. TMSCl, pyridin e, r.t., 1 h ; 2. 1.25 equiv. pen t-4-en oic an -
h ydride, pyridin e, r.t., 16 h ; 3. water, 0–5 °C, 5 m in ; 4. aq. NH3, r.t., 30 m in ; 5. Ac2O, pyridin e,
DMF, r.t., 16 h . b 4 equiv. 4-n itroben zyl brom ide, DMF, r.t., 60 h . c 3 equiv. tert-butyl
brom oacetate, DMF, 70 °C, 16 h. d 1. 4 equiv. Na2S2O4, aq. aceton e, pH 7.0, r.t., 30 m in ; 2. 70 °C,
16 h . β-D-Rf = β-D-ribofuran osyl, Ac3-β-D-Rf = 2′,3′,5′-tri-O-acetyl-β-D-ribofuran osyl
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Alkyl 2-(Guanin-9-yl)acetates
95
4-en oyl)guan in -9-yl]acetate (15) with n o N7 regioisom er form ation , solubil-
ity problem s or ch rom atograph ic purification (Sch em e 6) with th e sam e
36% overall yield as in th e case of Izawa et al. m eth od. Main advan tages of
our syn th esis are th e acylation of N2 am in o group at room tem perature due
to th e h igh solubility of th e startin g com poun d an d a reproducible rem oval
of th e 4-n itroben zyl group. Attem pts at th e con version of 15 to a guan in e
PNA m on om er are un der way. Th e exten t of th e in fluen ce of th e O- an d/or
N2-acyl groups on th e stability of N-glycosidic bon d an d reactivity of
2-am in o group is rem arkable. Th e site of th e alkylation in 7- an d 9-alkyl-
ated guan in e derivatives h as been corroborated by ch em ical sh ift differ-
en ces of 13C NMR spectra. Position of th e form yl group (N5) in th e rin g-
open ed derivative 12 form ed from guan in ium salt 4d un der alkalin e con di-
tion s (Sch em e 5) h as been un ravelled by 2D NMR.
EXPERIMENTAL
Th e followin g abbreviation s are em ployed: Bn (ben zyl), t-Bu (tert-butyl), TMSCl (ch loro-
trim eth ylsilan e), DDQ (2,3-dich loro-5,6-dicyan o-1,4-ben zoquin on e), Et2O (dieth yl eth er),
DMF (N,N-dim eth ylform am ide), DMSO (dim eth yl sulfoxide), ESI (electrospray ion ization ),
EtOAc (eth yl acetate), Fm oc (fluoren -9-ylm eth oxycarbon yl), Ibu (isobutyryl), MeOH (m eth a-
n ol), PMB (4-m eth oxyben zyl), PNB (4-n itroben zyl), Pn t (pen t-4-en oyl), PNA (peptide n ucleic
acid(s)), r.t. (room tem perature), TEA (trieth ylam in e).
Ch em icals were purch ased from Aldrich , Fluka, Merck or Rean al (Budapest, Hun gary).
Pen t-4-en oic an h ydride was altern atively syn th esized from pen t-4-en oic acid as described in
literature58,59. Com poun d 2a was prepared as described25. Com poun ds 2b an d 2c were pre-
pared in an an alogous way60. 4-Meth oxyben zyl brom ide is pron e to decom position an d it
was fresh ly prepared prior to use from th e correspon din g alcoh ol61. An h ydrous solven ts
were prepared as described62. Organ ic solution s were dried usin g an h ydrous MgSO4 an d
evaporated in Büch i rotary evaporators. TLC: Kieselgel 60 F254 (Merck); solven t system s:
CH2Cl2–MeOH 9:1 (S1), CH2Cl2–MeOH 95:5 (S2), CH2Cl2–iPrOH 5:0.25 (S3); visualization :
UV ligh t, H2SO4/eth an ol. All guan in ium salts gave oval spots with sign ifican t tailin g. M.p.:
Electroth erm al IA 8103 apparatus. Elem en tal an alysis: Perkin –Elm er CHN an alyzer m odel 2400.
UV: PE Lam bda 10 spectrom eter, λm ax in n m (log ε); sh , sh oulder. Stock solution s were m ade
in eth an ol except of com poun ds 3b an d 10, in wh ich cases addin g DMSO was n ecessary to
ach ieve clear solution s. In th e case of latter sam ples an d TEAc buffer th e region below
220 n m in UV spectra was un certain , because th ese com poun ds h ave h igh absorban ce in
th is region . NMR: Bruker Avan ce DRX 500 spectrom eter (1H: 500.13 MHz, 13C: 125.76 MHz),
DMSO-d6 solution s; δ (ppm ), J (Hz). Spectral pattern s: s, sin glet; d, doublet; dd, double dou-
blet; t, triplet; m , m ultiplet; br, broad; deut, deuterable. Th e superscripts *, # den ote in ter-
ch an geable assign m en ts. For th e 2D experim en ts (HMQC, HMBC), stan dard Bruker software
packages (INV4GSSW, INV4GSLRNDSW) were used. Mass spectrom etry: Fin n igan MAT TSQ
7000, electrospray (ESI) tech n ique. TLC/MS: th e an alyte solution h as been applied on to a
5 cm wide silica gel TLC plate as a ban d to obtain sufficien t m aterial. After developin g in a
solven t system , th e appropriate ban d was scraped off with a spatula, th e silica gel was
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

96
Ferenc, Forgó, Kele, Kovács:
suspen ded in MeOH (100 µl), son icated, cen trifuged an d th e supern atan t was used for MS
an alysis.
7-Ben zyl-N2-isobutyrylguan in e (3a)
Com poun d25 2a (8.393 g, 30.2 m m ol) suspen ded in an h ydrous DMF (90 m l) was treated
with isobutyric an h ydride (12.50 m l, 75.5 m m ol) an d TEA (8.43 m l, 60.4 m m ol) an d th e
m ixture was h eated with stirrin g at 150 °C for 3 h . Th e h om ogen eous solution was evapo-
rated in vacuo, coevaporated with MeOH (2×), triturated un der cold water, filtered an d
dried. Th e crude product (8.913 g) was boiled with eth an ol (200 m l), th e obtain ed solution
filtered an d th e filtrate was evaporated to dryn ess. Trituration un der cold eth er gave th e title
com poun d (6.663 g, 70.8%) as beige crystals, m .p. 176–178 °C. RF 0.65 (S1). For C16H17N5O2
(311.3) calculated: 61.7% C, 5.5% H, 22.5% N; foun d: 61.55% C, 5.4% H, 22.7% N. UV: λm ax
(50% (v/v) 1 M HCl in EtOH, pH 0) 202 sh (4.22), 267 (3.98); λm ax (50% (v/v) 1 M TEAc in
EtOH, pH 7) 267 (3.97); λm ax (50% (v/v) 0.1 M KOH in EtOH, pH 13) 272 (3.82). 1H NMR:
1.12 d, 6 H, J = 6.8 ((CH3)2CH); 2.75 pseudo t, 1 H, J = 6.8 ((CH3)2CH); 5.52 s, 2 H (CH2);
7.29–7.34 m , 5 H (C6H5); 8.33 s, 1 H (H-8); 11.49 s, 1 H (NH); 12.13 s, 1 H (NH). 13C NMR
(J-m od. spin -ech o): 18.65 ((CH3)2CH); 34.52 ((CH3)2CH); 49.08 (Ph CH2); 110.99 (C-5);
127.32, 127.64, 128.45 (arom . CHs); 137.09 (arom . Cq); 144.08 (C-8); 147.00, 152.49, 157.07
(C-2, C-4, C-6); 179.75 (iPrCO). MS (ESI), m/z (rel.%): 623 (30) [2 M + H]+), 312 (100) [M + H]+.
N2-Isobutyryl-7-(4-m eth oxyben zyl)guan in e (3b)
Prepared from 7-(4-m eth oxyben zyl)guan in e h ydroch loride60 (2b ; 7.8 m m ol scale) as de-
scribed for com poun d 3a (1.989 g, 74.7%). An an alytical sam ple was obtain ed by recrystal-
lizin g th e product from aceton itrile (4.6 g/100 m l), m .p. 185.9–186.6 °C (aceton itrile).
RF 0.48 (S1). For C17H19N5O3 (341.4) calculated: 59.8% C, 5.6% H, 20.5% N; foun d:
59.6% C, 5.65% H, 20.3% N. UV: λm ax (50% (v/v) 1 M HCl in EtOH, pH 0) 223 (4.13), 267
(4.08); λm ax (50% (v/v) 1 M TEAc in EtOH, pH 7) 267 (4.00); λm ax (50% (v/v) 0.1 M KOH in
EtOH, pH 13) 271 (3.90). 1H NMR: 1.10 d, 6 H, J = 6.8 ((CH3)2CH); 2.74 pseudo t, 1 H, J =
6.8 ((CH3)2CH); 3.71 s, 3 H (CH3O); 5.42 s, 2 H (CH2); 6.88 d, 2 H, J = 8.5 (ArH); 7.33 d, 2 H,
J = 8.5 (ArH); 8.31 s, 1 H (H-8); 11.51 s, 1 H (NH); 12.13 s, 1 H (NH). 13C NMR (J-m odulated
spin -ech o): 18.75 ((CH3)2CH); 34.60 ((CH3)2CH); 48.66 (ArCH2); 54.98 (CH3O); 110.92 (C-5);
113.92 (C-3′, C-5′); 129.12, 129.17 (C-2′, C-6′, C-1′); 143.97 (C-8); 147.02, 152.57, 157.18,
158.86 (C-2, C-4, C-6, C-4′); 179.84 (iPrCO). MS (ESI), m/z (rel.%): 683 (32) [2 M + H]+, 342
(100) [M + H]+.
N2-Isobutyryl-7-(4-n itroben zyl)guan in e (3c)
Prepared from 7-(4-n itroben zyl)guan in e h ydroch loride60 (2c; 8.6 m m ol scale) as described
for com poun d 3a (2.512 g, 81.8%). Th e substan ce was sufficien tly pure for furth er tran sfor-
m ation s, h owever, it can be recrystallized from MeOH (2.0 g/150 m l), m .p. > 260 °C. RF 0.68
(S1). For C16H16N6O4 (356.3) calculated: 53.9% C, 4.5% H, 23.6% N; foun d: 53.75% C, 4.6% H,
23.4% N. UV: λm ax (50% (v/v) 1 M HCl in EtOH, pH 0) 201 (4.22), 218 (4.05), 267 (4.11);
λm ax (50% (v/v) 1 M TEAc in EtOH, pH 7) 267 (4.15); λm ax (50% (v/v) 0.1 M KOH in EtOH,
pH 13) 272 (4.11). 1H NMR: 1.10 d, 6 H, J = 6.7 ((CH3)2CH); 2.72 pseudo t, 1 H, J = 6.7
((CH3)2CH); 5.66 s, 2 H (CH2); 7.53 d, 2 H, J = 8.3 (ArH); 8.18 d, 2 H, J = 8.3 (ArH); 8.38 s,
1 H (H-8); 11.55 s, 1 H (NH); 12.13 s, 1 H (NH). 13C NMR (J-m odulated spin -ech o): 18.77
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Alkyl 2-(Guanin-9-yl)acetates
97
((CH3)2CH); 34.64 ((CH3)2CH); 48.61 (ArCH2); 111.06 (C-5); 123.72, 128.43 (arom . CHs);
144.51 (C-8); 144.64, 146.97, 147.29, 152.51, 157.36 (C-2, C-4, C-6, C-1′, C-4′); 179.92
(iPrCO). MS (ESI), m/z (rel.%): 713 (5) [2 M + H]+, 357 (100) [M + H]+.
7-Ben zyl-9-[(tert-butoxycarbon yl)m eth yl]-N2-isobutyrylguan in ium Brom ide (4a)
Com poun d 3a (1.557 g, 5.0 m m ol) dissolved in warm DMF (20 m l) was allowed to react
with tert-butyl brom oacetate (0.813 m l, 5.5 m m ol) at 60 °C for 24 h . Th e solution was evap-
orated in vacuo, coevaporated with EtOAc (3×) an d EtOAc was added (20 m l). Th e resultin g
oil slowly crystallized in a refrigerator (1.801 g, 71%), m .p. 158 °C (dec., EtOAc). RF ca. 0.2
(S1). For C22H28BrN5O4 (506.4) calculated: 52.2% C, 5.6% H, 13.8% N; foun d: 52.0% C,
5.7% H, 13.65% N. UV: λm ax (50% (v/v) 1 M HCl in EtOH, pH 0) 201 (4.26), 271 (3.97); λm ax
(50% (v/v) 1 M TEAc in EtOH, pH 7) 278 (3.70); λm ax (50% (v/v) 0.1 M KOH in EtOH, pH 13)
243 (3.96). 1H NMR: 1.12 d, 6 H, J = 6.9 ((CH3)2CH); 1.44 s, 9 H (t-Bu); 4.02 pseudo t, 1 H,
J = 6.9 ((CH3)2CH); 5.19 s, 2 H (CH2COO); 5.82 s, 2 H (Ph CH2); 7.39–7.50 m , 5 H (C6H5);
9.87 s, 1 H (H-8); 12.10 br s, 1 H (NH); 12.70 s, 1 H (NH). 13C NMR (J-m odulated spin -ech o,
HSQC, HMBC experim en ts): 18.56 ((CH3)2CH); 27.48 ((CH3)3C); 34.72 ((CH3)2CH); 46.40
(CH2COO); 51.65 (Ph CH2); 83.57 ((CH3)3C); 109.61 (C-5); 128.16, 128.80 (arom . CHs);
134.04 (arom . Cq); 140.28 (C-8); 147.55 (C-4); 151.13 (C-2, C-6); 164.62 (COO); 180.52
(iPrCO). MS (ESI), m/z (rel.%): 426 (100) M+, guan in ium ion .
N2-Isobutyryl-7-(4-m eth oxyben zyl)-9-[(m eth oxycarbon yl)m eth yl]guan in ium Brom ide (4b)
Prepared from com poun d 3b (5.33 m m ol) as described for com poun d 4a (2.382 g, 90%),
m .p. 147.3–150.6 °C (EtOAc). RF ca. 0.2 (S1). For C20H24BrN5O5 (494.3) calculated: 48.6% C,
4.9% H, 14.2% N; foun d: 48.55% C, 5.0% H, 14.35% N. UV: λm ax (50% (v/v) 1 M HCl in
EtOH, pH 0) 200 sh (4.45), 270 (4.02); λm ax (50% (v/v) 1 M TEAc in EtOH, pH 7) 273 (3.83);
λm ax (50% (v/v) 0.1 M KOH in EtOH, pH 13) 273 (3.80). 1H NMR: 1.11 d, 6 H, J = 6.7
((CH3)2CH); 2.80 pseudoquin tet, 1 H, J = 6.7 ((CH3)2CH); 3.74 an d 3.76 each s, 6 H 2 ×
(CH3O); 5.30 s, 2 H (CH2COO); 5.72 s, 2 H (ArCH2); 6.97 d, 2 H, J = 8.3 (ArH); 7.49 d, 2 H,
J = 8.3 (ArH); 9.77 s, 1 H (H-8); 12.05 br s, 1 H (NH); 12.60 s, 1 H (NH). 13C NMR (J-m odu-
lated spin -ech o): 18.50 ((CH3)2CH); 34.70 ((CH3)2CH); 45.89 (CH2COO); 51.36 (ArCH2);
52.98, 55.13 (2 × CH3O); 109.63 (C-5); 114.21, 130.19 (arom . CHs); 125.56 (C-1′*); 141.03
(C-8); 147.60 (C-4′*); 151.04, 151.10, 159.64 (C-2, C-4, C-6); 166.23 (COO); 180.49 (iPrCO).
MS (ESI), m/z (rel.%): 414 (100) M+, guan in ium ion .
9-[(tert-Butoxycarbon yl)m eth yl]-N2-isobutyryl-7-(4-n itroben zyl)guan in ium Brom ide (4c)
an d Hexafluoroph osph ate (4d )
Prepared from com poun d 3c (10.0 m m ol) as described for com poun d 4a. Th e brom ide crys-
tallized very sluggish ly. It was very well soluble in EtOAc, th erefore a water-in soluble
h exafluoroph osph ate salt (4d ) was prepared as follows. Th e crude oily product from th e
above reaction was dissolved in aceton itrile (20 m l) an d am m on ium h exafluoroph osph ate
(1.793 g, 11.0 m m ol) in water (10 m l) was added. Th e solution was evaporated, th e residue
was triturated un der cold water an d th e precipitate was stored in vacuo over P2O5 (5.732 g,
93.0%). Th e title h exafluoroph osph ate dissolves well in cold MeOH, EtOAc, CH2Cl2 an d
aceton itrile but n ot in Et2O or water. Th e salt can be precipitated from aceton itrile solution
by addin g water. M.p. 126.8–127.5 °C (aqueous aceton itrile). RF (4c) ca. 0.10 (S1). For
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

98
Ferenc, Forgó, Kele, Kovács:
C22H27F6N6O6P (4d ; 616.4) calculated: 42.9% C, 4.4% H, 13.6% N; foun d: 42.65% C,
4.35% H, 13.4% N. UV (4c): λm ax (50% (v/v) 1 M HCl in EtOH, pH 0) 200 sh (4.50), 270
(4.31); λm ax (50% (v/v) 1 M TEAc in EtOH, pH 7) 271 (4.08); λm ax (50% (v/v) 0.1 M KOH in
EtOH, pH 13) 245 (4.11), 269 (4.06). 1H NMR (4c): 1.10 d, 6 H, J = 6.7 ((CH3)2CH); 1.45 s,
9 H (t-Bu); 2.81 pseudoquin tet, 1 H, J = 6.7 ((CH3)2CH); 5.22 s, 2 H (CH2COO); 5.99 s, 2 H
(ArCH2); 7.73 d, 2 H, J = 8.3 (ArH); 8.24 d, 2 H, J = 8.3 (ArH); 9.95 s, 1 H (H-8); 12.10 br s,
deut, 1 H (NH); 12.50 br s, deut, 1 H (NH). 13C NMR (4c; J-m odulated spin -ech o): 18.61
((CH3)2CH); 27.55 ((CH3)3C); 34.77 ((CH3)2CH); 46.54 (CH2COO); 50.99 (ArCH2); 83.68
((CH3)3C); 109.72 (C-5); 123.76, 129.29 (arom . CHs); 141.42, 147.52, 147.68, 151.04, 151.21
(C-1′, C-4′, C-2, C-4, C-6, C-8); 164.64 (COO); 180.58 (iPrCO). MS (ESI) (4c), m/z (rel.%): 471
(100) M+, guan in ium ion , 941 (10) [2 M – H+]+.
tert-Butyl (N2-Isobutyrylguan in -9-yl)acetate24 (5a)
Com poun d 4c (0.240 g, 0.435 m m ol) was dissolved in 50% (v/v) acetic acid (5 m l), zin c
powder (0.178 g, 2.72 m m ol, 6.0 equivalen ts) was added an d th e m ixture was stirred at
room tem perature for 18 h . A furth er portion of zin c powder (0.090 g) was added an d stir-
rin g was con tin ued for 6 h . Th e zin c alm ost com pletely dissolved an d a yellow precipitate
was form ed. After filtration th rough a Hyflo bed an d th orough wash in g with aceton itrile,
th e solution was evaporated an d dissolved in a m ixture of dich lorom eth an e (25 m l) an d
water (25 m l). Th e aqueous ph ase was extracted with dich lorom eth an e (25 m l) an d th e com -
bin ed organ ic ph ases were wash ed with 0.05 M EDTA disodium salt (2 × 10 m l), dried an d
evaporated in vacuo (0.077 g). Th e Hyflo bed was wash ed again with aceton itrile an d a com -
bin ed crop of 0.130 g was obtain ed. Ch rom atograph y (CH2Cl2–MeOH 98:2 to 94:6) yielded
0.064 g (43.8%), m .p. 203 °C. RF 0.13 (S2). Th e NMR an d MS data of th e product were in
full agreem en t with th ose previously publish ed24. In an oth er experim en t after th e work-up
as above, th e resultin g red oil was coevaporated with aceton itrile (3×) an d th e yellow solid
obtain ed (0.82 g, 60%) sh owed a sin gle spot on TLC. Furth er trituration un der eth er yielded
a purer product (0.60 g, 44%).
2′,3′,5′-Tri-O-acetyl-N2-(pen t-4-en oyl)guan osin e (8)
Guan osin e h ydrate (8.8 g, 31.1 m m ol) was suspen ded in aceton itrile (2 × 100 m l) an d evap-
orated to dryn ess. Ch lorotrim eth ylsilan e (30 m l, 234 m m ol) was added dropwise (20 m in ) to
th e suspen sion of dried guan osin e in an h ydrous pyridin e (150 m l) an d stirred for an oth er
40 m in . Pen t-4-en oic an h ydride (7.10 m l, 38.9 m m ol, 1.25 equivalen ts) was added an d th e
reaction was stirred at room tem perature for 16 h . Th e cooled reaction m ixture was diluted
with water (30 m l) an d treated with am m on ia solution (30 m l, 25%) for 30 m in . Th e residue
was dissolved in water (400 m l) an d extracted with a m ixture of Et2O an d EtOAc (1:1 v/v,
400 m l). Th e water ph ase was evaporated, th en coevaporated with aceton itrile (2 × 300 m l)
an d used for th e n ext step with out furth er purification .
2-(Pen t-4-en oyl)guan osin e (6) was dissolved in a m ixture of DMF (44 m l) an d pyridin e
(22 m l). Acetic an h ydride (18 m l) was added to th e m ixture, th e pyridin ium salts from th e
previous step were filtered off an d th e solution was set aside for 16 h . Eth an ol (10 m l) was
added to th e solution , th en th e residue was dissolved in EtOAc (400 m l) an d extracted with
1
M h ydroch loric acid (2 × 300 m l) an d saturated NaHCO3 solution (2 × 300 m l). Evapora-
tion in vacuo after dryin g gave th e product as a wh ite solid foam (10.9 g, 71%). RF 0.30 (S2),
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

Alkyl 2-(Guanin-9-yl)acetates
99
0.45 (S3). UV: λm ax (50% (v/v) 1 M HCl in EtOH, pH 0) 202 (4.20), 260 (3.97), 281 (3.84);
λm ax (50% (v/v) 1 M TEAc in EtOH, pH 7) 260 (4.00), 281 (3.87); λm ax (50% (v/v) 0.1 M KOH
in EtOH, pH 13) 266 (3.97). 1H NMR: 2.03 s, 3 H (CH3CO); 2.04 s, 3 H (CH3CO); 2.11 s, 3 H
(CH3CO); 2.34 m , 2 H (CH2CH=CH2); 2.58 t, 2 H, J = 7.2 (CH2CO); 4.29 m , 1 H (H-4′);
4.37 m , 2 H (H-5′); 4.99 d, 1 H, J = 10.2 (cis-CH2=CH); 5.06 d, 1 H, J = 17.2 (trans-CH2=CH);
5.48 dd, 1 H, J = 5.7, J = 3.7 (H-3′); 5.81 m , 2 H (H-1′, CH2=CH); 6.08 d, 1 H, J = 6.3 (H-2′);
8.23 s, 1 H (H-8); 11.60 br s, 1 H (NH); 12.06 br s, 1 H (NH). 13C NMR (decoupled an d
J-m odulated spin -ech o spectra): 20.03, 20.25, 20.39 (3 × CH3CO); 28.07 (CH2CH=CH2);
35.10 (CH2CO); 63.00 (C-5′); 70.28 (C-3′*); 72.16 (C-2′*); 79.83 (C-4′*); 84.56 (C-1′); 115.54
(CH2=CH); 120.37 (C-5); 136.73 (CH2=CH); 137.74 (C-8); 148.06 (C-2#); 148.57 (C-6#);
154.65 (C-4); 169.15, 169.35, 169.99 (3 × CH3CO); 175.46 (C4H7CONH). MS (ESI), m/z
(rel.%): 492.16 (100) [M + H]+.
7-(4-Nitroben zyl)-N2-(pen t-4-en oyl)guan in e (10)
Com poun d 8 (4.9 g, 10.0 m m ol) dissolved in an h ydrous DMF (60 m l) an d 4-n itroben zyl
brom ide (8.6 g, 40.0 m m ol) were stirred at room tem perature for 60 h . Wh en th e reaction
was com plete, pyridin e (6.4 m l, 80 m m ol) was added to scaven ge excess of th e alkylation re-
agen t an d set aside for 5 h . Th e reaction m ixture was h eated at 70 °C for 16 h to th erm olyse
th e guan in ium salt 9. Th e solution was evaporated in vacuo an d EtOAc (400 m l) an d water
(400 m l) were added to th e oily residue. Th e product (2.3 g, 62%) was precipitated an d fil-
tered off. A furth er crop (0.6 g, 16%) was precipitated wh en th e residue from th e evaporated
organ ic ph ase was treated with CH2Cl2 (10 m l). Overall yield of 10: 2.9 g (78%), am orph ous
solid. RF 0.29 (S2), 0.22 (S3). UV: λm ax (50% (v/v) 1 M HCl in EtOH, pH 0) 267 (4.15); λm ax
(50% (v/v) 1 M TEAc in EtOH, pH 7) 267 (4.14); λm ax (50% (v/v) 0.1 M KOH in EtOH, pH 13)
272 (4.11). 1H NMR: 2.32 m , 2 H (CH2CH=CH2); 2.53 t, 2 H, J = 7.2 (CH2CO); 4.97 d, 1 H, J =
10.2 (cis-CH2=CH); 5.04 d, 1 H, J = 17.1 (trans-CH2=CH); 5.65 s, 2 H (ArCH2); 5.77–5.85 m ,
1 H (CH2=CHCH2); 7.52 d, 2 H, J = 8.5 (ArH); 8.17 d, 2 H, J = 8.5 (ArH); 8.37 s, 1 H (H-8);
11.57 br s, 1 H (NH); 12.08 br s, 1 H (NH). 13C NMR (decoupled an d J-m odulated spin -ech o
spectra): 28.13 (CH2=CHCH2); 34.96 (CH2CO); 48.60 (ArCH2); 111.08 (C-5); 115.49
(CH2=CH); 123.68, 128.44 (arom . CH); 136.73 (CH2=CH); 144.49, 144.54, 147.00, 152.47,
157.33 (C-8, C-1′, C-4′, C-2, C-6, C-4); 175.26 (C4H7CONH). MS (ESI), m/z (rel.%): 368.97
(100) [M + H]+.
tert-Butyl ({2-Isobutyram ido)-5-[N-(4-n itroben zyl)form am ido]-
6-oxo-1,6-dih ydropyrim idin -4-yl}am in o)acetate (12)
To com poun d 4d (0.308 g, 0.5 m m ol) dissolved in aceton e (3 m l) was added sodium sulfide
n on ah ydrate (0.48 g, 2.0 m m ol) in water (1 m l) an d th e m ixture was stirred at room tem -
perature for 2 h . Th e reaction m ixture was diluted with water (20 m l) an d extracted with
CH2Cl2 (3 × 10 m l), th e organ ic layer was dried an d evaporated (0.178 g, 73%), m .p. 191 °C
(dec.). RF 0.39 (S2). For C22H28N6O7 (488.5) calculated: 54.1% C, 5.8% H, 17.2% N; foun d:
53.9% C, 5.95% H, 16.95% N. UV: λm ax (50% (v/v) 1 M HCl in EtOH, pH 0) 201 (4.24), 232
(4.42), 276 (4.00); λm ax (50% (v/v) 1 M TEAc in EtOH, pH 7) 276 (3.94); λm ax (50% (v/v)
0.1 M KOH in EtOH, pH 13) 245 (4.09), 270 (4.02). 1H NMR (HSQC, HMBC, see Fig. 1, m ajor
rotam er): 1.06 d, 6 H, J = 6.5 ((CH3)2CH); 1.37 s, 9 H (t-Bu); 2.73 pseudoquin tet, 1 H, J = 6.5
((CH3)2CH); 3.96 m , 2 H (CH2COO); 4.41, 4.97 2 × d, 2 × 1 H, J = 15.2 (ArCH2); 7.29 t, 1 H,
J = 6.0 (NH); 7.64 d, 2 H, J = 8.5 (ArH); 7.99 s, 1 H (HCO); 8.08 d, 2 H, J = 8.5 (ArH);
Collect. Czech. Chem. Commun. (Vol. 70) (2005)

100
Ferenc, Forgó, Kele, Kovács:
11.30 br s, 1 H (NH); 11.41 br s, 1 H (NH). 13C NMR (HSQC, HMBC, see Fig. 1, m ajor rota-
m er): 18.62 ((CH3)2CH); 27.65 ((CH3)3C); 34.58 ((CH3)2CH); 42.90 (CH2COO); 46.82
(ArCH2); 80.74 ((CH3)3C); 96.83 (C-5); 122.95 (C-3′, C-5′); 129.74 (C-2′, C-6′); 144.90 (C-1′);
146.60 (C-4′); 149.36 (C-2*); 158.15 (C-6*); 159.42 (C-4); 165.14 (HCO); 169.20 (COOt-Bu);
180.36 (iPrCO). MS (ESI), m/z (rel.%): 977.6 (48) [2 M + H]+, 489 (100) [M + H]+.
9-[(tert-Butoxycarbon yl)m eth yl-7-(4-n itroben zyl)-N2-(pen t-4-en oyl)guan in ium Brom ide (13)
tert-Butyl brom oacetate (1.2 m l, 8.1 m m ol) was added to a solution of com poun d 10 (1.0 g,
2.7 m m ol) in an h ydrous DMF (40 m l) an d h eated at 70 °C for 16 h . Th e residue obtain ed af-
ter evaporation was dissolved in a ph osph ate buffer (60 m l, pH 7.0) an d extracted with Et2O
(60 m l). Th e title salt 13 (1.30 g, 86%) precipitated as a wh ite am orph ous solid. RF 0.21 (S1).
UV: λm ax (50% (v/v) 1 M HCl in EtOH, pH 0) 201 sh (4.28), 270 (4.14); λm ax (50% (v/v) 1
M
TEAc in EtOH, pH 7) 272 (4.02); λm ax (50% (v/v) 0.1 M KOH in EtOH, pH 13) 245 (4.02), 269
(3.98). 1H NMR: 1.41 s, 9 H ((CH3)3C); 2.30 br s, 2 H (CH2CH=CH2); 2.63 br s, 2 H (CH2CO);
4.95 d, 1 H, J = 8.3 (cis-CH2=CH); 5.02 d, 1 H, J = 16.8 (trans-CH2=CH); 5.15 s, 2 H
(CH2COO); 5.81 br s, 1 H (CH=CH2); 5.96 s, 2 H (ArCH2); 7.72 d, 2 H, J = 7.9 (ArH); 8.19 d,
2 H, J = 7.9 (ArH); 9.81 s, 1 H (H-8); 11.32 br s, 1 H (NH); 12.20 br s, 1 H (NH). 13C NMR
(decoupled an d J-m odulated spin -ech o spectra): 27.45 (C(CH3)3); 28.22 (CH2CH=CH2); 35.29
(CH2CONH); 45.99 (CH2COO); 50.36 (ArCH2); 83.27 (C(CH3)3); 109.50 (C-5); 115.12
(CH2=CH); 123.62 (ArCH); 129.26 (ArCH); 137.11 (CH2=CH); 138.99 (C-8); 142.06 (C-1′,
C-4′); 147.37 (C-2*); 148.30 (C-6*); 153.83 (C-4); 164.92 (COOt-Bu); 173.75 (C4H7CONH).
MS (ESI), m/z (rel.%): 483 (100) [M+].
tert-Butyl [N2-(Pen t-4-en oyl)guan in -9-yl]acetate (15)
Sodium dith ion ite (0.790 g, 3.6 m m ol, 80%) was added to a solution of guan in ium salt 13
(0.530 g, 0.94 m m ol) in aceton e (15 m l) an d ph osph ate buffer (15 m l, pH 7.0). After 30 m in
stirrin g at room tem perature, th e solution was h eated at 70 °C for 16 h . Aceton e was evapo-
rated in vacuo an d th e water ph ase was extracted with EtOAc (50 m l). Th e title ester 15
(0.250 g, 76%) was obtain ed as a yellowish am orph ous solid. RF 0.19 (S2), 0.22 (S3). UV:
λm ax (50% (v/v) 1 M HCl in EtOH, pH 0) 203 (4.22), 261 sh (4.03); λm ax (50% (v/v) 1 M TEAc
in EtOH, pH 7) 260 (3.98), 280 (3.85); λm ax (50% (v/v) 0.1 M KOH in EtOH, pH 13) 267
(3.89). 1H NMR: 1.40 s, 9 H (C(CH3)3); 2.33 q, 2 H, J = 7.0 (CH2CH=CH2); 2.56 t, 2 H, J = 7.2
(CH2CO); 4.86 s, 2 H (CH2COO); 4.98 d, 1 H, J = 10.1 (cis-CH2=CH); 5.05 d, 1 H, J = 16.3
(trans-CH2=CH); 5.82 m , 1 H (CH=CH2); 7.94 s, 1 H (H-8); 11.66 br s, 1 H (NH); 12.04 br s,
1 H (NH). 13C NMR (decoupled an d J-m odulated spin -ech o spectra): 27.56 ((CH3)3C); 28.09
(CH2CH=CH2); 34.98 (CH2CO); 44.76 (CH2COO); 82.24 ((CH3)3C); 115.49 (CH2=CH); 119.54
(C-5); 136.74 (CH2=CH); 140.23 (C-8); 147.76 (C-2*); 148.85 (C-6*); 154.73 (C-4); 166.49
(COOt-Bu); 175.50 (C4H7CONH). MS (ESI), m/z (rel.%): 717.41 (38) [2 M + Na]+, 695.43 (68)
[2 M + H]+, 370 (18) [M + Na]+, 348 (100) [M + H]+.
This research has been supported by OTKA (grant T 22551) and EU FP6 (grant Targeting replica-
tion and integration of HIV, TRIoH 503480).
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