Synthesis and properties of 2-guanidinopurines

Article abstract of DOI:10.1135/cccc20061303

2-Guanidinopurines were prepared as derivatives of 2,6-diamino-9-[2- (phosphonomethoxy)ethyl]-9H-purine (PMEDAP) (1), which shows an important antiviral activity. It completes earlier described synthesis of 6-guanidinopurine derivatives. The title compounds were obtained by the reaction of the corresponding 2-chloropurines with guanidine. 2- And 6-guanidinopurines were used as model compounds for determination of dissociation constants (pK_a) of their ionogenic groups by capillary zone electrophoresis. The pK_a values of ionogenic groups of the above compounds were compared with those of the corresponding aminopurines. The pK_a of guanidino group at the purine moiety varies from 7.77 to 10.32. There is no protonation of N¹-position in contrast to aminopurines. None of these compounds showed any antiviral activity.

Full text of DOI:10.1135/cccc20061303

2-Guanidinopurines
1303
SYNTHESIS AND PROPERTIES OF 2-GUANIDINOPURINES
1,
2
3
Michal ČESNEK *, Milena MASOJÍDKOVÁ , Antonín HOLÝ ,
4
5
6
Veronika ŠOLÍNOVÁ , Dušan KOVAL and Václav KAŠIČKA
Gilead Sciences & IOCB Research Center; Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Prague 6,
1
2
Czech Republic; e-mail: cesnekm@uochb.cas.cz, masojid@uochb.cas.cz,
3
6
4
5
holy@uochb.cas.cz, veronika.solinova@uochb.cas.cz, koval@uochb.cas.cz,
kasicka@uochb.cas.cz
Received July 11, 2006
Accepted August 4, 2006
2-Guan idin opurin es were prepared as derivatives of 2,6-diam in o-9-[2-(ph osph on o-
m eth oxy)eth yl]-9H-purin e (PMEDAP) (1), wh ich sh ows an im portan t an tiviral activity. It
com pletes earlier described syn th esis of 6-guan idin opurin e derivatives. Th e title com poun ds
were obtain ed by th e reaction of th e correspon din g 2-ch loropurin es with guan idin e. 2- An d
6-guan idin opurin es were used as m odel com poun ds for determ in ation of dissociation con -
stan ts (pKa) of th eir ion ogen ic groups by capillary zon e electroph oresis. Th e pKa values of
ion ogen ic groups of th e above com poun ds were com pared with th ose of th e correspon din g
am in opurin es. Th e pKa of guan idin o group at th e purin e m oiety varies from 7.77 to 10.32.
Th ere is n o proton ation of N1-position in con trast to am in opurin es. Non e of th ese com -
poun ds sh owed an y an tiviral activity.
Keyw ord s: Purin es; Nucleosides; Guan idin es; Basicity; Dissociation con stan ts; PMEDAP an a-
logues; Acyclic n ucleoside ph osph on ates; Electroph oresis.
Th e an tiviral activity of 2,6-diam in o-9-[2-(ph osph on om eth oxy)eth yl]purin e
(PMEDAP)1 (1) is well kn own (Ch art 1). In th e last few years we carried out
a study of replacem en t of th e am in o group at th e position 6 in th e purin e
m oiety by th e guan idin o group an d studied th e effect of th is replacem en t
on an tiviral activity of th ese n ew an alogues. Th e con sequen ce of th is re-
placem en t was eith er a com plete loss of or a sign ifican t decrease in an tiviral
activity. On th e oth er h an d, several com poun ds bearin g guan idin o group
in th e purin e m oiety possess in terestin g im m un om odulatory activity2.
With in th e program m e of our structure–activity relation sh ip studies in th e
series of guan idin opurin e derivatives of acyclic aden in e n ucleotide an a-
logues we are n ow goin g to describe th e syn th esis an d properties of th e cor-
respon din g isom eric com poun ds, i.e. 2-guan idin opurin e derivatives an d
com parin g th eir an tiviral activity with th e correspon din g 2-am in opurin es.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 9, pp. 1303–1319
© 2006 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20061303

1304
Česnek, Masojídková, Holý, Šolínová, Koval, Kašička:
We attem pted to elucidate wh eth er an an tiviral effect would be preserved
on substitution of th e am in o group of 2,6-diam in opurin e at th e position 2
by th e guan idin o group. Th is paper describes th e syn th esis of
2-guan idin opurin e an alogues of PMEDAP an d guan idin o an alogues of
PMEMAP; both th ese paren t com poun ds sh ow an tiviral activity1a,1b
.
Th e an tiviral activity of acyclic n ucleotides an alogues depen ds on th e fol-
lowin g prin ciples: (i) Pen etration of th e active com poun d th rough th e cel-
lular m em bran e; (ii) activation by th e cellular en zym e by ph osph orylation
an d (iii) th e in h ibition of th e target viral en zym es by th ese an abolites. Evi-
den tly th e presen ce of th e guan idin o group itself does n ot preven t th e pas-
sage. It is eviden t from th e active tran sport of argin in e.
R6
N
N
N
O
R
N
2
O
P(OH)2
1, R2 = R6 = NH2, (PMEDAP)
2, R2 = H; R6 = NH2, (PMEA)
3, R2 = NH2; R6= H, (PMEMAP)
CHART 1
Hydrogen bon din g plays an im portan t role in th e latter two steps. On re-
placem en t by th e guan idin o group th e probability of h ydrogen bon d for-
m ation an d th ereby th e m utual in teraction of th e m olecules is even h igh er.
Th ese reason s lead us for th e syn th esis of guan idin opurin es.
Th e basicity of com poun d can also play th e sign iffican t role in th e sub-
strate–en zym e activity. Obviously th e replacem en t of am in o by guan idin o
group in creases th e overall basicity of th e purin e system . Th e size of th is
effect is so far un kn own . We h ave attem pted to elucidate wh eth er such a
replacem en t causes an y ch an ges in th e proton ation of th e purin e m oiety.
A determ in ation of pKa values of n ewly syn th esized am in o- an d (am i-
n o)guan idin opurin e n ucleotide an alogues3 as an alogues of PMEDAP by
poten tiom etric titration did n ot afford satisfactory results, th erefore we
h ave applied th e capillary zon e electroph oresis. We attem pted to elucidate
h ow th e acid-base dissociation con stan ts of guan idin opurin es differ from
th ose of am in opurin es.
Such a com parison of acid-base dissociation con stan ts between 2- an d
6-guan idin opurin es was n ot yet publish ed in th e literature. Th e data of dis-
sociation con stan ts of th e guan idin o-group-con tain in g com poun ds
described so far in th e literature con cern s arylguan idin es4 an d h etero-
arylguan idin es5.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 9, pp. 1303–1319

2-Guanidinopurines
1305
Aim in g at th e com parison of th e acid-base dissociation con stan ts of
guan idin opurin es with am in opurin es we h ave prepared th e correspon din g
guan idin opurin e com poun ds derived from PMEA (2) {6-am in o-9-[2-(ph os-
ph on om eth oxy)eth yl]-9H-purin e}, PMEMAP (3) {2-am in o-9-[2-(ph osph on o-
m eth oxy)eth yl]-9H-purin e} an d PMEDAP (1) (Ch art 1).
Furth erm ore, in order to com pare th e basicity of 2- an d 6-guan idin o
groups we h ave also syn th esized a spectrum of 2- an d 6-guan idin opurin es
bearin g at th e position 9 fun ction al groups oth er th an ph osph on ates.
RESULTS AND DISCUSSION
Synthesis
6-Guan idin opurin es 4 an d 5 n ecessary for com parison of basicity of th e 2-
an d 6-guan idin o regioisom ers were prepared from th e correspon din g
6-ch loropurin e derivatives accordin g to th e literature2. At th e position 9
th ere is n o fun ction al group in th ese com poun ds, wh ich could be proton -
ated at differen t pH. On th e oth er h an d, 6-guan idin opurin es 6 an d 7,
wh ich were prepared in accord with th e literature6 by sim ilar procedure can
be proton ated at th e side ch ain boun d at th e position 9 of th e purin e
m oiety. Apart from th e stron gly basic (cation ogen ic) guan idin o group,
com poun ds 5 an d 7 con tain also an oth er (cation ogen ic) am in o group sus-
ceptible to proton ation in position 2 (Ch art 2).
NH2
N
N
NH2
N
R6
N
iPr = CH(CH3)2
O
N
N
NH2
N
N
O
N
R9
N
R9
R2
H2N
P(OH)2
PME(OH)2
=
O
4, R2 = H; R9 = CH3
5, R2 = NH2; R9 = iPr
6, R2 = H; R9 = PME(OH)2,
12, R6 = NH2; R9 = PMEiPr2
13, R6 = NH2; R9 = PME(OH)2
15, R6 = NH2; R9 = iPr
O
PMEiPr2
=
P(OiPr)2
7, R2 = NH2; R9 = PME(OH)2,
20, R6 = H; R9 = iPr
24, R6 = H; R9 = PMEiPr2,
25, R6 = H; R9 = PME(OH)2,
Model compounds for the determination of basicity guanidinopurines
CHART 2
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 9, pp. 1303–1319

1306
Česnek, Masojídková, Holý, Šolínová, Koval, Kašička:
Experien ce from th e syn th esis of 6-guan idin opurin es was used for th e
preparation of 2-guan idin opurin es. It is well kn own th at th e reactivity of
C–Cl group in 6- an d 2-ch loropurin es differs sign ifican tly; th us, we h ave se-
lected th e syn th esis of 6-am in o-2-guan idin opurin e (10) to verify th e appli-
cability of our stan dard m eth od in th e preparation of th e guan idin o
derivatives, wh ich was origin ally developed for th e syn th esis of
6-guan idin opurin es6. Th is approach starts from th e easily available
6-am in o-2-ch loro-9-(tetrah ydropyran -2-yl)purin e7 (8). Th e reactivity of th e
2-ch loro atom at th e purin e m oiety in th e n ucleoph ilic attack by guan idin e
is m uch lower com pared to th e 6-ch loro atom 8. Wh ile th e 6-ch loro atom in
th e purin e m oiety un dergoes guan idin olysis already at room tem perature,
n o reaction durin g 24 h was observed in th e case of 2-ch loropurin e deriva-
tives at am bien t tem perature. Th erefore, we h ave applied th e con dition s
an alogous to th e reaction of 2-ch loro derivatives with am in es9. Th e
2-guan idin o derivative 9 was obtain ed by reaction of th e 6-am in o-
2-ch loropurin e derivative 8 with guan idin e in th e presen ce of DABCO
(1,4-diazabicyclo[2.2.2]octan e) as a base. Dim eth ylform am ide was n ot ap-
propriate as a solven t, th e h igh basicity of guan idin e solution an d h igh
tem perature caused its decom position an d subsequen t form ation of by-
products. Th erefore, N-m eth ylpyrrolidon e was used in stead DMF as a sol-
ven t. 6-Am in o-2-guan idin opurin e (10) was obtain ed by acid h ydrolysis,
wh ich cleavaged off th e tetrah ydropyran -2-yl protectin g group in th e in ter-
m ediate 9.
Th e tran sform ation of 6-am in o-2-ch loropurin e derivative1b 11 was used
for th e preparation of th e 2-guan idin opurin e an alogue of PMEDAP 1a 13 by
guan idin olysis with a guan idin e solution in th e presen ce of DABCO in
N-m eth ylpyrrolidon e. Th e in term ediate 12 gave, after rem oval of th e pro-
tectin g isopropyl groups with brom otrim eth ylsilan e in CH3CN, 6-am in o-
2-guan idin o-9-[2-(ph osph on om eth oxy)eth yl]-9H-purin e (13) in a satisfac-
tory yield.
Th e syn th esis of 6-am in o-2-guan idin o-9-isopropyl-9H-purin e (15) was
perform ed as follows: 2,6-dich loropurin e 16 was treated with isopropyl io-
dide in dim eth yl sulfoxide in th e presen ce of K2CO3 to give th e isopropyl
derivative 17 10. Its am m on olysis with m eth an olic am m on ia gave 6-am in o-
2-ch loro-9-isopropyl-9H-purin e8 (14) an d th e followin g guan idin olysis gave
th e desired com poun d 15 (Sch em e 1).
For th e preparation of 2-guan idin opurin es lackin g am in o group at th e
position 6 in th e purin e m oiety we h ave used several m eth ods. Th e first
m eth od con sisted in con version of th e 6-ch loro atom to th e 6-h ydrazin o
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 9, pp. 1303–1319

2-Guanidinopurines
1307
NH2
N
NH2
NH2
N
N
N
N
(i)
NH2
N
N
(ii)
N
NH2
N
N
N
N
N
H2N
Cl
N
H2N
R1
R1
R1
8, R1= O-THP
11, R1= PMEiPr2
14, R1= iPr
9, R1= O-THP
12, R1= PMEiPr2
15, R1= iPr
1. 10, R1= H
2. 13, R1= PME(OH)2
(i) guanidine in NMP, DABCO, 120 °C; (ii) 1. H2SO4, 60 °C for 9; 2. TMSBr,
CH3CN, r.t. for 12
SCHEME 1
group an d its subsequen t decom position by Ag2O. Th e startin g 2,6-dich loro-
purin e 16 was alkylated10 with isopropyl iodide in th e presen ce of K2CO3 in
DMSO. Th e th us obtain ed in term ediate 17 was tran sform ed in to
2-ch loro-6-h ydrazin o-9-isopropyl derivative 18 with h ydrazin e h ydrate in
m eth an ol an d subsequen tly by reaction with Ag2O in m eth an ol to
2-ch loro-9-isopropylpurin e derivative 19 as described in Experim en tal (pro-
cedure A). Th is com poun d gave th e desired 2-guan idin o derivative 20
(Sch em e 2) by th e procedure sim ilar to th at for oth er 2-guan idin o deriva-
tives.
Cl
R1
N
N
N
NHNH2
N
H
N
H
N
Cl
N
N
Cl
N
N
N
N
(iii)
(iv)
H2N
(ii)
(i)
N
N
NH2
N
N
N
Cl
N
N
N
N
N
H
Cl
Cl
Cl
N
R1
R1
R1
16
N
Cl
N
R1
17, R1 = iPr
21a, R1 = 9-PMEiPr2
21b, R1 = 7-PMEiPr2
18, R1= iPr
22, R1= PMEiPr2
19, R1= iPr
23, R1= PMEiPr2
20, R1= iPr
24, R1= PMEiPr2
25, R1= PME(OH)2
(v)
(i) 1. isopropyl iodide, K2CO3, DMSO, r.t. (7-regioisomer was not found);
2. 2-[(diisopropoxyphosphoryl)methoxy]ethyl 4-methylbenzene-1-sulfonate, NaH, DMF, 100 °C; (ii)
NH2NH2.H2O, MeOH, r.t.; (iii) Ag2O, dioxane, H2O, reflux; (iv) guanidine in NMP, DABCO, 120 °C; (v) TMSBr,
CH3CN, r.t.
SCHEME 2
Th e secon d m eth od was direct catalytic dech lorin ation with subsequen t
alkylation . Con trary to th e literature11, th e catalytic dech lorin ation of
2,6-dich loropurin e 16 on Pd/C led to purin e 26 as th e m ajor product. Also
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 9, pp. 1303–1319

1308
Česnek, Masojídková, Holý, Šolínová, Koval, Kašička:
th e m odification of th is m eth od usin g aqueous K2CO3 afforded purin e (26)
as a m ajor product. Th e desired product 19 was obtain ed by alkylation of a
m in or product 27 as described in Experim en tal (procedure B) (Sch em e 3).
Catalytic deh alogen ation s of 2,6-dich loropurin e 16 substituted with isopro-
pyl in position 9 gave solely 9-isopropylpurin e.
Due to th e better yields, reaction con dition s an d workup of th e reaction
m ixture, th e rem oval of ch lorin e atom by h ydrazin olysis an d subsequen t
decom position of th is com poun d by Ag2O, a sim ilar reaction sch em e as de-
scribed above for procedure A was used for th e syn th esis of 2-guan idin o-
purin e derivative 25 from th e protected diester 24.
H
N
N
N
N
N
N
H
N
H
Cl
Cl
N
28
and
N
and
26
Cl
H
(i)
N
N
(ii)
N
N
N
N
N
H
N
H
Cl
N
Cl
N
N
16
27
19
(i) H2, 5% Pd/C, CH3COONa, or H2, 5% Pd/C, K2CO3, H2O, r.t.; (ii) NaH, isopropyl bromide, DMF, r.t.
SCHEME 3
Th e startin g m aterial for th is syn th esis was prepared by th e reaction of
2,6-dich loro-9H-purin e (16) with 2-[(diisopropoxyph osph oryl)m eth oxy]-
eth yl 4-m eth ylben zen e-1-sulfon ate in th e presen ce of NaH in DMF. Th e
9-regioisom er 21a an d 7-regioisom er 21b were isolated by colum n ch rom a-
tograph y an d detem in ed by NMR spectroscopy. Th e th us obtain ed in term e-
diate 21a was treated with h ydrazin e h ydrate in m eth an ol to give
2-ch loro-6-h ydrazin o derivative 22 wh ich was furth er tran sform ed to th e
2-ch loro in term ediate 23 by th e sam e procedure as described for com poun d
19. Guan idin ation of th is 2-ch loro derivative was carried out usin g th e
sam e procedure as in th e case of 12 to obtain th e desired product 24. Th e fi-
n al step was rem oval of protectin g isopropyl ester groups by th e reaction
with brom otrim eth ylsilan e in CH3CN to give th e free 2-guan idin o deriva-
tive 25.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 9, pp. 1303–1319

2-Guanidinopurines
1309
Determination of pKa Values of Amino- and Guanidinopurines
and Their Analogues by Capillary Zone Electrophoresis
Th e determ in ation was carried out usin g th e capillary zon e electroph oresis
(CZE). Th e pKa values were determ in ed by n on -lin ear regression an alysis of
th e pH-depen den ce of th eir effective electroph oretic m obilities m easured
by CZE. Th e pKa values were determ in ed for com poun ds 4–7, 12, 13, 15, 20,
24, an d 25.
Com parison of 6-guan idin o derivatives with th eir 2-guan idin o coun ter-
parts sh ows th at com poun ds con tain in g 6-guan idin o group are less basic
com pared to th eir 2-isom ers (Ch art 3, Table I). In th e group of 6-guan idin o
derivatives, th e values stron gly depen d on th e presen ce of addition al fun c-
tion al groups (in particular th e am in o group on th e base an d ph osph on ate
group in th e aliph atic ch ain ). Th e lowest basicity sh owed com poun d 4
wh ich did n ot con tain am in o group in position 2 (pKa = 7.77), th e basicities
of com poun ds 5 an d 6 with am in o or an ion ic ph osph on ate group are com -
parable (pKa = 8.23 an d 8.29, respectively). Th e m ost basic com poun d in
th e 6-guan idin o series is 7 (pKa = 8.84).
NH2
N
NH2
N
NH2
N
NH2
N
H
N
H
N
H2N
H2N
H2N
H2N
H2N
N
N
N
N
N
N
N
N
N
N
NH2
N
N
NH2
N
N
N
N
N
N
N
N
H2N
H2N
N
H2N
PME(OH)2
N
N
N
PMEiPr2
Me
iPr
PME(OH)2
iPr
pKa = 9.26
pKa = 7.77
pKa = 8.23
pKa = 8.84
pKa = 9.16
pKa = 8.29
4
5
7
20
NH2
N
24
6
NH2
N
N
H
NH2
N
N
N
N
N
N
NH2
N
NH2
NH2
N
NH2
N
N
N
N
N
H2N
H2N
N
N
H2N
N
N
H2N
PMEiPr2
PME(OH)2
PME(OH)2
iPr
pKa = 9.64
pKa = 9.85
pKa = 9.80
pKa = 10.32
15
25
12
13
CHART 3
Con trary to th e 9-alkyl derivatives th e presen ce of free ph osph on ate
group in th e side ch ain in creases basicity of th e guan idin o group. It is prob-
ably con sequen ce of zwitterion form ation . In th e 2-guan idin opurin es th e
lowest value was foun d for com poun d 20 (pKa = 9.16), wh ich does n ot bear
an y oth er ion ogen ic group. Th e situation is differen t in com poun d 25
(pKa = 9.80). In spite of th e fact th at it does n ot con tain am in o group at po-
sition 6, it is m ore basic com pared with th e 6-am in o-9-alkyl derivative 15
(pKa = 9.64). Th e h igh est value of pKa was determ in ed in com poun d 13
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 9, pp. 1303–1319

1310
Česnek, Masojídková, Holý, Šolínová, Koval, Kašička:
(pKa = 10.32) with two ion ogen ic groups (6-am in o an d 2-guan idin o groups).
Whilst purine phosphonates with guanidino group at the position 2, i.e. com -
poun ds 25 (pKa = 9.80), 12 (pKa = 9.85) an d 13 (pKa = 10.32) are th e m ost
basic com poun ds, th e ph osph on ates bearin g guan idin o group at position 6
are less basic (6 (pKa = 8.29) an d 7 (pKa = 8.84)). Wh en com parin g basicity
of free acids an d th eir diisopropyl esters (with th e som e purin e m oiety is th e
sam e), th e lower basicity is always ch aracteristic of diisopropyl esters. Th e
correspon din g pairs of com poun ds are: 24 (pKa = 9.26) with 25 (pKa = 9.80)
an d 12 (pKa = 9.85) with 13 (pKa = 10.32). High er basicity was in both cases
observed in esters of ph osph on ic acids (24 an d 12) th an in th e correspon d-
in g alkyl derivatives (20 an d 15).
TABLE I
Th erm odyn am ic pKa values of ion ogen ic groups of an alyzed com poun ds (at 25 °C)
(N1)H+/(N1), dissociation of proton ated n itrogen
1
(N1) in th e purin e m oiety;
P(O)(OH)O–/P(O)(O)22–, dissociation of ph osph on ic acid group to th e secon d degree (–2);
(G)H+/(G), dissociation of proton ated n itrogen guan idin yl group; n .d., n ot determ in ed; n .p.,
n ot presen t.
pKa
Com pd
2–
(N1)H+/(N1)
P(O)(OH)O–/P(O)(O)2
(G)H+/(G)
4
n .d.
n .d.
n .d.
n .d.
7.77 ± 0.02
8.23 ± 0.03
8.29 ± 0.06
8.84 ± 0.05
9.16 ± 0.02
9.26 ± 0.02
n .d.
5
6
n .d.
6.86 ± 0.06
6.77 ± 0.04
n .d.
7
n .d.
20
n .d.
24
n .d.
n .d.
PMEMAP
PMEDAP
PMEG
15
4.13 ± 0.04
4.89 ± 0.05
n .d.
7.46 ± 0.06
7.37 ± 0.07
7.29 ± 0.06
n .d.
n .d.
10.02 ± 0.07 (en ol)a
9.64 ± 0.03
9.80 ± 0.08
9.85 ± 0.03
10.32 ± 0.11
n .d.
25
n .d.
6.64 ± 0.08
n .d.
12
n .d.
n .d.
6.77 ± 0.03
13
a
Th e value for th e dissociation of th e en ol h ydroxy group of PMEG (n o guan idin yl group is
presen t in PMEG), 9-[2-(ph osph on om eth oxy)eth yl]-9H-guan in e.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 9, pp. 1303–1319

2-Guanidinopurines
1311
Com parison of 6- or 2-am in o com poun ds with th eir guan idin opurin e
coun terparts sh owed a differen t m ech an ism of proton ation of th e purin e
m oiety. Th e proton ation of aden in es at N1 is well kn own 12. Obviously th e
proton ation of guan idin opurin e takes place at th e guan idin o group because
n o proton ation of N1 was observed.
It can be con cluded th at th e in troduction of guan idin o group led to an
in crease in basicity an d probably to th e total ch an ge of proton ation of th e
purin e m oiety. Con trary to th e am in opurin e ph osph on ates with zwitter-
ion ic ch aracter of m olecule, wh ich bear two n egative ch arges un der th e
weakly basic con dition s zwitterion ic purin e com poun ds with th e guan idin o
group are ch aracteristic by solely on e n egative ch arge in th e weakly basic
con dition s. Th is m ay be play im portan t role for th e bioavailability of guan i-
din opurin es.
Biological Activity
Non e of th e title 2-guan idin opurin es sh owed an y sign ifican t an tiviral activ-
ity again st DNA viruses, RNA viruses an d or retroviruses, n or exh ibited any
cytotoxicity in vitro in L929, L1210, HeLaS3 and CCRF CEM cells13 under
stan dard con dition s. Th ese tests sh ow th at th e substitution of th e am in o
fun ction by th e guan idin o group eith er in position 6 or in position 2 of th e
purin e m oiety, leads to a decrease in or com plete loss of an tiviral activity.
Conclusion
A gen eral m eth od for syn th esis of 2-guan idin opurin e derivatives was devel-
oped. Th e m on osubstituted 2-ch loropurin e derivatives were prepared by
th e tran sform ation of 6-ch loro fun ction in 2,6-dich loropurin e derivative
via h ydrazin e in term ediate. Th e catalytic deh ydroch lorin ation sh ows very
weak regioselectivity.
Basicity of th e guan idin o derivatives with pKa values in th e ran ge
7.77–10.32 is m uch h igh er (by th ree to six orders of m agn itude) th an th at
of th e am in o derivatives 3 an d 1 with pKa equal to 4.13 an d 4.89, respec-
tively. Th e stron ger basicity of th e guan idin o derivatives com pared to am i-
n o derivatives is reflected by th e in creased acidity of ph osph on ic acid
residue in th e form er derivatives. Basicity of th e derivatives with guan idin yl
group in position 2 of purin e is sign ifican tly larger (ca. by 1.5 un it differ-
en ce in pKa) th an th at of th e com poun ds bearin g guan idin yl group in posi-
tion 6 of purin e. Basicity of th e am in oguan idin o derivatives is h igh er th an
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 9, pp. 1303–1319

1312
Česnek, Masojídková, Holý, Šolínová, Koval, Kašička:
th at of th e guan idin o derivatives (ca. by 0.5 un it differen ce in pKa). Sim i-
larly, th e basicity of PMEDAP (1) with two am in o groups in position s 2 an d
6 of purin e is h igh er th an th at of PMEMAP (3) with sin gle am in o group at
position 2 of purin e.
CZE h as proved to be a suitable an d useful m eth od for determ in ation of
dissociation con stan ts (pKa) of am in o an d (am in o)guan idin opurin e n ucleo-
tide an alogues, such as acyclic n ucleoside ph osph on ate, acyclic n ucleoside
ph osph on ate diesters an d oth er related com poun ds, in a m icroscale, ap-
plyin g on ly few n an oliter sam ple volum es of 0.1 m M an alyte solution per
an alysis. It was proved th e ch an ge in proton ation in th e purin e m oiety
between guan idin opurin es an d am in opurin es. Th e bioavailability of th ese
basic com poun ds will be furth er exam in ed.
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 B-545
apparatus an d are un corrected. Ch rom atograph y system s S1: CHCl3–MeOH (95:5); S3:
EtOAc–EtOH–aceton e–H2O–NH3 (4:1:1:1:0.25)]; S4: EtOH–EtOAc (10:90) con tain in g 1% of
NH3; S5: EtOAc–EtOH–aceton e–H2O (6:1:1:0.5) with 1% of NH3; S6: EtOAc–EtOH–ace-
ton e–H2O (4:1:1:1) with 1% NH3; S7: EtOAc–EtOH–aceton e–H2O (4:1:1:1). Preparative TLC
was carried out on 40 × 17 × 0.4 cm loose layers of silica gel con tain in g a UV in dicator. Pa-
per electroph oresis was perform ed on Wh atm an paper No. 3 MM at 40 V/cm for 1 h in 0.05 M
trieth ylam m on ium h ydrogen carbon ate (TEAB) at pH 7.5 an d th e electroph oretic m obilities
(EUp) are referen ced to uridin e 3′-ph osph ate. Mass spectra were m easured on a spectrom eter
ZAB-EQ (VG An alytical) usin g FAB ion ization by Xe (acceleratin g voltage 8 kV, glycerol m a-
trix) or EI electron en ergy 70 eV tech n iques. NMR spectra (J, Hz; δ, ppm ) were m easured on
a Bruker DRX 500 (500 MHz for 1H, 125.7 MHz for 13C NMR spectra) in DMSO-d6.
Dim eth ylform am ide an d aceton itrile were distilled from P2O5 an d stored over m olecular
sieves (4Å). Preparative HPLC purification s were perform ed on colum n s packed with 7 µm
C18 reversed ph ase (Waters Delta 600 ch rom atograph colum n ), 17 × 250 m m , in ca. 200 m g
batch es of m ixtures usin g
a
lin ear gradien t of 0.025
M
t et raet h ylam m o n iu m
h yd ro gen carb o n at e bu ffer in H2O–CH3OH (0 to 100% CH3OH) or lin ear gradien t
MeOH–H2O (1:4 to 9:1) as eluen t.
Deion isation was perform ed on Dowex 50X8 (H+ form ) colum n s by th e followin g proce-
dure: after application of crude product th e colum n was wash ed with water un til th e UV ab-
sorption dropped. Th ereafter, th e colum n was eluted with 2.5% aqueous NH3 or with a
MeOH–Et3N–H2O (1:1:3) m ixture. Ch rom atograph y on Dowex 1X2 (acetate form ) was m ade
as follows: After application of th e aqueous solution of th e crude product on to th e colum n ,
it was wash ed with water un til th e UV absorption dropped. Th e colum n was th en eluted
with a gradien t of dilute acetic acid (0–1 m ol/l).
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 9, pp. 1303–1319

2-Guanidinopurines
1313
Guan idin e Solution
Guan idin e h ydroch loride (2.01 g, 21 m m ol) was added to a suspen sion of sodium h ydride
(60% suspen sion in paraffin oil; 0.84 g, 21 m m ol) in N-m eth ylpyrrolidon e (21 m l) an d th e
m ixture was stirred at room tem perature overn igh t un der exclusion of m oisture. Th e result-
in g slurry was directly used for tran sform ation s.
Cleavage of th e Ph osph on ate Esters. Gen eral Procedure
Th e ester (1 m m ol) was suspen ded in aceton itrile (30 m l) an d TMSBr (3 m l) was added
dropwise. Th e reaction m ixture was stirred at room tem perature overn igh t. After evapora-
tion of th e volatiles an d co-distillation with aceton itrile (20 m l), th e residue was treated
with water (20 m l) an d 35% aqueous NH3 (3 m l) for 5 m in an d evaporated. Th e residue was
dissolved in water an d applied on to a colum n of Dowex 50X8 (H+ form ). Th e colum n was
wash ed with water an d eluted with 2.5% aqueous NH3. Th e product-con tain in g
UV-absorbin g fraction s were evaporated, th e residue was dissolved in 35% aqueous NH3 an d
applied on to a colum n of Dowex 1X2 (acetate form ), wash ed with water an d eluted with a
lin ear gradien t of aqueous acetic acid (0–1 m ol/l). Th e product con tain in g fraction s were
evaporated, codistilled with water (5 × 30 m l) an d th e residue was crystallized to give a pure
free ph osph on ate. Th is procedure affords com poun ds 13 an d 25.
6-Am in o-2-ch loro-9-(tetrah ydropyran -2-yl)-9H-purin e (8)
Th e com poun d was prepared accordin g to lit.7, yield 95%.
6-Am in o-2-guan idin o-9-(tetrah ydropyran -2-yl)-9H-purin e (9)
A guan idin e solution prepared as above (18 m l, 18 m m ol) was added to a flask con tain in g
com poun d 8 (2.5 g, 9 m m ol) an d DABCO (1 g, 9 m m ol). Th e m ixture was stirred at room
tem perature for 24 h an d th en h eated at 120 °C for 8 h . Th e reaction m ixture was triturated
with eth er (160 m l), filtered an d th e precipitate was dissolved in MeOH, filtered th rough
Cellite an d evaporated in vacuo. Th e residue was extracted with a CHCl3–MeOH m ixture
(3:1), th e extract was adsorbed on silica gel an d purified on a colum n of silica gel (50 g,
CHCl3–MeOH, 88:12) to give com poun d 9 (1 g, 83%). Wh ite crystals; m .p. 217–220 °C
(EtOH). FAB MS, m/z (%): 277 (100), [M + H]. 1H NMR (DMSO-d6): 1.56 m , 2 H, 1.71 m , 1 H,
1.95 m , 2 H an d 2.23 m , 1 H (C-CH2); 3.64 m , 1 H an d 4.00 m , 1 H (O-CH2); 5.54 dd, 1 H,
J = 2.1 an d 10.7 (N-CH-O); 7.77 brs, 2 H (NH2); 8.20 s, 1 H (H-8); 8.37 br, 3 H (NH, NH2);
10.44 s, 1 H (NH). 13C NMR (DMSO-d6): 22.60, 24.68, 30.04, 67.80 an d 81.00 (N-THP);
115.73 (C-5); 138.80 (C-8); 149.49 (C-4); 152.43 (C-6); 155.62 an d 155.74 (C-2 a N-C). Exact
m ass (FAB HRMS) foun d: 277.1525; for C11H17N8O [M + H] calculated: 277.1469.
6-Am in o-2-guan idin opurin e (10)
Com poun d 9 (0.70 g, 2.5 m m ol) was dissolved in water (20 m l) an d treated with a 1
M
H2SO4 (5 m l) at room tem perature overn igh t. Th e reaction m ixture was n eutralized with
Ba(OH)2 an d boiled for a sh ort tim e. Th e precipitate was filtered off an d th e filtrate was
evaporated in vacuo to give com poun d 10 (0.34 g, 70%). Wh ite crystals; m .p. >318 °C (dec.)
(H2O). FAB MS, m/z (%): 193 (70), [M + H]. 1H NMR (DMSO-d6): 8.06 s, 1 H (H-8); 7.62 brs,
2 H, 8.20 br, 3 H, 10.15 br, 1 H an d 12.90 br, 1 H (NH, NH2). 13C NMR (DMSO-d6): 114.90
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 9, pp. 1303–1319

1314
Česnek, Masojídková, Holý, Šolínová, Koval, Kašička:
(C-5); 139.65 (C-8); 149.00 (C-4); 151.20 (C-6); 154.90 (C-2); 155.30 (N-C). Exact m ass (FAB
HRMS) foun d: 192.0836; for C6H9N8 [M + H] calculated: 192.0872.
2,6-Dich loro-9-{2-[(diisopropoxyph osph oryl)m eth oxy]eth yl}-9H-purin e (21a) an d
2,6-Dich loro-7-{2-[(diisopropoxyph osph oryl)m eth oxy]eth yl}-7H-purin e (21b)
Sodium h ydride (60% suspen sion in paraffin oil; 0.32 g, 8 m m ol) was added to a solution of
2,6-dich loropurin e (16; 1.5 g, 8 m m ol) in DMF (25 m l) an d th e reaction m ixture was stirred
at room tem perature for 1 h ; 2-[(diisopropoxyph osph oryl)m eth oxy]eth yl 4-m eth ylben zen e-
1-sulfon ate was added dropwise an d th e reaction m ixture was stirred at 100 °C for 13 h . Th e
solven t was rem oved in vacuo, th e residue was codistilled with toluen e (3 × 30 m l), ex-
tracted with ch loroform an d purified by colum n ch rom atograph y (50 g, CHCl3–MeOH, 97:3)
to give com pou n d 21a (2.09 g, 64%) as yellow oil. FAB MS, m/z (%): 411 (35), [M + H].
1H NMR (DMSO-d6): 1.09 d, 6 H, J(CH3,CH) = 6.1 an d 1.14 d, 6 H, J(CH3,CH) = 6.1 (CH3);
3.77 d, 2 H, J(P,CH) = 8.2 (P-CH2); 3.92 t, 2 H, J(2′,1′) = 4.9 (H-2′); 4.46 t, 2 H, J(1′,2′) = 4.9
(H-1′); 4.44 m , 2 H (POCH); 8.69 s, 1 H (H-8). 13C NMR (DMSO-d6): 23.68 d, 2 C, J(P,C) =
4.4 (CH3); 23.82 d, 2 C, J(P,C) = 4.4 (CH3); 43.78 (N-CH2); 64.78 d, J(P,C) = 164.1 (P-CH2);
69.90 d, J(P,C) = 11.7 (PO-CH2); 70.37 d, 2 C, J(P,C) = 6.3 (P-OCH); 130.49 (C-5); 148.85
(C-8); 149.68 (C-6); 151.10 (C-2); 153.75 (C-4). Exact m ass (FAB HRMS) foun d: 411.0747; for
C
14H22Cl2N4O4P [M + H] calculated: 411.0756.
Furth er elution of colum n gave th e 7-regioisom er 21b. FAB MS, m/z (%): 411 (50), [M + H].
1H NMR (DMSO-d6): 1.09 d, 6 H, J(CH3,CH) = 6.1 an d 1.14 d, 6 H, J(CH3,CH) = 6.1 (CH3);
3.76 d, 2 H, J(P,CH) = 8.1 (P-CH2); 3.93 t, 2 H, J(2′,1′) = 4.9 (H-2′); 4.44 m , 2 H (P-OCH);
4.67 t, 2 H, J(1′,2′) = 4.9 (H-1′); 8.81 s, 1 H (H-8). 13C NMR (DMSO-d6): 23.72 d, 2 C, J(P,C) =
4.4 (CH3); 23.82 d, 2 C, J(P,C) = 3.9 (CH3); 46.52 (C-1′); 64.78 d, J(P,C) = 164.1 (P-C); 70.20 d,
2 C, J(P,C) = 6.4 (P-OC); 70.89 d, J(P,C) = 11.2 (C-2′); 122.05 (C-5); 143.44 (C-6); 150.91
(C-2); 153.06 (C-8); 163.41 (C-4). Exact m ass (FAB HRMS) foun d: 411.0762; for
C
14H22Cl2N4O4P [M + H] calculated: 411.0756.
2-Ch loro-9-{2-[(diisopropylph osph oryl)m eth oxy]eth yl}-9H-purin e (23)
After dropwise addition of hydrazine hydrate to a solution of com pound 21a (1.86 g, 4.5 m m ol)
in m eth an ol (23 m l), th e reaction m ixture was stirred at room tem perature for 30 m in . Th e
solven t was evaporated in vacuo an d th e residue was codistilled with m eth an ol an d purified
by colum n ch rom atograph y on silica gel (40 g, S1). Th e desired product was dissolved in a
m ixture of dioxan e–water 1:1 (40 m l) an d Ag2O (1.1 g) was added. Th e reaction m ixture was
th en refluxed for 1 h . A th in layer of Cellite was used to filter off th e silver salts an d th e fil-
trate was evaporated in vacuo. Th e residue was dissolved in eth yl acetate an d extracted with
aqueous solution of EDTA (3 × 30 m l). Com bin ed organ ic layers were dried with an h ydrous
MgSO4. Purification of th e residue on a silica gel colum n (40 g, CHCl3–MeOH, 93:7) gave
com poun d 23 (0.9 g, 53%). FAB MS, m/z (%): 377 (75) [M + H]. 293 (100) [M – 2 iPr]. 1H NMR
(DMSO-d6): 1.07 d, 6 H, J(CH3,CH) = 6.1 an d 1.13 d, 6 H (CH3); 3.78 d, 2 H, J(P,CH) = 8.3
(P-CH2); 3.93 t, 2 H, J(2′,1′) = 5.0 (H-2′); 4.44 m , 2 H (P-OCH); 4.45 t, 2 H, J(1′,2′) = 5.0
(H-1′); 8.60 s, 1 H (H-8); 9.08 s, 1 H (H-6). 13C NMR (DMSO-d6): 23.74 d, 2 C, J(P,C) = 4.4
(CH3) an d 23.89 d, 2 C, J(P,C) = 3.9 (CH3); 43.15 (C-1′); 64.69 d, J(P,C) = 164.1 (P-C); 70.12 d,
J(P,C) = 11.8 (C-2′); 70.42 d, 2 C, J(P,C) = 6.3 (P-OCH); 133.21 (C-5); 148.57 (C-8); 149.91
(C-6); 152.97 (C-2); 153.47 (C-4). Exact m ass (FAB HRMS) foun d: 377.1130; for C14H23ClN4O4P
[M + H] calculated: 377.1145.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 9, pp. 1303–1319

2-Guanidinopurines
1315
9-{2-[(Diisopropylph osph oryl)m eth oxy]eth yl}-2-guan idin o-9H-purin e (24)
A guan idin e solution (10 m l, 10 m m ol) prepared as above was added to a flask con tain in g
com poun d 23 (0.8 g, 2.1 m m ol) an d DABCO (0.24 g, 2.1 m m ol). Th e m ixture was stirred at
120 °C for 2 h ; after th at th e reaction m ixture was evaporated in vacuo an d th e residue was
codistilled with toluen e (3 × 30 m l), dissolved in water, th e solution was n eutralized with
Dowex 50X8 (H+ form ), an d th e resin was applied on to a colum n of Dowex 50X8. Th e col-
um n was wash ed with H2O–MeOH (80:20), eluted with 2.5% aqueous am m on ia, an d th en
MeOH–Et3N–H2O (10:10:30). Th e com bin ed eluates were evaporated in vacuo an d th e resi-
due was purified on a colum n of silica gel (30 g, S5) to give com poun d 24 (0.54 g, 94%) as
yellow foam . FAB MS, m/z (%): 400 (100), [M + H]. 1H NMR (DMSO-d6): 1.10 d, 6 H,
J(CH3,CH) = 6.2 an d 1.15 d, 6 H (CH3); 3.78 d, 2 H, J(P,CH) = 8.3 (P-CH2); 3.89 t, 2 H,
J(2′,1′) = 5.0 (H-2′); 4.32 t, 2 H, J(1′,2′) = 5.0 (H-1′); 4.48 d, sept, 2 H, J(CH,CH3) = 6.2,
J(P,CH) = 7.6 (P-OCH); 7.10 brs, 4 H (NH); 8.14 s, 1 H (H-8); 8.74 s, 1 H (H-6). 13C NMR
(DMSO-d6): 23.74 d, 2 C, J(P,C) = 4.4 (CH3) an d 23.89 d, 2 C, J(P,C) = 3.9 (CH3); 42.51
(C-1′); 64.71 d, J(P,C) = 164.1 (P-C); 70.33 d, J(P,C) = 11.7 (C-2′); 70.38 d, 2 C, J(P,C) = 6.3
(P-OC); 127.63 (C-5); 144.50 (C-8); 147.73 (C-6); 152.53 (C-4); 158.39 (N-C); 161.91 (C-2).
For C15H26N7O4P (399.4) calculated: 45.11% C, 6.56% H, 24.55% N, 7.76% P; foun d: 44.90% C,
6.90% H, 24.34% N, 7.45% P.
2-Guan idin o-9-[2-(ph osph on om eth oxy)eth yl]-9H-purin e (25)
Workup viz. cleavage of th e ph osph on ate esters – gen eral procedure; startin g com poun d 24
(0.5 g, 1.25 m m ol); Dowex 1X2 eluted with 0.25 M AcOH; yield 0.3 g (76%); wh ite crystals;
m .p. >312 °C (dec.) (H2O). EUp = 0.66. FAB MS, m/z (%): 316 (100), [M + H]. 1H NMR (D2O +
NaOD): 3.53 d, 2 H, J(P,CH) = 8.5 (P-CH2); 3.95 t, 2 H, J(2′,1′) = 5.1 (H-2′); 4.36 t, 2 H,
J(1′,2′) = 5.1 (H-1′); 8.29 s, 1 H (H-8); 8.65 s, 1 H (H-6). 13C NMR (D2O + NaOD): 42.77
(C-1′); 69.09 d, J(P,C) = 149.4 (P-C); 70.03 d, J(P,C) = 9.7 (C-2′); 127.35 (C-5); 146.04 (C-8);
148.46 (C-6); 151.92 (C-4); 159.25 (N-C); 161.51 (C-2). Exact m ass (FAB HRMS) foun d:
316.0937; for C9H15N7O4P [M + H] calculated: 316.0923.
6-Am in o-2-ch loro-9-{2-[(diisopropylph osph oryl)m eth oxy]eth yl}-9H-purin e (11)
Com poun d 11 was prepared from 2-ch loroaden in e14 accordin g to lit.1b Yield 57%.
6-Am in o-9-{2-[(diisopropylph osph oryl)m eth oxy]eth yl}-2-guan idin o-9H-purin e (12)
A guan idin e solution prepared as above for guan idin ation (10 m l, 10 m m ol) was added to
th e flask con tain in g com poun d 11 (0.78 g, 2 m m ol) an d DABCO (0.22 g, 2 m m ol). Th e m ix-
ture was stirred at 120 °C for 24 h , th en triturated with eth er an d th e precipitate was dis-
solved in MeOH an d filtered th rough Cellite. Th e m eth an olic solution was adsorbed on
silica gel, th e crude com poun d was purified on a colum n of silica gel (30 g, EtOAc, th en S7)
to give com poun d 12 (0.77 g, 93%) as yellowish oil. FAB MS, m/z (%): 415 (100), [M + H].
1H NMR (DMSO-d6): 1.10 d, 6 H, J(CH3,CH) = 6.1 an d 1.15 d, 6 H (CH3); 3.77 d, 2 H,
J(P,CH) = 8.3 (P-CH2); 3.86 t, 2 H, J(2′,1′) = 5.0 (H-2′); 4.28 t, 2 H, J(1′,2′) = 5.0 (H-1′); 4.46
m , 2 H (P-OCH); 8.02 s, 1 H (H-8); 7.70 brs, 2 H, 8.40 brs, 3 H an d 10.45 brs, 1 H (NH,
NH2). 13C NMR (DMSO-d6): 23.77 d, 2 C, J(P,C) = 4.9 (CH3) an d 23.91 d, 2 C, J(P,C) = 3.9
(CH3); 42.75 (C-1′); 64.71 d, J(P,C) = 164.1 (P-C); 70.38 d, 2 C, J(P,C) = 6.4 (P-OC); 70.48 d,
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 9, pp. 1303–1319

1316
Česnek, Masojídková, Holý, Šolínová, Koval, Kašička:
J(P,C) = 11.2 (C-2′); 115.68 (C-5); 141.13 (C-8); 149.96 (C-4); 152.27 (C-6); 155.73 an d
155.79 (C-2 an d N-C). Exact m ass (FAB HRMS) foun d: 415.1982; for C15H28N8O4P [M + H]
calculated: 415.1971.
6-Am in o-2-guan idin o-9-[2-(ph osph on om eth oxy)eth yl]-9H-purin e (13)
Workup viz. cleavage of th e ph osph on ate esters – gen eral procedure. Startin g com poun d 12
(0.66 g, 1.6 m m ol); Dowex 1X2 eluted with 0.5 M AcOH; yield 0.17 g (36%); wh ite crystals;
m .p. 266–268 °C (H2O). EUp = 0.49. FAB MS, m/z (%): 331 (10), [M + H]. 1H NMR (D2O +
NaOD): 3.54 d, 2 H, J(P,CH) = 8.5 (P-CH2); 3.92 t, 2 H, J(2′,1′) = 5.1 (H-2′); 4.25 t, 2 H,
J(1′,2′) = 5.1 (H-1′); 7.96 s, 1 H (H-8). 13C NMR (D2O + NaOD): 42.74 (C-1′); 68.97 d, J(P,C) =
149.4 (P-C); 70.16 d, J(P,C) = 10.3 (C-2′); 113.51 (C-5); 141.28 (C-8); 150.46 (C-4); 155.34,
158.97 an d 161.57 (C-6, C-2 an d N-C). Exact m ass (FAB HRMS) foun d: 331.1015; for
C9H16N8O4P [M + H] calculated: 331.1032.
Catalytic Dech lorin ation of 2,6-Dich loropurin e. 2-Ch loropurin e (27) an d Purin e (26)
Com poun ds were prepared accordin g to literature11 with som e m odification s. Th e reaction
m ixture con tain in g 2,6-dich loropurin e (16; 1 g, 5.3 m m ol), CH3COONa (0.72 g, 8.7 m m ol)
an d 5% Pd/C (0.3 g) in H2O (50 m l) was treated with h ydrogen at room tem perature for 3 h .
Th e in soluble m aterial was filtered off from th e reaction m ixture with Cellite. Th e filtrate
was evaporated in vacuo an d purified on a colum n of silica gel (30 g, CHCl3–MeOH, 95:5) to
give two com poun ds:
Com poun d 27 (0.14 g, 17%); wh ite crystals; m .p. 232–234 °C (H2O). FAB MS, m/z (%):
155 (100), [M + H]. 1H NMR (DMSO-d6): 8.68 s, 1 H (H-8); 9.04 s, 1 H (H-6); 13.40 br, 1 H
(NH). 13C NMR (DMSO-d6): 129.04 br (C-5); 147.14 (C-6); 148.06 (C-8); 152.81 (C-2); 157.73 br
(C-4). Exact m ass (FAB HRMS) foun d: 155.0128; for C5H4ClN4 [M + H] calculated: 155.0124.
Com poun d 26 (0.37 g, 58%); wh ite crystals; m .p. 212–214 °C (H2O). FAB MS, m/z (%):
121 (25), [M + H]. 1H NMR (DMSO-d6): 8.61 s, 1 H (H-8); 8.91 s, 1 H (H-2); 9.12 s, 1 H (H-6);
13.45 br, 1 H (NH). 13C NMR (DMSO-d6): 133.05 br (C-5); 145.80 br (C-6); 147.12 br (C-8);
152.21 (C-2); 152.50 br (C-4). Exact m ass (FAB HRMS) foun d: 121.0518; for C5H5N4 [M + H]
calculated: 121.0514.
2-Ch loro-9-isopropyl-9H-purin e (19) an d 2-Ch loro-7-isopropyl-7H-purin e (28)
Preparation of com poun d 19. Procedure A: Hydrazin e h ydrate (0.26 m l) was added dropwise
10
to a solution of com poun d 17
(0.84 g, 3.6 m m ol) in MeOH (18 m l) an d th e resultin g m ix-
ture was stirred at room tem perature for 1 h . An oth er portion of h ydrazin e h ydrate (0.1 m l)
was added an d th e reaction m ixture was stirred at room tem perature for addition al 30 m in .
Th e solven t was rem oved un der reduced pressure, th e residue was codistilled with MeOH
an d purified on a sh ort colum n of silica gel (CHCl3–MeOH, 70:30). Th e product in 50%
aqueous dioxan e (40 m l) was treated with Ag2O (1 g) an d th e reaction m ixture was th en
h eated to reflux for 1 h . Th e in soluble m aterial was filtered off an d th e filtrate was taken
down to dryn ess in vacuo. Th e residue was dissolved in EtOAc (70 m l) an d extracted with a
saturated solution of EDTA (3 × 40 m l). Com bin ed organ ic layers were dried with an h ydrous
MgSO4, evaporated in vacuo an d purified on a colum n of silica gel (30 g, CHCl3) to give
com poun d 19 (0.48 g, 67%). Experim en tal data correspon d to th ose of th e product de-
scribed in procedure B.
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2-Guanidinopurines
1317
Procedure B: NaH (60% dispersion in paraffin oil; 0.04 g, 1 m m ol) was added to a solution
of com poun d 27 (0.15 g, 1 m m ol) in DMF (2 m l) an d th e resultin g m ixture was stirred at
room tem perature for 1 h . Isopropyl brom ide (0.18 m l, 2 m m ol) was added an d th e reaction
m ixture was stirred at room tem perature for 24 h . Th e solven t was rem oved in vacuo, th e
residue was codistilled with toluen e (3 × 10 m l) an d purified on a loose layer silica gel plate
(CHCl3–MeOH, 95:5) to give two com poun ds:
Com poun d 19 (80 m g, 41%); wh ite solid. FAB MS, m/z (%): 197 (100), [M + H]. 1H NMR
(DMSO-d6): 1.55 d, 6 H, J(CH3,CH) = 6.7 (CH3); 4.82 sept, 1 H, J(CH,CH3) = 6.7 (N-CH); 8.76 s,
1 H (H-8); 9.05 s, 1 H (H-6). 13C NMR (DMSO-d6): 22.02, 2 C (CH3); 47.53 (N-CH); 133.62
(C-5); 146.53 (C-8); 149.98 (C-6); 152.65 (C-2); 152.82 (C-4). Exact m ass (FAB HRMS) foun d:
197.0603; for C8H10ClN4 [M + H] calculated: 197.0594.
Com poun d 28 (30 m g, 16%); wh ite solid. FAB MS, m/z (%): 197 (100), [M + H]. 1H NMR
(DMSO-d6): 1.57 d, 6 H, J(CH3,CH) = 6.7 (CH3); 4.89 pen t, 1 H, J(CH,CH3) = 6.7 (N-CH);
8.87 s, 1 H (H-8); 9.25 s, 1 H (H-6). 13C NMR (DMSO-d6): 22.30, 2 C (CH3); 49.68 (N-CH);
124.12 (C-5); 143.77 (C-6); 149.41 (C-8); 153.03 (C-2); 162.55 (C-4). Exact m ass (FAB HRMS)
foun d: 197.0599; for C8H10ClN4 [M + H] calculated: 197.0594.
2-Guan idin o-9-isopropyl-9H-purin e (20)
Guan idin e solution prepared as above (12 m l, 12 m m ol) was added to th e flask con tain in g
com poun d 19 (0.48 g, 2.4 m m ol) an d DABCO (0.27 g, 2.4 m m ol) an d th e m ixture was
stirred at 120 °C for 2 h . Th e solven t was rem oved in vacuo, th e residue was codistilled with
toluen e (3 × 30 m l) an d dissolved in water. Th e solution was n eutralized with Dowex 50X8
(H+ form ) an d th e suspen sion was applied on to a colum n of Dowex 50X8 (20 m l). Th e col-
um n was wash ed with H2O an d eluted with 2.5% aqueous am m on ia, an d th en with
MeOH–Et3N–H2O (1:1:3). Th e com bin ed eluates were evaporated in vacuo an d th e residue
was purified on a colum n of silica gel (30 g, S3 an d S5) an d th e fin al purification was carried
out by HPLC (0.025 M TEAB in gradien t 7–100% MeOH) to give com poun d 20 (0.35 g, 65%);
wh ite crystals; m .p. 252–253 °C (aq u eou s EtOH). FAB MS, m/z (%): 220 (100), [M + H].
1H NMR (DMSO-d6): 1.50 d, 6 H, J(CH3,CH) = 6.7 (CH3); 4.71 sept, 1 H, J(CH,CH3) = 6.7
(N-CH3); 6.95 brs, 4 H (NH2); 8.27 s, 1 H (H-8); 8.72 s, 1 H (H-6). 13C NMR (DMSO-d6):
22.08, 2 C (CH3); 46.12 (N-CH); 127.92 (C-5); 142.12 (C-8); 147.72 (C-6); 152.13 (C-4);
158.72 (N-C); 162.45 (C-2). Exact m ass (FAB HRMS) foun d: 220.1313; for C9H14N7 [M + H]
2–
calculated: 220.1311. For C9H14N8·0.5CO3 (219.3) calculated: 49.30% C, 5.98% H, 44.72% N;
foun d: 49.20% C, 5.84% H, 44.50% N.
6-Am in o-2-guan idin o-9-isopropyl-9H-purin e (15)
A guan idin e solution prepared as decribed above (10.5 m l, 10.5 m m ol) was added to a flask
con tain in g 6-am in o-2-ch loro-9-isopropyl-9H-purin e10 (14; 0.46 g, 2.1 m m ol), DABCO (0.23 g,
2.1 m m ol) an d th e m ixture was stirred at 120 °C for 21 h . Th e solven t was rem oved in
vacuo; th e residue was codistilled with toluen e (3 × 30 m l), dissolved in water an d th e solu-
tion was n eutralized with Dowex 50X8 (H+ form ). Th e suspen sion was applied on to a col-
um n of Dowex 50X8 (20 m l). Th e colum n was wash ed with H2O an d th e product was eluted
with a m ixture of MeOH–Et3N–H2O (10:10:30). Th e eluate con tain in g UV-absorbin g frac-
tion s was evaporated in vacuo an d th e residue was purified on a colum n of silica gel (20 g,
S4–S6) an d th e fin al purification was carried out by HPLC (0.025 M TEAB in gradien t
7–100% MeOH) to give 0.27 g (53%) of wh ite solid; m .p. >206 °C (dec.). FAB MS, m/z (%):
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 9, pp. 1303–1319

1318
Česnek, Masojídková, Holý, Šolínová, Koval, Kašička:
235 (20), [M + H]. 1H NMR (DMSO-d6): 1.47 d, 6 H, J(CH3,CH) = 6.7 (CH3); 4.63 sept, 1 H,
J(CH,CH3) = 6.7 (N-CH); 7.52 brs, 2 H (NH2); 8.12 s, 1 H (H-8); 9.40 br, 4 H (NH). 13C NMR
(DMSO-d6): 22.25, 2 C (CH3); 46.40 (N-CH); 115.74 (C-5); 138.34 (C-8); 149.61 (C-4); 153.72
(C-6); 155.65 an d 156.64 (C-2 an d N-C); 176.42 (C=O). Exact m ass (FAB HRMS) foun d:
2–
235.1424; for C9H15N8 [M + H] calculated: 235.1420. For C9H14N8·0.5H2O·0.5CO3 (234.3)
calculated: 41.75% C, 5.60% H, 41.00% N; foun d: 41.53% C, 5.99% H, 39.82% N.
Capillary Zon e Electroph oresis – In strum en tation 3
CZE an alyses were carried out in th e capillary electroph oretic an alyzer P/ACE MDQ
(Beckm an Coulter, Fullerton (CA), U.S.A.) usin g th e software 32 Karat System , version 7.0
(Beckm an ) for data acquisition an d evaluation . Origin 6.1 (Origin Lab Corp., North am pton
(MA), U.S.A.) was used for data plottin g an d regression an alysis. Th e an alyzer was equipped
with th e in tern ally un coated fused silica capillary with outer polyim ide coatin g, total/effec-
tive len gth 394/292 m m , ID/OD 75/360 µm (Polym icro Tech n ologies, Ph oen ix (AR), U.S.A.).
Th e an alytes were detected by UV-VIS spectroph otom etric ph otodiode array detector
(190–600 n m ) set to operate in th e ran ge 190–300 n m . Absorban ce of an alyzed com poun ds
an d of electroosm otic flow m arkers (DMSO or isoph oron e) was m on itored at 225 an d 250 n m ,
respectively. Th e an alyses were perform ed at con stan t tem perature of th e BGE in side th e
capillary, 25 *C.
Th e n ew capillary was gradually flush ed with water, 0.1 M NaOH, water an d BGE, each
wash ed for 5 m in . Fin ally, th e capillary was con dition ed by a 20 m in application of th e
h igh voltage to equilibrate th e in n er surface an d to stabilize electroosm otic flow (EOF). Be-
tween run s un der th e sam e con dition s, th e capillary was rin sed with th e BGE for 1 m in .
Prior to an y ch an ge of th e BGE th e capillary was rin sed with 0.1 M NaOH for 5 m in an d
th en repeatedly stabilized. Th e sam ples were in jected h ydrodyn am ically, with pn eum atically
in duced pressure 6.9–13.8 m bar for 5–10 s. Th e sam ples were dissolved in con cen tration s of
0.1 m m ol/l in deion ized water or in deion ized water sligh tly alkalized by a sm all addition of
NaOH to en sure solubility of som e com poun ds.
BGE (backgroun d electrolytes) solution s with pH values coverin g a broad pH ran ge
(3.50–11.25) with 0.25 pH un it in crem en t were prepared by m ixin g th e appropriate am oun ts
of stock solution s an d th en diluted to th e con stan t ion ic stren gth 25 m m ol/l. Th e pH was
m easured at 25 °C by pH m eter CyberScan pH 2100 (Oakton In strum en ts, Vern on Hills (IL),
U.S.A.). Th e BGEs were filtered th rough
(Millipore, Bedford (MA), U.S.A.) before use.
a 0.45 µm pore n itrocellulose syrin ge filter
This work is a part of the research project No. Z40550506. 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), by the Gilead Sciences, Inc. (Foster City (CA),
U.S.A.), by the Grant Agency of the Czech Republic (grant No. 203/05/2539), and by the financial
support of the European Commission (René Descartes Prize-2001, grant. No. HPAW-2002-100096).
The authors’ thanks are also due to the staff of the mass spectrometry and analytical departments of
the Institute of Organic Chemistry and Biochemistry.
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