Polymemc conjugates of 9-[2-(phosphonomethoxy)ethyl]purine with potential antiviral and cytostatic activity

Article abstract of DOI:10.1135/cccc20061211

Syntheses and characterization of polymer conjugates of 9-[2-(phosphonomethoxy)ethyl] (PME) derivatives of adenine (PMEA), 2,6-diaminopurine (PMEDAP) and guanine (PMEG) are described. The phosphonate group of these acyclic nucleotide analogues was activated by reaction with triphenylphosphine and di(2-pyridyl) disulfide (TPP-PDS). The activated phosphonate reacted with a random copolymer containing N-(2-hydroxypropyl) methacrylamide (HPMA) and N-(3-methacrylamidopropanoyl)ethane-1,2-diamine (Ma-AP-ED) units. The phosphonamide bond between the nucleotide and polymer carrier proved to be relatively stable at physiological pH 7.4 while at pH 5.0 (corresponding to endosomal or lysosomal compartments of cells) underwent slow hydrolysis. The rate of hydrolysis (drug release) was shown to depend on the detailed structure of the heterocyclic base. The polymer-drug conjugates described in the paper represent a new family of antiviral and cytostatic drugs with potentially improved pharmacokinetics, sustained drug release and diminished non-specific toxicity.

Full text of DOI:10.1135/cccc20061211

Conjugates of 9-[2-(Phosphonomethoxy)ethyl]purine
1211
POLYMERIC CONJUGATES OF 9-[2-(PHOSPHONOMETHOXY)ETHYL]PURINE
WITH POTENTIAL ANTIVIRAL AND CYTOSTATIC ACTIVITY
a1,
a2
a3
a4
Michal PECHAR *, Alena BRAUNOVÁ , Vladimír ŠUBR , Karel ULBRICH and
b
Antonín HOLÝ
a
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic,
62 06 Prague 6, Czech Republic; e-mail: pechar@imc.cas.cz, braunova@imc.cas.cz,
subr@imc.cas.cz, ulbrich@imc.cas.cz
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
66 10 Prague 6, Czech Republic; e-mail: holy@uochb.cas.cz
1
2
1
3
4
b
1
Received May 31, 2006
Accepted July 10, 2006
The other authors M. P., A. B., V. Š. and K. U. wish to dedicate this paper to Professor Antonín Holý
on the occasion of his 70th birthday.
Syn th eses an d ch aracterization of polym er con jugates of 9-[2-(ph osph on om eth oxy)eth yl]
(
PME) derivatives of aden in e (PMEA), 2,6-diam in opurin e (PMEDAP) an d guan in e (PMEG) are
described. Th e ph osph on ate group of th ese acyclic n ucleotide an alogues was activated by
reaction with triph en ylph osph in e an d di(2-pyridyl) disulfide (TPP–PDS). Th e activated ph os-
ph on ate reacted with a ran dom copolym er con tain in g N-(2-h ydroxypropyl)m eth acrylam ide
(
HPMA) an d N-(3-m eth acrylam idopropan oyl)eth an e-1,2-diam in e (Ma-AP-ED) un its. Th e
ph osph on am ide bon d between th e n ucleotide an d polym er carrier proved to be relatively
stable at ph ysiological pH 7.4 wh ile at pH 5.0 (correspon din g to en dosom al or lysosom al
com partm en ts of cells) un derwen t slow h ydrolysis. Th e rate of h ydrolysis (drug release) was
sh own to depen d on th e detailed structure of th e h eterocyclic base. Th e polym er–drug con -
jugates described in th e paper represen t a n ew fam ily of an tiviral an d cytostatic drugs with
poten tially im proved ph arm acokin etics, sustain ed drug release an d dim in ish ed n on -specific
toxicity.
Keywords: Drug delivery system s; Polym er-supported drugs; Cytostatics; An tivirals; Hydrolyses;
Ph osph on am ide bon d; HPMA copolym er; Acyclic n ucleotide an alogues; Acyclic n ucleoside
ph osph on ates.
It h as becom e quite gen erally accepted th at water-soluble polym er con ju-
gates of som e th erapeutic agen ts (e.g., cytostatics or im m un osuppressives)
possess certain advan tages over th eir low-m olecular-weigh t coun terparts.
Prolon ged drug circulation in blood, im proved solubility of h ydroph obic
drugs, lower n on -specific toxicity, especially in case of cytostatics, sup-
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1211–1220
©
2006 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20061211

1
212
Pechar, Braunová, Šubr, Ulbrich, Holý:
pressed im m un ogen icity of polym er-m odified protein s or peptides an d pos-
sible targetin g are th e m ost sign ifican t ben efits.
1
–5
Hydroph ilic polym ers based eith er on poly(HPMA)
or poly(eth ylen e
glycol) (PEG)6
–10
h ave been described as carriers of various low-m olecular-
weigh t drugs. Th e HPMA-based copolym ers were successfully used as carri-
ers of th e an tican cer drug doxorubicin attach ed to th e polym er backbon e
1
1
12
via an en zym atically or h ydrolytically cleavable bon d. Som e of th ese
1
3
14
con jugates were also targeted by eith er polyclon al or m on oclon al an ti-
body to ach ieve a better tum our specificity an d, in con sequen ce, a h igh er
th erapeutic efficien cy. High an titum our efficien cy of th e con jugates was
1
5,16
verified
in m ice bearin g various m urin e tum our m odels, with 50–100%
lon g-term survivors depen din g on th e cell lin e an d sch em e an d dosin g of
polym er drug adm in istration .
In prin ciple, an y cytostatic drug with a suitable fun ction al group can be
covalen tly attach ed to a polym er carrier. A prerequisite for th e biological
activity of such a con jugate is usually degradability of th e bon d between
th e drug an d th e polym er in target tissue.
In th is work, we describe syn th esis an d ch aracterization of th ree
ph osph on ate n ucleotide an alogues lin ked to a prim ary am in e-con tain in g
polym er via a ph osph on am ide bon d kn own to be h ydrolyzed in acid m e-
1
7
dium , e.g. in side en dosom es or lysosom es of target cells.
Man y acyclic n ucleotide an alogues are kn own to sh ow both an tiviral an d
1
8
an titum our effects . 9-[2-(Ph osph on om eth oxy)eth yl) (PME) derivatives of
aden in e (PMEA), 2,6-diam in opurin e (PMEDAP) an d guan in e (PMEG) were
selected to verify feasibility of th eir covalen t attach m en t to copolym ers
based on HPMA bearin g prim ary am in o groups. We believe th at th e th era-
peutic activity of th ese drugs m igh t substan tially im prove on th eir m odifi-
cation with a h ydroph ilic polym er as a con sequen ce of prolon ged
circulation in blood, passive accum ulation of th e polym er con jugate in tu-
1
9
m our tissue due to th e en h an ced perm eability an d reten tion (EPR) effect
an d sustain ed release of th e active com poun d in th e target tissue. Addi-
tion al targetin g of th ese polym er drugs with a site-specific ligan d, e.g. an ti-
body, m igh t furth er en h an ce th eir th erapeutic poten tial.
EXPERIMENTAL
Meth acryloyl ch loride, 1-am in opropan -2-ol, 3-am in opropan oic acid, 2,2′-azobisisobutyro-
n itrile (AIBN), N,N-dim eth ylform am ide (DMF), N,N′-dicycloh exylcarbodiim ide (DCC),
4
,5-dih ydroth iazole-2-th iol (TT), dim eth yl sulfoxide (DMSO), eth an e-1,2-diam in e (ED) an d
pyridin e were purch ased from Fluka. 2,4,6-Trin itroben zen e-1-sulfon ic acid (TNBSA) was pur-
ch ased from Serva Fein bioch em ica Heidelberg. All ch em icals an d solven ts were of an alytical
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1211–1220

Conjugates of 9-[2-(Phosphonomethoxy)ethyl]purine
1213
grade. Solven ts were purified an d dried by stan dard procedures. Th e reagen ts were used
with out furth er purification .
NMR spectra were recorded on a Bruker spectrom eter (300 MHz). Determ in ation of m olec-
ular weigh ts was carried out by size-exclusion ch rom atograph y (SEC) on a Sh im adzu HPLC
system (Sh im adzu, Japan ) equipped with IR, UV an d m ulti-an gle ligh t scatterin g DAWN
DSP-F (Wyatt Tech n ology Corp., U.S.A.) detectors usin g 0.3 M acetate buffer (pH 6.5) an d
Superose 6 colum n (Ph arm acia) or TSK 3000 SWXL colum n (Tosoh Bioscien ce). Th e calcula-
tion of m olecular weigh ts from ligh t-scatterin g detector was based on th e kn own in jected
m ass presum in g 100% m ass recovery. Th e con ten t of ph osph on ate drugs PMEA, PMEDAP
an d PMEG boun d to th e polym ers was determ in ed by UV spectroph otom etry at 261 n m (ε =
–
1
–1
–1
–1
–1
–1
1
4 000 l m ol cm ), 281 n m (ε = 8 800 l m ol cm ), an d 252 n m (ε = 32 300 l m ol cm ),
in H O, respectively. Th e am oun t of th e ph osph on ate groups in th e polym ers was also veri-
2
fied by ph osph orus an alysis. Th e differen ce between th e two m eth ods was less th an 1%; for
th e average value, see Table I. Th e prim ary am in o group con ten t in th e copolym ers was
2
0
determ in ed usin g TNBSA assay as described in literature . Th e con ten t of TT groups in th e
–
1
–1
copolym ers was m easured spectroph otom etrically at 305 n m (ε = 10 880 l m ol cm ), in
2
1–23
DMSO. PMEA, PMEDAP an d PMEG were prepared accordin g to refs
. N-(2-Hydroxy-
3
propyl)m eth acrylam ide (HPMA; 1) was syn th esized as described previously . 3-Meth acryl-
am idopropan oic acid was prepared by th e reaction of m eth acryloyl ch loride with 3-am in o-
2
4
propan oic acid in aqueous alkalin e m edium accordin g to ref.
3
-(3-Meth acrylam idopropan oyl)th iazolidin e-2-th ion e (Ma-AP-TT) (2)
3
3
-Meth acrylam idopropan oic acid (5.0 g, 35 m m ol) an d 4,5-dih ydroth iazole-2-th iol (4.37 g,
7 m m ol) were dissolved in a m ixture of eth yl acetate (72 m ) an d tetrah ydrofuran (THF)
(
–
20 m l). DCC (8.64 g, 42 m m ol) was dissolved in eth yl acetate (11 m l). After coolin g to
15 °C, th e solution s were m ixed, DMAP (ca. 50 m g) was added an d th e resultin g solution
was kept at –15 °C for 1 h an d th en at 5 °C overn igh t. Th e reaction m ixture was stirred at
room tem perature for 1 h , an d th en 100 µl of acetic acid was added. After 1 h stirrin g th e
precipitated N,N′-dicycloh exylurea (DCU) was filtered off, solven ts were evaporated in vac-
uum an d th e oily residue was dissolved in eth yl acetate. A secon d crop of separated DCU
was rem oved by filtration an d th e product Ma-AP-TT was purified by crystallization from
eth yl acetate at –15 °C. Th e product was filtered off, wash ed with dieth yl eth er an d dried in
vacuum . Yield 6.18 g (69%) of yellow solid of m .p. 95–96 °C. For C10H14N O S (258.4) cal-
2
2 2
culated: 46.49% C, 5.46% H, 10.84% N, 24.82% S; foun d: 46.76% C, 5.26% H, 10.85% N,
TABLE I
Ch aracteristics of polym er–drug con jugates
Polym er
Drug
Mw
M /M
Drug loadin g, wt.%
w
n
7
7
7
a
b
c
PMEA
35400
49500
45000
2.28
1.92
1.81
4.7
20.6
3.4
PMEDAP
PMEG
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1211–1220

1
214
Pechar, Braunová, Šubr, Ulbrich, Holý:
2
4
4.46% S. 1H NMR ((CD ) SO): 1.83 s, 3 H (CH ); 3.30–3.43 m , 6 H (CH S, CH -β, CH -α);
3
2
3
2
2
2
.48 t, 2 H (CH N); 5.31 s, 1 H (CH =); 5.63 s, 1 H (CH =); 7.98 t, 1 H (NH).
2
2
2
Copolym er poly(HPMA-co-Ma-AP-TT) (3)
Th e title copolym er was prepared by solution radical copolym erization of HPMA (88 m ole %)
with Ma-AP-TT (12 m ole %) in DMSO at 60 °C for 6 h . Th e con cen tration of com on om ers in
polym erization m ixture was 13 wt.% an d th at of AIBN was 2 wt.%. Th en th e copolym er was
precipitated in to an aceton e–dieth yl eth er (3:1) m ixture, filtered off, wash ed with aceton e
an d dieth yl eth er an d dried in vacuum . Th e con ten t of TT groups was 0.71 m m ol/g.
Copolym er poly(HPMA-co-Ma-AP-ED) (4)
A solution of copolym er 3 (1 g, 0.71 m m ol of TT) in m eth an ol (10 m l) was added dropwise
to a solution of ED (0.4 m l, 7.4 m m ol) in m eth an ol (10 m l) un der stirrin g. Th e yellow solu-
tion of th iazolidin e-2-th ion e derivative decolorized im m ediately. Th e solven t was evapo-
rated un der reduced pressure; th e residue was dissolved in m eth an ol (10 m l); th e product
was precipitated in to an aceton e–dieth yl eth er (3:1) m ixture, filtered off, wash ed with ace-
ton e an d dieth yl eth er an d dried in vacuum . Th e polym er was dissolved in 50 m l of a m ix-
ture of m eth an ol an d 1 M aqueous NaCl (1:1), acidified with 0.1 m l of con cen trated HCl an d
purified by SEC on Seph adex LH 20 with m eth an ol as eluen t. Th e polym er fraction was
evaporated an d lyoph ilized from water yieldin g 920 m g of th e title copolym er. Th e con ten t
of NH2 groups determ in ed by TNBSA assay was 0.78 m m ol/g.
N,N′-Dicycloh exylm orph olin e-4-carboxim idam ide (6)
Th e title com poun d was prepared by a m odified procedure accordin g to literature25. A solu-
tion of N,N′-dicycloh exylcarbodiim ide (25 g, 0.12 m ol) an d distilled m orph olin e (16 m l,
0
.18 m ol) in 1,4-dioxan e (15 m l) was h eated un der reflux for 4 h . Th e solution was allowed
to cool overn igh t. Th e resultin g wh ite crystals were rem oved by filtration , wash ed with cold
dioxan e an d th en with dieth yl eth er. Th e yield was 34.5 g (84%) of wh ite crystals of m .p.
1
6
04–105 °C. For C17H31N O (293.5) calculated: 69.58% C, 10.65% H, 14.32% N; foun d:
9.76% C, 10.77% H, 14.41% N.
3
Polym eric Con jugates of PME–Purin e Derivatives (7a–7c)
A PME derivative (5a–5c) (345 m g, 1.2 m m ol) an d am idin e 6 (352 m g, 1.2 m m ol) were dis-
solved in a m ixture of water (1 m l) an d pyridin e (6 m l). Th e solven t was rem oved un der re-
duced pressure. Th e residue (am idin ium ph osph on ate salt) was dissolved in pyridin e (6 m l)
an d evaporated to dryn ess again . Th e solid dissolved in a m ixture of pyridin e (6 m l) an d
DMF (4 m l) was added to a solution of triph en ylph osph in e an d di(2-pyridyl) disulfide
(
TPP–PDS) in pyridin e (3 m l). Th e resultin g m ixture was added to a solution of copolym er
poly(HPMA-co-Ma-AP-ED) (800 m g, 0.62 m m ol of NH ) in pyridin e (5 m l) an d DMF (15 m l).
2
Th e reaction m ixture was kept at 25 °C for 6 days over an h ydrous Na SO (1 g) with occa-
2
4
sion al stirrin g. Th e progress of th e reaction was ch ecked by SEC followin g an in crease in
absorban ce of th e polym er at 280 n m . Th e m ixture was filtered, th e crude polym er was pre-
cipitated in to an aceton e–dieth yl eth er (3:1) m ixture, filtered off, dried in vacuum , dissolved
in 50 m l of a m ixture of m eth an ol an d 0.6 M ph osph ate buffer pH 7.4 (1:1) an d purified by
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1211–1220

Conjugates of 9-[2-(Phosphonomethoxy)ethyl]purine
1215
SEC on Seph adex LH 20 with m eth an ol as eluen t. Th e polym er fraction was con cen trated
an d th e product was isolated by precipitation in to dieth yl eth er yieldin g 900–920 m g of a
wh ite polym er 7a–7c.
Hydrolytic Release of Ph osph on ate Drugs from Polym er Carrier
A polym er–drug con jugate 7a–7c (5 × 10–4 M drug) was in cubated in 0.07 M ph osph ate
buffer pH 5.0 or pH 7.4 at 37 °C. Th e am oun t of released PME purin e 5a–5c were deter-
m in ed by SEC of th e reaction m ixture from th e area of th e correspon din g peak (TSK 3000
SWXL colum n in 0.3 M sodium acetate buffer, pH 6.5, flow rate 0.5 m l/m in , UV detection at
2
60 n m ).
RESULTS AND DISCUSSION
Th e aim of th is work was to verify th e feasibility of syn th esis of a con jugate
of a ph osph on ate n ucleotide an alogue with a h ydroph ilic syn th etic poly-
m er. Such a con jugate sh ould en able prolon ged drug circulation in blood
an d release of th e active com poun d at a con trolled rate in th e biological en -
viron m en t. Th e ph osph on am ide bon d between th e drug an d polym er car-
rier was ch osen as a h ydrolytically degradable pH-sen sitive lin kage.
Synthesis of Polymer Carrier
Polym er precursor 3, bearin g reactive th iazolidin e-2-th ion e groups, was pre-
pared by radical copolym erization of HPMA (1) and Ma-AP-TT (2) (Schem e 1)
in DMSO solution . Molecular weigh t of th e copolym ers (drug carriers) was
selected between 35–50 000 to ach ieve th e effective EPR effect (accum ula-
tion in solid tum ours) but still en surin g elim in ation of th e polym er from
body by glom erular filtration (th resh old for elim in ation of HPMA copoly-
m ers is ca. 50 000). Th e copolym ers con tain in g TT reactive groups sh ow
low susceptibility to h ydrolysis an d h igh rate of am in olysis in aqueous
2
6
solution s. Th e advan tage of usin g TT copolym ers for th e syn th esis of
polym eric drugs con sists in a possible on e-pot attach m en t of a drug an d
a targetin g ligan d (e.g., an tibody) to th e polym er carriers in aqueous
m edium . Recen tly, such syn th esis of a h um an im m un oglobulin -targeted
2
7
polym er–doxorubicin con jugate was perform ed in our laboratory .
Polym eric am in e 4 was obtain ed by am in olysis of 3 with large excess of
eth an e-1,2-diam in e. No eviden ce of bran ch in g or crosslin kin g of polym er
ch ain s was observed by SEC by com parison of m olecular weigh t distribu-
tion of th e paren t an d am in olyzed polym ers. It was foun d th at purification
of polym er 4 from excess of ED by preparative SEC was in evitable as we did
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1211–1220

1
216
Pechar, Braunová, Šubr, Ulbrich, Holý:
n ot succeed in rem ovin g free ED by sim ple precipitation or dialysis of th e
crude polym er.
Obviously, it would h ave been possible to prepare polyam in e 4 also by
direct copolym erization of HPMA with a suitable am in e-con tain in g m on o-
m er, (e.g. com m ercially available 3-am in opropylm eth acrylam ide). Th is ap-
proach sh ould lead to sim ilar results. However, we decided to use a versatile
polym er precursor 3 th at is used in our laboratory also for con jugation of
oth er biologically active com poun ds.
Th e ration ale for ch oice of β-alan yl spacer is based on its relative h ydro-
ph ilicity an d con ven ien t copolym erization param eters of m on om er 2
allowin g preparation of copolym ers with in a wide ran ge of com on om er
m ixture com position s.
SCHEME 1
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1211–1220

Conjugates of 9-[2-(Phosphonomethoxy)ethyl]purine
1217
Synthesis of the Polymer–Drug Conjugates
Polym er 4 was used for th e attach m en t of ph osph on ate n ucleotide an a-
logues 5a–5c (Sch em e 1). Th e ph osph on ate fun ction of th e drugs was acti-
2
8
vated with TPP–PDS adduct accordin g to th e procedure described for
syn th esis of ph osph oric esters an d ph osph oram ides of n ucleotides. Th e ad-
van tage of th is approach is its ch em oselectivity towards m on osubstituted
ph osph on ate derivatives avoidin g form ation of disubstituted ph osph on ates.
We h ave used th e sam e batch of polym er precursor 4 for preparation of
all con jugates 7a–7c (Mw = 28 000, M /M = 1.8). Th e differen t values in
w
n
Table I m ay be possibly explain ed by partial bran ch in g caused by form ation
of certain m in im al am oun t of disubstituted ph osph on am ides. Alth ough
form ation of disubstituted ph osph on ates usin g TPP–PDS activation is n ot
described in case of low-m olecular weigh t derivatives, th ere is n o in form a-
tion in literature regardin g sim ilar polym er an alogous reaction s. Presen ce of
even a very low am oun t of disubstituted ph osph on am ides would lead to a
substan tial in crease of Mw.
Our in itial attem pts to bin d PME purin es to polym er 4 in a h eteroge-
n eous reaction led eith er to n o or very low loadin g of th e drug. N,N′-di-
cycloh exylm orph olin e-4-carboxim idam ide (6), form in g am idin ium salt
with th e ph osph on ate group25, aided to sign ifican tly im prove solubility of
PME purin es in a DMF–pyridin e m ixture an d, con sequen tly, to en h an ce th e
overall con ten t of drugs in th e con jugates. Despite th e lon g reaction tim e
an d h om ogen eous reaction system , th e fin al loadin g of th e ph osph on ates
in polym er con jugates was far from th e th eoretical am oun t 20 wt.% with
th e exception of polym er 7b. In repeated experim en ts, PMEDAP proved to
bin d m uch m ore efficien tly to polym eric am in e 4 th an PMEA an d PMEG.
We can speculate th at th e reason m igh t con sist in som ewh at better solubil-
ity of PMEDAP leadin g to an equilibrium between th e free an d polym er-
boun d drug, sh ifted to th e latter.
Th e un boun d drugs h ad to be rem oved by preparative SEC in m eth an ol
as dialysis proved to be in efficien t an d led to partial release of ph os-
ph on ates from th e polym er.
Hydrolytic Release of Drugs from the Polymer Carrier
On e of m ajor advan tages of polym er-boun d drugs is, in m ost cases, th eir
low biological activity or even in activity durin g tran sport of th e con jugate
to th e target site in organ ism , e.g. to a tum our or tum our cells. Especially in
th e case of cytostatic drugs (with th eir usually h igh n on -specific toxicity),
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1211–1220

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Pechar, Braunová, Šubr, Ulbrich, Holý:
th is can m in im ize possible dam age caused by th e drug to h ealth y cells or
tissue com in g in to con tact with th e drug. Con sequen tly, a m ech an ism al-
lowin g drug release in th e target site m ust be con sidered before such a poly-
m er drug con jugate is design ed an d syn th esized. It h as been dem on strated
previously th at HPMA-based copolym ers, with th e an titum our drug doxo-
rubicin boun d via th e acid-labile h ydrazon e bon d, are capable of releasin g
doxorubicin in side en dosom es of m align an t cells wh ile th e con jugate is rel-
2
9
atively stable durin g blood tran sport. It h as been sh own usin g con focal
m icroscopy th at fluorescen tly labelled polym er con jugates rem ain in side
th e cells m ore th an 48 h th us en ablin g acidolytic release of th e drug.
In th is work, we h ave used a sim ilar strategy. We took advan tage of sus-
ceptibility of th e ph osph on am ide bon d to acid h ydrolysis. Figure 1 sh ows
th e release profiles of ph osph on ate drugs from polym ers 7a–7c. Th e
am oun t of drug released with in 48 h in ph osph ate buffer at pH 5.0 (corre-
spon din g to en dosom es or lysosom es) from polym ers 7a–7c was 47% of
PMEA, 35% of PMEDAP an d 61% of PMEG. At ph ysiological pH 7.4 (blood
pH), PMEA (7a) an d PMEG (7c) con jugates were practically stable (<2% of
th e drug released); on ly for th e PMEDAP con jugate 7b th e degree of h ydro-
lysis was 22%. Th is surprisin g differen ce in h ydrolytic stability of th e stud-
ied polym eric ph osph on am ides seem s to in dicate a sign ifican t in fluen ce of
FIG. 1
Hydrolytic release of drugs PMEA, PMEDAP an d PMEG from con jugates 7a–7c in cubated
–
4
in 0.07 M ph osph ate buffers pH 5.0 or 7.4 at 37 °C. Con cen tration s of th e drugs were 5 × 10
–
1
m ol l . Th e am oun t of released drug was determ in ed by SEC of th e reaction m ixture, from th e
correspon din g peak area (colum n TSK 3000 SWXL , 0.3 M sodium acetate buffer, pH 6.5, flow
rate 0.5 m l/m in , UV detector 260 n m ). ᭹ PMEA (pH 5.0), ᭺ PMEA (pH 7.4), ꢀ PMEDAP (pH
5
.0), ᭝ PMEDAP (pH 7.4), ᭿ PMEG (pH 5.0), ٗ PMEG (pH 7.4)
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 8, pp. 1211–1220

Conjugates of 9-[2-(Phosphonomethoxy)ethyl]purine
1219
th e detailed structure of th e h eterocyclic base on h ydrolysis. Obviously, th e
larger is th e differen ce in th e h ydrolytic rates at pH 5.0 an d 7.4, th e better
sh ould be th e tum our-versus-blood selectivity of th e polym eric drug. Un -
fortun ately, a h igh drug loadin g was ach ieved on ly with PMEDAP usin g th e
procedure described above. Neverth eless, th e ach ieved drug loadin g sh ould
be sufficien t for evaluation of biological properties of th e copolym ers.
CONCLUSION
Th e feasibility of preparation of polym er–drug con jugates con tain in g th ree
differen t ph osph on ate n ucleotide an alogues with cytostatic an d an tiviral
activity h as been dem on strated. Th e ph osph on am ide bon d between th e
polym eric carrier an d drug proved to be susceptible to m ild acid-catalyzed
h ydrolysis th us en ablin g con trolled release of th e free drug in m edia m odel-
lin g target cell en viron m en t. Th e con jugates in cubated in a buffer m odel-
lin g th e en viron m en t durin g blood tran sport were m ore resistan t to
h ydrolysis (PMEDAP) or even stable (PMEA, PMEG). Th e rate of drug release
sign ifican tly depen ded on th e detailed structure of th e h eterocyclic base in
th e drug m olecule. Cytostatic activities of th e con jugates with PMEDAP an d
PMEG are un der evaluation .
This research was supported by the Grant Agency of the Academy of Sciences of the Czech Republic
(
grant No. A4050201). The authors are indebted to Dr I. Rosenberg for his kind advice in phosphonate
chemistry.
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