Synthesis and cytostatic activity of N-[2-(phosphonomethoxy)alkyl] derivatives of N6-substituted adenines, 2,6-diaminopurines and related compounds

N-[2-Phosphonomethoxy)alkyl] Derivatives

1545

SYNTHESIS AND CYTOSTATIC ACTIVITY

OF N-[2-(PHOSPHONOMETHOXY)ALKYL] DERIVATIVES

OF N⁶-SUBSTITUTED ADENINES, 2,6-DIAMINOPURINES

AND RELATED COMPOUNDS

1,

2

Antonín HOLÝ *, Ivan VOTRUBA , Eva TLOUŠŤOVÁ and Milena MASOJÍDKOVÁ

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,

1

2

166 10Prague 6, Czech Republic; e-mail: holy@uochb.cas.cz, votruba@uochb.cas.cz

Received Septem ber 17, 2001

Accepted October 15, 2001

N⁶-Substituted aden in e an d 2,6-diam in opurin e derivatives of 9-[2-(ph osph on om eth oxy)-

eth yl] (PME), 9-[(R)-2-(ph osph on om eth oxy)propyl] [(R)-PMP] an d en an tiom eric (S)-PMP se-

ries were syn th esized by reaction s of prim ary or secon dary am in es with 6-ch loro-9-{[2-(diiso-

propoxyph osph oryl)m eth oxy]alkyl}purin es (26–28) or 2-am in o-6-ch loro-9-{[2-(diisopropoxy-

ph osph oryl)m eth oxy]alkyl}purin es (29–31) followed by treatm en t of th e diester in term edi-

ates 32 with brom o(trim eth yl)silan e an d h ydrolysis. Diesters 32 were also obtain ed by reac-

tion of N⁶-substituted purin es with syn th on s 23–25 bearin g diisopropoxyph osph oryl group.

Alkylation of 2-am in o-6-ch loropurin e (9) with dieth yl [2-(2-ch loroeth oxy)eth yl]ph osph on ate

(148) gave th e diester 149 wh ich was an alogously con verted to N⁶-substituted 2,6-diam in o-

9-[2-(2-ph osph on oeth oxy)eth yl]purin es 151–153. Alkylation of N⁶-substituted 2,6-diam in o-

purin es with (R)-[(trityloxy)m eth yl]oxiran e (155) followed by reaction of th us-obtain ed in -

term ediates 156 with dim eth ylform am ide dim eth ylacetal an d con den sation with diisopropyl

[(tosyloxy)m eth yl]ph osph on ate (158) followed by deprotection of th e in term ediates 159

gave N⁶-substituted 2,6-diam in o-9-[(S)-3-h ydroxy-2-(ph osph on om eth oxy)propyl]purin es

160–163. Th e h igh est cytostatic activity in vitro was exh ibited by th e followin g N⁶-deriva-

tives of 2,6-diam in o-9-[2-(ph osph on om eth oxy)eth yl]purin e (PMEDAP): 2,2,2-trifluoroeth yl

(53), allyl (54), [(2-dim eth ylam in o)eth yl] (68), cyclopropyl (75) an d dim eth yl (91). In

CCRF-CEM cells, th e cyclopropyl derivative 75 is deam in ated to th e guan in e derivative

PMEG (3) wh ich is th en con verted to its diph osph ate.

Keyw ord s: Purin es; Nucleotide an alogs; Ph osph on ates; Acyclic n ucleoside ph osph on ates;

ANP; Cytostatic activity; An tin eoplastic; An tican cer.

Acyclic n ucleoside ph osph on ates (ANP), isopolar an alogues of n ucleotides,

wh ich con tain ph osph on om eth oxy fun ction lin ked to an acyclic side ch ain

in stead of ph osph ate group boun d to th e n ucleoside sugar m oiety, attract

con siderable atten tion . An im portan t represen tative of th is class is 1-[(S)-3-

h ydroxy-2-(ph osph on om eth oxy)propyl]cytosin e (CAS 113852-37-2, HPMPC,

Cidofovir)¹sh owin g gen eral an ti-DNA-viral activity. Its paren teral form

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

doi:10.1135/cccc20011545

1546

Holý et al.:

Vistide was approved for treatm en t of cytom egalovirus retin itis in AIDS

patien ts². Cidofovir is also used in th e th erapy of acyclovir- an d foscarn et-

resistan t HSV-1 lesion s an d in oth er viral diseases, such as papillom avirus

in duced warts, n asoph aryn geal carcin om a, etc. (for a review, see ref.³).

An oth er im portan t ANP, 9-[2-(ph osph on om eth oxy)eth yl]aden in e (1)

(PMEA, Adefovir, ADV, CAS 106941-25-7) exh ibits an tiviral activity in vitro

directed again st som e DNA viruses⁴an d again st retroviruses^5,6. Also diseases

caused by oth er m am m alian retroviruses – MAIDS virus⁷, FLV (ref.⁸), Visn a

virus⁹an d sim ian im m un odeficien cy virus (SIV)¹⁰were successfully treated

with th is drug¹¹. Its bis[(trim eth ylacetoxy)m eth yl] ester (Bis-POM-PMEA,

Preveon ) sh ows an in creased oral resorption an d en h an ced tran sport in to

th e cell¹². It is active in vitro again st HIV-1 an d HIV-2. It was scrutin ized for

AIDS th erapy¹³but later with drawn due to certain n eph rotoxicity at a th er-

apeutic dosage. Presen tly, it is in ph ase III clin ical trials for ch ron ic h epati-

tis B virus (HBV) in fection s, in particular in patien ts with lam ivudin e-

resistan t HBV strain s¹⁴.

NH₂

OH

N

R

N

H₂N

N

O

P(O)(OH)₂

O

P(O)(OH)₂

1 R=H, PMEA

3 PMEG

2 R=NH₂, PMEDAP

NH₂

N

NH₂

N

R

N

R

N

CH₃

P(O)(OH)₂

CH₃

P(O)(OH)₂

O

4 R=H, (R)-PMPA

6 R=H,

(S)-PMPA

5 R=NH₂, (R)-PMPDAP

7 R=NH₂, (S)-PMPDAP

Th e an tiviral poten cy of th e 2,6-diam in opurin e derivative (PMEDAP, 2)

an d guan in e derivative (PMEG, 3) is h igh er com pared to PMEA. PMEDAP is

extrem ely active in vivo again st m am m alian retroviruses¹⁵. However, PMEG

is h igh ly cytotoxic an d th e in terest in an tiviral properties of th is com poun d

th us focused ch iefly on its effect on papillom aviruses¹⁶.

Th e structure of an oth er ANP group – PMP-derivatives¹⁷– is ch aracterized

by substitution with m eth yl group in position 2 of th e side-ch ain : 9-[(R)-2-

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

N-[2-Phosphonomethoxy)alkyl] Derivatives

1547

(phosphonom ethoxy)propyl]adenine [(R)-PMPA, 4] and 9-[(R)-2-(phosphono-

m eth oxy)propyl]-2,6-diam in opurin e [(R)-PMPDAP, 5] exh ibit h igh poten cy

again st HIV-1 an d HIV-2 in vitro¹⁸an d (R)-PMPA (Ten ofovir) sh ows th era-

peutic an d proph ylactic activity in sim ian m odel of im m un odeficien cy dis-

ease¹⁹. Ten ofovir or its oral prodrug Ten ofovir disoproxil (Viread ) reach ed

presen tly th e fin al clin ical ph ase for treatm en t of AIDS in both NRTI-n aïve

an d NRTI-resisten t (n ucleoside reverse tran scriptase in h ibitors) patien ts.

Th e activity in th e PMP series is strictly en an tiospecific. Both (S)-PMPA (6)

an d (S)-PMPDAP (7) are an tivirally in active. Replacem en t of th e m eth yl

group at th e side ch ain by oth er alkyl or cycloalkyl groups results in com -

plete loss of an tiviral activity²⁰

.

We h ave system atically in vestigated th e relation between th e structure of

th e h eterocyclic base an d an tiviral activity of PME derivatives²¹. Most of

our atten tion con cern ed th e C-substitution of th e h eterocyclic base²². Th ese

observation s led us to devise th e gen eral structure of th e h eterocyclic part

of th e ph arm acoph ore. It is ch aracterised by cum ulation of am in o groups

in th e pyrim idin e part of th e purin e system . In order to decide wh eth er th e

role of th e am in o groups con sists solely in th eir basicity or th eir

participation in h ydrogen bon d form ation , we h ave syn th esized th e

2-(am in om eth yl) an alogues of PMEG an d PMPG (ref.²³), as well as th e

6-(am in om eth yl) an alogues of PME-, PMP- an d HPMP-derivatives of ade-

n in e an d 2,6-diam in opurin e²⁴. Non e of th em exerted an y an tiviral activity.

Nor was an y activity observed with 6-(C-h etaryl)²⁵, or 6-[1-(N-alkylam in o)-

ethyl²⁶derivatives. The quaternary PME derivatives of 2,4-diam inopyrim idine

an d 4,6-diam in opyrim idin e²⁷wh ich are also relevan t to th e h ypoth etical

ph arm acoph ore, were devoid of an y activity as well.

We h ave also studied th e in fluen ce of N-substitution of th e 6-am in o

group in th e PME an d PMP derivatives of aden in e an d 2,6-diam in opurin e

on th eir an tiviral activity. In th e origin al con tribution s, we reported on ex-

trem ely h igh an tiviral activity of n um erous PME derivatives of th ese groups

again st DNA viruses, in particular CMV an d VZV (ref.²⁸). Later, oth er papers

described th eir sign ifican t in vitro activity again st Epstein –Barr virus²⁹. Al-

ready in th e first ph ase of th e SAR study, several com poun ds (e.g.

N⁶-cyclopropyl, N⁶-allyl an d N⁶-dim eth yl derivatives of PMEDAP), sh owed

m arkedly h igh er an tiviral activities com pared to th e oth er m em bers of th e

group.

Adefovir exh ibits also cytostatic activity in vivo in rat an d m ouse carcin o-

m as an d sarcom as³⁰. Th e th erapeutic effect is still m ore pron oun ced in

PMEDAP³¹, both in m on oth erapy an d in com bin ation with oth er an ti-

can cer drugs (taxotere)³². Cytostatic activity was reported also for ANPs in

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

1548

Holý et al.:

N⁶-substituted-purin e series: we h ave reported on an an tiproliferative effect

of th ese com poun ds on m urin e lym ph ocytes. Th is study revealed a parallel

between an tiviral an d an tiproliferative activities, at least in som e of th e

PME an d PMP series derived from aden in e an d/or 2,6-diam in opurin e. Som e

of th e com poun ds sh owed extrem ely h igh in vitro activities³³. Cytostatic ac-

tivity of th e N⁶-cyclopropyl derivative of PMEDAP (cypr-PMEDAP) was fur-

th er in vestigated by oth er auth ors³⁴; th is com poun d is believed to act as a

prodrug of PMEG (3) form in g th is h igh ly cytotoxic an tican cer com poun d³⁵

by th e action of cellular AMP deam in ase³⁶. We h ave in vestigated an ti-

tum our activity of cypr-PMEDAP an d several of its con gen ers selected on

th e basis of th eir in vitro cytostatic activity (vide infra), in an in vivo m odel

of h aem atological m align an cy of in bred Sprague–Dawley rats. However, in

th is m odel, th ey were less poten t or exh ibited th e sam e an tin eoplastic ef-

fect as PMEDAP (ref.³⁷).

Bioch em ical studies³⁸dem on strated ph osph orylation of ANP to th eir

m on o- an d diph osph ates. Th us, PMEA is con verted to its m on o- an d di-

ph osph ate (dADP an d dATP an alogues) un der catalysis of n ucleotide kin -

ases³⁹. Th e diph osph ate (PMEApp) selectively in h ibits cellular DNA

polym erases⁴⁰, actin g as a substrate/in h ibitor for reverse tran scriptases⁴¹,

an d its in corporation in th e growin g DNA ch ain leads to ch ain term in a-

tion . PMEDAP an d PMEG are also ph osph orylated by cellular n ucleotide

kin ases^39,42. In addition to th e action of th eir diph osph ates on DNA

polym erases an d reverse tran scriptases, PMEG an d its an abolites efficien tly

in h ibit purin e n ucleoside ph osph orylases⁴³; th is activity m igh t explain

h igh toxicity of guan in e derivatives in all in vestigated ANP series.

As already m en tion ed, our open in g SAR studies on an tiviral an d an ti-

proliferative effects provided th ree lead ANP structures with cyclopropyl-

am in o, allylam in o an d dim eth ylam in o substituen ts in position 6 of th e

purin e rin g. Variation of substituen ts in com poun ds of th is study th us leads

m ain ly to (a) alkyl, alken yl an d alkyn ylam in o derivatives, (b) cycloalkyl-

am in o derivatives, (c) dialkylam in o, dialken ylam in o, dicycloalkylam in o an -

alogues an d (d) N-h etaryl (pyrrolidin -1-yl, piperidin -1-yl, m orph olin -4-yl,

etc.) con gen ers. Substituen ts bearin g on e or two h ydroxyalkyl, alkoxyalkyl,

am in oalkyl or dialkylam in oalkyl groups were also in troduced.

In th is paper we describe (i) th e syn th esis of N⁶-substituted purin e ANP

derivatives, (ii) detailed SAR in vestigation of th e effect of substituen ts at th e

6-am in o group in th e aden in e an d 2,6-diam in opurin e derivatives on th e

cytostatic activity in vitro an d (iii) in troductory bioch em ical studies with

special atten tion to th e m etabolism of N⁶-substituted ANPs, th eir

ph osph orylation an d deam in ation to th e 6-oxopurin e com poun ds. Th e ul-

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

N-[2-Phosphonomethoxy)alkyl] Derivatives

1549

tim ate objective was th e selection of optim um can didates for in vestigation

of th eir an tin eoplastic activity in vivo.

Th e SAR study was perform ed in several series of ANP type in cludin g

PME, (R)-PMP, (S)-PMP derivatives an d was com plem en ted by related

N⁶-substituted (S)-HPMPDAP derivatives, N⁶-substituted 9-[2-(2-ph osph on o-

eth oxy)eth yl] (PEE) an alogues⁴⁴(h om ologs of PMEDAP) an d by regio-

isom eric N⁶-substituted 7-PMEDAP derivatives. Th e lim ited ch oice of

N⁶-substituen ts in two last m en tion ed series followed th e structure of th ose

substituen ts wh ich gave optim um effects in th e basic series of ANPs.

Synthesis

Th e preparation of 9-PME or 9-PMP derivatives substituted at th e 6-am in o

fun ction of aden in e or 2,6-diam in opurin e em ployed two routes (Sch em e 1):

Tran sform ation of 6-ch loro-9-{2-[(diisopropoxyph osph oryl)m eth oxy]eth yl}-

purin e (26) an d 2-am in o-6-ch loro-9-{2-[(diisopropoxyph osph oryl)m eth oxy]-

eth yl}purin e (29) to th e in term ediate N-substituted 6-am in o derivatives 32

by th e action of excess prim ary or secon dary am in e in eth an ol solution .

Th e crude reaction product was desalted an d cleaved with brom o(tri-

m eth yl)silan e (Me₃SiBr) to afford, after deion ization an d ion exch an ge

ch rom atograph y th e free acids of PME derivatives of th e aden in e series

35–50 or th e 2,6-diam in opurin e series (51–104). Th e required startin g m a-

terials 26 an d 29 are easily available by alkylation of 6-ch loropurin e (8) or

2-am in o-6-ch loropurin e (9) with diisopropyl [(2-ch loroeth oxy)m eth yl]-

ph osph on ate (23).

En an tiom eric (R)- an d (S)-PMP-derivatives were prepared an alogously start-

ing from the 6-chloro-9-{2-[(diisopropoxyphosphoryl)m ethoxy]propyl}purines

(27, 28) and -2-amino-6-chloro-9-{2-[(diisopropoxyphosphoryl)methoxy]propyl}-

purin es (30, 31). Th e deprotection of th e correspon din g in term ediate

diesters 32 an d purification of th e free ANPs was perform ed an alogously to

afford N⁶-substituted (R)-PMP derivatives of aden in e 105–115 or 2,6-di-

am in opurin e 116–130, or, th e en an tiom eric N⁶-substituted derivatives of

(S)-PMPA 131–137 or (S)-PMPDAP 138–142. Also in th is case, th e startin g

6-ch loro derivatives are accessible by alkylation of 6-ch loropurin es 8, 9

with 2-[(diisopropoxyph osph oryl)m eth oxy]propyl tosylates 24, 25 (ref.^17b).

Reaction s with am in es were usually perform ed in refluxin g eth an olic so-

lution un der exclusion of CO₂, except for low-boilin g am in es (m eth yl-

am in e, dim eth ylam in e, 2,2,2-trifluoroeth ylam in e, etc.); wh ere th e reaction s

were perform ed in an autoclave. (In altern ative preparation of

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

1550

Holý et al.:

6-(dim eth ylam in o)purin e derivatives 32 (R¹= R²= CH₃), dim eth ylam in e

was replaced by dim eth ylam m on ium N,N-dim eth ylcarbam ate; in th is case,

th e reaction of com poun ds 26, 29 proceeded sm ooth ly in boilin g

aceton itrile.)

Th e oth er gen eral route for th e syn th esis of N⁶-substituted ANPs m akes

use of th e alkylation of N⁶-substituted aden in es (10–17) or 2,6-diam in o-

purin es 18–22 with appropriate syn th on s 23–25 un der th e earlier described

con dition s^17b,22. Th e th us-obtain ed in term ediary ph osph on ate diesters 32

were subsequen tly con verted to th e free ph osph on ic acids by th e above

R¹R²

N

R¹

Cl

N

HN

R²

N

H

N

H

R

N

8 R=H

10-17 R=H

9 R=NH₂

18-22 R=NH₂

R³

*

X

O

P(O)(iPr)₂

R³config.

R²

R¹

N

R¹

HN

Cl

N

X

N

R²

N

23 Cl

H

-

R

N

24 OTs CH₃(R)

25 OTs CH₃(S)

R³

*

R³

*

O

P(O)(OiPr)₂

26-31

32

O

P(O)(OiPr)₂

R

H

R³config.

1. TMSBr

2. H₂O

26

27

28

H

-

CH₃(R)

CH₃(S)

29 NH₂

30

H

-

R¹R²

N

R¹R²

N

R¹R²

N

NH₂CH₃(R)

31 NH₂CH₃(S)

N

R

N

R

N

R

CH₃

P(O)(OH)₂

CH₃

O

P(O)(OH)₂

O P(O)(OH)₂

33-50 R=H

51-104 R=NH₂

105-115 R=H

116-130 R=NH₂

131-137 R=H

138-142 R=NH₂

For list of substituents R¹and R²see Table I.

SCHEME 1

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

N-[2-Phosphonomethoxy)alkyl] Derivatives

1551

procedure. Th e startin g h eterocyclic bases 10–22 were obtain ed from

6-ch loropurin e (8) or its 2-am in o con gen er 9 by th e action of an appropri-

ate am in e followed by deion isation . Th e alkylation was perform ed in DMF

in th e presen ce of eith er Cs₂CO₃or, preferably, DBU (1,8-diazabicyclo-

[5.4.0]un dec-7-en e). Th is route was used particularly in th ose cases wh ere a

larger quan tity of th e ph osph on ate was required.

7-[2-(Ph osph on om eth oxy)eth yl] derivatives 144–147 were prepared by

reactin g 6-ch loropurin e (143a) or 2-am in o-6-ch loropurin e in term ediate

143b with excess prim ary or secon dary am in e in eth an ol followed by

deion isation , deprotection with Me₃SiBr an d purification (Sch em e 2). Th e

startin g m aterials are accessible as by-products of th e alkylation of

6-ch loropurin es 8, 9 with com poun d 23 (ref.²²).

O

P(O)(OH)₂

O

P(O)(OiPr)₂

R¹

N

R²

N

Cl

N

R

143 a, R=H

b, R=NH₂

144-147

For list of substituents R¹and R²see Table I.

SCHEME 2

Hom ologues of PMEDAP derivatives, N⁶-substituted 2-am in o-9-[2-(2-

ph osph on oeth oxy)eth yl]purin es (PEE derivatives) were syn th esized by th e

procedure described in Sch em e 3: Reaction of 2-am in o-6-ch loropurin e (9)

with dieth yl [2-(2-ch loroeth oxy)eth yl]ph osph on ate (148) gave th e diester

in term ediate 149; th is com poun d was tran sform ed by reaction with am in es

to com poun ds 150 wh ich in turn gave th e free ph osph on ates 151–153 by

tran ssilylation with Me₃SiBr followed by h ydrolysis. Alkalin e h ydrolysis of

com poun d 149 followed by ester cleavage afforded th e guan in e derivative

154 (PEEG) (Sch em e 3).

Th e last type of ANP derivatives in cluded in th is study, is th e 9-[3-

h ydroxy-2-(ph osph on om eth oxy)propyl] derivatives (HPMP derivatives).

With respect to our previous kn owledge of th eir biological activity²¹, we

h ave syn th esized solely th e (S)-en an tiom ers related to (S)-HPMPDAP (164)

or (S)-HPMPG (165). Owin g to th e laborious m ultistep syn th esis, th e ch oice

of 6-substituen ts was lim ited to th ose groups wh ich gen erally sh ow th e

m ost prom isin g effects (dim eth ylam in o, allylam in o, cyclopropylam in o,

[(2-dim eth ylam in o)eth yl]am in o group). Treatm en t of N⁶-substituted

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

1552

Holý et al.:

NH₂

N

O

N

HN

H₂N

OH

O

P(O)(OH)₂

O

P(O)(OH)₂

164 (S)-HPMPDAP

165 (S)-HPMPG

2,6-diam in opurin es 18–21 with (S)-tritylglycidol (155) gave 9-[(S)-2-

h ydroxy-3-(trityloxy)propyl] derivatives 156. Th e 2-am in o group in th e pu-

rin e base was protected by reaction with (dim eth oxym eth yl)dim eth ylam in e

(dim eth ylform am ide dim eth ylacetal) an d th e resultin g form am idin e deriva-

tives 157 were directly reacted with diisopropyl [(tosyloxy)m eth yl]ph os-

ph on ate (158)^1can d excess NaH. After alkalin e work-up, th e resultin g crude

O

N

HN

N

H₂N

N

154

O

P(O)(OH)₂

Cl

N

Cl

N

Cl

148

N

O

N

P(O)(OEt)₂

N

H

H₂N

DBU

9

149

O

P(O)(OEt)₂

R¹

R²

NH

R¹

R²

R¹

R²

N

1. TMSBr

2. H₂O

N

H₂N

N

H₂N

N

O

P(O)(OH)₂

P(O)(OEt)₂

151-153

150

For list of substituents R¹and R²see Table I.

SCHEME 3

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

N-[2-Phosphonomethoxy)alkyl] Derivatives

1553

trityl-con tain in g diester 159 was treated with Me₃SiBr to deprotect sim ulta-

n eously trityl an d ph osph on ate ester groups. Th is procedure afforded

N⁶-substituted (S)-HPMPDAP derivatives 160–163. Ion exch an ge ch rom a-

tograph y of th e deion ised crude m aterials was used for th e ultim ate purifi-

cation (Sch em e 4).

Free acyclic n ucleoside ph osph on ic acids prepared in th is study were usu-

ally crystallized from water or aqueous eth an ol givin g frequen tly m on o- or

dih ydrates. Th eir structure was routin ely con firm ed by ¹H NMR wh ich

sh owed, in addition to ch aracteristic sign als of side-ch ain proton s (an d

m eth yl group in th e PMP-series), C-8 (an d C-2) proton s, also th e proton s

correspon din g to N⁶-substituen t(s). Th eir electroph oretic m obility was gen -

erally sligh tly lower com pared to th e paren t ph osph on ates, except for th e

N⁶-(ω-am in oalkyl)am in o derivatives wh ich sh owed a substan tially de-

creased m obility.

R¹R²

N

R¹R²

N

OTr

O

N

155

N

H

H₂N

N

H₂N

N

OTr

156

19-21

OH

R¹R²

N

R¹R²

N

TsOCH₂P(O)(OiPr)₂

158

N

NaH

N

(CH₃)₂N

N

OTr

P(O)(OiPr)₂

OTr

157

159

O

OH

BrSiMe₃

R¹R²

N

OH

P(O)(OH)₂

For list of substituents R¹and R²see Table I.

160-163

O

SCHEME 4

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

1554

Cytostatic Activity

Holý et al.:

Th e evaluation of cytostatic activity of N⁶-substituted aden in e an d 2,6-di-

am in opurin e ANPs in vitro was perform ed with four cell lin es: two of th em

(L1210, L929) were m ouse cell lin es, two (HeLaS3 an d CCRF-CEM) were of

h um an origin . Th e procedure con sisted of two steps: in th e first approxim a-

tion , th e cells were grown in th e presen ce of tested com poun ds at a con -

stan t drug con cen tration an d, after a certain period of tim e, th e cells were

coun ted an d th e in h ibition expressed as percen tage of cell coun t in a con -

trol culture grown in th e culture m edium alon e. Essen tially all ANPs pre-

pared in th is study were in cluded in th is assay (Table I). Com poun ds wh ich

passed th e first screen sh owin g sign ifican t in h ibitory activity (usually

50–70% of th e con trol, at least in two cell lin es) were selected for th e sec-

on d evaluation step, an d th eir IC₅₀values for in dividual cell cultures were

estim ated usin g stan dard m eth odology (Table II). Th e results lead to th e

followin g con clusion s:

a) Th e h um an lym ph oblastoid cell lin e CCRF-CEM is gen erally m ost sen -

sitive to th e action of ANPs, followed by m ouse leukem ia L929, wh ile

L1210 an d HeLaS3 are gen erally less sen sitive to th e drug action in vitro.

b) Th e N-substitution of 6-am in o group in PMEA with alkyl, alken yl or

cycloalkyl group as well as with alkyl groups ω-substituted by am in o,

dim eth ylam in o or h ydroxy fun ction (33–46) results in in active com -

poun ds. On ly som e N,N-disubstituted derivatives, e.g. N,N-dieth ylam in o-

(48) an d N-(piperidin -1-yl)purin e (50) derivatives sh ow in dication of sligh t

growth in h ibition wh ich , h owever, was n ot con firm ed in th e secon d scru-

tin y.

c) Th e N-substitution of 6-am in o group in PMEDAP led to n um erous

com poun ds with cytostatic activity: (i) in th e N-m on oalkyl group, som e ef-

fect was observed in all com poun ds; N⁶-m eth yl (51), butyl (56) an d

sec-butyl (57) derivatives were m odestly active, but th e stron gest activity

was observed in N⁶-propyl (52) an d isobutyl (58) derivatives. (ii) Th e m ost

active com poun d in th e N⁶-alken yl-PMEDAP series is th e allyl derivative 54;

it is closely followed by th e structurally related but-2-en -1-yl, but-3-en -2-yl

an d 2-m eth ylallyl derivatives (59, 60 an d 62, respectively) all of wh ich con -

tain th e structural elem en t of th e N-allyl group. Th e isom eric but-3-en -1-yl

(h om oallyl) derivative 61 is m uch less active. Cytostatic activity was ob-

served also for N⁶-propargyl-PMEDAP (55).

d) Substitution in th e N⁶-alkyl ch ain in th e PMEDAP series with h ydroxy

(63–66, 71) or m eth oxy group (67) as well as th e presen ce of an am in o

group (69) in activates th e com poun ds. On th e con trary, th e in troduction of

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

N-[2-Phosphonomethoxy)alkyl] Derivatives

1555

TABLE I

Cytostatic activity^aof N⁶-substituted 6-am in o-9-[2-(ph osph on om eth oxy)alkyl]purin es an d

2,6-diam in o-9-[2-(ph osph on om eth oxy)alkyl]purin es

% Con trol

Com -

poun d

R¹

R²

CCRF-

CEM

L-1210

L-929

HeLaS3

N⁶-Substituted 6-am in o-9-[2-(ph osph on om eth oxy)eth yl]purin es

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

Allyl

H

85

100

96

91

101

98

94

–

68

90

93

81

83

86

92

–

85

97

88

91

Cyclopropyl

Isopropyl

H

103

100

101

94

97

Isobutyl

H

95

2-Hydroxyeth yl

2-Meth oxyeth yl

3-Hydroxypropyl

2-Hydroxypropyl

1-Hydroxypropan -2-yl

2-Am in oeth yl

2-(Dim eth ylam in o)eth yl

3-Am in opropyl

4-Am in obutyl

Cycloh exyl

H

94

H

93

H

96

97

H

96

H

–

90

H

97

91

89

87

92

70

94

95

86

92

74

85

78

69

75

68

96

79

H

92

–

H

104

101

94

92

H

108

89

H

Meth yl

m eth yl

eth yl

92

101

87

Eth yl

96

–(CH₂)₄–

–(CH₂)₅–

97

106

100

N⁶-Substituted 2,6-diam in o-9-[2-(ph osph on om eth oxy)eth yl]purin es

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

Meth yl

H

89

57

63

58

83

72

70

83

90

70

66

89

77

78

73

46

26

15

69

67

84

25

38

85

70

40

84

105

94

72

62

58

43

41

42

63

59

55

82

48

86

41

93

81

82

Propyl

2,2,2-Trifluoroeth yl

Allyl

50

48

Propargyl

83

Butyl

78

sec-Butyl

91

Isobutyl

65

But-2-en -1-yl

But-3-en -2-yl

But-3-en -1-yl

2-Meth ylallyl

2-Hydroxyeth yl

3-Hydroxypropyl

2-Hydroxypropyl

71

59

92

81

100

105

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

1556

Holý et al.:

TABLE I

(Continued)

% Con trol

L-1210 L-929 HeLaS3 CCRF-CEM

Com -

poun d

R¹

R²

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

1-Hydroxypropan -2-yl

2-Meth oxyeth yl

2-(Dim eth ylam in o)eth yl

3-Am in opropyl

3-(Dim eth ylam in o)propyl

3-Am in o-2-h ydroxypropyl

6-Am in oh exyl

Carboxym eth yl

5-Carboxypen tyl

Cyclopropyl

H

93

94

60

89

68

83

87

85

50

66

72

89

78

97

67

100

96

73

85

82

73

97

81

73

28

98

28

95

100

86

95

39

53

55

63

80

87

43

81

82

68

60

69

72

63

61

100

105

75

88

75

71

72

70

81

66

86

94

89

97

36

42

55

87

81

93

47

101

93

73

84

80

99

100

101

93

40

46

84

80

89

62

81

85

96

95

96

89

67

101

92

108

83

67

68

106

105

78

82

64

100

93

97

85

101

67

Cyclobutyl

Cyclopen tyl

Cycloh exyl

Cycloh eptyl

Cyclooctyl

Cyclopropylm eth yl

Cycloh exylm eth yl

Ben zyl

4-Am in oben zyl

Ph en yl

1-Naph tyl

2-Pyridylm eth yl

3-Pyridylm eth yl

4-Pyridylm eth yl

Diph en ylm eth yl

Meth yl

Allyl

2-Hydroxyeth yl

2-Meth oxyeth yl

–(CH₂)₄–

81

H

m eth yl

eth yl

allyl

2-h ydroxyeth yl 102

2-m eth oxyeth yl

70

58

88

54

31

84

93

87

90

41

56

75

77

82

41

80

67

63

100

91

98

91

81

95

120

99

92

57

65

86

75

73

92

81

80

81

–(CH₂)₅–

–(CH₂)₂–O–(CH₂)₂–

–(CH₂)₂–NH–(CH₂)₂–

–(CH₂)₂–N(CH₃)–(CH₂)₂–

–(CH₂)₂–NH–(CH₂)₃–

Cyclopropyl

1-(Cyclopropyl)eth yl

Ben zyl

cyclopropyl

H

m eth yl

73

63

97

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

N-[2-Phosphonomethoxy)alkyl] Derivatives

1557

TABLE I

(Continued)

% Con trol

L-929 HeLaS3 CCRF-CEM

N⁶-Substituted 6-am in o-9-[(R)-2-(ph osph on om eth oxy)propyl]purin es

Com -

poun d

R¹

R²

L-1210

105

106

107

108

109

110

111

112

113

114

115

Isopropyl

H

91

82

73

81

84

73

98

85

68

84

95

57

70

93

57

84

74

66

80

73

87

107

102

92

102

94

74

92

96

74

Allyl

H

Cyclopropyl

Isobutyl

H

Cycloh exyl

2-(Dim eth ylam in o)eth yl

3-(Dim eth ylam in o)propyl

Meth yl

H

86

H

102

96

H

m eth yl

eth yl

83

78

80

81

92

Meth yl

112

83

Eth yl

–(CH₂)₄–

88

N⁶-Substituted 2,6-diam in o-9-[(R)-2-(ph osph on om eth oxy)propyl]purin es

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

Meth yl

H

92

74

110

84

86

87

75

80

73

86

82

75

63

92

96

112

89

Cyclopropyl

Butyl

H

101

105

84

116

96

94

sec-Butyl

H

90

Isobutyl

H

94

92

2,2,2-Trifluoroeth yl

Cyclopen tyl

Cycloh exyl

Ben zyl

H

80

105

96

95

H

71

51

H

91

89

102

118

90

H

100

82

95

Ph en eth yl

Meth yl

H

85

m eth yl

91

120

82

–(CH₂)₄–

–(CH₂)₅–

99

85

99

–(CH₂)₂–O–(CH₂)₂–

96

65

83

18

87

65

103

2-(Dim eth ylam in o)eth yl

N⁶-Substituted 6-am in o-9-[(S)-2-(ph osph on om eth oxy)propyl]purin es

131

132

133

134

135

136

137

Allyl

H

100

99

88

82

80

95

91

93

82

108

100

105

98

91

86

Isobutyl

H

Isopropyl

H

100

96

90

Cyclopropyl

2-(Dim eth ylam in o)eth yl

Meth yl

H

92

H

96

103

98

95

m eth yl

99

–(CH₂)₄–

100

105

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

1558

Holý et al.:

TABLE I

(Continued)

% Con trol

L-929 HeLaS3 CCRF-CEM

N⁶-Substituted 2,6-diam in o-9-[(S)-2-(ph osph on om eth oxy)propyl]purin es

Com -

poun d

R¹

R²

L-1210

138

139

140

141

142

Allyl

90

75

84

61

81

79

100

74

Cyclopropyl

Isopropyl

Meth yl

90

m eth yl

74

89

69

78

76

79

76

–(CH₂)₄–

95

N⁶-Substituted 6-am in o-7-[2-(ph osph on om eth oxy)eth yl]purin es

144

145

146

Cyclopropyl

H

90

82

89

Meth yl

m eth yl

90

96

103

89

99

–(CH₂)₄–

100

N⁶-Substituted 2,6-diam in o-7-[2-(ph osph on om eth oxy)eth yl]purin es

m eth yl 89 99 98

N⁶-Substituted 2,6-diam in o-9-[2-(2-ph osph on om eth oxy)eth yl]purin es

147

Meth yl

151

152

153

154

Allyl

H

101

103

99

89

85

95

83

94

91

Cyclopropyl

–(CH₂)₄–

9-[2-(2-phosphonoethoxy)ethyl]guanine

98

N⁶-Substituted 2,6-diam ino-9-[(S)-3-hydroxy-2-(phosphonom ethoxy)propyl]purines

160

161

162

163

Meth yl

H

90

85

100

92

95

100

79

96

89

95

94

Cyclopropyl

2-Dim eth ylam in oeth yl

Meth yl

H

93

75

m eth yl

101

98

100

a

In h ibition of th e cell growth at c = 10 µm ol l^–1

.

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

N-[2-Phosphonomethoxy)alkyl] Derivatives

1559

TABLE II

Cytostatic ch aracteristics of selected com poun ds in vitro

IC₅₀, µm ol l^–1

L929 HeLaS3 CCRF-CEM

N⁶-Substituted 2,6-diam in o-9-[2-(ph osph on om eth oxy)eth yl]purin es

Com -

poun d

R¹

R²

L1210

52

53

54

55

56

57

58

59

60

61

62

68

70

75

76

77

81

84

91

92

96

97

98

102

103

Propyl

H

15

2

15

3.3

222-Trifluoroeth yl

Allyl

H

15

25

20

H

11.8

1

3

12

31

40

Propargyl

H

Butyl

H

sec-Butyl

H

Isobutyl

H

2.5

30

4.5

But-2-en -1-yl

But-3-en -2-yl

But-3-en -1-yl

2-Meth ylallyl

2-(Dim eth ylam in o)ewth yl

3-(Dim eth ylam in o)propyl

Cyclopropyl

Cyclobutyl

H

7.5

H

6

H

>50

32.5

5

H

35

6

24

30

2

H

16.5

5

H

5

H

7.5

25

2

18.5

>50

H

10

9.8

7.6

12

10

40

4.5

35

Cyclopen tyl

Cyclopropylm eth yl

4-Am in oben zyl

Meth yl

H

10

47

H

50

m eth yl

2.5

4.0

25

26

Eth yl

8.8

30

–(CH₂)₄–

20

–(CH₂)₅–

20

5

3.8

20

–(CH₂)₂–O–(CH₂)₂–

Cyclopropyl cyclopropyl

1-(Cyclopropyl)eth yl

H

20

N⁶-Substituted 6-am in o-9-[(R)-2-(ph osph on om eth oxy)propyl]purin es

106

110

Allyl

H

5

2-(Dim eth ylam in o)eth yl

4.2

N⁶-Substituted 2,6-diam in o-9-[(R)-2-(ph osph on om eth oxy)propyl]purin es

122

139

Cyclopen tyl

Cyclopropyl

H

22

5

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

1560

Holý et al.:

th e dim eth ylam in o fun ction in th e N⁶-alkyl m oiety substan tially en h an ces

th e cytostatic effect (68, 70). N⁶-(2,2,2-Trifluoroeth yl)-PMEDAP (53) ran ks

am on g th e m ost efficien t cytostatics of th e ANP series.

e) In troduction of N⁶-cycloalkyl substituen ts in to th e PMEDAP m olecule

en h an ces th e activity in th e case of cyclopropyl (75) an d cyclobutyl deriva-

tives (76), less so in th e cyclopen tyl con gen er 77. A sligh t effect is still ob-

servable in th e cycloh exyl derivative 78, furth er rin g expan sion to seven - or

eigh t-m em bered cycloalkan e leads to in active com poun ds 79 an d 80. Th e

order of activity is as follows: cyclopropyl > cyclobutyl >> cyclopen tyl >>

cycloh exyl. Com poun d 75 ran ks am on g th e m ost efficien t ANP in h ibitors

of th e cell growth . Special features of th e cyclopropyl substituen ts are also

illustrated by th e rath er sign ifican t activity of th e N⁶-(cyclopropylm eth yl)-

PMEDAP (81) an d by th e N⁶-(1-cyclopropyleth yl)-PMEDAP (103).

f) Substitution of PMEDAP at N⁶with ben zyl, ph en yl or 1-n aph th yl group

(83–86) does n ot sign ifican tly en h an ce th e cytostatic activity in vitro. In

con trast to N-substitution with regioisom eric C-pyridylm eth yl fun ction is

m ore effective: m edium activity was registered for com poun ds 87–89.

g) Approxim ately th e sam e pattern of N-substitution effects was en coun -

tered in th e N,N-disubstituted derivatives of PMEDAP: th e N,N-dim eth yl

(91) an d N-eth yl-N-m eth yl derivative (92) are stron gly active, followed by

N,N-dicyclopropyl com poun d 102. Substitution of th e N-alkyl group by

h ydroxyl or alkoxyl (94, 95) in activates th e system . 6-Pyrrolidin -1-yl (96),

6-(piperidin -1-yl) (97) an d 6-(m orph olin -4-yl) (98) derivatives are active,

wh ile piperazin e (99) an d 4-m eth ylpiperazin e (100) derivatives are n ot.

h) In th e series of N⁶-substituted (R)-PMPA an alogues, th e allyl (106) an d

2-(dim eth ylam in o)eth yl (110) derivatives are active (in th e L929 cell lin e),

cyclopropyl con gen er (107) an d th e N⁶,N⁶-dialkyl derivatives 112 an d113

also sh ow som e activity. In troduction of oth er substituen ts also did n ot

lead to active com poun ds. Th is observation is very sign ifican t in th e ligh t

of th e prodrug h ypoth esis (vide supra). In th ese cases, deam in ation would

lead to h ypoxan th in e derivatives, wh ich are in all ANP series devoid of an y

biological activity.

i) Con trary to th e N⁶-substituted (R)-PMPA or PMEDAP an alogues, th e

N⁶-substitution in th e series of (R)-PMPDAP is m uch less useful. In th is case,

th e h ypoth etical deam in ation would lead to cytostatic (R)-PMPG. However,

of all alkyl, cycloalkyl or N-h etaryl derivatives tested (116–130) on ly th e

cyclopropyl (117) an d cyclopen tyl derivative (122) sh ow certain cytostatic

effects.

j) Th e (S)-en an tiom ers of N⁶-substituted PMPA (131–137) or PMPDAP

(138–142) were in active in th e test system .

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

N-[2-Phosphonomethoxy)alkyl] Derivatives

1561

k) Also in active were th e 7-isom ers of N⁶-substituted PMEA (144–146)

an d N⁶,N⁶-dim eth yl derivative of 7-PMEDAP (147), as well as th e side-ch ain

h om ologues of PMEDAP with CH₂CH₂group in serted between th e eth er

oxygen an d ph osph orus atom s (151–154).

l) All four (S)-HPMPDAP derivatives substituted at th e 6-am in o group by

selected substituen ts (160–163) were devoid of an y cytostatic activity in our

system .

Metabolic Transformation of N⁶-Cyclopropyl-PMEDAP (75)

As m en tion ed above, we were in terested in verifyin g th e h ypoth esis th at

cypr-PMEDAP (75) acts as a prodrug for PMEG in wh ich it is con verted by

cellular AMP deam in ase. Th erefore, we h ave exposed CCRF-CEM cells in

vitro to [³H]-labelled com poun d 75 an d an alyzed th e cell pool for th is com -

poun d, PMEDAP (2), PMEG (3) an d th eir poten tial m etabolites by HPLC.

Kin etics of in tracellular m etabolism of com poun d 75 is sh own in Table III.

Th e con cen tration of N⁶-cyclopropyl-PMEDAP (75) an d its m etabolite

PMEGpp in th e cellular pool rem ain ed approxim ately th e sam e after 24 h

in cubation period, but after an oth er 24 h th e PMEGpp con cen tration com -

pared to N⁶-cyclopropyl-PMEDAP (75) in creased twice. Durin g th e sam e pe-

riod, th e am oun t of prim ary deam in ation product – PMEG – in creased

TABLE III

Metabolism of N⁶-cyclopropyl-[8-³H]PMEDAP (75) in T-lym ph oblastoids CCRF-CEM

In tracellular level^a

N⁶-Cyclopropyl-PMEDAP

24 h of treatm en t

pm ol/10⁷cells µm ol l^–1

48 h of treatm en t

pm ol/10⁷cells µm ol l^–1

Metabolite

N⁶-Cyclopropyl-PMEDAP (75)

3.53

0.23

1.43

3.16

1.045

0.067

0.423

0.934

3.04

0.65

3.08

5.97

0.898

0.195

0.913

1.765

PMEG (3)

PMEGp

PMEGpp

a

Extracellular con cen tration of N⁶-cyclopropyl-[8-³H]PMEDAP (75) was 3.83 µM.

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

1562

Holý et al.:

th ree tim es an d th at of th e PMEGpp precursor – PMEGp – twice. Th e in cre-

m en t of N⁶-cyclopropyl-PMEDAP (75) an d its m etabolites in th e cellular

pool after 24 h am oun ted to 8.35 pm ol/10⁷cells an d after 48 h to

12.7 pm ol/10⁷cells (Table III). Th is observation reflects th e saturation ch ar-

acter of in tracellular uptake of [8-³H]cypr-PMEDAP with respect to tim e.

Data from th e in vitro deam in ation assay (Table IV) un equivocally dem -

on strate th e en zym e-catalyzed con version of N⁶-cyclopropyl-[8-³H]PMEDAP

to [8-³H]PMEG. Th is deam in ation is m ost probably catalyzed by AMP

deam in ase or AMP deam in ase-like en zym e. Our study con firm s th e previ-

ously publish ed fin din gs^34,36th at com poun d 75 beh aves in CCRF-CEM cells

as a prodrug of PMEG. HPLC an alysis of th e acid soluble pool of CCRF-CEM

cells h as sh own th at th e m ain m etabolite is PMEGpp. Th e in tracellular con -

cen tration of PMEGpp reach ed 1.7 µm ol l^–1after 48 h at 3.83 µM (≈IC₆₄

,

IC₅₀= 1.7–2 µm ol l^–1) con cen tration in th e cell culture m edium . PMEG is

easily ph osph orylated by GMP kin ase isoen zym es^39,42an d PMEGpp is a

very efficien t com petitive in h ibitor of DNA polym erase α an d ε (ref.^40b

)

with K_ivalues 0.55 an d 0.059 µm ol l^–1, respectively. Th erefore, th e stron g

cytostatic effect of com poun d 75 is due to th e in h ibition of DNA syn th esis

by PMEGpp. Th e rate-lim itin g step of th is process is th e in tracellular

deam in ation to PMEG wh ich is m ost probably catalyzed by AMP

deam in ase. However, it is n ot yet clear wh ich isoen zym e of AMP deam in ase

is respon sible for th e in tracellular form ation of PMEG. Hatse et al.³⁴dem on -

strated th at com poun d 75 is n ot deam in ated by com m ercial rabbit m uscle

en zym e even at extrem ely h igh en zym e con cen tration . In vitro on ly th e

crude cell-free extracts^34,36were effective. Th e in vitro N⁶-cyclopropyl-

PMEDAP (75) deam in ation assay in th is study m ade use of an extract pre-

pared in th e presen ce of a n on -ion ic detergen t (Non idet NP-40) an d treated

with streptom ycin . It seem s to be th e m ost efficien t source of en zym e activ-

TABLE IV

N⁶-Cyclopropyl-[8-³H]PMEDAP (75) deam in ation in vitro^a

N⁶-Cyclopropyl-[8-³H]PMEDAP (75)

µm ol l^–1

[8-³H]PMEG (3) form ed

pm ol m l^–1m g^–1of protein

10

50

14.63

58.35

a

After 120 m in at 37 °C.

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

N-[2-Phosphonomethoxy)alkyl] Derivatives

1563

ity (58.35 pm ol m l^–1m g^–1of protein , Table IV) an d in dicates m em bran e lo-

calization of AMP deam in ase isoen zym e⁴⁵

.

In con clusion , we h ave syn th esized 123 n ew N⁶-substituted aden in e an d

2,6-diam in opurin e derivatives of th e PME-, (R)-PMP, (S)-PMP, PEE an d

(S)-HPMP series of acyclic n ucleoside ph osph on ates an d tested th eir

cytostatic activity in vitro on four cell-lin es. Based on th ese data, th e follow-

in g group of PMEDAP derivatives dem on strated th e h igh est activity:

N⁶-(2,2,2-trifluoroeth yl) (53), N⁶-allyl (54), N⁶-[2-(dim eth ylam in o)eth yl]

(68), N⁶-cyclopropyl (75) an d N⁶,N⁶-dim eth yl (91). Th ese com poun ds were

selected for furth er biological evaluation . Th ey are followed by less, but sig-

n ifican tly active N⁶-isobutyl-PMEDAP (58), N⁶-(but-2-en -1-yl)-PMEDAP (59)

an d N⁶-m orph olin o-PMEDAP (98) an d, also, by N⁶-allyl-(R)-PMPA (106),

N⁶-[2-(dim eth ylam in o)eth yl]-(R)-PMPA (110) an d N⁶-cyclopropyl-(R)-

PMPDAP (139).

In CCRF-CEM cells, th e cyclopropyl derivative 75 is deam in ated to PMEG

(3) wh ich is th en con verted to its diph osph ate.

EXPERIMENTAL

Un less oth erwise stated, solven ts were evaporated at 40 °C/2 kPa an d com poun ds were dried

overn igh t at 2 kPa over P₂O₅. Meltin g poin ts were determ in ed on a Büch i Meltin g Poin t

B-545 apparatus. TLC was perform ed on Silufol UV254 plates (Kavalier Votice, Czech Repub-

lic) in system s S1 (ch loroform –eth an ol 95 : 5) an d S2 (ch loroform –eth an ol 9 : 1). Paper elec-

troph oresis was perform ed on a Wh atm an No. 3 MM paper at 40 V/cm for 1 h in 0.05

trieth ylam m on ium h ydrogen carbon ate, pH 7.5.

M

¹H NMR spectra were taken on Varian UNITY-200 (at 200 MHz) or Varian UNITY-500 (at

500 MHz) in strum en ts in CD₃SOCD₃, D₂O or D₂O + NaOD solution s with tetram eth ylsilan e

(TMS) or sodium disilapen tan esulfon ate (DSS) as th e respective in tern al stan dards. ¹H ch em -

ical sh ifts (δ, ppm ) an d couplin g con stan ts (J, Hz) were obtain ed by th e first-order an alysis

of th e spectra. Num berin g system for assign m en t of NMR sign als is outlin ed in Fig. 1. Optical

rotation was determ in ed with a Perkin –Elm er type 241 polarim eter in 0.01 M HCl solution s.

1"

3"

R

4"

N

2"

N

4"

6

5"

6"

3"

2"

N

8

N

2

N

X

2'

O

P(O)(OR)₂

1'

3'

FIG. 1

Gen eral n um berin g sch em e for assign m en t of NMR sign als

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

1564

Holý et al.:

Materials

Brom o(trim eth yl)silan e, 6-ch loropurin e, all am in es except for isom eric buten ylam in es, so-

dium h ydride an d cesium carbon ate were purch ased from Aldrich (Prague, Czech Republic),

2-am in o-6-ch loropurin e from Mack (Germ an y). Dowex 50 X 8 an d Dowex 1 X 2 were ob-

tain ed from Fluka (Switzerlan d). Dim eth ylform am ide was distilled from P₂O₅in vacuo. Eth a-

n ol was absolutised by stan dard procedure. Aceton itrile was refluxed with CaH₂an d

distilled. All solven ts were stored over m olecular sieves (4 Å). Buten ylam in es were prepared

usin g th e procedures described in th e literature⁴⁶. (R)-[(Trityloxy)m eth yl]oxiran e was ob-

tain ed from Raylo (Can ada). Diisopropyl [(2-ch loroeth oxy)m eth yl]ph osph on ate was pre-

pared as described in ref.²², diisopropyl [(tosyloxy)m eth yl]ph osph on ate accordin g to ref.^1c

.

Deion isation of th e Reaction Mixtures

Th e solution of reaction products in water (20–25 m l) was applied to a colum n of Dowex

50 X 8 (H⁺form ) (100 m l if n ot stated oth erwise) an d th e colum n was wash ed with water

(20% aqueous m eth an ol for deion isation of ph osph on ate diesters) un til th e UV-absorption

(254 n m ) an d acid reaction of th e eluate dropped (stan dard elution rate, 3 m l/m in ). Th e elu-

tion con tin ued with 2.5% am m on ia (in water or 20% aqueous m eth an ol) an d th e UV-

absorbin g eluate was collected an d evaporated in vacuo.

Purification of Ph osph on ates by Dowex 1 X 2 Colum n Ch rom atograph y

Un less stated oth erwise, 100 m l colum n s of Dowex 1 X 2 (100–200 m esh , acetate form ) were

used. Th e deion ized sam ple in water (20–25 m l) was alkalin ized with con cen trated aqueous

am m on ia to pH 9–10 an d applied on to th e colum n . Elution with water (3 m l/m in ) was con -

tin ued un til th e in itial UV-absorption (254 n m ) of th e eluate dropped. Th e colum n was th en

eluted (3m l/m in , 30-m l fraction s) eith er with lin ear gradien t of acetic acid or with 1 M acetic

acid as in dicated.

N⁶-Substituted 6-Am in opurin es. Gen eral Procedure

A m ixture of 6-ch loropurin e (8) (2.0 g, 13 m m ol) an d prim ary or secon dary am in e (6 m l) in

eth an ol (70 m l) was refluxed for 6 h . Th e course of th e reaction was ch ecked with TLC in

system S2. Th e m ixture was evaporated to dryn ess in vacuo, deion ized on a Dowex 50 col-

um n (see above) an d th e am m on ia eluate evaporated to dryn ess. Th e residue was crystallized

from eth an ol (eth er added to turbidity). Th e followin g com poun ds were prepared by th is

procedure:

6-(Isopropylamino)purine (10). Yield 1.9 g (82.5%), m .p. 199 °C. For C₈H₁₁N₅(177.2) calcu-

lated: 54.22% C, 6.26% H, 39.52% N; foun d: 53.97% C, 6.13% H, 37.73% N.

6-(Cyclopropylamino)purine (12). Yield 2.1 g (100%), m .p. 250 °C. For C₈H₉N₅(175.2) calcu-

lated: 54.85% C, 5.18% H, 39.98% N; foun d: 54.67% C, 5.10% H, 39.96% N. ¹H NMR

(DMSO-d₆): 11.50 br, 1 H an d 8.15 br, 1 H (NH); 8.23 s, 1 H an d 8.11 s, 1 H (H-2 + H-8);

3.04 m , 1 H (N-CH); 0.71 m , 2 H an d 0.60 m , 2 H (C-CH₂).

6-Isobutylaminopurine (13). Yield 2.2 g (88.5%), m .p. 217 °C. For C₉H₁₃N₅(191.2) calcu-

lated: 56.53% C, 6.85% H, 36.62% N; foun d: 56.38% C, 6.77% H, 36.66% N.

6-[(2-Hydroxyethyl)amino]purine (14). Crystallized from water; yield 2.3 g (89.5%), m .p.

253 °C. For C₇H₉N₅O·H₂O (197.2) calculated: 42.64% C, 5.62% H, 35.51% N; foun d:

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

N-[2-Phosphonomethoxy)alkyl] Derivatives

1565

42.51% C, 5.42% H, 35.62% N. ¹H NMR (DMSO-d₆): 12.80 br, 1 H (N-H); 8.18 br s, 1 H

(H-8); 8.10 s, 1 H (H-2); 7.45 brs, 1 H (N-H); 4.80 br, 1 H (OH); 3.58 m , 4 H (CH₂).

6-{2-[(Dimethylamino)ethyl]amino}purine (15). Precipitated from eth an ol with eth er; yield

1.7 g (63.5%), m .p. 163 °C. For C₉H₁₄N₆(206.2) calculated: 52.41% C, 6.84% H, 40.75% N;

foun d: 52.50% C, 6.75% H, 40.82% N.

6-(Dimethylamino)purine (16) [with dim eth ylam in e solution in eth an ol (33%, 70 m l) in an

autoclave at 110 °C for 14 h ]. Yield 84.5%, m .p. > 280 °C. For C₇H₉N₅(163.2) calculated:

51.52% C, 5.56% H, 42.92% N; foun d: 51.69% C, 5.47% H, 42.67% N.

6-(Pyrrolidin-1-yl)purine (17). Yield 5.25 g (85.5%), m .p. > 300 °C. For C₉H₁₁N₅(189.2) cal-

culated: 57.13% C, 5.86% H, 37.01% N; foun d: 57.02% C, 5.85% H, 36.93% N.

6-(Allylam in o)purin e (11)

Th e com poun d was prepared by th e sam e procedure from com poun d 8 with allylam in e.

After evaporatin g to dryn ess in vacuo, an d application of th e residue on to a Dowex 50 X 8

(H⁺form ) colum n (150 m l), th e colum n was eluted with water un til th e acidity of th e eluate

dropped. Th e resin was th en suspen ded in water (200 m l), th e suspen sion was alkalin ized

with am m on ia an d, after 15 m in stirrin g, filtered. Th e resin was extracted with boilin g water

(four 250-m l portion s) an d th e com bin ed eluates were evaporated in vacuo. Crystallization

from water afforded com poun d 11 (1.6 g, 70.0%), m .p. 216 °C. For C₈H₉N₅(175.2) calcu-

lated: 54.85% C, 5.18% H, 39.98% N; foun d: 54.53% C, 5.10% H, 39.73% N.

N⁶-Substituted 2,6-Diam in opurin es. Gen eral Procedure

A m ixture of 2-am in o-6-ch loropurin e (9) (5.09 g, 30 m m ol), eth an ol (80 m l) an d prim ary

am in e (10 m l) was refluxed un der exclusion of CO₂for 8 h . Th e course of th e reaction was

ch ecked by TLC in system S2. Th e m ixture was filtered off from in soluble m aterial an d th e

filtrate was taken down to dryn ess in vacuo. Th e residue in water (70 m l) was dissolved on

acidification by addition of Dowex 50 X 8 resin (H⁺form ). Th is suspen sion was applied to a

colum n (150 m l) of th e sam e ion exch an ge resin an d th e colum n was eluted with water un -

til th e acidity an d UV-absorption of th e eluate dropped (th e elution was followed by con tin -

uous detection of UV-absorption of th e eluate). Th e product was th en eluted with dilute

(2.5%) aqueous am m on ia, th e UV-absorbin g eluate was evaporated in vacuo an d th e residue

was stirred with th e m ixture of eth an ol an d eth er (1 : 1, 150 m l). Th e crystallin e product

was filtered off, wash ed with th e sam e m ixture an d dried in vacuo. Th e followin g com -

poun ds were prepared by th is procedure:

2-Amino-6-(cyclopropylamino)purine (19). Yield 5.0 g (87.6%) of com poun d 19, m .p.135 °C.

For C₈H₁₀N₆(190.2) calculated: 50.52% C, 5.30% H, 44.18% N; foun d: 50.26% C, 5.24% H,

43.98% N.

6-(Allylamino)-2-aminopurine (18). Yield 4.8 g (84%), m .p. 163–164 °C. For C₈H₁₀N₆

(190.2) calculated: 50.52% C, 5.30% H, 44.18% N; foun d: 50.35% C, 5.24% H, 43.89% N.

2-Amino-6-{[2-(dimethylamino)ethyl]amino}purine (20). Yield 5.8 g (86.9%), m .p. 217 °C. For

C₉H₁₅N₇(221.3) calculated: 48.86% C, 6.83% H, 44.31% N; foun d: 48.83% C, 6.68% H,

44.46% N. MS (m/z): 222.1 (100) MH⁺; 177 (27) (M – Me₂N).

2-Amino-6-(dimethylamino)purine (21) [with dim eth ylam in e solution in eth an ol (33%,

80 m l) in an autoclaveat 110 °C for 16 h ]. Yield 5.2 g (97%), m .p. 263 °C. For C₇H₁₀N₆

(178.2) calculated: 47.18% C, 5.66% H, 47.16% N; foun d: 47.41% C, 5.55% H, 46.90% N.

MS (m/z): 179.0.

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

1566

Holý et al.:

2-Amino-6-(pyrrolidin-1-yl)purine (22). Yield 1.82 g (68.6%), m .p. 267 °C. For C₉H₁₂N₆

(204.2) calculated: 52.93% C, 5.92% H, 41.15% N; foun d: 52.97% C, 5.87% H, 40.98% N.

6-Ch loro-9-{2-[(diisopropoxyph osph oryl)m eth oxy]eth yl}purin e (26) an d

6-Ch loro-7-{2-[(diisopropoxyph osph oryl)m eth oxy]eth yl}purin e (143a)

Th e com poun ds were prepared by alkylation of 6-ch loropurin e (8) with diisopropyl

2-ch loroeth oxym eth ylph osph on ate (23) in th e presen ce of NaH as described in ref.²²

.

2-Am in o-6-ch loro-9-{2-[(diisopropoxyph osph oryl)m eth oxy]eth yl}purin e (29) an d

2-Am in o-6-ch loro-7-{2-[(diisopropoxyph osph oryl)m eth oxy]eth yl}purin e (143b)

Th e com poun ds were prepared by alkylation of 2-am in o-6-ch loropurin e (9) with diisopropyl

[(2-ch loroeth oxy)m eth yl]ph osph on ate (23) in th e presen ce of DBU as described in ref.²²

.

6-Ch loro-9-{(S)-2-[(diisopropoxyph osph oryl)m eth oxy]propyl}purin e (28) an d

6-Ch loro-9-{(R)-2-[(diisopropoxyph osph oryl)m eth oxy]propyl}purin e (27)

Th e com poun ds were prepared as described in ref.¹⁷

2-Am in o-6-ch loro-9-{(S)-2-[(diisopropoxyph osph oryl)m eth oxy]propyl}purin e (31) an d

2-Am in o-6-ch loro-9-{(R)-2-[(diisopropoxyph osph oryl)m eth oxy]propyl}purin e (30)

Th e com poun ds were prepared as described in ref.¹⁷

Preparation of N⁶-Mon o- an d Disubstituted

6-Am in o-9-[2-(ph osph on om eth oxy)eth yl]purin es 33–50

Method A. A prim ary or secon dary am in e (5 m l) was added to a solution of com poun d 26

(1.9 g, 5 m m ol) in eth an ol (50 m l) an d th e solution was refluxed for 6–8 h . Th e course of

th e reaction was m on itored by TLC in system s S1 an d S2. After com pletion , th e m ixture was

taken down in vacuo an d th e residue codistilled twice with eth an ol (20 m l each ). Th e residue

was treated with aqueous m eth an ol (1 : 4, 50 m l) an d Dowex 50 X 8 (H⁺form ) un til dissolu-

tion an d persisten t acid reaction . Th is suspen sion was applied on to a colum n (150 m l) of

th e sam e ion exch an ger equilibrated in aqueous m eth an ol (1 : 4) an d th e colum n was

wash ed with th e sam e eluen t. Th e elution was con tin ued un til th e UV-absorption of th e

eluate (254 n m ) dropped to th e origin al value. Th e colum n was th en wash ed with 2.5% am -

m on ia in aqueous m eth an ol (1 : 4) an d th e UV-absorbin g am m on ia fraction collected. After

evaporation in vacuo, th e residue of com poun d 32 was codistilled with eth an ol (2 × 25 m l)

an d dried overn igh t in vacuo over ph osph orus pen toxide. Aceton itrile (40 m l) an d Me₃SiBr

(4 m l) were added an d th e solution was left to stan d overn igh t at am bien t tem perature. Th e

m ixture was evaporated in vacuo, codistilled with aceton itrile (20 m l) an d water (50 m l) was

added. After 10 m in , th e acid solution was alkalin ized with con cen trated aqueous am m on ia

an d evaporated. Th e residue in water (20–30 m l) was applied on to a colum n (100 m l) of

Dowex 50 X 8 (H⁺form ) equilibrated in water an d th e described desaltin g procedure was re-

peated. Th e residue from th e am m on ia eluate was dissolved in water (20 m l) by addin g con -

cen trated aqueous am m on ia to pH 10–10.5 an d th e solution was applied on to a colum n

(100 m l) of Dowex 1 X 2 (acetate form ) wash ed with 0.02 M acetic acid. Th e colum n was

wash ed first with 0.02 M acetic acid un til th e UV-absorption dropped an d th en with a lin ear

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

N-[2-Phosphonomethoxy)alkyl] Derivatives

1567

gradien t of acetic acid (0.02–0.30 M, 1 l each ). Th e m ain UV-absorbin g fraction was taken

down to dryn ess an d th e residue was codistilled with water (3 × 25 m l). Th e product was ob-

tain ed by recrystallization of th e residue from water. After stan din g overn igh t in a refrigera-

tor, th e product was collected, wash ed with eth an ol an d eth er, an d dried. Th e followin g

com poun ds were obtain ed:

6-(Allylamino)-9-[2-(phosphonomethoxy)ethyl]purine (33). Yield 1.15 g (69.4%), m .p. 225 °C.

For C₁₁H₁₆N₅O₄P·H₂O (331.4) calculated: 39.88% C, 5.48% H, 21.14% N, 9.35% P; foun d:

40.09% C, 5.22% H, 21.18% N, 9.26% P. ¹H NMR (D₂O + NaOD): 8.12 s, 1 H an d 8.04 s, 1 H

(H-2 + H-8); 6.03 ddt, 1 H, J(2",1") = 4.9, J(2",3"cis) = 10.5, J(2",3"trans) = 17.3 (H-2");

5.26 dq, 1 H, J(3"trans,1") = J(gem ) = 1.5, J(3"trans,2") = 17.6 (H-3"trans); 5.21 dq, 1 H,

J(3"cis,1") = J(gem ) = 1.5, J(3"cis,2") = 10.7 (H-3"cis); 4.36 t, 2 H, J(1′,2′) = 5.0 (H-1′); 4.11 m ,

2 H (H-1"); 3.97 t, 2 H, J(2′,1′) = 5.0 (H-2′); 3.65 d, 2 H, J(P,CH) = 8.5 (P-CH₂).

6-(Cyclopropylamino)-9-[2-(phosphonomethoxy)ethylpurine (34). Yield 1.30 g (83%), m .p.

242 °C. For C₁₁H₁₆N₅O₄P (313.2) calculated: 42.18% C, 5.15% H, 22.36% N, 9.89% P; foun d:

41.90% C, 5.18% H, 22.19% N, 9.59% P. ¹H NMR (D₂O + NaOD): 8.13 s, 2 H (H-2 + H-8);

4.39 t, 2 H, J(1′,2′) = 5.0 (H-1′); 3.99 t, 2 H, J(2′,1′) = 5.0 (H-2′); 3.69 d, 2 H, J(P,CH) = 8.8

(P-CH₂); 2.79 m , 1 H (H-1"); 0.95 m , 2 H an d 0.70 m , 2 H (H-2" + H-3").

6-(Isobutylamino)-9-[2-(phosphonomethoxy)ethyl]purine (36). Yield 1.42 g (81.8%), m .p.

137 °C. For C₁₂H₂₀N₅O₄P·H₂O (347.3) calculated: 41.50% C, 6.38% H, 20.16% N, 8.92% P;

foun d: 41.84% C, 6.03% H, 20.10% N, 8.95% P. ¹H NMR (D₂O + NaOD): 8.12 s, 1 H an d

8.08 s, 1 H (H-2 + H-8); 4.38 t, 2 H, J(1′,2′) = 5.0 (H-1′); 4.00 t, 2 H, J(2′,1′) = 5.0 (H-2′);

3.72 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 3.26 m , 2 H (H-1"); 1.92 m , 1 H (H-2"); 1.00 d, 6 H,

J(2",3") = 6.6 (H-3").

6-[(2-Hydroxyethyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (37). Yield 0.80 g (50.4%),

m .p. 208 °C. For C₁₀H₁₆N₅O₅P (317.2) calculated: 37.86% C, 5.08% H, 22.08% N, 9.76% P;

foun d: 37.75% C, 4.97% H, 21.91% N, 10.06% P. ¹H NMR (D₂O + NaOD): 8.40 s, 1 H an d

8.38 s, 1 H (H-2 + H-8); 4.52 t, 2 H, J(1′,2′) = 4.9 (H-1′); 3.99 t, 2 H, J(2′,1′) = 4.9 (H-2′);

3.89 t, 2 H, J(1",2") = 4.9 (H-2"); 3.77 m , 2 H (H-1"); 3.65 d, 2 H, J(P,CH) = 8.8 (P-CH₂).

6-[(2-Methoxyethyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (38) (th e product was eluted

with reten tion from Dowex 50 colum n with water). Yield 1.2 g (72.4%), m .p. 182 °C (water).

For C₁₁H₁₈N₅O₅P (331.3) calculated: 39.88% C, 5.48% H, 21.14% N, 9.35% P; foun d:

39.95% C, 5.45% H, 20.81% N, 9.44% P. ¹H NMR (D₂O + NaOD): 8.41 s, 1 H an d 8.39 s, 1 H

(H-2 + H-8); 4.53 t, 2 H, J(1′,2′) = 5.0 (H-1′); 3.99 t, 2 H, J(2′,1′) = 5.0 (H-2′); 3.80 m , 4 H

(H-1" + H-2"); 3.65 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 3.42 s, 3 H (OCH₃).

6-[(3-Hydroxypropyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (39). Yield 1.04 g (59.5%),

m .p. 165 °C. For C₁₁H₁₈N₅O₅P·H₂O (349.4) calculated: 37.83% C, 5.77% H, 20.05% N,

8.87% P; foun d: 37.80% C, 5.49% H, 20.21% N, 8.89% P. ¹H NMR (D₂O + NaOD): 8.41 s,

1 H an d 8.38 s, 1 H (H-2 + H-8); 4.54 t, 2 H, J(1′,2′) = 4.9 (H-1′); 4.02 t, 2 H, J(2′,1′) = 4.9

(H-2′); 3.78 t, 2 H, J(3",2") = 6.4 (H-3"); 3.70 brt, 2 H, J(1",2") = 6.4 (H-1"); 3.68 d, 2 H,

J(P,CH) = 8.8 (P-CH₂); 2.03 brpen t, 2 H, J = 6.4 (H-2").

6-(2-Hydroxypropyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (40). Yield 1.20 g (68.7%),

m .p. 163 °C. For C₁₁H₁₈N₅O₅P·H₂O (349.4) calculated: 37.83% C, 5.77% H, 20.05% N,

8.87% P; foun d: 38.08% C, 5.54% H, 20.12% N, 8.97% P. ¹H NMR (D₂O + NaOD): 8.00 s,

1 H (H-2); 7.93 s, 1 H (H-8); 4.25 t, 2 H, J(1′,2′) = 4.9 (H-1′); 4.05 m , 1 H (H-2"); 3.91 t, 2 H,

J(2′,1′) = 4.9 (H-2′); 3.68 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 3.50 dd, 1 H, J = 4.6 an d 13.9;

3.35 dd, 1 H, J = 8.0 an d 13.9 (H-1"); 1.24 d, 3 H, J(3",2") = 6.4 (H-3").

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Holý et al.:

6-[(1-Hydroxypropan-2-yl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (41). Yield 1.00

g

(57.2%), m .p. 142 °C. For C₁₁H₁₈N₅O₅P·H₂O (349.4) calculated: 37.83% C, 5.77% H,

20.05% N, 8.87% P; foun d: 37.61% C, 5.72% H, 20.05% N, 8.62% P. ¹H NMR (D₂O +

NaOD): 8.42 s, 1 H an d 8.39 s, 1 H (H-2 + H-8); 4.54 t, 2 H, J(1′,2′) = 4.9 (H-1′); 4.29 m , 1 H

(H-1"); 4.01 t, 2 H, J(2′,1′) = 4.9 (H-2′); 3.86 dd, 1 H, J = 4.3 an d 11.9; 3.73 dd, 1 H, J = 6.8

an d 11.9 (H-2"); 3.68 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 1.38 d, 3 H, J(1",CH₃) = 6.7 (CH₃).

6-[(2-Aminoethyl)amino] 9-[2-(phosphonomethoxy)ethyl]purine (42). Yield 0.92 g (58%), m .p.

298 °C. For C₁₀H₁₇N₆O₄P (316.3) calculated: 37.98% C, 5.42% H, 26.57% N, 9.79% P; foun d:

37.78% C, 5.67% H, 26.62% N, 9.50% P. ¹H NMR (D₂O + NaOD): 8.20 s, 1 H an d 8.15 s, 1 H

(H-2 + H-8); 4.40 t, 2 H, J(1′,2′) = 5.0 (H-1′); 3.95 t, 2 H, J(2′,1′) = 5.0 (H-2′); 3.63 brt, 2 H,

J(1",2") = 6.1 (H-1"); 3.49 d, 2 H, J(P,CH) = 8.1 (P-CH₂); 2.90 t, 2 H, J(2",1") = 6.1 (H-2").

6-{[(2-(Dimethylamino)ethyl]amino}-9-[2-(phosphonomethoxy)ethyl]purine (43). Yield 1.50 g

(79.7%), m .p. 154 °C. For C₁₂H₂₁N₆O₄P·2H₂O (376.4) calculated: 37.90% C, 6.63% H,

22.10% N, 8.14% P; foun d: 38.06% C, 6.81% H, 22.30% N, 8.40% P. ¹H NMR (D₂O +

NaOD): 8.19 s, 2 H (H-2 + H-8); 4.41 t, 2 H, J(1′,2′) = 5.0 (H-1′); 4.02 t, 2 H, J(2′,1′) = 5.0

(H-2′); 3.95 brt, 2 H, J(1",2") = 5.2 (H-1"); 3.69 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 3.50 t, 2 H,

J(2",1") = 5.2 (H-2"); 3.01 s, 6 H (N-CH₃).

6-[(3-Aminopropyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (44). Yield 1.3 g (74.6%)

(elution from Dowex

1

colum n with 0.02

M

acetic acid), m .p. 233–234 °C. For

C

₁₁H₁₉N₆O₄P·H₂O (348.4) calculated: 37.93% C, 6.08% H, 24.13% N, 8.89% P; foun d:

37.70% C, 5.89% H, 24.34% N, 9.12% P. ¹H NMR (D₂O + NaOD): 8.13 s, 1 H an d 8.05 s, 1 H

(H-2 + H-8); 4.33 t, 2 H, J(1′,2′) = 5.1 (H-1′); 3.90 t, 2 H, J(2′,1′) = 5.1 (H-2′); 3.51 m , 2 H

(H-1"); 3.46 d, 2 H, J(P,CH) = 8.5 (P-CH₂); 2.75 t, 2 H, J(3",2") = 7.1 (H-3"); 1.82 m , 2 H

(H-2").

6-[(4-Aminobutyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (45). Yield 1.3 g (75.5%) (elu-

tion from Dowex 1 colum n with 0.02 M acetic acid), m .p. 265 °C. For C₁₂H₂₁N₆O₄P (344.3)

calculated: 41.86% C, 6.15% H, 24.41% N, 9.00% P; foun d: 4165% C, 6.23% H, 24.14% N,

8.89% P. ¹H NMR (D₂O + NaOD): 8.14 s, 1 H an d 8.09 s, 1 H (H-2 + H-8); 4.36 t, 2 H,

J(1′,2′) = 5.1 (H-1′); 3.91 t, 2 H, J(2′,1′) = 5.1 (H-2′); 3.48 m , 2 H (H-1"); 3.46 d, 2 H, J(P,CH) =

8.6 (P-CH₂); 2.63 t, 2 H, J(4",3") = 7.1 (H-4"); 1.67 m , 2 H an d 1.52 m , 2 H (H-2" + H-3").

6-[Cyclohexylamino]-9-[2-(phosphonomethoxy)ethyl]purine (46). Yield 1.25 g (70.3%), m .p.

140–142 °C. For C₁₄H₂₂N₅O₄P (355.4) calculated: 47.32% C, 6.24% H, 19.71% N, 8.72% P;

foun d: 47.67% C, 6.23% H, 19.72% N, 8.75% P. ¹H NMR (D₂O + NaOD): 8.32 s, 1 H an d

8.27 s, 1 H (H-2 + H-8); 4.49 t, 2 H, J(1′,2′) = 4.9 (H-1′); 4.01 t, 2 H, J(2′,1′) = 4.9 (H-2′);

3.82 m , 1 H (N-CH); 3.69 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 2.02 m , 2 H an d 1.82 m , 2 H an d

1.68 m , 1 H an d 1.45 m , 4 H an d 1.26 m , 1 H (C-CH₂).

6-(Dimethylamino)-9-[2-(phosphonomethoxy)ethyl]purine (47) [prepared with dim eth ylam in e

solution in eth an ol (30%, 70 m l) in an autoclave at 100 °C for 8 h ]. Yield 1.25 g (78.3%),

m .p. 205–207 °C (water). For C₁₀H₁₆N₅O₄P·H₂O (319.3) calculated: 37.62% C, 5.68% H,

21.94% N, 9.70% P; foun d: 37.83% C, 5.59% H, 22.01% N, 10.08% P. ¹H NMR (D₂O +

NaOD): 8.33 s, 1 H an d 8.26 s, 1 H (H-2 + H-8); 4.49 t, 2 H, J(1′,2′) = 4.8 (H-1′); 3.99 t, 2 H,

J(2′,1′) = 4.8 (H-2′); 3.68 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 3.55 br, 6 H (N-CH₃).

6-(Diethylamino)-9-[2-(phosphonomethoxy)ethyl]purine (48). Yield 1.48 g (85.2%), m .p.

189 °C. For C₁₂H₂₀N₆O₄P·H₂O (347.4) calculated: 41.50% C, 6.38% H, 20.16% N, 8.92% P;

foun d: 41.77% C, 6.12% H, 20.33% N, 8.91% P. ¹H NMR (D₂O + NaOD): 8.02 s, 1 H an d

7.95 s, 1 H (H-2 + H-8); 4.31 t, 2 H, J(1′,2′) = 5.0 (H-1′); 3.91 t, 2 H, J(2′,1′) = 5.0 (H-2′);

3.65 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 3.65 m , 4 H (N-CH₂); 1.12 t, 6 H, J(CH₃,CH₂) = 7.1 (CH₃).

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N-[2-Phosphonomethoxy)alkyl] Derivatives

1569

9-[2-(Phosphonomethoxy)ethyl]-6-(pyrrolidin-1-yl)purine (49). Yield 1.43 g (82.8%), m .p.

261 °C. For C₁₂H₁₈N₅O₄P·H₂O (345.3) calculated: 41.74% C, 5.84% H, 20.28% N, 8.97% P;

foun d: 41.84% C, 5.56% H, 20.36% N, 9.04% P. ¹H NMR (D₂O + NaOD): 8.03 s, 1 H an d

7.91 s, 1 H (H-2 + H-8); 4.32 t, 2 H, J(1′,2′) = 5.1 (H-1′); 3.93 t, 2 H, J(2′,1′) = 5.1 (H-2′);

3.72 m , 2 H (H-2"); 3.56 d, 2 H, J(P,CH) = 8.6 (P-CH₂); 3.42 m , 2 H (H-5"); 2.04 m , 2 H an d

1.99 m , 2 H (H-3" + H-4").

9-[2-(Phosphonomethoxy)ethyl]-6-(piperidin-1-yl)purine (50). Yield 1.38 g (80.8%), m .p.

175 °C. For C₁₃H₂₀N₅O₄P (341.3) calculated: 45.75% C, 5.91% H, 20.52% N, 9.08% P; foun d:

46.08% C, 5.91% H, 20.22% N, 8.89% P. ¹H NMR (D₂O + NaOD): 8.07 s, 1 H an d 7.98 s, 1 H

(H-2 + H-8); 4.34 t, 2 H, J(1′,2′) = 5.0 (H-1′); 3.95 t, 2 H, J(2′,1′) = 5.0 (H-2′); 3.89 m , 4 H

(H-2" + H-6"); 3.66 d, 2 H, J(P,CH) = 8.6 (P-CH₂); 1.69 m , 2 H an d 1.57 m , 4 H (H-3" + H-4").

Method B. Com poun d 23 (5.8 m l, 25 m m ol) was added to th e m ixture of th e N⁶-substi-

tuted aden in e derivative 10, 14 or 17 (20 m m ol) an d cesium carbon ate (4 g, 12.3 m m ol) in

DMF (80 m l), stirred at 100 °C. Th e stirrin g an d h eatin g was con tin ued for 16 h , th e suspen -

sion was filtered an d th e solven t was stripped down in vacuo. Th e residue was extracted with

h ot ch loroform (3 × 100 m l) an d, after evaporation of th e solven t, purified by ch rom atogra-

ph y on silica gel in ch loroform solution . Oily product 32 was dried in vacuo an d treated

with aceton itrile (150 m l) an d Me₃SiBr (15 m l) overn igh t. Th e m ixture was evaporated to

dryn ess an d taken up in water (100 m l). After 10 m in , th e solution was alkalin ized with

aqueous NH₃an d evaporated. Th e desaltin g was perform ed on a Dowex 50 X 8 colum n

(200 m l) as described in Method A. Th e crude product was purified on a Dowex 1 X 2 col-

um n (200 m l) as described in Method A. Th e product was crystallized from water. Th e fol-

lowin g com poun ds were prepared:

6-(Isopropylamino)-9-[2-(phosphonomethoxy)ethyl]purine (35). Yield 3.80 g (60.2%), m .p.

193 °C. For C₁₁H₁₈N₅O₄P (315.4) calculated: 41.91% C, 5.75% H, 22.21% N, 9.82% P; foun d:

41.90% C, 5.80% H, 22.05% N, 9.57% P. ¹H NMR (D₂O + NaOD): 8.36 s, 1 H an d 8.32 s, 1 H

(H-2 + H-8); 4.51 t, 2 H, J(1′,2′) = 5.0 (H-1′); 4.18 m , 1 H (H-1"); 4.01 t, 2 H, J(2′,1′) = 5.0

(H-2′); 3.67 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 1.39 d, 6 H, J(1",2") = 6.6 (H-2").

6-[(2-Hydroxyethyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (37). Yield 3.95 g (62%), m .p.

208 °C (water). ¹H NMR spectrum is iden tical with th at of th e com poun d described un der

Method A.

9-[2-(Phosphonomethoxy)ethyl]-6-(pyrrolidin-1-yl)purine (49). Yield 5.30 g (76.8%), m .p.

259 °C. ¹H NMR spectrum is iden tical with th at of th e com poun d prepared by Method A.

Preparation of N⁶-Mon o- an d Disubstituted

2,6-Diam in o-9-[2-(ph osph on om eth oxy)eth yl]purin es 51–104

Method C. A prim ary or secon dary am in e (5 m l) was added to a stirred solution of com -

poun d 29 (1.95 g, 5 m m ol) in eth an ol (70 m l), an d th e m ixture was refluxed un der stirrin g

for 6–8 h . Th e reaction course was m on itored by TLC in system s S1 an d S2. After com ple-

tion , th e m ixture was worked up as described in Method A an d th e desalted product purified

by Dowex 1 X 2 ch rom atograph y with lin ear gradien t of acetic acid (0.02–0.3 M, 1 l each )

an d fin ally crystallized from water. Th e followin g com poun ds were syn th esized:

2-Amino-6-(propylamino)-9-[2-(phosphonomethoxy)ethyl]purine (52). Yield 1.2 g (74%), m .p.

239 °C. For C₁₁H₁₉N₆O₄P (330.3) calculated: 40.00% C, 5.80% H, 25.45% N, 9.38% P; foun d:

40.32% C, 5.74% H, 25.78% N, 9.45% P. ¹H NMR (D₂O + NaOD): 7.87 s, 1 H (H-8); 4.24 t,

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1570

Holý et al.:

2 H, J(1′,2′) = 5.2 (H-1′); 3.90 t, 2 H, J(2′,1′) = 5.2 (H-2′); 3.50 d, 2 H, J(P,CH) = 8.4 (P-CH₂);

3.44 m , 2 H (H-1"); 1.66 sext, 2 H, J = 7.3 (H-2"); 0.98 t, 3 H, J(CH₃,CH₂) = 7.4 (H-3").

2-Amino-6-[(2,2,2-trifluoroethyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (53) [prepared

with 2,2,2-trifluoroeth ylam in e in an autoclave at 100 °C for 16 h ]. Yield 1.6 g (84%), m .p.

235 °C. For C₁₀H₁₄F₃N₆O₄P·H₂O (388.2) calculated: 30.94% C, 4.15% H, 14.68% F, 21.65% N,

7.98% P; foun d: 30.89% C, 3.98% H, 14.72% F, 21.87% N, 7.57% P. ¹H NMR (D₂O + NaOD):

7.91 s, 1 H (H-8); 4.34 br q, 2 H, J(H,F) = 8.8 (H-1"); 4.24 t, 2 H, J(1′,2′) = 5.2 (H-1′); 3.90 t,

2 H, J(2′,1′) = 5.2 (H-2′); 3.51 d, 2 H, J(P,CH) = 8.4 (P-CH₂).

6-(Allylamino)-2-amino-9-[2-(phosphonomethoxy)ethyl]purine (54). Yield 1.35 g (82.2%), m .p.

239 °C. For C₁₁H₁₇N₆O₄P (328.3) calculated: 40.25% C, 5.22% H, 25.60% N, 9.44% P; foun d:

40.02% C, 5.20% H, 25.43% N, 9.72% P. ¹H NMR (D₂O + NaOD): 7.83 s, 1 H (H-8); 6.00 m ,

1 H (H-2"); 5.26 dt, 1 H, J(3"trans,1") = 1.0, J(gem ) = 1.5, J(3"trans,2") = 17.3 (H-3"trans);

5.19 dt, 1 H, J(3"cis,1") = 1.0, J(gem ) = 1.5, J(3"cis,2") = 10.5 (H-3"cis); 4.23 brt, 2 H (H-1′);

4.13 m , 2 H (H-1"); 3.97 t, 2 H, J(2′,1′) = 5.0 (H-2′); 3.64 d, 2 H, J(P,CH) = 8.5 (P-CH₂).

2-Amino-9-[2-(phosphonomethoxy)ethyl]-6-(propargylamino)purine (55). Yield 1.3 g (78%),

m .p. 262 °C. For C₁₁H₁₅N₆O₆P (326.3) calculated: 40.50% C, 4.63% H, 26.76% N, 9.49% P;

foun d: 40.41% C, 4.74% H, 26.61% N, 9.53% P. ¹H NMR (DMSO-d₆): 7.74 s, 1 H (H-8); 7.63 br,

1 H (NH); 6.05 br, 2 H (NH₂); 4.22 m , 2 H (H-1"); 4.14 t, 2 H, J(1′,2′) = 5.4 (H-1′); 3.80 t, 2 H,

J(2′,1′) = 5.4 (H-2′); 3.58 d, 2 H, J(P,CH) = 8.7 (P-CH₂); 3.02 t, 1 H, J = 2.4 (H-3").

2-Amino-6-(butylamino)-9-[2-(phosphonomethoxy)ethyl]purine (56). Yield 1.55 g (90%), m .p.

217–220 °C. For C₁₂H₂₁N₆O₄P (344.3) calculated: 41.86% C, 6.15% H, 24.41% N, 9.00% P;

foun d: 41.61% C, 6.34% H, 24.52% N, 8.84% P. ¹H NMR (D₂O + NaOD): 7.87 s, 1 H (H-8);

4.24 t, 2 H, J(1′,2′) = 5.2 (H-1′); 3.90 t, 2 H, J(2′,1′) = 5.2 (H-2′); 3.50 d, 2 H, J(P,CH) = 8.3

(P-CH₂); 3.46 m , 2 H (H-1"); 1.62 brpen t, 2 H (H-2"); 1.40 sext, 2 H (H-3"); 0.93 s, 3 H, J =

7.3 (H-4").

2-Amino-9-[2-(phosphonomethoxy)ethyl]-6-(propylamino)purine (57). Yield 1.17 g (68%), m .p.

262 °C. For C₁₂H₂₁N₆O₄P (344.3) calculated: 41.86% C, 6.15% H, 24.41% N, 9.00% P; foun d:

41.68% C, 5.94% H, 24.18% N, 9.24% P. ¹H NMR (D₂O + NaOD): 7.92 s, 1 H (H-8); 4.28 brt,

2 H (H-1′); 3.97 brt, 2 H, J(1′,2′) = 5.0 (H-2′); 3.73 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 3.27 m , 2 H

(H-1"); 1.92 m , 1 H (H-2"); 0.97 d, 6 H, J(2",3") = 6.6 (H-3").

2-Amino-6-(isobutylamino)-9-[2-(phosphonomethoxy)ethyl]purine (58). Yield 1.30 g (57.2%),

m .p. 262 °C. For (C₁₂H₂₁N₆O₄P) (344.3) calculated: 41.86% C, 6.15% H, 24.41% N, 9.00% P;

foun d: 42.07% C, 5.94% H, 24.31% N, 8.83% P. ¹H NMR (D₂O + NaOD): 7.90 s, 1 H (H-8);

4.26 t, 2 H, J(1′,2′) = 5.2 (H-1′); 3.91 t, 2 H, J(2′,1′) = 5.2 (H-2′); 4.16 m , 1 H (H-1"); 3.50 d,

2 H, J(P,CH) = 8.5 (P-CH₂); 1.62 brpen t, 2 H, J = 7.3 (H-2"); 1.25 d, 3 H, J(1",CH₃) = 6.6

(1"-CH₃); 0.94 t, 3 H, J(2",3") = 7.3 (H-3").

2-Amino-6-(but-2-en-1-ylamino)-9-[2-(phosphonomethoxy)ethyl]purine (59). Yield 1.4 g (81%),

m .p. 244 °C. For C₁₂H₁₉N₇O₄P (342.3) calculated: 42.11% C, 5.59% H, 24.55% N, 9.05% P;

foun d: 42.20% C, 5.70% H, 24.70% N, 9.04% P. ¹H NMR (D₂O + NaOD): 7.65 s, 1 H (H-8);

5.52 brdq, 1 H, J(3",1") = 1.0, J(3",4") = 6.5, J(3",2") = 15.4 (H-3"); 5.40 dtq, 1 H, J(2",4") = 1.2,

J(2",1") = 5.6, J(2",3") = 15.4 (H-2"); 4.02 t, 2 H, J(1′,2′) = 5.2 (H-1′); 3.81 m , 2 H (H-1");

3.68 t, 2 H, J(2′,1′) = 5.2 (H-2′); 3.29 d, 2 H, J(P,CH) = 8.5 (P-CH₂); 1.47 d, 3 H, J(4",3") = 6.5

(H-4").

2-Amino-6-(but-3-en-2-ylamino)-9-[2-(phosphonomethoxy)ethyl]purine (60). Yield 1.16

g

(68%), m .p. 265 °C. For C₁₂H₁₉N₇O₄P (342.3) calculated: 42.11% C, 5.59% H, 24.55% N,

9.05% P; foun d: 42.02% C, 5.60% H, 24.40% N, 9.16% P. ¹H NMR (D₂O + NaOD): 8.00 s,

1 H (H-8); 6.15 ddd, 1 H, J(3",2") = 4.6, J(3",4"a) = 17.3, J(3",4"b) = 10.5 (H-3"); 5.34 ddt, 1 H,

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N-[2-Phosphonomethoxy)alkyl] Derivatives

1571

J(4"a,2") = 1.5, J(gem ) = 1.5, J(4"a,3") = 17.3 (H-4"a); 5.26 dt. 1 H, J(4"b,2") = 1.5, J(gem ) =

1.5, J(4"b,3") = 10.5 (H-4"b); 4.92 m , 1 H (H-2"); 4.36 t, 2 H, J(1′,2′) = 5.1 (H-1′); 4.03 t, 2 H,

J(2′,1′) = 5.1 (H-2′); 3.65 d, 2 H, J(P,CH) = 8.5 (P-CH₂); 1.50 d, 3 H, J(1",2") = 6.8 (H-1").

2-Amino-6-(but-3-en-1-ylamino)-9-[2-(phosphonomethoxy)ethyl]purine (61). Yield 1.32

g

(77%), m .p. 216 °C. For C₁₂H₁₉N₆O₄P (342.3) calculated: 42.11% C, 5.59% H, 24.55% N,

9.05% P; foun d: 41.98% C, 5.67% H, 24.30% N, 9.21% P. ¹H NMR (D₂O + NaOD): 7.85 s,

1 H (H-8); 5.92 ddt, 1 H, J(3",2") = 6.8, J(3",4"a) = 17.2, J(3",4"b) = 10.3 (H-3"); 5.18 ddt, 1 H,

J(4"a,2") = 1.6, J(gem ) = 2.0, J(4"a,3") = 17.2 (H-4"a); 5.14 ddt, 1 H, J(4"b,2") = 1.1, J(gem ) =

2.0, J(4"b,3") = 10.3 (H-4"b); 4.23 t, 2 H, J(1′,2′) = 5.4 (H-1′); 3.91 t, 2 H, J(2′,1′) = 5.4 (H-2′);

3.53 m , 2 H (H-1"); 3.52 d, 2 H, J(P,CH) = 8.4 (P-CH₂); 2.41 qt, 2 H, J(2",4") = 1.3, J(2",1") =

J(2",3") = 6.8 (H-2").

2-Amino-6-(2-methylallylamino)-9-[2-(phosphonomethoxy)ethyl]purine (62). Yield 1.33

g

(78%), m .p. 233 °C. For C₁₂H₁₉N₆O₄P (342.3) calculated: 42.11% C, 5.59% H, 24.55% N,

9.05% P; foun d: 41.96% C, 5.77% H, 24.37% N, 9.17% P. ¹H NMR (D₂O + NaOD): 8.92 s,

1 H (H-8); 4.91 m , 2 H (C=CH₂); 4.29 t, 2 H, J(1′,2′) = 5.4 (H-1′); 4.05 m , 2 H (N-CH₂);

3.98 t, 2 H, J(2′,1′) = 5.4 (H-2′); 3.62 d, 2 H, J(P,CH) = 8.6 (P-CH₂); 1.83 s, 3 H (CH₃).

2-Amino-6-[(2-hydroxyethyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (63). Yield 1.25 g

(75.2%), m .p. 234 °C. For C₁₀H₁₇N₆O₅P (332.3) calculated: 36.13% C, 5.16% H, 25.29% N,

9.34% P; foun d: 36.38% C, 5.08% H, 25.11% N, 9.31% P. ¹H NMR (D₂O + NaOD): 7.83 s,

1 H (H-8); 4.21 t, 2 H, J(1′,2′) = 5.0 (H-1′); 3.94 t, 2 H, J(2′,1′) = 5.0 (H-2′); 3.83 t, 2 H,

J(2",1") = 5.4 (H-2"); 3.68 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 3.65 m , 2 H (H-1").

2-Amino-6-[(3-hydroxypropyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (64). Yield 1.38 g

(79.7%), m .p. 219 °C. For C₁₁H₁₉N₆O₅P (346.3) calculated: 38.15% C, 5.53% H, 24.27% N,

8.94% P; foun d: 38.04% C, 5.47% H, 23.97% N, 8.67% P. ¹H NMR (D₂O + NaOD): 7.85 s,

1 H (H-8); 4.23 t, 2 H, J(1′,2′) = 5.1 (H-1′); 3.90 t, 2 H, J(2′,1′) = 5.1 (H-2′); 3.71 t, 2 H, J = 6.3

(H-3"); 3.58 m , 2 H (H-1"); 3.54 d, 2 H, J(P,CH) = 8.6 (P-CH₂); 1.89 m , 2 H (H-2").

2-Amino-6-[(2-hydroxypropyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (65). Yield 1.40 g

(38.4%), m .p. 246 °C. For C₁₁H₁₉N₆O₅P·H₂O (364.3) calculated: 36.27% C, 5.81% H,

23.07% N, 8.50% P; foun d: 36.65% C, 5.46% H, 23.32% N, 8.49% P. ¹H NMR (D₂O +

NaOD): 7.91 s, 1 H (H-8); 4.26 t, 2 H, J(1′,2′) = 5.1 (H-1′); 4.10 m , 1 H (H-2"); 3.90 t, 2 H,

J(2′,1′) = 5.1 (H-2′); 3.63 m , 1 H; 3.51 m , 1 H (H-2"); 3.49 d, 2 H, J(P,CH) = 8.4 (P-CH₂);

1.25 d, 3 H, J(3",2") = 6.4 (H-3").

2-Amino-6-[(1-hydroxypropan-2-yl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (66). Yield

1.45 g (83.7%), m .p. 200 °C. For C₁₁H₁₉N₆O₅P (346.3) calculated: 38.15% C, 5.53% H,

24.27% N, 8.94% P; foun d: 38.34% C, 5.59% H, 24.48% N, 8.07% P. ¹H NMR (D₂O +

NaOD): 7.83 s, 1 H (H-8); 4.40 m , 1 H (H-2"); 4.22 t, 2 H, J(1′,2′) = 4.9 (H-1′); 3.93 t, 2 H,

J(2′,1′) = 4.9 (H-2′); 3.76 dd, 1 H, J = 4.9 an d 11.6 an d 3.68 dd, 1 H, J = 7.0 an d 11.6 (H-1");

3.65 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 1.29 d, 3 H, J = 6.7 (H-3").

2-Amino-6-[(2-methoxyethyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (67). Yield 1.39 g

(80%), m .p. 192 °C. For C₁₁H₁₉N₆O₅P (346.3) calculated: 38.15% C, 5.53% H, 24.27% N,

8.94% P; foun d: 38.27% C, 5.48% H, 23.96% N, 8.85% P. ¹H NMR (D₂O + NaOD): 7.95 s,

1 H (H-8); 4.33 t, 2 H, J(1′,2′) = 5.0 (H-1′); 3.96 t, 2 H, J(2′,1′) = 5.0 (H-2′); 3.75 m , 4 H

(H-1" + H-2"); 3.71 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 3.42 s, 3 H (OCH₃).

2-Amino-6-{[2-(dimethylamino)ethyl])amino}-9-[2-(phosphonomethoxy)ethyl]purine (68). Yield

1.55 g (75%), m .p. 237 °C. For C₁₂H₂₂N₇O₄P·3H₂O (413.3) calculated: 34.87% C, 6.83% H,

23.72% N, 7.49% P; foun d: 35.06% C, 6.52% H, 23.43% N, 7.44% P. ¹H NMR (D₂O +

NaOD): 7.86 s, 1 H (H-8); 4.25 t, 2 H, J(1′,2′) = 4.8 (H-1′); 3.98 t, 2 H, J(2′,1′) = 4.8 (H-2′);

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3.91 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 3.90 brt, 2 H, J(1",2") = 5.7 (H-1"); 3.46 t, 2 H, J(1",2") =

5.7 (H-2"); 3.00 s, 6 H (N-CH₃).

2-Amino-6-[(3-aminopropyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (69). Yield 1.30 g

(75.3%). Am orph ous precipitate. For C₁₁H₂₀N₇O₄P (345.3) calculated: 38.26% C, 5.84% H,

28.40% N, 8.97% P; foun d: 38.11% C, 5.95% H, 28.21% N, 9.14% P. ¹H NMR (D₂O +

NaOD): 7.88 s, 1 H (H-8); 4.25 t, 2 H, J(1′,2′) = 5.2 (H-1′); 3.90 t, 2 H, J(2′,1′) = 5.2 (H-2′);

3.56 t, 2 H, J(1",2") = 7.0 (H-1"); 3.48 d, 2 H, J(P,CH) = 8.5 (P-CH₂); 2.71 t, 2 H, J(3",2") = 7.1

(H-3"); 1.80 pen t, 2 H, J = 7.0 (H-2").

2-Amino-6-{[3-(dimethylamino)propyl]amino}-9-[2-(phosphonomethoxy)ethyl]purine (70). Yield

1.50 g (70.2%), m .p. 115 °C. For C₁₃H₂₄N₇O₄P·3H₂O (427.4) calculated: 36.53% C, 7.07% H,

22.94% N, 7.25% P; foun d: 36.66% C, 7.08% H, 22.68% N, 7.25% P. ¹H NMR (D₂O +

NaOD): 7.85 s, 1 H (H-8); 4.24 t, 2 H, J(1′,2′) = 4.4 (H-1′); 3.98 t, 2 H, J(2′,1′) = 4.4 (H-2′);

3.73 d, 2 H, J(P,CH) = 8.8 (P-CH₂); 3.72 brt, 2 H, J = 7.0 (H-1"); 3.226 t, 2 H, J = 7.6 (H-3");

2.96 s, 6 H (N-CH₃); 2.14 m , 2 H (H-2").

2-Amino-6-[(3-amino-2-hydroxypropyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (71). Yield

1.63 g (90%), m .p. 275 °C. For C₁₁H₂₀N₇O₅P (361.3) calculated: 36.57% C, 5.58% H,

27.14% N, 8.57% P; foun d: 36.91% C, 5.71% H, 27.25% N, 8.75% P. ¹H NMR (D₂O +

NaOD): 7.95 s, 1 H (H-8); 4.30 t, 2 H, J(1′,2′) = 5.4 (H-1′); 3.95 t, 2 H, J(2′,1′) = 5.4 (H-2′);

3.92 tt, 1 H, J(2",1") = J(2",3") = 4.5 an d 7.0 (H-2"); 3.70 m , 1 H an d 3.61 m , 1 H (H-1");

3.54 d, 2 H, J(P,CH) = 8.6 (P-CH₂); 2.80 dd, 1 H, J(3"a,2") = 4.5, J(gem ) = 13.4 (H-3"a); 2.70 dd,

1 H, J(3"b,2") = 7.0, J(gem ) = 13.4 (H-3"b).

2-Amino-6-[(6-aminohexyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (72). Yield 1.70 g

(84%), m .p. 153 °C. For

C₁₄H₂₆N₇O₄P·H₂O (405.4) calculated: 41.48% C, 6.96% H,

24.19% N, 7.64% P; foun d: 41.64% C, 7.05% H, 24.10% N, 7.59% P. ¹H NMR (D₂O +

NaOD): 7.88 s, 1 H (H-8); 4.28 t, 2 H, J(1′,2′) = 5.0 (H-1′); 3.98 t, 2 H, J(2′,1′) = 5.0 (H-2′);

3.72 d, 2 H, J(P,CH) = 8.6 (P-CH₂); 3.51 m , 2 H an d 3.02 m , 2 H (N-CH₂); 1.68 m , 4 H an d

1.43 m , 4 H (C-CH₂).

2-Amino-6-[(carboxymethyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (73). Yield 1.19 g

(69%), m .p. 273 °C. For C₁₀H₁₅N₆O₆P (346.2) calculated: 34.69% C, 4.37% H, 24.27% N,

8.95% P; foun d: 34.49% C, 4.49% H, 24.09% N, 9.13% P. ¹H NMR (D₂O + NaOD): 7.86 s, 1 H

(H-8); 4.24 t, 2 H, J(1′,2′) = 5.3 (H-1′); 4.02 brs, 2 H (N-CH₂); 3.86 t, 2 H, J(2′,1′) = 5.3 (H-2′);

3.48 d, 2 H, J(P,CH) = 8.4 (P-CH₂).

2-Amino-6-[(5-carboxypentyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (74). Yield 1.64 g

(81%), m .p. 168 °C. For C₁₄H₂₃N₆O₆P·H₂O (405.4) calculated: 40.00% C, 5.99% H, 19.99% N,

7.37% P; foun d: 40.14% C, 6.08% H, 20.11% N, 7.53% P. ¹H NMR (D₂O + NaOD): 7.83 s, 1 H

(H-8); 4.19 t, 2 H, J(1′,2′) = 5.3 (H-1′); 3.91 t, 2 H, J(2′,1′) = 5.3 (H-2′); 3.51 d, 2 H, J(P,CH) =

8.4 (P-CH₂); 3.46 m , 2 H (H-1"); 2.22 t, 2 H, J(5",4") = 7.5 (H-5"); 1.67 m , 2 H an d 1.62 m ,

2 H an d 1.41 m , 2 H (H-3" + H-2" + H-4").

2-Amino-6-(cyclopropylamino)-9-[2-(phosphonomethoxy)ethyl]purine (75). Yield 1.40 g (85.3%),

m .p. 263 °C. For C₁₁H₁₇N₆O₄P (328.3) calculated: 40.25% C, 5.22% H, 25.60% N, 9.44% P;

foun d: 40.41% C, 5.33% H, 25.70% N, 9.18% P. ¹H NMR (D₂O + NaOD): 7.86 s, 1 H (H-8);

4.24 t, 2 H, J(1′,2′) = 4.6 (H-1′); 3.91 t, 2 H, J(2′,1′) = 4.6 (H-2′); 3.54 d, 2 H, J(P,CH) = 8.6

(P-CH₂); 2.82 m , 1 H (H-1"); 0.89 m , 2 H; 0.67 m , 2 H (H-2" + H-3").

2-Amino-6-(cyclobutylamino)-9-[2-(phosphonomethoxy)ethyl]purine (76). Yield 1.59 g (88%),

m .p. 262 °C. For C₁₂H₁₉N₆O₄P·H₂O (360.3) calculated: 40.00% C, 5.87% H, 23.32% N,

8.60% P; foun d: 40.19% C, 5.84% H, 23.46% N, 8.36% P. ¹H NMR (D₂O + NaOD): 7.86 s,

1 H (H-8); 4.52 m , 1 H (N-CH); 4.22 t, 2 H, J(1′,2′) = 5.0 (H-1′); 3.90 t, 2 H, J(2′,1′) = 5.0

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(H-2′); 3.53 d, 2 H, J(P,CH) = 8.4 (P-CH₂); 2.42 m , 2 H an d 2.04 m , 2 H an d 1.78 m , 2 H

(C-CH₂).

2-Amino-6-(cyclopentylamino)-9-[2-(phosphonomethoxy)ethyl]purine (77). Yield 1.55 g (82.8%),

m .p. 267 °C. For C₁₃H₂₁N₆O₄P·H₂O (374.3) calculated: 41.71% C, 6.19% H, 22.45% N,

8.27% P; foun d: 42.03% C, 6.02% H, 22.39% N, 8.32% P. ¹H NMR (D₂O + NaOD): 7.89 s,

1 H (H-8); 4.29 t, 2 H, J(1′,2′) = 5.5 (H-1′); 4.32 m , 1 H (H-1"); 3.97 t, 2 H, J(2′,1′) = 5.5 (H-2′);

3.62 d, 2 H, J(P,CH) = 8.6 (P-CH₂); 2.01 m , 2 H an d 1.71 m , 2 H an d 1.62 m , 2 H an d 1.55 m ,

2 H (H-2" + H-3" + H-4" + H-5"); 1.17 d, 3 H, J(3′,2′) = 6.3 (H-3′).

2-Amino-6-(cyclohexylamino)-9-[2-(phosphonomethoxy)ethyl]purine (78). Yield 1.60 g (86.4%),

m .p. 264 °C. For C₁₄H₂₃N₆O₄P (370.4) calculated: 45.40% C, 6.26% H, 22.69% N, 8.36% P;

foun d: 44.99% C, 6.09% H, 22.77% N, 8.09% P. ¹H NMR (D₂O + NaOD): 7.87 s, 1 H (H-8);

4.25 brt, 2 H, J(1′,2′) = 5.2 (H-1′); 3.95 m , 1 H (H-1"); 3.90 t, 2 H, J(1′,2′) = 5.2 (H-2′); 3.52 d,

2 H, J(P,CH) = 8.6 (P-CH₂); 1.98 m , 2 H an d 1.75 m , 2 H an d 1.62 m , 1 H an d 1.35 m , 4 H

an d 1.22 m , 1 H (C-CH₂).

2-Amino-6-(cycloheptylamino)-9-[2-(phosphonomethoxy)ethyl]purine (79). Yield 1.63 g (85%),

m .p. 257 °C. For C₁₅H₂₅N₆O₄P (384.4) calculated: 46.87% C, 6.56% H, 21.86% N, 8.06% P;

foun d: 46.64% C, 6.58% H, 21.77% N, 8.11% P. ¹H NMR (D₂O + NaOD): 7.85 s, 1 H (H-8);

4.24 t, 2 H, J(1′,2′) = 5.1 (H-1′); 4.10 m , 1 H (N-CH); 3.91 t, 2 H, J(2′,1′) = 5.1 (H-2′); 3.58 d,

2 H, J(P,CH) = 8.4 (P-CH₂); 1.93 m , 2 H an d 1.64–1.40 m , 10 H (C-CH₂).

2-Amino-6-(cyclooctylamino)-9-[2-(phosphonomethoxy)ethyl]purine (80). Yield 1.53 g (77%),

m .p. 250 °C. For C₁₆H₂₇N₆O₄P (398.4) calculated: 48.24% C, 6.83% H, 21.09% N, 7.77% P;

foun d: 48.04% C, 6.88% H, 20.87% N, 7.80% P. ¹H NMR (D₂O + NaOD): 7.89 s, 1 H (H-8);

4.14 m , 1 H (N-CH); 4.25 t, 2 H, J(1′,2′) = 5.1 (H-1′); 3.90 t, 2 H, J(2′,1′) = 5.1 (H-2′); 3.51 d,

2 H, J(P,CH) = 8.4 (P-CH₂); 1.83 m , 2 H an d 1.62 m , 4 H an d 1.52–1.43 m , 8 H (C-CH₂).

2-Amino-6-[(cyclopropylmethyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (81). Yield 1.62 g

(90%), m .p. 234–235 °C. For C₁₂H₁₉N₆O₄P·H₂O (360.3) calculated: 40.00% C, 5.87% H,

23.32% N, 8.60% P; foun d: 39.98% C, 5.92% H, 23.11% N, 8.71% P. ¹H NMR (D₂O +

NaOD): 7.87 s, 1 H (H-8); 4.24 t, 2 H, J(1′,2′) = 5.2 (H-1′); 3.90 t, 2 H, J(2′,1′) = 5.2 (H-2′);

3.50 d, 2 H, J(P,CH) = 8.4 (P-CH₂); 3.35 m , 2 H (N-CH₂); 1.15 m , 1 H (C-CH); 0.57 m , 2 H

an d 0.31 m , 2 H (cyclopropyl-CH₂).

2-Amino-6-[(cyclohexylmethyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (82). Yield 1.39 g

(69%), m .p. 219 °C. For

C₁₅H₂₅N₆O₄P·H₂O (402.4) calculated: 44.77% C, 6.76% H,

20.89% N, 7.70% P; foun d: 44.54% C, 6.74% H, 20.60% N, 7.80% P. ¹H NMR (D₂O +

NaOD): 7.89 s, 1 H (H-8); 4.26 t, 2 H, J(1′,2′) = 5.2 (H-1′); 3.91 t, 2 H, J(2′,1′) = 5.2 (H-2′);

3.52 d, 2 H, J(P,CH) = 8.4 (P-CH₂); 3.26 m , 2 H (N-CH₂); 1.65 m , 6 H an d 1.12 m , 3 H an d

0.90 m , 2 H (C-CH).

2-Amino-6-(benzylamino)-9-[2-(phosphonomethoxy)ethyl]purine (83). Yield 1.55 g (82%), m .p.

149–150 °C. For C₁₅H₁₉N₆O₄P (378.3) calculated: 47.62% C, 5.06% H, 22.21% N, 8.19% P;

foun d: 47.81% C, 5.05% H, 22.23% N, 8.32% P. ¹H NMR (D₂O + NaOD): 7.72 s, 1 H (H-8);

7.03 m , 5 H (arom .); 4.41 s, 2 H (CH₂-Ph ); 4.08 t, 2 H, J(1′,2′) = 5.0 (H-1′); 3.80 t, 2 H,

J(1′,2′) = 5.0 (H-2′); 3.65 d, 2 H, J(P,CH) = 8.3 (P-CH₂).

2-Amino-6-[(4-aminobenzyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (84). Yield 1.73 g

(88%), m .p. 177 °C. For C₁₅H₂₀N₇O₄P (393.3) calculated: 45.80% C, 5.12% H, 24.93% N,

7.87% P; foun d: 45.58% C, 5.07% H, 24.72% N, 7.67% P. ¹H NMR (D₂O + NaOD): 7.79 s,

1 H (H-8); 7.07 d, 2 H an d 6.69 d, 2 H (arom .); 4.48 m , 2 H (Ph -CH₂); 4.18 t, 2 H, J(1′,2′) =

5.4 (H-1′); 3.87 t, 2 H, J(2′,1′) = 5.4 (H-2′); 3.51 d, 2 H, J(P,CH) = 8.6 (P-CH₂).

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2-Amino-6-(phenylamino)-9-[2-(phosphonomethoxy)ethyl]purine (85). Yield 1.60 g (87.8%),

m .p. 240–245 °C. For C₁₄H₁₇N₆O₄P (364.3) calculated: 46.16% C, 4.70% H, 17.57% N,

8.50% P; foun d: 46.29% C, 4.76% H, 17.82% N, 8.38% P. ¹H NMR (D₂O + NaOD): 7.77 s,

1 H (H-8); 7.48 d, 2 H an d 7.32 t, 2 H an d 7.12 t, 1 H (arom .); 4.11 t, 2 H, J(1′,2′) = 4.5

(H-1′); 3.86 t, 2 H, J(1′,2′) = (H-2′); 3.64 d, 2 H, J(P,CH) = 8.8 (P-CH₂).

2-Amino-6-(1-naphthylamino)-9-[2-(phosphonomethoxy)ethyl]purine (86). Yield 1.25 g (58%),

m .p. 209 °C. For C₁₉H₂₃N₆O₄P (430.3) calculated: 53.02% C, 5.39% H, 19.53% N, 7.20% P;

foun d: 53.17% C, 5.51% H, 19.81% N, 6.97% P. ¹H NMR (D₂O + NaOD): 7.77 s, 1 H (H-8);

7.88 + 7.87 + 7.74 + 7.65 4 × d, 4 × 1 H; 7.50 + 7.45 + 7.44 3 × t, 3 × 1 H (arom .); 4.13 t,

2 H, J(1′,2′) = 5.5 (H-1′); 3.86 t, 2 H, J(1′,2′) = 5.5 (H-2′); 3.51 d, 2 H, J(P,CH) = 8.3 (P-CH₂).

2-Amino-9-[2-(phosphonomethoxy)ethyl]-6-[(2-pyridylmethyl)amino]purine (87). Yield 1.59 g

(84%), m .p. 256 °C. For C₁₄H₁₈N₇O₄P (379.3) calculated: 44.33% C, 4.78% H, 25.85% N,

8.17% P; foun d: 44.57% C, 5.00% H, 25.82% N, 8.21% P. ¹H NMR (D₂O + NaOD): 8.39 m ,

2 H an d 7.36 m , 2 H (Py); 7.93 s, 1 H (H-8); 4.77 m , 2 H (N-CH₂-Py); 4.26 t, 2 H, J(1′,2′) =

5.2 (H-1′); 3.91 t, 2 H, J(2′,1′) = 5.2 (H-2′); 3.50 d, 2 H, J(P,CH) = 8.4 (P-CH₂).

2-Amino-9-[2-(phosphonomethoxy)ethyl]-6-[(3-pyridylmethyl)amino]purine (88). Yield 1.40 g

(74%), m .p. 133 °C. For C₁₄H₁₈N₇O₄P (379.3) calculated: 44.33% C, 4.78% H, 25.85% N,

8.17% P; foun d: 44.45% C, 4.90% H, 25.71% N, 8.26% P. ¹H NMR (D₂O + NaOD): 8.60 brs,

1 H an d 8.48 brd, 1 H, J = 5.6 an d 8.26 brd, 1 H, J = 8.0 an d 7.72 dd, 1 H, J = 5.6 an d 8.0

(Py); 7.77 s, 1 H (H-8); 4.75 m , 2 H (N-CH₂-Py); 4.16 t, 2 H, J(1′,2′) = 5.0 (H-1′); 3.85 t, 2 H,

J(2′,1′) = 5.0 (H-2′); 3.62 d, 2 H, J(P,CH) = 8.8 (P-CH₂).

2-Amino-9-[2-(phosphonomethoxy)ethyl]-6-[(4-pyridylmethyl)amino]purine (89). Yield 1.52 g

(80%), m .p. 259 °C. For C₁₄H₁₈N₇O₄P (379.3) calculated: 44.33% C, 4.78% H, 25.85% N,

8.17% P; foun d: 44.62% C, 4.98% H, 26.06% N, 8.06% P. ¹H NMR (D₂O + NaOD): 8.37 d,

2 H an d 7.34 d, 2 H (Py); 4.75 m , 2 H (N-CH₂-Py); 4.25 t, 2 H, J(1′,2′) = 5.1 (H-1′); 3.90 t,

2 H, J(2′,1′) = 5.1 (H-2′); 3.51 d, 2 H, J(P,CH) = 8.4 (P-CH₂).

2-Amino-6-[(diphenylmethyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (90). Yield 1.35 g

(57%), m .p. 167 °C. For C₂₁H₂₃N₆O₄P·H₂O (472.4) calculated: 53.39% C, 5.33% H, 17.79% N,

6.56% P; foun d: 53.10% C, 5.18% H, 17.41% N, 6.38% P. ¹H NMR (D₂O + NaOD): 7.50 s,

1 H (H-8); 7.04 m , 4 H an d 6.90–6.75 m , 6 H (arom .); 6.49 brs, 1 H (H-1"); 3.99 m , 2 H

(H-1′); 3.70 t, 2 H, J(2′,1′) = 5.0 (H-2′); 3.49 m , 2 H (P-CH₂).

2-Amino-6-[ethyl(methyl)amino]-9-[2-(phosphonomethoxy)ethyl purine (92). Yield 1.44

g

(83%), m .p. 142 °C. For ₁₁H₁₉N₆O₄P·H₂O (348.4) calculated: 37.93% C, 6.08% H,

C

24.13% N, 8.89% P; foun d: 38.20% C, 5.89% H, 24.09% N, 9.09% P. ¹H NMR (D₂O +

NaOD): 7.81 s, 1 H (H-8); 4.24 t, 2 H, J(1′,2′) = 5.2 (H-1′); 3.91 t, 2 H, J(2′,1′) = 5.2 (H-2′);

3.80 brq, 2 H, J(1",2") = 7.1 (H-1"); 3.53 d, 2 H, J(P,CH) = 8.3 (P-CH₂); 3.17 brs, 3 H (N-CH₃);

1.17 t, 3 H, J(2",1") = 7.1 (H-2").

2-Amino-6-(diallylamino)-9-[2-(phosphonomethoxy)ethyl]purine (93). Yield 1.30 g (70.5%),

m .p. 177 °C. For C₁₄H₂₁N₆O₆P (368.3) calculated: 45.65% C, 5.75% H, 22.82% N, 8.41% P;

foun d: 45.77% C, 5.80% H, 22.97% N, 8.26% P. ¹H NMR (D₂O + NaOD): 7.83 s, 1 H (H-8);

5.90 ddt, 2 H, J(2",1") = 5.1, J(2",3"cis) = 10.5, J(2",3"trans) = 17.1 (H-2"); 5.18 dq, 2 H, J(3",1") =

J(gem ) = 1.5, J(3",2") = 10.5 (H-3"cis); 5.15 dq, 2 H, J(3",1") = J(gem ) = 1.5, J(3",2") = 17.1

(H-3"trans); 4.39 m , 4 H (H-1"); 4.25 t, 2 H, J(1′,2′) = 5.2 (H-1′); 3.90 t, 2 H, J(2′,1′) = 5.2

(H-2′); 3.60 d, 2 H, J(P,CH) = 8.6 (P-CH₂).

2-Amino-6-[bis(2-hydroxyethyl)amino]-9-[2-(phosphonomethoxy)ethyl]purine (94). Yield 1.50 g

(79.7%), m .p. 211 °C. For C₁₁H₂₁N₆O₆P·H₂O (394.3) calculated: 36.55% C, 5.88% H,

21.31% N, 7.85% P; foun d: 36.34% C, 5.57% H, 21.48% N, 7.91% P. ¹H NMR (D₂O +

Collect. Czech. Chem. Commun. (Vol. 66) (2001)

Article Doi

Synthesis and cytostatic activity of N-[2-(phosphonomethoxy)alkyl] derivatives of N6-substituted adenines, 2,6-diaminopurines and related compounds

DOI: 10.1135/cccc20011545

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Article abstract of DOI:10.1135/cccc20011545

Full text of DOI:10.1135/cccc20011545

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