Vol. 27, No. 2ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1985, p. 197-2020066-4804/85/020197-06$02.00/0Copyright C 1985, American Society for Microbiology

Structure-Activity Studies on PhosphonoacetateJAMES C.-H. MAO,* ELLEN R. OTIS, ANNE M. VON ESCH, THOMAS R. HERRIN, JOHN S. FAIRGRIEVE,

NATHAN L. SHIPKOWITZ, AND RONALD G. DUFFPharmaceutical Discovery Division, Abbott Laboratories, North Chicago, Illinois 60064

Received 7 May 1984/Accepted 25 October 1984

Phosphonoacetic acid is a selective antiherpesvirus agent. More than 100 congeners of phosphonoacetic acidwere evaluated in vitro and in vivo to understand structure-activity relationships in the hope of designing asuperior analog. Results showed that the antiherpesvirus activity had highly specific structural requirements.Neither the carboxylic nor the phosphono groups could be replaced. The distance between these two groups isimportant. Increase of this distance caused complete loss of activity. However, if this distance was maintained,the addition of groups to the methylene carbon resulted in a reduction, but not loss, of activity. On the otherhand, decrease of the carbon chain to formic acid did not deteriorate its antiherpes activity. All analogs testedhad lower activity than the parent compound. However, some compounds with decreased activity in vitroappeared to have favorable pharmacological properties in vivo.

Phosphonoacetic acid (PAA) has been previously shownto suppress replication of herpes simplex virus (1, 19), avianherpesvirus (11), cytomegalovirus (9), Epstein-Barr virus(16, 23, 27), vaccinia virus, equine abortion virus, andvaricella-zoster virus (1, 18) in tissue culture. Promisingresults in the treatment of ocular, cutaneous, and encepha-litic herpesvirus infections in mice, hamsters, and rabbitshave been documented (4, 5, 14, 17, 21, 25).PAA at a level of 100 ,ug/ml was effective against herpes-

viruses and nontoxic to cells (24). The lack of cellulartoxicity was accounted for by the preferential inhibition ofherpesvirus-induced DNA polymerase by the drug (3, 13,20). Since PAA is not a substrate analog, it cannot beincorporated into host DNA; and since PAA interacts di-rectly with viral DNA polymerase, but not with DNA, it hashigh selectivity (9, 12, 13). However, there are severalopportunities for improving the efficacy of the drug in vivo,as follows.

Increase of therapeutic index. Although PAA is very potentas an inhibitor of a viral DNA polymerase and usuallyrequires less than 0.2 ,ug/ml for 50% inhibition (18), a100-fold-greater concentration is required to be effective intissue culture and animals. This suggests that penetration ofhighly charged PAA through biomembranes is difficult.

Decrease of side effects. PAA frequently causes skin irrita-tion in preparations containing PAA at a concentrationgreater than 2%. Anorexia and emesis have been observed inanimals if the drug is administered orally at levels of 1 g/kgper day (Shipkowitz, unpublished data).Improvement of absorption and distribution. PAA is poorly

absorbed and is rapidly excreted via the kidney whenadministered orally to animals. When PAA was adminis-tered intravenously to rats, it appeared to be deposited in thebone (B. A. Bopp, C. B. Estep, and D. J. Anderson, Fed.Proc. 36:939, 1977). Whether the deposited PAA would havepathological effect is not known at present.

Inhibition of resistant strains. The appearance of PAA-re-sistant herpesvirus has been previously reported (10). Re-sistance was traced to the occurrence of PAA-resistant viralDNA polymerase (6, 18). The drug does not induce mutants,but strains with relatively insensitive polymerases are se-lected from heterologous virus populations.

* Corresponding author.

The above properties of PAA are related to its chemicalstructure. It is an acidic molecule, with pKa values of 2.6,5.0, and 8.2 determined by titration. Irritation to skin andintestines is most likely related to this highly acidic property.Likewise, slow penetration of PAA through biomembraneand rapid excretion may be attributable to low lipophilicityand the ionic nature of the molecule.More than 100 PAA analogs, synthesized or obtained from

commercial sources, were tested in three systems: (i) PAA-sensitive and PAA-resistant viral DNA polymerases of her-pesviruses, (ii) virus-infected tissue cultures, and (iii) virus-infected animals. The structural requirements for activity arediscussed in this paper.

MATERIALS AND METHODS

Purification and assay of herpesvirus-induced DNA polymer-ase. Herpes simplex virus type 2 (HSV-2) strain MS-infectedWI-38 cells were grown as formerly described (19) and har-vested when 25% of the cells showed cytopathology. Herpes-virus-induced DNA polymerase was purified by column chro-matography on DEAE-cellulose and phosphocellulose asreported previously (13). It had a specific activity of 313U/mg of protein. One unit was defined as the amount cata-lyzing the incorporation of 1 nmol of dTTP into the acid-precipitable material in 30 min at 37°C. The DNA polymerasereaction mixture (0.15 ml) contained 10 ,uM each of dATP,dCTP, and dGTP, 2.5 ,uM [3H]dTTP (880 cpm/pmol), 10 p.gof activated calf thymus DNA, 50 mM Tris-hydrochloride(pH 8.0), 3 mM MgC92, 100 mM KCI, and 1 mM dithiothr-eitol. The amount of enzyme was selected to give a linearrate of synthesis for at least 30 min. The reaction mixtureswere incubated at 37°C for 30 min and terminated by theaddition of 3 ml of cold 5% trichloracetic acid in 10 mM PP1.The acid-precipitable material was then collected and washedon a glass filter disk (Reeve Angel 984-Hi), and radioactivitywas determined by liquid scintillation spectrometry.

Isolation of PAA-resistant strains. Laboratory stocks ofHSV-1 strain Patton were grown in the presence of 100 p,g ofPAA per ml in primary rabbit kidney cells. The survivingviruses were inoculated onto fresh rabbit kidney cells forthree additional passages in the presence of 100 ,ug of PAAper ml. The resistant strain was found to be geneticallystable for at least 20 generations when grown in the absence

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of PAA (18). DNA polymerase, induced by the resistantstrain, was isolated as above and assayed under the sameconditions. It had a specific activity of 220 U/mg of protein.

Antiherpesvirus activity in tissue culture. The inhibitoryeffect of PAA and its analogs upon HSV replication wasanalyzed by the plaque reduction assay. Confluent monolay-ers of rabbit kidney cells grown in 60-mm plastic dishes wereinfected with HSV-1 strain Patton by adsorption for 1 h atroom temperature. The appropriate concentration of com-pound was added at the time of initial virus infection. After,virus adsorption, the cells were overlaid with Eagle basalmedium supplemented with 10% fetal bovine serum, 0.225%NaHCO3, and 0.5% methylcellulose. The appropriate con-centration of the test compound was also added to thisoverlay; 3 to 4 days after virus infection, each culture wasstained with crystal violet, and the herpesvirus-inducedplaques were counted. Inhibition of virus replication wasdetermined by comparing the number of herpesvirus plaquesin the presence and absence of the compound. Each com-pound was assayed twice in triplicate.

Determination of efficacy of PAA analogs for mouse derma-titis. Female 20-g CF mice, under light ether anesthesia, hada 2-cm2 area of their backs plucked free of hair. InfectiousHSV-2 strain MS (0.05 ml; 107 50% tissue culture infectivedose per ml) was applied topically to the denuded skin. A27-gauge sterile hypodermic needle was used to prick thesurface of the skin beneath the drop. In control untreatedmice, a small lesion developed in 3 to 5 days, and the lesionextended across the denuded area in the following 3 to 4days. At approximately day 10, the mice developed a flacid,posterior paralysis. Once paralyzed, death usually followedin 24 h.The PAA analogs, as a 2% aqueous solution, were applied

to the site 2 h after the virus infection. It was recognized thata 2-h interval between infection and treatment favored thedrug. However, since the purpose of this study was tocompare the activity of various compounds, the earliertreatment could enable detection of those compounds havingweak activity. Medication was applied twice daily for 5consecutive days. A single application of the 2% solutiondelivered ca. 2 mg of the compound. The test period was 17days. Ten mice were used for each compound. The Mann-Whitney U Test (22) was used to statistically analyze theherpes infection in mice by making paired comparisonsbetween infected treated mice and infected untreated mice.Ranks were evaluated on the basis of severity of infectionfor each mouse according to a severity scale (lesion, paral-ysis, death) and the time when the sign occurred. Only thosetreated groups that showed statistically significant differ-ences from the infected untreated control group were con-sidered active.

Chemicals. PAA, phosphonopropionic acid, and triethylphosphonoformate were synthesized according to Nylen(15). The procedures for synthesis of carboxylic esters (8),amides (A. von Esch, U.S patent 4,138,432, February 1979),amino acid derivatives (A. von Esch et al., U.S. patent4,272,528, June 1981), and lipid derivatives (T. Herrin and J.Fairgrieve, U.S. patent 4,150,125, April 1979) of PAA havebeen previously described. Phosphonic esters, methylene-modified analogs of PAA, and other phosphonic derivativeswere prepared by the method of Worms and Schmidt-Dunker (26). Sodium sulfonoacetate and sodium arsenoace-tate were from Pfaltz and Bauer Inc., Stamford, Conn.Sodium PP1, sodium methylene diphosphonate, sodiumcarbamyl phosphate, cyclophosphamide, and deoxynucleo-tide triphosphates were from Sigma Chemical Co., St.

Louis, Mo. [3H]dTTP was purchased from New EnglandNuclear Corp., Boston, Mass.

RESULTS

Structure-activity relationship of PAA analogs as inhibitorsof HSV-induced DNA polymerase. PAA analogs were classi-fied according to chemical structure into 13 classes. Theirability to inhbit HSV-induced DNA polymerase is presentedas the 50% inhibition concentration (IC50) in Table 1.

Class I: salts of PAA. Sodium, ammonium, calcium, andzinc salts of PAA were equally active. This was expectedsince salts ionize rapidly in solution.

Class II: carboxylic esters. Low-molecular-weight esters,such as methyl, ethyl, and n-propyl [5, 6, 7] were aboutequally as potent as PAA. Higher-molecular-weight esters,such as n-octyl and t-butyl [8, 9] were less active. Benzyl[10], cyclohexyl [11], and 2-adamantyl [12] esters were muchless active.

Class III: monoesters of phosphono group. Mono esterifi-cation of the phosphono acid significantly decreased po-tency. n-Propyl ester [14] was more active than the methylester [13], but n-hexyl [15] had much lower activity.

Class IV: diesters of phosphono group. This type of com-pound was inactive.

Class V: triesters of PAA. This group of compounds wasinactive because phosphono diesters were inactive.

Class VI: carboxylic amides. Most amides, as withcarboxylic esters, retained appreciable activity, particularlycyclohexylamide [28], azide [32], and N-hydroxy phospho-noacetamide [33].

Class VII: amino acid derivatives. Amino acid derivativesrepresented a special group of carboxylic amides. Theyusually had lower activity than the simple amides, except fora dipeptidyl PAA (PAA-L-Asp-Gly) [51] which had an IC50 of3.3 p.g/ml. It is of some interest that PAA-L-Asp [38] hasbeen shown to be an inhibitor of aspartate transcarbamylase(2), and it was found here to also inhibit HSV-induced DNApolymerase. The inhibition of aspartate transcarbamylase isdue to the structural similarity of PAA-L-Asp to the transi-tional substrate of that enzyme. The measurable inhibition ofDNA polymerase was probably due to the residual activityof the PAA moiety of the compound.

Class VIII: methylene-modified analogs. A methylene car-bon separates the carboxylic and phosphono groups ofPAA.Increasing the separation by two and three carbons [52, 53]caused complete loss of activity. In contrast, compoundswhich maintained the one carbon separation of the twogroups but added branches to the methylene carbon retainedsome activity. Among the alkyl derivatives such as methyl,ethyl, and butyl [54 to 56], the ethyl derivative was the mostactive. All aryl derivatives [57, 58] had extremely lowactivity, and the cyclohexyl [59] had no activity. If themethylene group of PAA was replaced by p-phenylene [60],all activity was lost, probably due to a combination ofincreased separation of the carboxylic and phosphono groupsand the size of the group. 2-Bromophosphonoacetic acid [61]retained considerable activity, and this suggests furtherstudy of other 2-halo derivatives.

Class IX: gross modification of acetic acid moiety. This classof compounds [62 to 81] had either low or no activity againstHSV-induced DNA polymerase.

Class X: phosphono group-modified analogs. Diphe-nylphosphinylacetic acid [87], which had no ionic group onphosphorus, was completely inactive. Several phosphonicacids [84 to 86] which retained one ionic group showed a

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very low activity. Sulfonoacetic acid [82] was virtuallyinactive. On the other hand, arsenoacetic acid [83] retainedsome activity against HSV DNA polymerase.

Class XI: lipid derivatives. These derivatives have highlipophilicity and low acidity and should, therefore, be moreeasily transported through biomembranes. They can beregarded as analogs of high-molecular-weight esters of PAAand, therefore, should have low activity against the polymer-ase. Indeed, they had low antipolymerase activity, yet werehighly active in the mouse dermatitis test (Table 1).

Class XII: phosphono esters and carboxylic amide. Thisclass has an amide group at the carboxyl position and eitherone or two phosphono esters. All had very minimal activity.

Class XIII: miscellaneous compounds. Leinbach and col-leagues (12) proposed that phosphonoacetate inhibitedherpesvirus-induced DNA polymerase by interacting withthe enzyme at the PP, binding site. PP, is a general inhibitorof DNA polymerase because elongation of the growingpolynucleotide requires elimination of PP1 from substratetriphosphates as they are coupled to the 3'-hydroxyl. Wefound that PP1 inhibited HSV-induced DNA polymerase butwas much less potent than PAA. Methylene diphosphonate[98], an analog of PP1, was inactive. In addition, 75 com-pounds not listed in Table 1 which were distantly related tothe PAA structure were tested and found to be inactiveagainst HSV-induced DNA polymerase.

Antiherpesvirus activity of PAA analogs in tissue cultureand mouse dermatitis. The potency of PAA analogs in tissueculture was determined by the plaque reduction method asdescribed above. The concentration of compounds used was100 ,ug/ml in each case. Selected compounds were alsotested against herpes-induced dermatitis in mice by theprocedure outlined above. Only those that showed statisti-cally significant differences from virus controls at the P <0.1 level were defined as active. Results are shown in Table1. Compounds inactive in tissue culture were also inactive inmice. However, some weakly active compounds such as 13,which reduced plaques by 35%, and 16, which reducedplaques by 30%, had no measurable activity against mousedermatitis. On the other hand, some very weak compoundssuch as 24, 26, 51, and 89, which reduced plaques less than10%, had significant activity against mouse dermatitis.Potency of PAA derivatives against sensitive and resistant

HSV-induced DNA polymerases. As reported previously (18),PAA-resistant HSV strains contained a DNA polymerasewhich was about 10-fold less sensitive to PAA than that ofthe wild strain. PAA-resistant DNA polymerase was purifiedaccording to the same procedure employed for the purifica-tion of the sensitive enzyme. Inhibition of sensitive andresistant polymerases by selected analogs of PAA wascompared (Table 2). The resistant polymerase was 14-foldless sensitive to PAA than the sensitive polymerase. Allderivatives tested were less active against the resistantpolymerase. Only one compound [8] showed a favorableratio toward the resistant enzyme. Further study is neededto determine whether there are some PAA derivatives whichwill significantly inhibit a PAA-resistant DNA polymerase.

DISCUSSION

The results of testing more than 100 analogs against theHSV-induced DNA polymerase suggest the following rulesof structure-activity relationships.

(i) Structural requirements are very rigid. Each of 103modifications decreased activity.

(ii) Blockage of the carboxylic group by small alkyl esters,such as methyl, ethyl, and propyl [5, 6, 7], only slightlydecreased activity. This suggests that an ionizable carboxylicacid is not an absolute requirement for inhibitory activity.However, larger esters, such as benzyl, cyclohexyl, andadamantyl [10, 11, 12], were much less active, due perhapsto steric hindrance or increased hydrophobicity. Blockage ofthe carboxylic groups by amides [22 to 34], including aminoacid derivatives [35 to 51], significantly decreased activity.Eriksson et al. (3) have studied the inhibitory effect of propylester [7] and amide [22] of PAA on HSV-1 DNA polymeraseand reported they had much lower activity than is shown inthis study. We cannot fully explain this discrepancy. Per-haps HSV-2 DNA polymerase under our experimental con-ditions was more sensitive than HSV-1 DNA polymerase.Nevertheless, both studies showed a trend that carboxylicester has more activity than carboxylic amide.

(iii) Monoesters of the phosphono group, such as methyland propyl esters of PAA [13, 14], retained some activity.Eriksson et al. (3) found ethyl ester of PAA had no signifi-cant activity. This suggests that the ionic property of thephosphono group is more important than that of thecarboxylic group and that at least one ionizable group onphosphono must be available for activity. As expected,diesters [16, 17] and triesters [18 to 21] of PAA were notactive.

(iv) The distance between the phosphono and thecarboxylic groups is important. The acetic group could bereplaced by the formic group without significantly changingthe antiviral activity (7). Elongation of the carbon chain,such as phosphonopropionic acid and phosphonobutyricacid [52, 53], on the other hand, inevitably resulted in a totalloss of activity. Similar conclusions were previously re-ported (3, 12). However, if the separation was not changed,some branching of the methylene group [54 to 57] waspossible with retention of activity, corroborating the earlierfinding for HSV-1 DNA polymerase (3).

(v) The phosphono group is very specific. It cannot bereplaced by a sulfono group [82]. This agrees with earlierresults (3, 12). Replacement of the phosphono group byphosphinic [84 to 86] or by phosphine [87] led to almost totalloss of activity. However, arsenoacetic acid [83] showedstrong inhibition of polymerase at high concentration, butthe inhibition decreased rapidly as concentrations werereduced. The arseno analog was not active in vivo.The antiherpesvirus activity of compounds in tissue cul-

ture and in vivo mouse dermatitis did not completely coin-cide with antipolymerase activity. Tissue culture and partic-ularly animal models are more complex and may involvemetabolism of drug and delivery and penetration to the virusreplication site. Therefore, some compounds [8, 9, 28, 42,61] had an IC50 of less than 10 ,ug/ml for DNA polymerasebut had no activity either in tissue culture or in vivo. On theother hand, some compounds [24, 25, 26, 45, 48] showed lowactivity against polymerase but were relatively more activein tissue culture and in animals.PAA is a small, highly charged molecule, and we have

studied many possible types of modification. Unfortunately,all analogs had lower activity both in vitro and in vivo. Inaddition, no analogs were found to be particularly potentagainst HSV-induced DNA polymerase resistant to PAA.Further work is required to assess the potential of com-pounds with decreased activity in vitro but favorable inhib-iting properties in vivo, such as the lipid PAA [89]. Thiscompound had low activity in the enzyme assay and in tissueculture, yet was quite effective for mouse dermatitis.

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TABLE 1. Inhibitory effect of PAA analogs on HSV DNA polymerase, virus replication, and mouse dermatitisIC50 of DNA Y Mouse

Compound polymerase" Inhibition dermatitis'(~tgIrn) of PFU5

Class l. Salts of phosphonoacetic acid1. 2 Na' 0.16 99.5 ++2. 2 NH4+ 0.17 99.5 ++3. Ca2+ 0.18 75.0 NT4. Zn2+ 0.18 99.3 NT

Class II. Carboxylic esters: ROCO-CH2-PO3H2R

5. CR3- 0.20 77.0 ++6. CH3CH2- 0.23 44.0 ++7. CH3CH,CH2- 0.20 72.0 ++8. CH3(CH2)7 2.6 Toxic N9. (CH3)3C- 3.9 0 NT

10. C6H5CH2- 40.0 011. Cyclohexyl- 49.0 16 NT12. 2-Adamantyl 164.0 33 NT

Class III. Monoesters of phosphono group: HOCO-CH,-PO(OR)OHR

13. CH3- 20.0 3514. CH3CH2CH2- 3.0 1615. CH3(CH,)5- 170 NT-

Class IV. Diesters of phosphono group: HOCO-CH2-PO3R2R

16. CH3CH,- >200 3017. C6H3- >200 0 NT

Class V. Triesters: ROCO-CH2-PO3R'2R R'

18. CH3- CH3- >200 019. CH3CH,- CH3CH,- >200 620. C6H5CH2- CH3- >200 0 NT21. C,6H5CH,- CH3CH2 >200 NT NT

Class VI. Carboxylic amides: R-CO-CH2-PO3H2R

22. NH2- 16.6 023. CH3NH- 40.0 024. CH3(CH2)2NH- >200 4 ++25. CH3(CH-)3NH- >200 NT ++26. CH3(CH2-)7NH- >200 10 +27. (CH3)3CNH- 100 028. C6H11NH- 5.9 029. C6H,,CH2NH- >200 0 _30. Adamantyl- 16.3 0 N31. C6H5CH,NH- >200 NT -

32. H2N-NH- 7.5 0 NT33. HONH- 9.3 NT NT34. C6H5CH2NHO- 140 0 NT

Class VII. Amino acid derivatives: H203PCH2CORR

35. -D-Ala 6.6 2036. -n-Ala 10.6 0 NT37. -L-Arg 69.0 0-38. -L-Asp 36.0 58 +39. -D-Asp 170 8-40. -L-Asn >200 0-41. -L-diethyl Asp >200 NT NT42. -Gly 5.7 0-43. -L-Glu >200 NT-44. -L-Leu 180 NT NT45. -D-Leu >200 16 ++46. -L-Leu-NH2 160 NT47. -L-Lys >200 048. -L-Phe 50 25 ++49. -L-Pro 150 1-5.--l450.-L-Ala14251. -L-Asp-Gly 3.3 9 +

Continued on following page

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TABLE 1-ContinuedIC50 of DNA Y Mouse

Compound polymerase" Inhibition dermatitis([pg/mi) ofPFUbdemtis

Class VIII. Methylene-modified analogs: HO2C-R-PO3H252. -CH2CH2- >200 NT NT53. -CH2CH2CH2- >200 NT NT54. -CH(CH3)- 33 NT NT55. -CH(CH2CH3)- 4.7 NT ++56. -CH(CH2CH2CH2CH3)- 86 42 ++57. -CH(C6H5)- 150 0 -58. p-ClC6H4CH >200 0 NT59. (CH2)5C >200 0 -60. -C6H4- >200 NT NT61. -CHBr- 8.6 NT

Class IX. Gross modification of acetic acid moiety: R-PO3H2R

62. CH3- >200 2 -63. CH3CH2- >200 0 NT64. C6H5CH2- >200 765. CH3CH(OH)- >200 NT NT66. H2NCH2- 95 NT NT67. H2NCH2CH2- >200 068. H2NCH2CH2CH2- >200 9 NT69. H2NCH(CH3)- >200 NT70. ClCH2- >200 NT NT71. ClCH2CH2- >200 NT72. BrCH2CH2- >200 3173. C5H4NCONHCH2- >200 NT NT74. 1-NH2-4-CH3-C6H3- >200 0 NT75. 4-CH3C6H4- >200 NT NT76. 4-Cl-3-NO2C6H3- >200 0 NT77. 3-NO2C6H4- 100 NT78. 4-ClC6H4SCH2- >200 NT NT79. C6H5SCH2- >200 NT NT80. 3-NH2C6H4- >200 NT81. Cl3C- >200 NT NT

Class X. Phosphono group-modified analogs: HO2C-CH2-RR

82. -SO3H >200 NT NT83. -As03H2 45 NT84. -PO(CH3)OH 140 NT NT85. -PO(C6H5)OH >200 NT NT86. -PO(p-4-CH3C^,H4)OH 150 NT NT87. -PO(C6H5)2 >200 NT NT

Class XI. Lipid derivatives88. P-Phosphonoacetyl-dipalmitoyl glycerate 50 NT NT89. ,-Phosphonoacetyl-diacetyl glycerate 100 7 + +

Class XII. Phosphono esters and carboxylic amide:R-CO-CH2-PO3R'R"

R R' R"90. NH2- CH3- H- >200 0 NT91. NH2- CH3CH2- H- >200 892. NH2- CH3(CH2)3- H- >200 3893. NH2- CH3- CH3- >200 094. CH3(CH2)3NH- CH3- H- >200 8 NT95. CH3NH- CH3- H- >200 096. L-Asp- C6H5- C6H5- >200 0 NT

Class XIII. Miscellaneous compounds97. Sodium ppi 34 NT NT98. Sodium methylene diphosphonate >200 NT NT99. Sodium carbamyl phosphate >200 NT NT

100. Cyclophosphamide >200 NT NT101. Trimethyl phosphonopropionate >200 NT NT102. Triethyl phosphonoformate >200 NT NTa IC50 of each compound was determined by using five concentrations performed in duplicate.bPercent inhibition was determined by the plaque reduction technique performed in triplicate in the presence of 100 ,ug of compounds per ml.c++, Active at P <-0.05; +, active at P < 0.10; -, inactive; NT, not tested.

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TABLE 2. Comparison of inhibitory effect of PAA analogs onPAA-sensitive and -resistant HSV-induced DNA polymerases

IC50 (,g/ml) Resistant/Class Compound

no. Sensitive Resistant sensitivepolymerase polymerase ratio

I 1 0.16 2.2 13.7

II 5 0.20 7.0 35.06 0.23 3.9 17.07 0.20 4.7 23.58 2.6 15.0 5.810 40.0 >200

VI 22 16.6 >20025 >200 >20032 7.5 >200

IX 65 >200 >20079 >200 >200

XII 90 >200 >200

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