J. Med. Chem. 1994,37, 1857-1864
1857
Synthesis, Oral Bioavailability Determination, and in Vitro Evaluation of Prodrugs of the Antiviral Agent 9-[2-(Phosphonomethoxy)ethyl]adenine(PMEA) John E. Starrett, Jr.,. David R. Tortolani, John Russell, Michael J. M. Hitchcock,? Valerie Whiterock, John C. Martin,? and Muzammil M. Mansuri Bristol-Myers Squibb Company, 5 Research Parkway, Wallingford, Connecticut 06492-7660 Received January 26, 1994'
A series of phosphonate prodrugs were evaluated in an attempt to increase the oral bioavailability of the anti-HIV agent 9-[2-(phosphonomethoxy)ethylladenine (PMEA; 1). The majority of the bis(alky1 ester) and bis(alky1 amide) prodrugs were prepared by alcohol or amine displacement of dichlorophosphonate 2. Basic hydrolysis of the bis(esters) or bis(amides) provided the corresponding monoesters or monoamides. Synthesis of bis[ (acy1oxy)alkyll phosphonates loa-c was accomplished by alkylation of PMEA with the appropriate chloromethyl ether in the presence of N,N'-dicyclohexylmorpholinecarboxamidine. The systemic levels of PMEA following oral administration of a PMEA prodrug to rats were determined by measuring the concentration of PMEA in the urine for 48 h after administration of the prodrug. The oral bioavailability of PMEA employing this method was determined to be 7.8 % . Oral dosing with bis(alky1) phosphonates 3a,b resulted in apparent absorption of the prodrugs (140%), although neither of the esters were completely cleaved to liberate the parent phosphonate PMEA. The mono(alky1 esters) 7a-e and 8a,b exhibited poor oral bioavailability (55 % ). Phosphonamides 5,6, and 9 were unstable under acidic conditions and provided levels of PMEA comparable to the parent compound after oral administration. Bis[(acyloxy)alkyll phosphonates loa-c demonstrated significantly improved oral bioavailabilities of 17.6%,14.6%,and 15.476, respectively. When evaluated in vitro against HSV-2, (acy1oxy)alkylphosphonates loa-c were greater than 200-fold more active than PMEA. Introduction
The adenine phosphonate 9- [(2-phosphonomethoxy)ethylladenine (PMEA; 1) has demonstration broad antiviral activity against human immunodeficiency virus (HIV),' Rauscher murine leukemia virus (R-MuLV),~ herpes simplex virus (HSV),3 murine cytomegalovirus (MCMV),2 Simian immunodeficiency virus (SIV),4 and feline immunodeficiency virus (FIV).5 In addition to in vitro activity, in vivo efficacy has been demonstrated when administered intravenously, intraperitoneally, or intramuscularly. Because of the interesting antiviral activity of PMEA, it is currently undergoing phase 1/11 trials for the evaluation of its toxicity and/or efficacy in AIDS patients. Recently presented clinical data indicate that PMEA has activity against HIV-1 in uiuo.6 However, the oral bioavailability of PMEA has been reported to be 4 % in monkeys4 and 11% in rats.' This very low oral bioavailability could limit the potential therapeutic uses of PMEA; current clinical trials with PMEA employ daily iv infusion.6 We therefore undertook a study to prepare and evaluate prodrugs of PMEA in an attempt to increase its oral bioavailability. Relatively few examples of phosphonate prodrugs or prodrugs of closely related analoguesof phosphonates have appeared in the literature. Farquhar and co-workershave reported the use of (acy1oxy)alkyl prodrugs of organophosphates to increase permeation across biological membranes.' The acyloxy alkyl ester of phosphonoformate has been ~ r e p a r e dand , ~ Krapcho et al. have employed (acy1oxy)alkylprodrugs to improve the bioavailability of phosphinates.'O A prodrug of PMEA has been synthesized by linking a synthetic polymer bearing mannosylated t Present address: Gilead Sciences, 353 Lakeside Dr., Foster City, CA 94404. e Abstract published in Advance ACS Abstracts, May I, 1994.
residues to PMEA." In contrast to phosphonates, amuch wider range of prodrugs have been successfully employed for preparation of carboxylicacid prodrugs. Acyloxy alkyl esters, as well as glycolamide esters, alkyl esters, and amides, have been extensively used.12 The goal of the present study was to build on the carboxylicacid experience and evaluate a wide array of structural types as potential prodrugs of the phosphonate functionality. Preliminary results describing in vitro antiviral activity of the bis[(pivaloyloxy)methyll prodrug of PMEA (loa) against HIV, HCMV (human cytomegalovirus), HSV-1, and HSV-2 have recently been published in communication form." We herein report on the synthesis, oral bioavailability, and antiviral activity of several different classes of phosphonate-derived prodrugs of PMEA. Chemistry
For purposes of rapidly evaluating a diverse number of PMEA prodrugs, an easily accessible intermediate was desired. Stowell demonstrated that dichlorophosphonates, generated by oxalyl chloride treatment of phosphonic acids, react with alcohols or amines to give the corresponding bis(alky1 esters) or bis(alkylamines).14 Quast has treated methylphosphonic acid with PC15 to prepare the corresponding di~hlorophosphonate.~~ We found that dichlorophosphonate 2 can be conveniently prepared by refluxing PMEA (1) with thionyl chloride and a catalytic amount of DMF followed by cooling and concentration of the mixture to remove volatiles (Scheme 1). Intermediate 2 was very versatile and allowed us to prepare a majority of the prodrugs described in this study. Alcohols and amines which boil at 1150O C were employed as both the solvent and nucleophile and added directly to crude dichlorophosphonate 2 and heated (Table 1). With the higher boiling alcohols and amines (bp 2150 "C), stoichiometric amounts were added to a suspension of
0022-2623/94/1837-1857$04.50/0 0 1994 American Chemical Society
Starrett, Jr., et al.
1858 Journal of Medicinal Chemistry, 1994, Vol. 37, No. 12
Scheme 1. Synthetic Scheme for Cyclic and Acyclic Ester and Amide Prodrugs of PMEA
Scheme 2. Synthetic Scheme for (Acy1oxy)alkyl Prodrugs of PMEA
0 HO-blnO+ OH
1 I OH
[ J;.+ ]
RCOCYO'
Y
II
N
2
Scheme 3. Synthetic Scheme for Mono(pivaloy1oxy)methylProdrug of PMEA
I
ROH
+
+ 4 ab
I
1)NaOH 2)HCI
I
5
1)NaOH 2)HCI
A:
chlorophosphonate 2 in either dichloromethane or acetonitrile and then heated. (This eliminated the need to remove large volumes of high-boiling liquids.) Unless otherwise noted, the products were isolated by cooling the reaction, evaporating the mixture to dryness, and purifying the residue on a chromatography column. In general, there were large differencesin Rfbetweenthe desired compounds and the unwanted side products. The bis(alky1) phosphonate esters 3a-i, cyclic esters 4a,b, bis(alky1amide)5, and cyclic amide 6 were all prepared in this manner. Saponification of esters 3a-c,f-i with an excess of aqueous sodium hydroxide and heating (Table 1)followed by acidification and reverse-phase chromatography to remove the inorganic salts provided monoesters 7a-c and 7d-7g, respectively. These monoesters were remarkably resistant to further hydrolysis; little, if any PMEA was detected under the reaction conditions outlined. Similarly, cyclic esters 4a,b provided monohydroxypropyl esters 8a,b. Bis(NJV-dialkylamide) 5 proved to be extremely stable to basic conditions, and the corresponding mono-NJVdialkylamide could not be prepared. In contrast to the bis(NJV-dialkylamide),cyclic N-alkylamide 6 was easily cleaved to mono-N-alkylamide 9. Similar to monoesters 7 and 8, mono-N-alkylamide 9 was stable to further hydrolysis to PMEA. A variety of unsuccessful methods were employed in an attempt to synthesize bis[(pivaloyloxy)methyll PMEA (loa). These attempts have been previously described in detail, as well as the successful method shown in Scheme 2.13 Briefly, homogeneous reaction of PMEA (1) with chloromethyl pivalate in the presence of the hindered base N,"-dicylohexylmorpholinecarboxamidine provided bis(pivaloyl ester) 10a in 40 % yield. Bis[(isobutyryloxy)methyl1 PMEA (lob) and bis[(propionyloxy)methyll
Nk> I;,
I
PMEA (1Oc) were prepared in a corresponding manner. The monopivaloyl ester of bis(ester) 10a could not be prepared by simple saponification since it was not stable to the reaction conditions and was quickly cleaved to afford PMEA. As a result, an alternate approach was employed (see Scheme 3). Ester exchange of diphenyl PMEA (3h) with the sodium salt of benzyl alcohol produced unstable dibenzyl PMEA (1 1) which cleaved under the reaction conditions to give monobenzyl ester 12. Alkylation of the monoester with chloromethyl pivalate in the presence of triethylamine provided mixed alkyl phosphonate 13. Subsequent removal of the benzyl group by hydrogenation with catalytic palladium on carbon provided monopivaloyl ester 14. The NJV-diethylacetamidewas prepared as outlined in Scheme 4. In addition to determining the prodrug potential of the acetamide, we also studied the effects of mixed phosphonates containing acetamides,acyloxy alkyl esters, and alkyl esters. Acid chloride 15 was treated with sodium acetate to provide ester 16. Hydrolysis of the ester with sodium methoxide gave hydroxyacetamide 17. Acylation of dichloride 2 with alcohol 17 gave bis(ester) 18 which could not be isolated in pure form. The crude bis(ester) was saponified directly to provide mono-NJVdialkylacetamide ester 19. To prepare mixed (pivaloyloxy)methyl alkyl phosphonates, monoesters 19 and 7b were alkylated with chloromethyl pivalate to give phosphonates 20a,20b, respectively. Cyclic esters 4a,b were poor PMEA prodrugs (vide infra), possibly because of the high stability of the alkyl ester functionality. Studies with cyclic phosphates have shown that introduction' of fluorine to give the cyclic difluorophosphate increased the rate of hydrolysis as compared to the unsubstituted cyclic phosphate.16 In the present study, cyclic difluoro phosphonates were prepared to investigate the effect of fluorine on the stability of cyclic phosphonates. Fluorination of ketone 2 1 with (diethylamino)sulfur trifluoride (DAST) gave difluoride 22 which was hydrolyzed to afford dihydroxy difluoride 23 (Scheme
Journal of Medicinal Chemistry, 1994, Vol. 37, No. 12 1859
Evaluation of Phosphonate Prodrugs of PMEA
Table 1. Yields, Synthetic Methods, and Oral Bioavailability of PMEA Prodrugs compd no. 1 3a 3b 3c 3d 30 3f 3g 3h 3i 4a 4b 5 6
Ra
yield* (%)
synthetic methode
time (h)
temp ("C)
PMEA CH3 CHsCHz (CHsIzCH (CH3)zCHCHz (CH3)zCHCHzCHz n-CsH17 Cl3CCHz Ph p-NOz-PhCHz H CH3
oral bioavailability 7.8 0 (421d (44.4)'
47 A 4 60 69 A 3 60 83 A 3 60 75 A 3 60 0 77 A 1.5 80 0.5 40 Bf 16 40 NDS 44 B 20 82 10.6 38 B 20 22 11.1 15 B 1 60 1.7 9 Ch 16 22 0.7 56 C 24 40 0 18 A 16 55 7.2 C 24 40 5.8 46 7a CH3 78 D 2 60 1.4 CHsCHz 52 D 24 22 0 7b 7c (CHs)zCH 68 D 2 60 5.0 7d n-CsH1.1 80 E' 1.5 60 0.8 7e ClsCCHz 69 E 1 60 2.5 7f Ph 76 E 1 22 14.0 P-NOz-PhCHz 78 E 20 60 1.9 7g H 50 D 2 60 0 8a 8b CH3 80 E 1.5 60 0 9 61 D 1.5 60 10.6 108 32 17.3 10b 9 14.6 1oc 9 15.4 14 12 6.5 19 19 B,D',k 20; 0.3 0; 22 3.5 20a 4 2.2 20b 17 (34.9)' B 1 82 5.8 21 44 B, Ei,"' 1;l 82;22 3.5 26 18 a See schemes for general structures. Yields are unoptimized starting from PMEA (1). Yields for compounds 7a-g,8a,b, and 9 are from bis(ester) or bis(amide). Synthetic methods are described in the Experimental Section of this paper. Where no method is given, see the Experimental Section for specificexamples. d Detected as the diethyl ester. Oral bioavailabilityof PMEA was 0. e Detected as the monoisopropyl ester. Oral bioavailability of PMEA was 0. f The crude product obtained from column chromatography was recrystallized from CH3CN.g The oral bioavailability could not be determined due to lack of solubility. The impure product obtained from column chromatography was recrystallized from 25% MeOH/CHsCN. i The crude product from acidification was recrystallized from water. j The crude product obtained from method B was employed directly in method E. Temperature and time are given for methods B and E, respectively. k See detailed Experimental Section for synthesis of hvdroxvacetamide 17. I Detected as the monoethyl ester. Oral bioavailability of PMEA was 0. See detailed Experimental Section for synthesis df difluoro alcohol 23.
*
Scheme 4. Synthetic Scheme for Glyoxamide Prodrugs of PMEA
Aco&oAc
0
0 15
17
J
18
19 R E12NC(O)CHz 7b R ICH3CHp
A c o x o A c
N~OH_ , o x o ,
21
22
23
19
20a R = EtzNC(0)CY 20b R = CHiCY
.%)), NH?
A=
DAST_
0
16
L
Scheme 5. Synthetic Scheme for Difluoropropyl Ester Prodrug of PMEA
N '
5). Crude cyclic difluoro ester 24 was obtained upon reaction of dichloride 2 with diol 23. Introduction of the fluorine groups activated the phosphonate functionality to nucleophilic attack (as compared to cyclic ester 4a) to such an extent that methoxy ester 25 was obtained upon column chromatography of the crude product when
methanol was employed as an eluting solvent. Hydrolysis of crude cyclic difluoro ester 24 with sodium hydroxide gave monoester 26. Biological Evaluation and Discussion
To estimate the oral bioavailability of the prodrugs relative to that of PMEA, urinary concentrations of PMEA in male rats after oral administration of PMEA and each prodrug were compared to that of PMEA after iv administration a t 30 mg/kg. Urine samples were collected for 48 h, and the amount of PMEA excreted in the urine
Starrett, Jr., et al.
1860 Journal of Medicinal Chemistry, 1994, Vol. 37, No. 12
was measured by an HPLC method as previously reported.17 As shown in Table 1,the absolute oral bioavailability of PMEA is estimated to be 7.8% in the rat. This value compares favorably with the previously reported oral bioavailability of 11% in the rat, which was obtained by measuring the concentration of PMEA in rat plasma after oral and iv administration of PMEAs7In the present study, the urinary method was chosen in preference to the plasma method because of the ease of sample analysis, thereby allowing rapid screening of a large number of compounds. Table 1 shows that the oral bioavailability of PMEA after administration of bis(alky1esters) 3a-e was less than 1% , indicating that the bis(alky1 esters) were either not absorbed or absorbed but not cleaved completely to PMEA. The bioavailability value shown in parentheses after oral administration of diethyl ester 3b (42%)is an estimate of the amount of diester 3b excreted in the urine, based upon HPLC retention times. Similarly, the 44.4% value shown in parentheses after oral administration of bis(isopropy1 ester) 3b represents the amount of monoester 7c detected in the urine, based upon HPLC retention times. These data suggest that both of these bis(alky1esters) were well absorbed (>40%)but that the diethyl ester was excreted unchanged and that the bis(isopropy1ester) was cleaved to the mono(isopropy1) ester after absorption. Similar effects may be occurring with bis(secbuty1 ester) 3d and bis(isoamy1ester) 3e (oral bioavailability of 0 % and 0.5 % , respectively) as well as with cyclic esters 4a,b (oral bioavailability of PMEA of 0.7% and 0%);however, no attempt was made to assay for any of the mono- or bis(esters). In the case of mixed alkyl (acy1oxy)alkyl esters, following administration of ethyl (pivaloy1oxy)methylester 20b, a peak was observed that was consistent with the retention time of monoethyl ester 7b (34.9% recovery). PMEA was not observed in this sample. This result indicates that mixed ester 20b was absorbed but underwent incomplete hydrolysis to give the monoethyl ester. Bis(trichloroethyl ester) 3g was unique among the alkyl esters, demonstrating an oral bioavailability of 10.6%. The electron-withdrawing effect of the chlorines activate the phosphorus-oxygen bond and make it more susceptible to cleavage than that in the unactivated esters, such as diethyl ester 3b. In general, the monoesters 7a-g, 8a,b, 19, and 26 exhibited very poor oral bioavailability, probably due to the polar nature of these compounds. The increased polarity relative to that of the diesters restricts their ability to permeate the gastrointestinal membrane. If monoesters 7b,7c were absorbed but not cleaved, they would have been detected under the assay conditions (vide supra). In contrast to the low bioavailability of the alkyl esters, monophenyl ester 7f provided an oral bioavailability of 14%. The reason for the elevated oral bioavailability of monophenyl ester 7f is unclear at this time. The phenyl ester is less stable than the alkyl esters, and it may be cleaved to PMEA in vivo; however, this does not fully account for the 14% oral bioavailability of 7f. It is also not well understood why PMEA is able to achieve oral bioavailability levels of 7.8%. The bioavailability data from the monoesters show that phosphonates bearing a negative charge are poorly absorbed ( 1 5 % 1, and therefore, PMEA would be expected to have negligible bioavailability levels. It is possible that different mechanisms of absorption are used by the monoesters and PMEA.
Table 2. In Vitro Activity of Selected PMEA Prodrugs against HSV-2 (Gstrain) comDd no. ICm (uM)O 1 (PMEA) 119 3b >300 >300 7b I1 3h 7f 95 0.6 10a c0.2 10b 1oc 14
0.4
22 a In HSV-infected vero cells, the concentration which gives a 50% reduction of plaque formation.
Amides 5,6, and 9 were found to be extremely unstable under acidic conditions (tlp at pH 2 < 10 min; data not shown) and therefore, would be expected to be cleaved to PMEA before absorption. This conclusion is supported by the oral bioavailability values (7.2 % ,5.8 % ,and 10.6 % , respectively). The (acy1oxy)alkylesters 10a-c are the most promising PMEA prodrugs prepared to date. The oral bioavailability in rats of bis[(pivaloyloxy)methyll PMEA (loa; 17.6 % ) more than doubled the oral bioavailability of PMEA (7.8%). Bis[(isobutyryloxy)methyl] PMEA (lob) and bis[(propionyloxy)methyll PMEA (1Oc) also demonstrated improved oral bioavailabilities with values of 14.6% and 15.4%, respectively. The correct balance between lipophilicity and stability is obviously important, as mono(pivaloy1oxy)methyl ester 14 had an oral bioavailability of PMEA of 6.5%. This value is lower than that of bis(ester) 10a (17.3 % ) yet higher than that of any of the monoalkyl esters and higher than all but two of the bis(esters) (trichloroethyl ester 3g and amide 5). The in vitro activity of selected PMEA prodrugs against HSV-2 is given in Table 2. Both diethyl ester 3b and monoethyl ester 7b did not inhibit plaque reduction at doses less than 300 pM, compared to PMEA which had an ICw (dose which gives a 50% reduction of plaque formation) of 119 pM. The lack of activity with ethyl ester 3b probably arises from the stability of the alkyl ester, which is then unable to undergo phosphorylation to the active diphosphate form of PMEA. Both diphenyl ester 3h (IC50 = 77 pM) and monophenyl ester 7f (ICs0 = 95 pM) demonstrated similar efficacy to PMEA. This activity may reflect the instability of the esters, resulting in conversion to PMEA during the course of the assay. Mono(pivaloy1oxy)methyl PMEA (14) had a 5-fold lower ICS0than PMEA (22 vs 119pM), possibly due to improved cell penetration. The efficacy of PMEA was further enhanced through the masking of both charges with the use of bis[(acyloxy)alkyll esters to give 10a-c (ICs0 = 0.6,
