`
`ELSEVIER
`
`
`
`advanced
`drug delivery
`reviews
`Advanced Drug Delivery Reviews, 19 (1996) 287-310
`
`Review
`
`Prodrugs of phosphates, phosphonates, and phosphinates
`Jeffrey P. Krise, Valentino J. Stella*
`Department of Pharmaceutical Chemistry and the Center for Drug Delivery Research, The University of Kansas,
`2095 Constant Avenue, Lawrence, KS 66047, USA
`
`Received 1 August 1995; accepted | October 1995
`
`
`
`Abstract
`
`The objective of this paper is to review the literature on the use of prodrugs to overcome the drug delivery
`obstacles associated with phosphate, phosphonate and phosphinate functional group-containing drugs. This is an
`important area of research because, although we have been successful at identifying numerous phosphate and
`phosphonate functional group-containing drugs as antiviral and anticancer agents, as well as for other uses, our
`ability to orally deliver these drugs and to target
`them to desired sites has led to limited success. Various
`acyloxymethyl- and aryl-ester prodrugs have shown promise. Alternative and imaginative approaches may be
`necessary before complete success is realized. It
`is our hope that this review will stimulate further innovative
`prodrug research into overcoming the barriers to the delivery of these important drugs.
`
`Keywords: Nucleotide analog; Nucleotide prodrug; Bioavailability; Ester; Permeability
`
`
`Contents
`
`2. Why prodrugs:ofphosphates,‘phosphonatesandphosphine.fetetetennnetinea208
`
`3, How can prodrugs overcome these* Problems?. s—-2B8
`
`boceeeeesssesueeseeesessaueeeesasceseseseeesseesssesseseaestoesesaesneeseeseeeeeseeeeeseteeeneseteeetteeeeeesettieececeeee
`4. Specific examples 0.000.000.
`290)
`4.1. Nucleotide analog prodrugs...
`vo
`lites 290
`4.1.1. Simple and substituted alkylanddaryesesterprodrugofphosphatesaandnd phosphonatessettee290
`295
`4.1.2, Acyloxyalkyl esters..
`seveeeteaereaseneneenees
`ceneneees
`seceeeeeeeesttesneseeeterteeesarettenetnaseeneeneterseeesree
`4.1.3. Phospholipid derivatives.
`297
`
`-
`4.1.4. Cyclic prodrugs...
`298
`4.1.5. Nucleotide analog conjugate systems.
`vo
`299
`
`4.1.6. Carbohydrate derivatives..........cccccccessersuseessssssneereesscsteeceeescsnsueeeesensastscessnsustaeesenensetessessnaseserceneaaestertnaeareeseseeree
`QOD
`4.1.7. ‘SATE’ and STE? ecsctscsssseissssvesnersennsevenne (eenOL
`4.2. Miscellaneous prodrug applications...
`.
`301
`
`4.2.1. Fosinopril: An ACE inhibitor...
`ve
`seeeeeeeeetaeeseceeceessuseatgecsctesseegeereeseteetsereetetttrmerttteeeeeee
`SOL
`4.2.2. CGS 24562: A neutral endopeptidase inhibitor.
`vse sence eesaseeteeeeeceessesneceeseeseeenieseetetettessntetttecssees
`402
`
`4.2.3. L-690,330: A inhibitorof inositol monophosphatase.... feetitieeeee—303ceeetesceenseeeneeseeaeaseesseeseeatenees
`
`
`4.2.4. (Hydroxy2--naphthalenyimethy!)phosphonicaacid: an IRTK inhibitor..
`seneeesteeeeteerees tee 304
`ceeeeeguuceseetedenaseeteeeeeayes
`-
`5. Conclusions..
`304
`
`RefEKENCEScesseecsscceeevnssevssnerevsevsevesesvesusetsneseesssnsesnisnsssveseessvesunetnsesvettetsnsgunstnntsnesnsttusesnsinsisusissnstussnsetiseue
`305
`
`“Corresponding author. Fax: + 1 913 8425612.
`
`© 1996 Elsevier Science BV. All rights reserved
`0169-409X /96/ $32.00
`SSDI 0169-409X(95)00111-5
`
`1
`
`GIL2019
`I-MAK, INC. V GILEAD PHARMASSETLLC
`IPR2018-00121
`
`1
`
`GIL2019
`I-MAK, INC. V GILEAD PHARMASSET LLC
`IPR2018-00121
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`
`288
`
`J.P. Krise, VJ, Stella | Advanced Drug Delivery Reviews 19 (1996) 287-310
`
`OR’
`
`Phosphate
`
`OR’
`
`Phosphonate
`
`OR"
`
`Phosphinate
`
`Drug:
`
`R= organic residue; R' = H or (-)
`
`Prodrug:
`
`R= organic residue; R' = promoeity
`
`|. General structure of phosphate, phosphonate, and
`Fig.
`phosphinate drugs. R represents an organic residue while R°
`represents H or anionic charge for the parent drug or the
`promoiety for the prodrug.
`
`1. Phosphate, phosphonate and_phosphinate
`
`oO
`0
`RO—P—oR'
`R—-P—oR' pt
`groups impart an anionic charge (mono- or
`di-) at nearly all physiological pH values
`making them very polar. This high polarity
`can be the basis for many deficiencies in the
`efficacy of drug delivery. Specifically, highly
`ionized species do not readily undergo passive
`diffusion across cellular membranes.
`2. Because of the increasedpolarity, these agents
`often exhibit a low volumeof distribution and
`therefore tend to be subject to efficient renal
`clearance as well as possibly biliary excretion.
`in addition to renal clearance, phosphates,
`particularly those of primary alcohols and
`phenols, are known to be substrates for many
`phosphorylases present
`in the body which
`readily clip the phosphate group from the
`drug. The rapid dephosphorylation results in a
`short duration of action. Phosphonates and
`phosphinates have the advantage of being
`more chemically stable and showing essential-
`ly no enzymatic lability.
`
`1. Introduction
`
`to review the
`The intent of this paper is
`literature on the use of prodrugs to overcome
`drug delivery obstacles associated with phos-
`phate, phosphonate and phosphinate functional
`group containing drugs. The general structures of
`phosphate, phosphonate, and phosphinate-con-
`taining drugs are shown in Fig.
`1 where R
`represents an organic residue, and R’ represents
`either an anion charge or a hydrogen atom in the
`case of the parent compound,or R’ is a neutral
`ester in the case of the prodrug.
`Initial
`research on the problems associated
`with the delivery of phosphate functiona] group-
`containing drugs began in the early 1960s with
`phosphate-containing nucleoside analogs used in
`cancer and viral chemotherapy. Effort continued
`to expand and has recently increased in interest
`with the advent of the variety of drug classes
`utilizing the phosphate, phosphonate or phosphi-
`nate functionalities, especially antiviral agents
`and various molecules designed to alter cell
`signaling processes.
`
`2. Why prodrugs of phosphates, phosphonates
`and phosphinates?
`
`Although the range of drugs containing either
`a phosphate, phosphonate or phosphinate-con-
`taining functional has changed,
`the underlying
`problem with the delivery of such drugs has not.
`The shortcomings in the delivery of these drugs
`can be broken down into two basic problems:
`
`3. How can prodrugs overcome these
`problems?
`
`In an attempt to overcome these shortcomings,
`the ionizable phosphate, phosphonate and phos-
`phinate groups have been neutralized via chemi-
`cal derivatization. This generally involves de-
`rivatization of the phosphorus-coupled oxygen(s)
`to form neutral ester(s). If the intention of the
`ester is for it to breakdownin the body to release
`the parent drug, then such a derivative would be
`a prodrug. The neutralization of the charge(s)
`has been proposed to serve a number of pur-
`poses:
`
`1. The first is to decrease the polarity by increas-
`ing the lipophilicity of the drug molecule thus
`allowing access to cells and tissues that might
`not be available to the non-modified specie
`and possibly altering the distribution/elimina-
`tion pattern of the parent drug.
`2. Another use of the neutral ester prodrugs is
`particularly important for phosphates which
`are substrates for nonspecific serum phos-
`phohydrolases, such as alkaline and acid phos-
`
`2
`
`
`
`J.P. Krise, V.J. Stella | Advanced Drug Delivery Reviews 19 (1996) 287~310
`
`289
`
`it has been pro-
`phatases. With these drugs,
`posed that the neutral ester serves to disguise
`the phosphate from the enzymes thereby
`altering the apparent elimination and half-life
`as illustrated in Scheme 1. A flaw in this
`thinking is that once deprotected, the intrinsic
`properties of the drug should not be dramati-
`cally altered. Nevertheless, entrapment of the
`parent drug in tissues in which it
`is not
`normally accessible could lead to apparent
`changes in its pharmacokinetic/pharmacody-
`namic properties.
`
`Although alterations in apparent clearance
`rates may be important, the principal goals of
`most prodrug modification efforts on phosphate,
`phosphonate and phosphinate drugsis alteration
`of membrane permeability to improve oral (GI
`permeability), brain, tumor and cellular delivery
`(mainly to virally infected cells) of these agents.
`When these prodrugs are used for improving
`oral bioavailability, various issues dealing with
`GI absorption of drugs must be considered. The
`ability to address these issues will ultimately
`determine the proper selection of the prodrug
`system and its
`likely success. The optimal
`scenario for enhanced systemic delivery of pro-
`drugs after oral dosing is as follows:
`
`1. The prodrug must display adequate chemical
`stability for formulation purposes as well as
`stability in the variable pH environmentofthe
`GI tract.
`2. The prodrug should have adequate solubility
`in the GI
`tract environment
`to allow for
`dissolution.
`
`oO
`Il
`:
`—P~o
`CD0-F-08
`08
`DAUG
`
`Prodrug
`Neutralization
`
`Phosphatase
`
`i
`+ HO-P—OH
`OA
`
`a
`METABOLITE
`
`3. Once dissolved, the prodrug should also dis-
`play enzymatic stability to lumenal contents as
`well as the enzymes found in the brush border
`membrane.
`4. The prodrug should have properties that allow
`for good permeability (generally associated
`with an adequate log P value).
`5. After permeation of the lumenal membrane,
`the prodrug could revert to the parent drug
`either in the enterocyte or once absorbed into
`systemic circulation. Post-enterocyte reversion
`is desired because conversion in the en-
`terocyte would also allow for back diffusion
`into the GI lumen, a problem which is not
`generally recognized.
`
`When the prodrug is formulated to increase
`cellular permeability into viral-infected cells,
`tumorcells or across barriers like the blood brain
`barrier, the desired characteristics might change.
`Replacing the desire for complete and rapid post
`absorption reversion,
`is a need for balance in
`lability. The most optimal scenario, however
`unrealistic, would be for the prodrug to have
`complete enzymatic and chemicalstability during
`the absorption process and in blood but readily
`revert
`to the parent compound once it has
`permeated the targeted cell,
`thereby ‘trapping’
`the drug in the cell (Scheme 2).
`Considering both of these scenarios, prodrugs
`for
`improved oral delivery and prodrugs for
`improved cell
`targeted delivery,
`the rate of
`bioreversion is a very important process that
`
`SYSTEMIC CIRCULATION
`
`o
`
`—P-08
`de
`DRUG
`
`Neutralization
`Prodrug
`
`7
`Coen
`OF
`recor
`
`cell
`membrane
`
`N
`S
`NS
`
`N
`s
`NS
`
`;
`Ni
`
`TARGET CELL
`
`o
`
`<_»-F-00
`de
`DRUG
`
`@
`Cen
`OF
`rronnu
`
`. 2 (ROH)
`
`i
`i
`Phi
`a
`—P—
`Doon PRE Tm + oho
`OF
`OH
`PRODRUG
`
`se
`
`—
`
`METABOLITE
`
`Scheme 1, Scheme showing the possible advantages of pro-
`drugs in altering the phosphatase cleavage of phosphate-
`containing drugs.
`
`Scheme 2. Proposed scheme illustrates the potential advan-
`tages of prodrugs over the parent molecule for intracellular
`targeting.
`
`3
`
`
`
`290
`
`JP. Krise, VJ. Stella | Advanced Drug Delivery Reviews 19 (1996) 287-310
`
`must be considered in detail when designing
`prodrug systems. For example, if bioreversion is
`very fast and non-specific, prodrug reversion may
`take place before the limiting barrier is over-
`come. On the other hand, if reversion is slow and
`inefficient at all sites, the prodrug may readily
`reach the site of action but never release enough
`parent drug to elicit a pharmacological response.
`With these factors in mind, choosing a suitable
`bioreversible protective group for phosphates,
`phosphonates and phosphinates presents a major
`challenge.
`
`4. Specific examples
`
`4.1, Nucleotide analog prodrugs
`
`Purine and pyrimidine nucleoside analogs have
`found great utility in treatment of neoplastic and
`viral diseases [1-8]. Please note, the word nu-
`cleoside will be equated with nucleoside analog
`for the remainder of this manuscript for simplici-
`ty purposes. These nucleosidic drugs mostly rely
`on viral or kinase-mediated (i.e., thymidine ki-
`nase) activation step(s)
`to produce the phos-
`phorylated nucleotide, necessary to display bio-
`logical activity i.e.,
`the nucleosides are them-
`selves prodrugs. Unfortunately, dependency on
`kinase mediated synthesis can lead to the de-
`velopment of resistance [9-11]. The first step in
`the phosphorylation of a nucleoside to the mono-
`phosphate is known to be highly specific and
`often causes the development of resistance [12-
`
`NH
`
`15]. Therefore, it can be argued that an approach
`to circumventing the resistance development
`problem is
`to administer the monophosphate-
`containing nucleoside drug. This strategy has two
`flaws:
`
`1. The highly polar monophosphate has limited
`passive absorption properties and therefore,
`transcellular transport is very restricted [16-
`19]. This fiaw is supported by the work of
`Leibmanetal. [18] who demonstrated the lack
`of passive as well as the absence of an active
`transport mechanism for ara-CMP(the mono-
`phosphate derivative of ara C; Fig. 2).
`. Rapid in vivo dephosphorylation of the mono-
`phosphate is observed with this class of drugs
`[20-22].
`
`bh
`
`Could prodrugs be used to overcome these
`problems? The aim of this portion of the review
`is to focus on phosphorus-coupled oxygen pro-
`drug esters of nucleotides, namely the mono-
`phosphates and their phosphonate analogs. An
`effort has been made to group the various
`prodrug strategies according to the type of neu-
`tral ester chosen. The interested reader may wish
`to refer to other reviews on this subject that take
`a little different approach from those taken here
`(23-25].
`
`4.1.1. Simple and substituted alkyl and aryl ester
`prodrugs of phosphates and phosphonates
`Rosowsky et al.
`[26] have examined several
`mono-5'-(alkyl phosphate) esters of ara-C (Fig.
`
`yo
`
`ook
`
`R,O-P =O
`dr
`
`a)
`
`i
`
`OH
`
`(1) Ry, Ry =CyHs, H
`(2) Ry, Ry =n-CyHy, H
`(3) Ry, Ry =n-C,Hy3, H
`(4) Ry, R; = n-CgH)>, H
`(5) Ry, Rp =n-CygHy3, H
`(6) Ry. Ro = C3Hs, C3iHs
`(7) Ry, Ry =n-CyHo, n-CyHo
`(8) Ry. Ry = n-CgHj7. n-CgHy7
`(9) Ry, Ry = n-CygHyg, n-C) gH;
`
`HO
`
`no
`
`aA N
`
`s)
`
`HO,
`
`OH
`
`ara-C
`
`Fig. 2. Structures of ara-~C, ara~-CMP, and araCMPalkyl prodrugs. Structures (1)—(4) are mono-alkyl prodrugs [26] while (5)-(9)
`are dialkyl prodrugs [30].
`
`ara-CMP (R, = H, R; = H)
`
`4
`
`
`
`J.P. Krise, V.J. Stella | Advanced Drug Delivery Reviews 19 (1996) 287-310
`
`291
`
`2, 1-5) in an effort to deliver ara~-CMPto cancer
`cells. The cytotoxicity of the proposed prodrugs
`toward cultured L1210 leukemia
`and B16
`melanoma cells appeared to obey an inverse
`structure-activity relationship with respect
`to
`alkyl chain length. The n-butyl and n-hexyl
`prodrugs were approximately half as active as the
`ethyl prodrug. The structure-activity relationship
`seemedto plateau as chain length became longer
`as exemplified by n-octyl and n-C,, H,, esters
`having nearly the same [D6 y,jye.. Similarly,
`Mullah and co-workers [27] produced a 5’-O
`methyl and 5’-O phenylesters of 2’,3’-didehydro-
`2',3'-dideoxyadenosine and 2',3’-didehydro-2',3’-
`dideoxycytosine which displayed similar in vitro
`results to the parent nucleosides. When incu-
`bated in serum containing medium, the phenyl
`prodrug produced parent nucleoside along with
`nucleoside monophosphate; the methyl prodrug
`was not evaluated in this manner, In an in vivo
`experiment, assessing activity against adenocar-
`cinoma 755 in mice, Montgomeryetal. [28] has
`evaluated the mono- and diethyl, butyl, and
`phenyl
`esters
`of 6-mercaptopurine
`ribonu-
`cleotide. The monoester prodrugs were of the
`same order of effectiveness as 6-mercaptopurine
`ribonucleotide, whilst the diesters were markedly
`less effective.
`In general, the mono-alkyl/aryl ester analogs
`of phosphates failed to act as efficient prodrugs
`for the delivery of nucleoside-monophosphate
`analogs intracellularly. The poor activity of the
`monoalkyl/aryl esters can be attributed to:
`
`1. High degree of polarity. Due to presence of
`the mono-anionic
`charge,
`limited passive
`transport across cells can be expected.
`2. The relative ease of in vivo conversion of the
`monoalkyl esters in vivo back to the parent
`nucleoside before the cell barrier has been
`overcome. This could be a possible explana-
`tion for the monoalkyl prodrugs and parent
`nucleoside sharing similar activities.
`
`McGuigan etal. [29] recently synthesized and
`evaluated, in vitro, a series of alkyl prodrugs of a
`hydrogen-phosphonate derivative of AZT (Fig.
`3) in an attempt to increase its antiviral activity.
`They showedthat the short chain (C1—C7) alkyl
`
`NH3
`
`v7
`
`1 |
`°
`"
`
`0
`
`~"
`
`R=Me
`R - mHept
`Ran,Fy
`
`1
`RO-P—0
`
`Ng
`
`AZT H-phosphonate analog (R = H)
`
`Fig. 3. Structures of an AZT H-phosphonate analog and its
`prodrugs [29].
`
`hydrogen phosphonates to be more active than
`the long-chain (C18) ester prodrugs in HIV-1-
`infected C8166 cells. The short chain phospho-
`nate prodrugs were 5-10 times more potent than
`the parent phosphonate; however, all prodrugs
`were poorly active in an infected JM cell
`line,
`which is thought
`to lack the kinase mediated
`phosphorylation to produce the active nucleotide
`analog. This demonstrated lack of activity in the
`JM cell line suggested that these prodrugs were
`acting as depot forms of the nucleoside rather
`than the nucleotide.
`To cause a further reduction in polarity, inves-
`tigators have also synthesized diesters of phos-
`phates/phosphonates and evaluated their effec-
`tiveness as prodrugs in in vitro and in in vivo
`tests. The results were not consistent with the
`observation with the monoesters. For example,
`Colin et al.
`[30] showed a clear
`relationship
`between inhibition of thymidine incorporation by
`mammalian epithelial cells and lipophilicity (in-
`creased alkyl chain length) of diester prodrugs of
`ara-CMPutilizing a similar series of protecting
`groups to those studied by Rosowskyetal. (Fig.
`2, 6-9). The in vitro activity was lowest for the
`ethyl ester and highest
`for
`the hexyl ester.
`Similar work performed on ara-AMP gave a
`correlation between an in vitro inhibition of
`DNA synthesis and lipophilicity; however, no
`anti-viral activity at concentrations up to 100
`ug/ml was detected against a range of viruses
`[31,32]. AZTMPalkyl esters were also employed
`to improve membrane permeability [33,34].
`In
`vitro,
`the triesters showed a complete lack of
`
`5
`
`
`
`292
`
`JP. Krise, V.J. Stella | Advanced Drug Delivery Reviews 19 (1996) 287-310
`
`inhibition of HIV, which was attributed to the
`high stability of the simple alkyl esters with little
`or no conversion to the active 5’-phosphate.
`
`NH2
`
`¢ “
`0.
`R,0 P
`N 2
`QO
`;0—
`ud A ANS
`9-[2-(Phosphonomethoxyethoxy]adenine (R), Ry =H)
`
`Fig. 4. Structure of 9-[2-(phosphonomethoxy )ethoxy]adenine
`(R1, R2=H). Refer to Table 1 for representations of R1 and
`R2 [35].
`
`Serafinowska and co-workers [35] have made a
`series of dialkyl (among others) prodrugs of 9-[2-
`(phosphonomethoxy)ethoxyJadenine
`(Fig.
`4,
`Table 1)
`in an effort
`to improve the poor
`bioavailability, 7% in rats [36], <1% in monkeys
`[37].
`After oral administration to mice, the inves-
`tigators monitored blood concentrations of the
`diester, monoester, and free acid (1-5, Table 1).
`In all cases,
`the diester was well absorbed;
`however, subsequent conversion to the monoes-
`ter and free acid was more dependent upon the
`nature of the alkyl ester. Short chain diesters,
`being chemically (and probably enzymatically)
`
`Table 1
`Concentration of 9-[2-(phosphonomethoxy)ethoxy]adenine 1 (see Fig. 4) and its mono- and dialkyl esters in the blood following
`oral administration of esters to mice (Reprinted with permission from [35] American Chemical Society.)
`Number
`Rl
`R2
`Total AUC 15-180 min (4M)
`
`1
`Monoester
`Diester
`Bioavailability
`
`of 1(%S
`
`2
`-
`-
`-
`H
`H
`1
`0
`39
`9
`0
`Me
`Me
`2
`8
`9
`35
`4
`Et
`Et
`3
`10
`10
`29
`5
`i-Pr
`i-Pr
`4
`0
`2
`69
`0
`n-Bu
`n-Bu
`5
`30
`0
`0
`15
`Me,CCO,CH,
`Me,CCO,CH,
`6
`0
`0
`9
`0
`Me
`Me,CCO,CH,
`7
`0
`0
`40
`0
`Et
`Me,CCO,CH,
`8
`0
`0
`8
`0
`i-Pr
`Me,CCO,CH,
`9
`0
`0
`11
`0
`n-Bu
`Me,CCO,CH,
`10
`0
`0
`0
`0
`Me,C(CH,CI)CO,CH,
`Me,C(CH,CI)CO,CH,
`11
`24
`0
`0
`12
`Me,CHCO,CH(Me)
`Me,CHCO,CH(Me)
`12
`74
`0
`0
`37
`Me,CCO,CH(Me)
`Me,CCO,CH(Me)
`13
`4
`0
`18
`2
`C,H,CH,O
`C,H;CH,O
`14
`2
`9
`3
`I
`4-BrC,H,CH,O
`4-BrC,H,CH,O
`15
`8
`31
`12
`4
`4-CIC,H,CH,O
`4-CIC,H,CH,O
`16
`1
`0
`0
`0.5
` 4-Me,CHCO,C,H,CH,O
`4-Me,CHCO,C,H,CH,O
`17
`8
`0
`0
`4
`4-AcOC,H,CH,
`4-AcOC,H,CH,
`18
`8
`0
`0
`4
`Br(CH,),O0
`Br(CH,),0
`19
`6
`15
`13
`3
`Cl,CHCH,O
`CI,CHCH,O
`20
`0
`13
`22
`0
`EtO(CH,),O
`EtO(CH,),0
`21
`26
`0
`|
`13
`C,H,O
`C,H,O
`22
`50
`0
`0
`25
`C,H,O
`C,H,O
`23
`6
`0
`0
`3
`4-BrC,H,O
`4-BrC,H,O
`24
`22
`0
`0
`Il
`2-MeC,H,O
`2-MeC,H,O
`25
`10
`0
`16
`5
`2-AcOC,H,O
`2-AcOC,H,O
`26
`8
`0
`5
`4
`4-AcOC,H,O
`4-AcOC,H,O
`27
`0
`0
`1
`0
`4-AcOCH,C,H,O
`4-AcOCH,C,H,O
`28
`i0
`0
`14
`5
`4-MeOC,H,O
`4-MeOC,H,O
`29
`
`30 0 4-Me,CCO,C,H,O 4-Me,CCO,C,H,O 0 0 0
`
`
`
`
`
`“The bioavailability of 1 after oral administration of prodrugs was calculated from the equation: % bioavailability = (AUC1)/(iv
`AUC1) x 100, where iv AUC 1=50 uM h.
`
`6
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`
`293
`
`stable, were predominantly detected unchanged
`in the serum after oral administration, As the
`alkyl diester size increased the prodrugs tended
`to break down moreefficiently to the monoester;
`however, the monoester tended to build up in
`the blood and failed to be converted to the
`parent phosphonate. Starrett et al.
`[38] also
`explored the effectiveness of dialkyl prodrugs to
`improve
`the
`bioavailability
`of
`9-[2-(phos-
`phonomethoxy)ethyljadenine (PMEA) and ob-
`tained similar results.
`Investigators also described the utility of the
`aryl functionality in prodrug design, both with
`unsubstituted and substituted aryl phosphate
`esters. When these were designed to increase
`cellular permeability, the ability of the prodrug
`to deliver
`the phosphate-containing drug in-
`tracellularly was minimal [39-43] probably due
`to removal of one or both of the esters prior to
`reaching the target cell. However, when the aryl
`functionality was used to increase GI tract per-
`meability several authors reported dramatic in-
`creases in bioavailability [35,38,44,45] (also refer
`to section 4.3.2 of this review). Table 1 contains
`oral bioavailability data for a number of aryl
`prodrugs of 9-[2-(phosphonomethoxy)ethoxy]-
`adenine (22-30). The most promising aryl pro-
`drug was the phenyl prodrug 23 (23 is a hydro-
`chloride salt of 22) which gave 50% oral bio-
`availability of the parent phosphonate.
`In an effort
`to produce more chemically/
`metabolically labile prodrugs, Mitchell et al. [46]
`synthesized the dibenzyl ester of methoxycar-
`bonylphosphonate. The hydrolysis
`in pH 7.4
`buffers (36.4°C) was rapid and resulted in a
`complex product profile. The major complication
`with this potential prodrug was that P-C cleavage
`occurred, yielding the dibenzyl phosphite. In an
`attempt to shift the hydrolysis more toward P-O,
`or O-benzyl group bond cleavage possibly yield-
`ing parent monophosphate-containing drug after
`hydrolysis,
`para-substituents
`on
`the
`benzyl
`groups were employed [47]. As expected, para-
`electron withdrawing substituents yielded esters
`that hydrolyzed to the monobenzyl ester but
`little or no parent drug was formed. Only hydro-
`lytic data was provided with these studies. A
`potential draw back in the clinical usefulness of
`benzyl esters with para electron donating sub-
`
`stituents is the potential formation of quinone
`methide intermediates which are known to be
`toxic.
`By what mechanism do these alkyl esters of
`monophosphates exert their potential biological
`effect? The prodrugs could either act as depot
`for the nucleoside in which the P-O-nucleoside
`link would be broken to provide the nucleoside
`(pathway 1, Scheme 3), or act as an intracellular
`source of
`the nucleoside monophosphate by
`cleavage of
`the P-O-alkyl bond (pathway 2,
`Scheme3).
`The first possibility (pathway 1), although
`potentially useful [48], would fail to overcome
`the resistance seen with manyof these drugsie.,
`it still delivers the nucleoside rather than its
`monophosphate.
`[31]
`To assess this issue, McGuigan et al.
`prepared compounds analogous to conventional
`dialkyl phosphate prodrugs through the use of
`methylene bridges from the phosphorus to the
`alkyl chain (phosphinate linkage). The phosphi-
`nate (P-C)
`linkage is well known to be bio-
`logically stable [49]. The activity of di-n-butyl
`phosphinate ((C,H,),PO-nucleoside) and di-n-
`propyl
`phosphate
`((C,H,O),PO-nucleoside)
`were compared (assumes similar Van der Waals
`radii for methyl and oxygen). Similar biological
`activities of di-n-butyl phosphinate and di-n-pro-
`pyl phosphate could be expected if the mecha-
`nism of action was that both acted as depot
`prodrugs of the parent nucleoside. However, the
`phosphate was nearly twice as effective as the
`phosphinate. The fact that the phosphinate dis-
`played activity (albeit
`low) provides some evi-
`dence that
`the prodrugs may act,
`in part, as
`depots for the nucleoside. On the other hand,
`since the phosphates were consistently more
`active than their phosphinate counterparts, it was
`possible to infer that an increase in the phos-
`phate-to-alkyl link lability would produce more
`efficient prodrugs.
`In an effort
`to increase the lability of the
`phosphate-promoiety bond (pathway 2, Scheme
`3), several halo alkyl prodrug esters have been
`made and evaluated [50,51]. The halo substitu-
`tion was thought
`to favor
`the formation of
`nucleoside monophosphate andincrease biologi-
`cal activity. Bis(2,2,2-trihaloethyl) phosphate de-
`
`7
`
`
`
`294
`
`JP. Krise, V.J. Stella | Advanced Drug Delivery Reviews 19 (1996) 287-310
`
`+
`
`RO-P—OH
`
` I
`
`+
`
`(ROH)
`
`Scheme3. Illustration of the possible decomposition pathways for a generic nucleotide prodrug. Pathway | represents cleavage of
`the P-O-nucleoside link and results in nucleoside release. Pathway 2 represents cleavage of the P-O-alkyl linkage and results in
`accumulation of nucleotide inside the cell.
`
`2’,3'-dideoxycytidine
`of AZT and
`rivatives
`(Fig. 5) have been synthesized and
`(ddCD)
`evaluated [52].
`The in vitro anti-HIV activities of these com-
`pounds were evaluated and it was found that
`none of the prodrugs were as active as the parent
`nucleosides. The trichloroethyl moiety was found
`to be more efficacious than the trifluoroethyl
`moiety for ddCDMP; however, the two halogen
`prodrugs were
`nearly equally effective
`as
`AZTMP. Whenthis promoeity was subsequently
`applied to ara-AMP and ara-CMP[53], in vitro
`testing showedthe trichloro-containing prodrugs
`were consistently more active than the trifluoro-
`
`containing prodrugs. For the ara-CMP prodrug,
`the activity was found to be greater than that of
`the parent nucleoside. These observations were
`not consistent with the expected lability of the
`prodrugesters, as one would expectthe trifluoro
`analogs to be the most labile. The observed trend
`in activity, however, was found to correlate with
`lipophilicity as exemplified by the larger octanol/
`water partition coefficient for the trichloro-con-
`taining prodrug. Subsequently, experiments were
`conducted to discover the potential utility of
`other related halo-alkyl prodrugs [54]. Mono-,
`di-, and trichloro substituted alkyl prodrugs of
`AZTMP (Fig. 5) were synthesized and com-
`
`NH»
`
`n= )
`0 pe
`
`N
`
`i
`RO-P—O
`OR
`
`0,
`
`H
`
`Ng
`
`Me
`
`R=F,CCH,
`R=Cl,CCH,
`
`R = Ci,CHCH,
`
`R = CICH,CH;
`
`NH
`
`n>
`
`2 ASN
`
`RO-P—O
`oR
`
`©.
`
`R = Cl,CCH)
`
`R= F,CCH,
`
`AZTMP (R = H)
`
`ddCDMP (R = H)
`
`Fig. 5. Structures of AZT monophosphate and ddCD monophosphate along with their corresponding haloalkyl prodrugs [52,54].
`
`8
`
`
`
`J.P. Krise, V.J. Stella | Advanced Drug Delivery Reviews 19 (1996) 287-310
`
`295
`
`pared. The expected lability of these prodrugs
`would be in the order
`trichloro > dichloro >
`monochloro. The observed activity, however, was
`in
`the
`order
`of
` trichloro>monochloro >
`dichloro. When the haloalkyl prodrugs were used
`to increase bioavailability, only marginal
`in-
`creases were observed (see Table 1) [35,38].
`The results on alkyl, haloalkyl, benzyl and aryl
`prodrugs tend to imply that ability of
`these
`prodrugs to biorevert from the diester to mono--
`ester prodrug to the parent phosphate or phos-
`phonate does not solely rely on their chemical
`lability. For example, prodrugs with good leaving
`groups were not always the most efficient pro-
`drugs. What mechanism(s) are involved in the in
`vivo reversion of these prodrugs? The conversion
`of the diester prodrug to the monoester prodrug
`is not well understood and the in vivo rate of
`conversion of the diester prodrug to the monoes-
`ter prodrugis too rapid to be explained solely by
`chemical hydrolysis. On the other hand, no one,
`to our knowledge,has identified a true phosphot-
`niesterase in the mammalian system. One could
`speculate either non-specific enzymes or micro
`environmental pH changes are responsible for
`the conversion. Conversion of the monoester
`
`prodrug to the parent phosphate/phosphonate
`drug can be explained by the presence of phos-
`phodiesterases (and phosphonodiesterases) [55-
`57].
`In addition, phosphonate monoesters and
`phosphate diesters with systematically varied
`Jeaving groups were tested as substrates for 5’-
`nucleotide phosphodiesterase [58]. Although ali-
`phatic and benzyl esters were poor substrates,
`the aryl ester derivatives were shown to be good
`substrates for their diesterases. It was suggested
`that the geometry of the aryl group may be more
`important than the inductive nature of the aro-
`matic group.
`
`4.1.2. Acyloxyalkyl esters
`One approach to overcome the hydrolytic
`resistance observed with many of the prodrugs
`previously reviewed would be to utilize an en-
`zymatically labile promoiety; acyloxymethyl pro-
`drugs could serve as neutral lipophilic prodrugs.
`In theory, these prodrugs could transverse cell
`membranes by passive diffusion and revert, in-
`tracellularly,
`to the parent
`ionic phosphate or
`phosphonate after cleavage of the acyl group by
`esterases and rapid elimination of formaldehyde
`spacer group (Scheme 4).
`
`OH
`
`- CH,O
`Fast
`
`os
`NN7
`0
`KN
`
`HO-P—O
`
`0.
`
`Need
`
`oe
`NN2
`A 0
`NN
`
`—_——_—_-—_—_
`
`R—C—0
`
`O-P—0
`
`0.
`
`Neel
`
`Scheme 4. General mechanism for the bioreversion of acyloxymethyl phosphate prodrugs. The acyl group is cleaved through
`esterase activity to yield the hydroxymethyl analog. This analog quickly decomposes to formaldehyde and the monoester prodrug.
`The second group is subsequently cleaved by the same mechanism but possibly a different enzyme.
`
`9
`
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`
`296
`
`JP. Krise, V.J. Stella | Advanced Drug Delivery Reviews 19 (1996) 287-310
`
`Investigators have utilized this strategy and
`In addition to increasing antiviral activity,
`evaluated the acyloxymethyl ester prodrugs for a
`investigators have also evaluated acyloxyalkyl
`variety of antiviral agents [59-61,139] and cAMP
`
`ester prodrugs of PMEA and_9-[2-(phos-
`[60]. Starrett et al.
`[62] have synthesized the
`phonomethoxy ethoxy] adenine in order to in-
`bis(pivaloyloxymethyl) prodrug of the antiviral
`crease their poor oral bioavailabilities [35,38].
`agent PMEAtoincrease antiviral activity. Star-
`Table 1, compounds 6-13, showed dramatically
`rett’s studies showed the prodrug to have sub-
`increased oral bioavailability for some acyloxy
`stantially increased anti-herpes
`simplex virus
`alkyl prodrugs. The bis(pivaloyloxymethyl) pro-
`activity in vitro compared with PMEA.Similarly,
`drug (6) had reached an oral bioavailability of
`Sastry et
`al.
`[63] have produced the bis-
`30%; however, methyl substitution at the alpha
`(pivaloyloxymethyl) prodrug of 2’,3'-dideoxy-
`carbon of6, led to an oral bioavailability of 74%
`for 13. Shaw etal. [66] tested alkyl, aryl, alkox-
`uridine 5’-monophosphate (ddUMP). Addition-
`yalkyl, and alkyl amino acid phosphoramidate
`ally, metabolism studies in two human T cell
`esters. Based upon an in vitro stability and
`lines lead to the formation of 5’-mono-, di-, and
`triphosphates of ddU after exposure to_ bis-
`transport
`screen,
`the
` bis(pivaloyloxymethyl)
`PMEAwasselected as a potential oral prodrug
`(pivaloyloxymethyl) ddUMP.
`In contrast,
`these
`for further in vivo animal studies. The bioavail-
`phosphorylated metabolites were not observed in
`cells treated with ddU or ddUMP alone, sug-
`ability of bis(pivaloyloxymethyl)PMEA utilizing
`gesting that this prodrug was able to intracellu-
`three different
`formulations was subsequently
`larly release ddUMP. Freed et al.
`[64] utilized
`evaluated in monkeys [67]. The three formula-
`tions (hydroxypropyl-B-cyclodextrin, PEG, and
`this same prodrug strategy for 5-fluorodeoxy-
`uridine monophosphate (SdUMP) and obtained
`aqueous suspension) were used to explore any
`dissolution limitations with the poorly water
`results consistent with intracellular delivery of
`soluble prodrug. The prodrug was shown to
`the monophosphate. Bis(pivaloyloxymethyl) pro-
`deliver an acceptable amount (~ 24%bioavail-
`drugs of 9-(2-phosphonylmethoxyethyl)adenine
`(PMEA),
`—_9-(2-phosphonylmethoxypropyl)ad-
`ability on average) of PMEA bythe oral route
`enine (PMPA), and 9-(2-phosphonylmethoxy-
`using any formulation. The choice of formula-
`propyl)diaminopurine
`(PMPDAP) were
`also
`tion,
`therefore, was not
`limited by the low
`aqueous solubility of the prodrug, although the
`synthesized and compared with the unmodified
`analogs. The bis(pivaloyloxymethyl) derivatives
`bioavailability may have been limited by the
`hydrolytic instability of the prodrug in the GI
`were biologically more active and showed en-
`tract.
`hanced antiviral activities in vitro [65], most
`