Phosphoryl Prodrugs: Characteristics to Improve Drug Development

Abstract

Phosphoryl prodrugs are key compounds in drug development. Biologically active phosphoryl compounds often have negative charges on the phosphoryl group, and as a result, frequently have poor pharmacokinetic (PK) profiles. The use of lipophilic moieties bonded to the phosphorus (or attached oxygen atoms) masks the negative charge of the phosphoryl group, cleavage releasing the active molecule. The use of prodrugs to improve the PK of active parent molecules is an essential step in drug development. This review highlights promising trends in terminal elimination half-life, C_max, clearance, oral bioavailability, and cLogP in phosphoryl prodrugs. We focus on specific prodrug families: esters, amidates, and ProTides. We conclude that moderating lipophilicity is a key part of prodrug success. This type of evaluation is important for drug development, regardless of clinical application. It is our hope that this analysis, and future ones like it, will play a significant role in prodrug evolution.

Introduction

Phosphoryl moieties are commonly found in metabolic pathways ranging from isoprenoids to nucleotides.[1] Often, the active molecule will bear one or more negative charge at biological pH, and, as a result, this negative charge can inhibit uptake into cells and tissues, resulting in a poor PK profile, even if the molecule is shown to be impressively potent against the target[1, 2]. The use of prodrugs, a strategy to side-step use of the active/parent compounds, was first popularized by Adrien Albert in 1958[3]. Prodrugs are defined as inactive molecules that, when subjected to metabolism, are converted into the active molecule.[3] The goal of phosphoryl prodrugs is to mask the negative charge found on the active compound by increasing the lipophilicity of the molecule—ultimately improving the PK profile. Evaluation of the PK profile is typified by following parameters such as oral bioavailability, terminal elimination half-life, plasma C_max, plasma clearance, and cLogP[4].

This review strives to compile and highlight PK data of a variety of phosphoryl prodrugs, illuminating the impact of the prodrug moiety on PK. Interestingly, but not surprising, these prodrugs have found a variety of uses, including the treatment of hepatitis B, diabetes mellitus, and several types of cancers. Figure 1 shows the general structures of the prodrugs discussed. The families of interest are the esters, amidates, and ProTides. Wherever possible, we discuss human PK data. If not available, we rely on primate or other higher order mammalian data for comparisons. With a history in phosphoryl—specifically phosphonate—chemistry([5–9]). We hope this review will serve as a guide to those interested in phosphoryl prodrugs, as well as those, like us, who are in pursuit of compounds with improved PK characteristics. Thus, this endeavor aims to aid in synthetic decision making. All cLogP values were calculated using the Consensus cLogP from SwissADME[10].

Figure 1.

Structures of the phosphonate prodrugs discussed: esters, amidates, and ProTides.

Esters

The ester family of prodrugs is comprised of molecules where the prodrug has phosphor(mono)ester or phosphodiester bonds connecting the pro-moiety to the rest of the molecule (Figure 1). In this section, we chose to examine PK data for Adefovir, Tenofovir, and Cidofovir (Table 1). These parent compounds have extensive PK data available in the literature, and researchers have taken great interest in improving the PK profile through prodrugs. The use of POM (pivaloyloxymethyl) and POC (propargyloxycarbonyl) groups (Figure 2) are popular and simple lipophilic moieties that produce profound changes in the PK parameters of the drugs, as highlighted in this review. Cidofovir, however, has a monoester prodrug Brincidofovir. Brincidofovir is a mono-alkoxyalkyl phosphoester with equally promising improvements in the PK profile. The PK data compiled for each parent compound and prodrug of the mono- and diesters is summarized in Table 1.

Table 1.

Mono and Diesters

Compound	Structure	Half-lifea	Cmaxb	Clearancec	cLog Pd	% Fe
Adefovir		38±15 hr[11] (HepG2 cells)	0.28±0.33 L/kg[12] (Monkeys, PO)		−1.29	15.9±11.5%[13] (mice) 4.0%[12] (Monkeys)
Adefovir Dipivoxil		< 5 mins[14] (Human serum)	0.35±0.15 μg-eq/mL[15] (Monkeys, PO)		1.77	52.8±11.4%[13] (Mice) 22.2±15.6[15] (Monkeys)
Tenofovir (TFV)		6.64 hr[16] (Humans, IV) 7.31 hr [17] (Dogs, PO)	2.50 μg/mL [16] (Humans, IV) 1.29 μg/mL [17] (Dogs, PO)	217±55.8 mL/hr/kg [16] (Humans, IV) 0.28 6 0.05 liter/h/kg[17] (Dogs, PO)	−1.01	18.5±6.79 %[17] (Dogs)
Tenofovir Bis-POM		14.8±2.9[18] hr (dogs, PO) <10mins[19] (Dog plasma, PO)	2.7±0.6[18] μg/mL (Dogs, PO)		2.03	37.8±5.1[18] (Dogs)
Tenofovir Bis-POC (Disoproxil Fumarate)		12–13 hr[20] (Fasted Humans, PO)	111 μg/L[20] (Fasting Humans, PO)	247 mL/hr [20] (Fasted H uman, PO)	1.49	20%[21] (Mice) 31% [22] (Monkeys) 25% (TFV)[20] (Fasted humans)
Cidofovir (CDV)		63.0±11.0 hr[23] (Humans, PO)	31.1±7.00 ng/mL[23] (Humans, PO)	148±20 mL/hr/kg[24] (Humans, PO)	−2.01	2.2%[25] (Rats) <5.3±2.9%[24] (HIV patients)
Brincidofovir (BCV)		24.0±0.705 hr[23] (Humans, PO)	350±119 ng/mL[23] (Humans, PO)		3.94	88%[26] (Mice)

^a)Plasma half-life: time required for the concentration of the drug in the plasma to be reduced to 50%,

^b)C_max: maximum concentration of the drug in plasma during a dosing interval,

^c)clearance: renal clearance rate of drug,

^d)cLogP: calculated partition coefficient,

^e)systemic bioavailability in % after oral administration. [27]

Figure 2.

Molecular structure of Bis-POC and Bis-POM groups

Adefovir

Developed by Gilead Sciences, Adefovir is a reverse transcriptase inhibitor used to treat chronic hepatitis B. The active metabolite, Adefovir diphosphate, is formed after the parent compound Adefovir is phosphorylated twice [28]. The structure of Adefovir is an adenine analog with an ethyloxymethyl linker to the phosphoryl moiety. The PK profile of Adefovir is unfavorable. Despite a 38 hour half-life[11], the C_max and oral bioavailability are only 0.28 L/kg and 4.0%, respectively, in monkeys[12]. Adefovir has a very low cLogP of −1.29.

The parent compound Adefovir was transformed to the bis-POM analog, Adefovir Dipivoxil[29]. The structure of Adefovir Dipivoxil is the identical to adefovir, except that the diacid has been esterified to create two pivaloyloxymethyl (POM) esters. This prodrug displays significant changes in the PK profile. Adefovir Dipivoxil has a half-life in human serum of less than 5 minutes[14], but a C_max and oral bioavailability improvement to 0.35 μg-eq/mL and 22.2%, respectively, in monkeys[15]. The improvements in C_max and oral bioavailability are correlated with a significant increase in cLogP from −1.29 to 1.77. The increase in lipophilicity likely contributes to the marked increase in these two PK parameters. Adefovir Dipivoxyl is quickly cleaved on first-pass metabolism in the intestines and the liver to form Adefovir. Any intact Adefovir Dipivoxyl that reaches cells is readily absorbed, rapidly cleaved, and ultimately phosphorylated twice to produce the active metabolite[15]. Optimization of Adefovir Dipivoxyl would allow for an increased plasma half-life and C_max, which would ultimately allow for increased absorption and conversion to the active metabolite by cells. Despite Adefovir Dipivoxyl having extraordinarily lipophilic POM groups to promote cellular penetration, one drawback to the use of POM groups is how readily they are cleaved by esterases during metabolism[30].

Tenofovir

The reverse transcriptase inhibitor Tenofovir is used to treat chronic hepatitis B as well as treat and prevent AIDS/HIV[31]. Tenofovir is a nucleoside analog of adenine with an aliphatic hydrocarbon chain linking the nucleoside to the diacid phosphoryl group. The parent compound Tenofovir was found by Deeks et al. to have a half-life of 6.64±2.1 hours, a C_max of 2.50 μg/mL, and a clearance of 217 mL/hr/kg in humans[16]. It was found to have an oral bioavailability of 17.1±1.88 % in dogs[17]. The cLogP of Tenofovir is −1.01.

The first Tenofovir prodrug we consider is Tenofovir bis-POM, or Tenofovir Dipivoxil. The modification to the structure of the parent drug to reach this prodrug was the esterification of the diacid to create a pivaloyloxymethyl ester. The bis-POM prodrug had a great increase in lipophilicity with a cLogP of 2.03 compared to the parent compound’s value of −1.01. In dogs, Tenofovir Dipivoxil had a half-life of 14.8 hours, resulted in a C_max of Tenofovir of 2.7 μg/mL, and an oral bioavailability of Tenofovir of 37.8%[18]. Despite esterases being found throughout the blood and tissues of dogs, the highest esterase activity was found to be in the liver. This indicates that the liver may be the primary location of prodrug cleavage[18]. As a result, a drug with a longer half-life and greater C_max value would be greatly beneficial to prevent all of the prodrug being eliminated in the liver before being distributed throughout the body, as the prodrug is more likely to penetrate cells. Intracellular enzymatic cleavage to the diacid followed by two phosphorylation events would result in the active metabolite formation[30].

A second Tenofovir prodrug is Tenofovir bis-POC, or Tenofovir Disoproxil Fumarate (TDF). The structure of TDF is the same as tenofovir, but instead of the diacid, the molecule is esterified to give the propargyloxycarbonyl (POC) diester. Dosing with TDF resulted in a 12–13-hour half-life of the parent compound Tenofovir. The C_max was found to be 111 μg/mL of Tenofovir after dosing with TDF in humans, and Tenofovir clearance was found to be 247 mL/hr after a single dose of TDF. Finally, in a fasted state, oral bioavailability was determined to be 25%[20]. Similar to Tenofovir Dipivoxil, TDF had a great increase in lipophilicity with a cLogP of 1.49. Preliminary in vitro studies found that TDF was metabolized first by non-specific carboxylesterase enzymes found in the blood, organs, and tissues with the monoester as the major circulating metabolite[20]. A long half-life and high C_max are found in human serum, which are essential in increasing the effectiveness of the active metabolite. Ultimately, any circulating TDF, monoester, or TFV would enter the cells, and the esters, if present, would be fully cleaved. The TFV is then phosphorylated twice to produce the active metabolite. Not only was TDF found to have a favorable half-life and C_max in the serum, but was also found to have a long intracellular half-life[32]. A long intracellular half-life is favorable as HIV inhibition will remain high long after drug administration is discontinued.

Cidofovir

Cidofovir (CDV) is a broad-spectrum antiviral agent used against herpesviruses, papillomaviruses, and poxviruses[33]. The structure of cidofovir is that of a cytosine with an aliphatic linker with a hydroxymethyl group on the second carbon of the linker. The cytosine is linked to the phosphonic acid moiety. In humans, Cidofovir was found to have a half-life of 63 hours, a C_max of 31.1 ng/mL, a clearance of 148 mL/hr/kg, an oral bioavailability of <5%, and a very low cLogP of −2.01[23, 34]. The prodrug of interest for Cidofovir is Brincidofovir. The modification to the structure is addition of a lipid alkoxyalkyl ester. This prodrug was found to have a half-life 24 hours and a C_max of 150 μM, as well as an oral bioavailability of 88% and a cLogP of 3.94[23, 26]. This impressive change in bioavailability is even more impressive due to the fact that Brincidofovir is a monoester, compared with the diester prodrugs above. BVC is taken into cells using endogenous lipid uptake pathways where BVC resembles the naturally occurring lipid lysophosphatidylcholine. Once inside the cell, the lipid moiety can be cleaved by esterases to release CDV which is phosphorylated twice to produce the active metabolite. A high serum half-life and C_max is advantageous with BCV as cleavage of BCV in the serum will result in the potentially nephrotoxic CDV [35]. Therefore, BCV is able to accumulate in high concentrations, remaining in the body long enough to be transported into the cells where cleavage and phosphorylation can occur.

Amidates

Another option for masking the negative charges of the phosphoryl group is to use phosphoramide bonds, usually where the phosphorus atom is bonded to an amino acid (Table 2). Bisamidates have not been as popular as esters (or ProTides, discussed below) as a prodrug strategy. However, it is a powerful technique when applied to particular molecules and has been shown to have lower toxicity and higher plasma stability while still masking the charged phosphonate, similar to esters or ProTides[36].

Table 2.

Bisamidates

Compound	Structure	Half-life	Cmax	Clearance	cLog P	% F
Adefovir		1.6±0.5 hr[37] (Humans, IV)	10.6±2.27 mg/L[37] (Humans, IV)	220±52 mL/hr/kg[37] (Humans, IV)	−1.29	<12%[37] (Humans)
GS-9219		<30 min[38] (Dogs, IV) 0.33±0.20 hr[39] (Dogs, IV)	4280±1550 nmol/L[39] (Dogs, IV)	2.25±0.23 L/hr/kg[38] (Dogs, IV)	1.04
MB05032					1.19	2%[40] (Rats)
CS-917		>7 days [41] pH 3.0–7.4 (t90)			2.72	22%[40] (Rats)

Adefovir

As previously mentioned, Adefovir is a reverse transcriptase inhibitor used to treat chronic hepatitis B. Developed by Gilead Sciences, the adenine analog has a terminal half-life of 1.6±0.5 hours, a Cmax of 10.6±2.27 mg/L, and a clearance of 220±52 mL/hr/kg in humans. Adefovir has a low cLog P value of −1.29 which is accompanied by a low oral bioavailability of <12% in humans[37]. A bisamidate prodrug of an Adefovir analog is GS-9219. Also developed by Gilead, this prodrug is Adefovir with an N⁶-cyclopropyl group and a phosphoryl group added to the primary alcohol. The phosphoryl group has two amide bonds, each bonded to an ethyl ester of (L)-alanine. It was found that in dogs, GS-9219 had a half-life of about 30 minutes, a C_max of 4280±1550 nmol/L, and a clearance of 2.25±0.23 L/hr/kg. The cLog P is 1.04. With such a great increase in lipophilicity, we would expect to also see an overall trend of increasing oral bioavailability. The clearance of GS-9219 was relatively high, however. For any GS-9219 that did penetrate the cells, the molecule was quickly hydrolyzed to cPrPMEDAP and then deaminated to PMEG. Phosphorylation of PMEG twice results in the active metabolite PMEGpp[38]. The high clearance and short half-life of GS-9219 are concerning. Optimization of GS-9219 would seek to maintain lipophilicity and C_max, but increase the half-life and decrease the clearance rate so that GS-9219 has more time to penetrate cells.

MB05032

MB05032 and its prodrug CS-917 are used to inhibit fructose 1,6-bisphosphatase, an enzyme that is crucial for gluconeogenesis. Therefore, inhibition of fructose 1,6-bisphosphatase is an attractive enzymatic target for small molecule treatment of type 2 diabetes mellitus[41]. The structure of MB05032 is comprised of a thiazole ring with 2-amino and 5-methylpropyl groups branching and connected to the phosphonic acid through a furanyl ring. MB05032 has extremely low oral bioavailability in rats of only 2% when dosed at 30 mg/kg, and the polar parent molecule has a cLogP value of 1.19. CS-917 is a bisamidate of MB05032 bearing two phosphoramidate substituents: ethyl esters of L-alanine. Prodrug CS-917 displays an 11-fold increase in bioavailability, up to 22%. Such a large jump in bioavailability is expected as the cLogP increased by roughly 1.5 log units, to 2.72. In addition, CS-917 was found to be extremely stable at pH2.5–7 at 37°C with a t₉₀ of greater than 7 days. Erion et al. also found that the ethyl esters of L-alanine are quickly cleaved from the phosphoryl group in liver s9 fractions of rats and humans, but did not report quantitative values for this experiment[40, 41]. Although half-life, C_max, and clearance data on CS-917 and its parent MB05032 are limited, the increase in lipophilicity of CS-917 allows for greater penetration into cells as shown by the increased bioavailability. Once inside the cell, the prodrug moieties can be cleaved to produce the active metabolite MB05032 to inhibit fructose 1,6-bisphosphatase[41].

ProTides

ProTides were first invented and launched to stardom by Christopher McGuigan from Cardiff University in Wales[42]. The name comes from the structure—a prodrug form of a nucleotide. ProTide prodrugs consist of a mixed ester and phosphoryl amidate typically using an aryloxy ester and an esterified amino acid[42]. ProTides were found to be an extremely effective prodrug strategy and have resulted in several refined ProTides becoming FDA approved, some of which will be explored in this review[43, 44]. Sofosbuvir and Tenofovir Alafenamide are two FDA approved ProTides. These molecules were chosen to discuss because of their prevalence in medicine, and because of the amount of PK data available. More recently, research groups are seeking to make ProTide versions of active drugs in order to improve the PK profile. The data is summarized in Table 3.

Table 3.

ProTides

Compound	Structure	Half-life	Cmax	Clearance	cLog P	% F
Tenofovir (TFV)		6.64 hr[16] (Humans, IV) 7.31hr[17] (Dogs, PO)	2.50 μg/mL [16] (Humans, IV) 1.29 μg/mL[17] (Dogs, PO)	217±55.8 mL/hr/kg [16] (Humans, IV) 0.28 6 0.05 liter/h/kg[17] (Dogs, PO)	−1.01	18.5±6.79 %[17] (Dogs)
Tenofovir Alafenamide (TAF)		35.95 hr[45] (Humans, as TVF, PO)	5.74±0.62 μM[46] (Dogs, PO)		1.79	17% of TVF and >70% of TAF[47] (Dogs)
PSI-6130		5.64±1.13 hr[48] (Monkeys, PO)	9.64±1.13 μM[48] (Monkeys, PO)		−0.59	24.0%±14.3[48] (Monkey)
PSI 6206
Sofosbuvir (PSI7977)		0.4 hr[49] (Humans, PO)	622±56.1 ng/mL[49] (Humans, PO)		1.24	9.89 %[50] (Dogs)
Gemcitabine		32.72±2.28 min[51] (Mice) 13.4±4.0 min[52] (Humans, IV)	2.3 ng/mL [53] (Humans, IV) 124μM[54] (Humans, IV)	1.173±0.059 mL/min[51] (Mice, IV)	−0.64	10%[53] (Human)
Acelarin		139 min[55] (Human hepatocytes) 8.3 hr[54] (Humans, IV)	26,910 μM[54] (Humans, IV)		1.79

Tenofovir

As described above, Tenofovir is a nucleoside analog of adenine used to treat chronic hepatitis B and treat/prevent AIDS and HIV. Tenofovir is the parent compound of the Tenofovir ProTide: Tenofovir Alafenamide Fumarate (TAF). TAF has the same structure as tenofovir except that it has been prodrugged by the modification of the phosphonic acid to a phosphoramidate. The phosphoramidate consists of a phenoxy ester and an amide bond to L-alanine that has been esterified with isopropanol. TAF is used to treat chronic Hepatitis B and HIV by acting as a reverse transcriptase inhibitor[45, 56]. Cundy et al. found that, in dogs at 10 mg/kg, Tenofovir has a half-life of 7 hours in plasma, a C_max of 1 μg/mL, plasma clearance of 0.28 L/hr/kg, and oral bioavailability of 17%[17]. As mentioned previously, Tenofovir has a cLog P of −1.01. The cLog P for TAF jumps to 1.79. Such an increase in lipophilicity was accompanied by a plasma half-life of TAF of 0.30 hours, a C_max of 4.51 μM, and resulted in an oral bioavailability of greater than 70% for TAF[56]. TAF metabolism is unique. Once the lipophilic TAF has been absorbed by the intestines and eventually goes from the blood to cells, the prodrug is hydrolyzed by cathepsin A (CatA) or carboxylesterase 1 to produce TFV-Alanine. The molecule is then further hydrolyzed in lysosomes to produce TFV. TFV is finally phosphorylated twice to produce the active metabolite TFV-DP[57]. TAF has a long half-life and relatively high C_max which affords high oral bioavailability and time for TAF to penetrate cells and continue on to the active metabolite.

PSI-6130

The compound PSI-6130 is the parent compound of PSI-6206. PSI-6130 was found to be readily deaminated to PSI-6206. A prodrug of PSI-6206 is the clinically approved Sofosbuvir. Sofosbuvir, originally discovered by Pharmasett (then acquired by Gilead), is a ProTide used to treat chronic hepatitis C, working as a polymerase inhibitor[58]. PSI-6130, PSI-6202 and Sofosbuvir have fluoro and methyl groups on the 5’ carbon of the deoxyribose ring. However, PSI-6130 has a cytosine while PSI-6206 and Sofosbuvir are uridine analogs. The prodrug Sofosbuvir has a phosphoryl group attached to the hydroxyl of the 5’ carbon containing a phenyl ester as well as an amide of (L)-alanine isopropyl ester. PSI-6130 was dosed in monkeys at 33.3 mg/kg orally and found to have a half-life of 5 hours and a C_max of 9 μM with a cLog P of −0.59, and oral bioavailability of 24%[48]. On the other hand, Sofosbuvir was found by Wang et al. to have a half-life of 1.9 hours, and a C_max 0.058 μM in monkeys at 5 mg/kg PO[59]. These changes in half life and C_max are accompanied by a cLog P increase to 1.24. Once Sofosbuvir is taken into cells, it is first hydrolyzed by human carboxylesterase 1 or CatA to remove the phenoxy moiety of the prodrug. Then, histidine triad nucleotide-binding protein 1 (HINT1) cleaves the amide bond to release the diacid-Sofosbuvir metabolite. Finally, this diacid is phosphorylated twice to produce the active metabolite[60]. The unique metabolic pathway of Sofosbuvir is best supported with long plasma half-life and C_max to allow for as much time as possible for Sofosbuvir to penetrate cells and begin its breakdown.

Gemcitabine

Gemcitabine is an anti-tumor medication used for many different cancers including ovarian, pancreatic, and bladder cancer[61]. This parent compound is an analog of deoxycytidine with two fluorine atoms attached to the 2’-carbon of the deoxyribose ring. Gemcitabine is typically administered intravenously, and the half-life and C_max values shown in Table 3 are from intravenous administration. From a study in which Gemcitabine was administered orally, oral bioavailability was reported as 10%[53]. Gemcitabine is not particularly lipophilic with a cLog P of −0.64. It also has a short half-life of 13 minutes[52] and C_max of 64 μmol/L[54]. Comparatively, the prodrug Acelarin is much more lipophilic. This prodrug has the same structure as Gemcitabine except that it has a prodrugged phosphoramide group attached. This ProTide is comprised of a phenyl ester and an amide bond to (L)-alanine benzyl ester. The prodrug moiety is attached to the 5’-carbon of the deoxycytidine ring by a phosphorester bond. The prodrug has a cLog P of 1.79, a half-life of 8.3 hours and a C_max of 26,910 μM, which is a 217-fold increase in C_max from gemcitabine[54]. Acelarin is able to avoid the equilibrative nucleoside transporter 1 (hENT1), which is normally how gemcitabine enters the cell, because of its lipophilic nature. Once inside the cell, esterases remove the ester and amide linked moieties of the prodrug to release gemcitabine, which is ultimately phosphorylated twice to release the active metabolite[54]. Acelarin has a relatively long half-life and high C_max which is advantageous as the prodrug has ample time to enter cells and undergo its unique metabolism to produce the active metabolite.

Concluding Remarks

The compilation of molecules in this review are meant to highlight the PK parameters that are essential to an effective phosphoryl drug. Increasing the lipophilicity of the molecule, as reflected in cLogP, through the use of pro-moieties bound by phosphoester, phosphoramide, or a mixture of phosphoester/phosphoramide (aka ProTide) is key to the drug’s success. Using molecules with a variety of clinical applications has shown the power of phosphoryl prodrugs and cemented their use in drug development. Phosphoryl prodrug design can be continued through the use of this review as it tabulates, and therefore, emphasizes where and how increasing lipophilicity can directly relate to improvements in half-life, C_max, and oral bioavailability.