Discovery of Orally Available Prodrugs of the Glutamate

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Discovery of orally available prodrugs of the Glutamate Carboxypeptidase II (GCP II) inhibitor 2-phosphonomethyl pentanedioic acid (2-PMPA) Pavel Majer, Andrej Jan#arík, Marcela Kre#merová, Tomáš Tichý, Lukáš Tenora, Krystyna Wozniak, Ying Wu, Elie Pommier, Dana V. Ferraris, Rana Rais, and Barbara S. Slusher J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00062 • Publication Date (Web): 01 Mar 2016 Downloaded from http://pubs.acs.org on March 9, 2016

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Journal of Medicinal Chemistry

Discovery of orally available prodrugs of the Glutamate Carboxypeptidase II (GCPII) inhibitor 2-phosphonomethyl pentanedioic acid (2-PMPA) Pavel Majer,† Andrej Jančařík,† Marcela Krečmerová,† Tomáš Tichý,† Lukáš Tenora,† Krystyna Wozniak‡, Ying Wu,‡ Elie Pommier,‡ Dana Ferraris,§ Rana Rais,‡,¥* Barbara S. Slusher‡,¥* †

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

v.v.i., Prague, Czech Republic ‡

Johns Hopkins Drug Discovery, Johns Hopkins University, Baltimore, Maryland 21205 U.S.A.

¥

Johns Hopkins Department of Neurology, Johns Hopkins University, Baltimore, Maryland

21205, U.S.A. §

Department of Chemistry, McDaniel College, Westminster, Maryland, 21157, U.S.A.

KEYWORDS: Glutamate Carboxypeptidase, NAAG, Glutamate, mGlu3, NMDA, Prodrugs

ACS Paragon Plus Environment


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ABSTRACT 2-phoshonomethyl-pentanedioic acid (1, 2-PMPA) is a potent inhibitor of glutamate carboxypeptidase II which has demonstrated robust neuroprotective efficacy in many neurological disease models. However, 1 is highly polar containing a phosphonate and two carboxylates, severely limiting its oral bioavailability. We strategized to mask the polar groups via a prodrug approach, increasing the likelihood of passive oral absorption. Our initial strategy was to cover the phosphonate with hydrophobic moieties such as PivaloylOxyMethyl (POM) and isoPropylOxyCarbonyloxymethyl (POC) while keeping the α- and γ-carboxylates unsubstituted. This attempt was unsuccessful due to the chemical instability of the bis-POC/POM derivatives. Addition of α,γ-diesters and α-monoesters enhanced chemical stability and provided excellent oral exposure in mice, but these mixed esters were too stable in vivo, resulting in minimal release of 1. By introducing POC groups on both the phosphonate and a-carboxylate we synthesized trisPOC-2-PMPA (21b), which afforded excellent release of 1 following oral administration in both mice and dog.


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INTRODUCTION Glutamate

Carboxypeptidase

II

(GCPII,

EC

3.4.17.21),

also

known

as

N-

acetylaspartylglutamate (NAAG) peptidase and NAALADase, is a type I membrane zinc metallopeptidase that hydrolyzes synaptically released NAAG to produce glutamate in the nervous system.1  Excess extracellular glutamate is pathogenic and has been shown to play a crucial role in many neurological disorders. GCPII activity is enhanced following synaptic activation2,

3

and inhibition of GCPII under these conditions both enhances NAAG4,

5

and

reduces extracellular glutamate levels4 providing neuroprotection, and is therefore considered an attractive target for therapeutic intervention.6 Over the last 20 years several GCPII inhibitors with distinct chemical scaffolds have been synthesized and found to be efficacious in several neurological models wherein excess glutamatergic transmission is presumed pathogenic. These include animal models of neuropathic pain,7 peripheral neuropathy,8,

9

stroke,4 amyotrophic

lateral sclerosis,10 multiple sclerosis11, schizophrenia,12 epilepsy,13 traumatic brain injury

14, 15

,

addiction16, and cognition17. Potent GCPII inhibitors identified to date have required two primary functionalities – a glutarate or dicarboxylic acid moiety that binds the C-terminal glutamate recognition site of GCPII, and a zinc-binding group to engage one or both of the zinc atoms at the active site.18 Although inclusion of these functionalities has led to highly potent inhibitors, the compounds suffer from being exceedingly hydrophilic, resulting in low membrane permeability and low oral bioavailability. Figure 1 outlines examples of each class of GCPII inhibitors including phosphonates (e.g. 2-(PhosphonoMethyl)-Pentanedioic Acid, 2-PMPA, 1),19 thiols (e.g. 2-(3MercaptoPropyl)Pentane-dioic

Acid;

2-MPPA,

2)20


ureas

(e.g.

(N-[N-[(S)-1,3-


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Dicarboxypropyl]Carbamoyl]-Methyl-L-Cysteine; DCMC, 3)21, and hydroxamates (e.g. 2(hydroxycarbamoylmethyl)pentanedioic acid 4)22. Of these classes of GCPII inhibitors, only the thiol-based inhibitors have exhibited oral bioavailability; with compound 2 advancing into clinical studies.23 Although 2 was safe and well tolerated in two Phase 1 studies (up to 14 day dosing), subsequent immunological toxicities were observed in chronic GLP primate studies, halting its development. The primate toxicology seen with 2 was immune complex formation in the kidneys and was attributed to the thiol zinc-binding group, not its GCPII inhibitory activity. It is well documented that thiol-containing drugs have a propensity to illicit immune hypersensitivity reactions,24, 25 a fact that prohibited further development of the only bioavailable series of GCPII inhibitors. Thus, the next logical step towards an orally bioavailable GCPII inhibitor was to design a prodrug of a potent, efficacious compound from another class. Compound 1 has played a prominent role as one of the most potent (Ki = 0.3 nM) and selective prototype GCPII inhibitors for in vivo efficacy studies. In fact, 1 has shown neuroprotective and analgesic efficacy in over 20 preclinical models conducted by multiple independent laboratories.26 However, the compound is highly polar with two carboxylates and a phosphonate that are ionized at physiological pH, with a cLogP of -1.5, resulting in low intestinal permeability and low oral exposure. This fact necessitates administration at very high systemic intraperitoneal or intravenous doses or via direct brain injection to observe efficacy, severely limiting its potential use as a drug, especially for chronic diseases where the oral route is preferred. For all the reasons stated above, an orally bioavailable prodrug of compound 1 would pave the way for development of this promising GCPII inhibitor and others in its class. While developing a prodrug for a phosphonate moiety can be challenging, there are multiple examples of successful, FDA approved drugs that have used this strategy.27 While


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simple esterification is the most often used method to prodrug carboxylates, this method fails in the case of phosphonates, as the in vivo conversion of their simple alkyl esters is very slow in most tissues.27 Thus, a common and successful strategy for increasing the rate of hydrolysis of phosphonate prodrugs is to use a spacer group to connect the phosphonate with a lipophilic carboxylate ester. Carboxylesterase mediated hydrolysis of the carboxylate ester then triggers a cascade reaction providing an effective way to release the phosphonate.28 Two examples of such prodrug moieties are PivaloylOxyMethyl (POM) and isoPropylOxyCarbonyloxymethyl (POC) that have been utilized in FDA approved phosphonate-base drugs such as Adefovir dipivoxil, and Tenofovir disoproxyl. It is important to note that none of the clinically successful phosphonate prodrugs contain another acidic functionality within the molecule such as the two carboxylic acids in compound 1, making our prodrug strategy particularly challenging in this case. Herein we report the synthesis and biological evaluation of new prodrugs of compound 1. The lead prodrug, tris-POC-2-PMPA 21b, was determined to be chemically stable, yet exhibited excellent release of 1 in both mice and dog following oral administration. This represents the first report of an orally available prodrug of 1 that can potentially be employed in the clinic as a therapeutic for disorders requiring chronic dosing.

CHEMISTRY The bis-POC prodrug of compound 1 was synthesized as outlined in Scheme 1. The phosphonic acid 5a was acylated by chloromethyl isopropyl carbonate (POC-chloride) to afford compound 6 in 39% yield. The resulting fully protected compound 6 was then subjected to hydrogenation in THF to deprotect the benzyl esters yielding prodrug 7. Despite the clean deprotection of 6, compound 7 was found to be chemically unstable. Immediate and significant



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decomposition was observed and removal of the POC group was observed even in deuterated acetone solution (i.e. the NMR sample) within several hours. The synthesis of the bis-POM and bis-POC dialkyl esters 9-11 is outlined in Scheme 2. The dialkyl diethylphosphonates 8a (R1 = Me) and 8b (R1 = Et) were synthesized according to the literature. 1 Partial deprotection with trimethylsilyl bromide afforded the free the phosphonic acids 5b-c followed by acylation with POM-chloride or POC-chloride to afford prodrugs 9-11, respectively. The bis-POM prodrugs with the free γ–carboxylate were synthesized as outlined in Scheme 3. The orthogonally protected 2-methylenepentanedioates 12a-b were synthesized according to the literature29,30 These acrylates reacted with diethyl phosphonate to afford phosphonates 13a-b in excellent yield. After selectively removing the phosphonate ethyl esters, the POM and POC groups were introduced using the same method as described above to yield 14a-c. Removal of benzyl ester by catalytic hydrogenation afforded prodrugs 15a-c in good yields. The tris-POM and tris-POC prodrugs 21a and 21b were prepared according to Scheme 4. Meldrum’s acid derivative 1619 was converted to 5-benzyl 1-tert-butyl 2-methylenepentanedioate 17. Phosphonation, followed by deprotection of the t-butyl ester resulted in the carboxylate 18. Acylation by either POM-chloride or POC-chloride afforded compounds 19a and 19b in 70% and 72% yield respectively.

Removal of phosphonate esters rendered phosphonic acids

intermediates that were subsequently acylated with POM-chloride and POC-chloride to afford 20a-b. Final deprotection by catalytic hydrogenation afforded prodrugs 21a and 21b in excellent yield.


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RESULTS AND DISCUSSION Prodrug strategy is often employed to enhance oral availability and tissue penetration of poorly permeable molecules. Indeed, prodrug strategies are common in drug development as 5– 7% of the approved worldwide drugs are prodrugs31. Conceptually, the less polar prodrugs will have enhanced intestinal permeability and once in plasma, will be cleaved to release the active drug via plasma or liver enzymes. Our initial prodrug strategy involved masking the phosphonate of compound 1 with two POC groups.

We rationalized that derivatization of the two

carboxylates may not be necessary since thiol-based GCPII inhibitor 2 (Figure 1) with two free carboxylates is orally bioavailable in several mammalian species including humans.7 Unfortunately, the phosphonate masked with two POC groups (7, Scheme 1) was chemically unstable as immediate decomposition occurred upon deprotection of the benzyl esters of compound 6. While this result was unexpected, POC groups are known to be acid labile and the close proximity of the α-carboxylate of compound 7 may have contributed to this instability. Also, the α-carboxylate could reasonably displace the POC group by hydrolysis of the phosphonate ester through a 5-membered intramolecular cyclization. Either one of these mechanisms, or a combination of both, would result in the lability of the POC ester. Although less energetically favorable due to the fact it is further away from the POC group, the γcarboxylate may also account in part, for chemical instability of 7. Postulating that the carboxylates were responsible for chemical instability of compound 7, we made a series of simple bis-POM and bis-POC dialkyl esters 9-11 (Scheme 2). Simple alkyl esters are commonly used to prodrug carboxylic acids and are readily cleaved in vivo by a host of carboxylesterases32. As predicted, the chemical stability of compounds 9-11 improved dramatically over compound 7. Furthermore, compounds 15a-c containing alkyl esters only on



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the α-carboxylate displayed robust chemical stability as well, strongly indicating that the αcarboxylate was responsible for the chemical instability of compound 7. The improved stability of compounds 9-11 and 15a-c prompted in vivo evaluation to assess their potential to enhance the exposure of 1 after oral administration. Compounds 9-11 and 15a-c were initially tested in a single 30-minute time point pharmacokinetic study in mice (Figure 2). As detailed in the experimental section, our protocol was designed to analyze plasma for parent prodrug, compound 1 and all other metabolites resulting from partial hydrolysis of the POM, POC or alkyl esters (Figure 2A) or selectively for released compound 1 (Figure 2B). As demonstrated in Figure 2A, compounds 9-11 and their metabolites displayed good oral exposure achieving total concentrations of 20-40 mM in plasma after 30 min regardless of the nature of the carboxylate ester or the POM/POC group. Furthermore, similar plasma levels were achieved with oral doses of compounds 15a-c indicating that the additional masking of the γ-carboxylate did not improve oral absorption. Although the prodrugs and their metabolites showed good overall plasma exposure, likely due to better penetration through the gastrointestinal tract, none showed ability to release 1 to any appreciable extent (Figure 2B). This result was surprising since there is a breadth of data supporting the rapid hydrolysis of POM and POC esters as well as basic alkyl esters in vivo

27, 31

. Given the

chemical lability of compound 7 when the α-carboxylate was free, we hypothesized that the carboxylate esters were likely too stable. If the carboxylate esters were hydrolyzed, particularly the α-carboxylate ester, they would almost certainly contribute to the hydrolysis of the POM and POC groups in a similar manner to compound 7. To further evaluate this hypothesis, we tested the metabolic lability of dialkyl esters 5b-c with a free phosphonic acid moiety. The dimethyl ester 5b was found to be surprisingly stable to mouse and human plasma and liver microsomes


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with almost 100% remaining after 60 min (Table 1). Indeed, the diethyl ester 5c was also stable under these conditions with 83% and 88% remaining in mouse and human microsomes, respectively. These results support the hypothesis that the diesters are not readily hydrolyzed by ubiquitous carboxylesterases. On the other hand, all compounds containing POM or POC esters (e.g. 9-11, 15a-c and 21a-b) were rapidly and in many cases completely metabolized in mouse plasma and microsomes. To a lesser extent, the same POC and POM based prodrugs were metabolized in human microsomes and plasma (Table 1). These results suggest that POM and POC esters may be more susceptible to metabolism versus the carboxylate alkyl esters. Therefore in attempt to circumvent the stability of the alkyl carboxylic esters, we introduced POM and POC promoeities on the α-carboxylate, resulting in the synthesis of trisPOM and tris-POC prodrugs 21a-b (Scheme 4). In mice, oral administration of compound 21a resulted in a significant increase in the concentration of 1 in the plasma after 30 min (15.8 ±0.35mM) versus the corresponding methyl ester 15a (0.74 ±0.23 mM) or ethyl ester 15b (0.54 ±0.27mM, Figure 3).

Similarly, 21b showed a ~20-fold increase in release of 1 (15.66

±2.82mM) over the ethyl ester 15c (0.49± 0.2mM, Figure 2B). To our knowledge, these prodrugs are the first to deliver high levels of 1 in plasma following oral administration. Given that trisPOM has three pivaolyloxylmethyl esters that form pivalic acid upon hydrolysis, a compound known to cause serum carnitine depletion, all subsequent pharmacokinetic studies were conducted with the Tris-POC prodrug 21b. The main cleavage products of POC promoeity in 21b are carbon dioxide, formaldehyde and isopropyl alcohol which on chronic administration have had no impact on carnitine homeostasis. While some concerns have been previously reported with formaldehyde generation from the POM and POC moeities, FDA-approved drugs such as Fosphenytoin, Pivampicillin, Tenofovir disoproxil, Adefovir dipivoxil and Fospropofol



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contain these promoeities, and daily formaldehyde load from these is generally considered insignificant with no safety issues reported.33 In mice, oral administration of prodrug 21b enabled approximately 20-fold enhancement in the plasma exposure of 1 based on AUC0-t when compared to oral administration of compound 1 itself. (Figure 3A). Importantly, the prodrug also afforded sustained levels of liberated 1 for >4 hours. Since the metabolism of prodrugs is known to exhibit species specificity32,

34

we

confirmed the oral exposure of 21b in a preliminary single dose oral pharmacokinetic study in a beagle dog (Figure 3B). Compound 21b demonstrated excellent oral exposures of 1 with a Cmax of 51µM, and AUC0-t 126.4 µmole/L/h. Although oral data for 1 in dogs for direct comparison is not currently available, these exposures are >100 fold higher compared to 1 administered orally at the same dose in mice (Cmax of 0.28 µM, and AUC0-t 0.6 µmole/L/h). In addition, in a preliminary pharmacokinetic study in rhesus monkey, oral doses of 10mg/kg 1 provided very low exposures with plasma 1 concentrations ranging from 0.18 to 0.5 uM at 0.25 to 2 h post dose (data not shown). Overall the excellent exposure of 1 following oral dosing with 21b in both dog and mice are very encouraging and substantiate the feasibility and translatability of our prodrug strategy. CONCLUSION A systematic approach was undertaken to determine the optimal combination of phosphonic ester and carboxyl ester based prodrugs for the potent and efficacious GCPII inhibitor 2-PMPA, 1. Simply masking the phosphonic acid of 1 with POC groups led to a compound with chemical instability, likely due to the close proximity of the α-carboxylate. By masking the α-carboxylate with simple alkyl esters, a dramatic improvement in oral absorption was noted in mice, but with minimal release of the active compound 1. Changing the α-carboxyl ester to a POC group


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resulted in the lead compound 21b. This prodrug was orally bioavailable in mice providing sustainable levels of compound 1 for over 4 hours, with >20 fold enhancement of total exposure when compared to orally administered 1 at a molar equivalent dose. The substantial oral exposure was subsequently confirmed in a beagle dog. The results provide the first examples of orally bioavailable prodrugs of phosphonate based GCPII inhibitors and a roadmap for the design and development of other prodrugs from this potent class of compounds.

EXPERIMENTAL SECTION General information The commercially available HPLC grade methanol, catalysts and reagent grade materials were used as received. The diisopropylamine and triethylamine were distilled from calcium hydride under argon. The THF was freshly distilled from sodium/benzophenone under nitrogen. TLC was performed on Silica gel 60 F254‐coated aluminum sheets (Merck) and spots were detected by the solution of Ce(SO4)2. 4H2O (1%) and H3P(Mo3O10)4 (2%) in sulfuric acid (10%). Flash

chromatography was performed on Silica gel 60 (0.040‐0.063 mm, Fluka) or on Biotage® KP‐ C18‐HS or KP‐Sil® SNAP cartridges using the Isolera One HPFC systém (Biotage, Inc.). All chemicals were purchased from Sigma-Aldrich and were used without further purification. The 1

H NMR spectra were measured at 400.1 MHz, 500.1 MHz or 600.1 MHz, 13C NMR spectra at

100.8 MHz, 125.7 MHz or 150.9 MHz, 31P NMR spectra at 162.3 MHz or 202.4 MHz in CDCl3, CD3COCD3 or CD3OD as indicated in 5 mm PFG probe. For standardization of 1H NMR spectra the internal signal of TMS (δ 0.0, CDCl3) or residual signals of solvents (δ 7.26 for CDCl3, δ 2.05 for CD3COCD3 and δ 3.31 for CD3OD) were used. In the case of

13

C spectra the residual

signals of solvents (δ 77.00 for CDCl3, δ 29.84 and δ 206.26 for CD3COCD3 and δ 49.00 for



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CD3OD) were used. The chemical shifts are given in δ‐scale, the coupling constants J are given in Hz. The ESI mass spectra were recorded using ZQ micromass mass spectrometer (Waters) equipped with an ESCi multimode ion source and controlled by MassLynx software. Alternatively, the low resolution ESI mass spectra were recorded using a quadrupole orthogonal acceleration time‐of‐flight tandem mass spectrometer (Q‐Tof micro, Waters) and high resolution ESI mass spectra using a hybrid FT mass spectrometer combining a linear ion trap MS and the Orbitrap mass analyzer (LTQ Orbitrap XL, Thermo Fisher Scientific). The conditions were optimized for suitable ionization in the ESI Orbitrap source (sheat gas flow rate 35 a.u., aux gas flow rate 10 a.u. of nitrogen, source voltage 4.3 kV, capillary voltage 40 V, capillary temperature 275 °C, tube lens voltage 155 V). The samples were dissolved in methanol and applied by direct injection. As a mobile phase was used 80% methanol (flow rate 100 μl/min). Optical rotations were measured in CHCl3 or acetone using an Autopol IV instrument (Rudolph Research Analytical). Purity for each compound was established using HPLC (Jasco Inc.) equipped with a Reprosil 100 Watrex C18, 5 mm, 250 x 4 mm column. The analysis was performed using a gradient of 2% CH3CN/98%:H2O with 0.1% TFA  100% CH3CN, with UV detection, λ=210 nm. All compounds were deemed >95% purity by this method and lead compounds 20b and 21b were >99% pure. General Procedure for the deprotection of phosphonate ethyl esters. Bromotrimethylsilane (9.3 mL; 70 mmol) was added at 0 °C to a solution of appropriate phosphonate diester (17.5 mmol) in acetonitrile (100 mL) and kept at 0 °C for 24 h. The solution was evaporated, the residue co-evaporated with acetonitrile, followed by water and toluene. The crude product was purified by chromatography on silica gel in system chloroform – ethyl acetate – methanol (2:2:1).


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(5-Methoxy-2-(methoxycarbonyl)-5-oxopentyl)phosphonic acid (5b). Yield: 3.2g (73%) of a colorless syrup; 1H NMR (MeOD): 1.77 – 1.91 (m, 1H), 1.87 – 2.05 (m, 2H), 2.08 – 2.21 (m, 1H), 2.29 – 2.42 (m, 2H), 2.71 – 2.86 (m, 1H), 3.66 (s, 3H), 3.69 (s, 3H);

13

C NMR (MeOD):

29.53 (d, JC,P = 12.8), 30.33 (d, JC,P = 140.4), 32.10, 40.89 (d, JC,P = 3.4), 52.43, 52.47, 174.83, 176.44 (d, JC,P = 8.1); 31P NMR (MeOD): 27.09; ESI MS: 253 ([M - H]-); HR ESI MS: calcd for C8H14O7P 253.04826; found 253.04794. (5-Ethoxy-2-(ethoxycarbonyl)-5-oxopentyl)phosphonic acid (5c). Yield: 3.0 g (60%) of a colorless syrup; 1H NMR (CDCl3): 1.29 (m, 6H), 2.06 (m, 3H), 2.27 (m, 3H), 2.83 (m, 1H), 4.14 (m, 4H);

31

P NMR (CDCl3): 26.70; ESI MS: 281.1 ([M - H]-); HR ESI MS: calcd for

C10H19O7P 281.07956; found 281.07958. General procedure for addition of POC or POM group. DBU (2 mmol) was added to a solution of appropriate phosphonic acid (1 mmol) in dry dioxane (10 mL) and then heated with POC-Cl (20 mmol, 80 °C, 4 h) or POM-Cl (4 mmol, reflux, 6 h). The reaction course was monitored by TLC in toluene:acetone (4:1); detection was performed by spraying of the TLC plate with a solution of phosphomolybdenic acid and heating. Reaction mixture was evaporated and the residue chromatographed on silica gel using toluene:acetone (4:1) as the eluent. Dibenzyl 2-((bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl) pentanedioate (6). Yield: 247 mg (39 %) of a colorless oil. 1H NMR (CDCl3) δ1.34 (4xd, 12H), 2.04 (m, 3H), 2.37 (m, 3H), 2.90 (m, 1H), 4.93 (m, 2H), 5.11 (s, 2H), 5.14 (d, 2H), 5.63 (m, 4H), 7.35 (m, 10H); 13C NMR (CDCl3): 21.59 (4C), 28.14 (d, J = 13.2), 28.41 (d, J = 143.2), 31.21, 38.76 (d, J =

3.5), 66.37, 66.99, 73.27 (2C), 83.95 (d, J = 6.2), 83.97 (d, J = 6.2), 128.21 (2C), 128.23 (2C),

128.31, 128.37, 128.53 (2C), 128.54 (2C), 135.52, 135.77, 153.10, 153.12, 172.09, 173.18 (d, J



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= 9.1);

31

Page 14 of 44

P NMR (CDCl3): 29.85; ESI MS: 661 ([M + Na]+); HR ESI MS: calc for For

C30H40O13P [M+H]+ 639.22010; found: 639.22034. 2-((Bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)

pentanedioic

acid

(7).

Compound 6 (60 mg) was hydrogenated in acetone (3 mL) in the presence of 5% Pd/C (10 mg) at room temperature for 1 h. Catalyst was then filtered off and the solvent was evaporated yielding 42 mg (97%). 1H NMR (CD3COCD3) δ 1.29 (d, 12H, J = 6.2), 1.92 – 2.13 (m, 3H), 2.29 – 2.46 (m, 3H), 2.81 (m, 1H), 4.86 – 4.94 (m, 2H), 5.62 – 5.70 (m, 4H);

13

C NMR

(CD3COCD3): 21.75 (4C), 28.68 (d, J = 11.7), 28.72 (d, J = 142.3), 31.36, 39.10 (d, J = 3.3), 73.63 (2C), 84.84, 84.96, 153.99 (2C), 173.97, 175.08 (d, J = 10.5);

31

P NMR (CD3COCD3):

30.91 (s); ESI MS: 457 ([M - H]-); HR ESI MS: calcd for C16H26O13P 457.11165; found 457.11090. General procedure for the esterification of phosphonic acids. DBU (2 mmol) was added to a solution of the appropriate phosphonic acid (1 mmol) in dry dioxane (10 mL) and then heated with POC-Cl (20 mmol, 80 °C, 4 h) or POM-Cl (4 mmol, reflux, 6 h). The reaction course was monitored by TLC in system toluene – acetone (4:1); detection was performed by spraying of the TLC plate with a solution of phosphomolybdenic acid and heating. Reaction mixture was evaporated and the residue chromatographed on a silica gel column (200 mL) in toluene – acetone (4:1) for POM and POC esters. Dimethyl 2- ((bis{[(pivaloyloxy)methoxy]phosphoryl}methyl)pentanedioate (9). Yield: 195 mg (40 %) of a colorless syrup; 1H NMR (CDCl3): δ 1.22 (2 × s, 2 × 9H), 1.91-2.03 (m, 3H), 2.26-2.38 (m, 3H), 2.80 (dtt, 1H, J = 13.5, 8.4, 5.5), 3.65 (s, 3H), 3.70 (s, 3H), 5.61-5.67 (m, 4H);

13

C NMR (CDCl3): 26.79, 28.14 (d, JC,P = 13.3), 28.69 (d, JC,P = 142.8), 31.07 (d, JC,P =


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1.1), 38.70, 38.71 (d, JC,P = 3.5), 51.71, 52.17, 81.36, 81.39 (2 × d, JC,P = 6.2), 172.72, 173.87 (d, JC,P = 9.1), 176.80, 176.81; HR ESI MS: calcd for C20H35O11PNa 505.18092; found 505.18099. Dimethyl

2-((bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)-pentanedioate

(10). Yield: 177 mg (37 %) of a colorless syrup; 1H NMR (CDCl3): δ 1.31 (4 × d, 12H), 1.87 – 2.08 (m, 2H), 2.00 (ddd, 1H, J = 19.1, 15.6, 5.4), 2.25 – 2.38 (m, 2H), 2.37 (ddd, 1H, J = 19.0, 15.6, 8.6), 2.73 – 2.90 (m, 1H), 3.65 (s, 3H), 3.70 (s, 3H), 4.92 (2 × sept), 5.58 – 5.68 (m, 4 H); 13

C NMR (CDCl3): 21.58 (4C), 28.13 (d, JC,P = 13.5), 28.45 (d, JC,P = 143.3), 31.09 (d, JC,P =

1.1), 38.62 (d, JC,P = 3.6), 51.69, 52.17, 73.28, 83.91, 83.92 (2 × d, JC,P = 6.2), 153.10, 153.12, 172.74, 173.83 (d, JC,P = 8.9);

31

P NMR (CDCl3): 29.92;ESI MS: 509 ([M + Na]+); HR ESI

MS: calcd for C18H31O13PNa 509.13945; found 509.13938. Diethyl 2-({bis[(pivaloyloxy)methoxy]phosphoryl}methyl)pentanedioate (11). Yield: 429 mg (84 %) of a yellowish syrup; 1H NMR (CDCl3): δ 1.25 (m, 24H), 1.99 (m, 3H), 2.34 (m, 3H), 2.80 (m,1H), 4.17 (m, 4H), 5.67 (m, 4H); 31P NMR (101 MHz, CDCl3): 29.64; ESI MS: 533.1 ([M + Na]+); HR ESI MS: calcd for C22H39O11PNa 533.21222; found 533.21221. 5-Benzyl 1-methyl 2-((diethoxyphosphoryl)methyl)pentanedioate (13a). Diethyl phosphite (2.6 mL, 20.14 mmol, 1 equiv.) was dissolved in absolute dichloromethane (57 mL) under argon and cooled to 0 °C. A solution of trimethyl aluminium (2 M in hexanes, 10 mL, 20.14 mmol, 1.0 equiv.) was added dropwise and the solution was stirred at 0 °C for 30 min. Solution of compound 12a (5 g, 20.14 mmol, 1.0 equiv) in dichloromethane (171 mL) was added and the cooling bath was removed. The reaction mixture was then stirred at room temperature overnight. The reaction was quenched with 2 N hydrochloric acid (40 mL). Then it was extracted with diethyl ether (3 x 40 mL), the combined organic layers were washed with water (40mL), brine



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(40mL), and dried over anhydrous MgSO4. The evaporation of the solvents afforded an oil, which was filtered through pad of silica gel (hexane-ethyl acetate 3:1 to 1:1) to afford the desired product (7.3g, 94%) as an oil. 1H NMR (CDCl3): δ 1.22 – 1.26 (6H, m), 1.75 – 1.85 (1H, m), 1.88 – 2.02 (2H, m), 2.14 – 2.24 (1H, m), 2.27 – 2.40 (2H, m), 2.72 – 2.82 (1H, m), 3.63 (3H, s), 3.97 – 4.07 (4H, m), 5.05 (2H, s), 7.24 – 7.33 (5H);

13

C NMR (CDCl3): 16.29 (d, JC,P = 2.2),

16.35 (d, JC,P = 2.1), 27.82 (d, JC,P = 142.6), 28.32 (d, JC,P = 12.9), 31.42, 39.12 (d, JC,P = 3.7), 51.93, 61.69 (d, JC,P = 6.67), 61.76 (d, JC,P = 6.6), 66.33, 128.19, 128.21, 128.51, 135.76, 172.19, 174.34 (d, JC,P = 8.1); 31P NMR (CDCl3): 31.00; ESI MS: 409 ([M + Na]+); HR ESI MS: calcd. for C18H28O7P 387.15672; found 387.15678. 5-Benzyl 1-ethyl 2-((diethoxyphosphoryl)methyl)pentanedioate (13b). The reaction was carried out in a similar manner to 13a above to afford the desired product (7.5g, 94%) as an oil. 1

H NMR (CDCl3): δ 1.20 – 1.28 (9H, m), 1.76 – 1.85 (1H, m), 1.90 – 2.04 (2H, m), 2.16 – 2.26

(1H, m), 2.30 – 2.43 (2H, m), 2.71 – 2.81 (1H, m), 3.99 – 4.07 (4H, m), 4.08 – 4.13 (2H, m), 5.07 (2H, s), 7.26 – 7.35 (5H); 13C NMR (CDCl3): 14.17, 16.35 (d, JC,P = 2.2), 16.41 (d, JC,P = 2.2), 27.79 (d, JC,P = 142.6), 28.38 (d, JC,P = 12.6), 31.47, 39.23 (d, JC,P = 3.7), 60.92, 61.73 (d, JC,P = 6.4), 61.79 (d, JC,P = 6.3), 66.38, 128.23, 128.26, 128.56, 135.82, 172.30, 173.93 (d, JC,P = 8.5);

31

P NMR (CDCl3): 31.20; ESI MS: 423 ([M + Na]+); HR ESI MS: calcd for C19H30O7P

401.17237; found 401.17229. 5-Benzyl-1-methyl-2-({bis[(pivaloyloxy)methoxy]phosphoryl}methyl)pentanedioate

(14a).

This compound was synthesized using a similar procedure to compound 6. Yield: 195 mg (40 %) of a colourless syrup; 1H NMR (CDCl3): δ 1.22 (2 × s, 2 × 9H), 1.91-2.03 (m, 3H), 2.26-2.38 (m, 3H), 2.80 (dtt, 1H, J = 13.5, 8.4, 5.5), 3.65 (s, 3H), 3.70 (s, 3H), 5.61-5.67 (m, 4H);

13

C

NMR (CDCl3): 26.79 (6C), 28.14 (d, J = 13.3), 28.69 (d, J = 142.8), 31.07 (d, J = 1.1), 38.70,


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38.71 (d, J = 3.5), 51.71, 52.17, 81.36, 81.39 (2 × d, J = 6.2), 172.72, 173.87 (d, J = 9.1), 176.80, 176.81;

31

P NMR (CDCl3): 29.88; ESI MS: 505 ([M + Na]+); HR ESI MS: calcd for

C20H35O11PNa 505.18092; found 505.18099. 5-Benzyl-1-ethyl-2-({bis[(pivaloyloxy)methoxy]phosphoryl}methyl)pentanedioate

(14b).

Yield: 183 mg (32 %) of a colorless syrup; 1H NMR (CDCl3): δ 1.25 (m, 21H), 2.01 (m, 3H), 2.39 (m, 3H), 2.82 (m, 1H), 4.17 (q, 2H, J = 7.1), 5.13 (s, 2H), 5.66 (d, 2H, J = 13.1), 5.67 (d, 2H, J = 13.1), 7.36 (m, 5H);

31

P NMR (CDCl3): 29.40; ESI MS: 595.3 ([M + Na]+); HR ESI

MS: calcd for C27H41O11PNa 595.22787; found 595.22783. 5-Benzyl-1-ethyl-2-((bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)pentanedioate (14c). Yield: 217 mg (38%) of a colorless syrup. 1H NMR (CDCl3): δ 1.27 (t, 3H, J = 7.1), 1.33 (d, 6H, J = 1.6), 1.34 (d, 6H, J = 1.6), 2.04 (m, 3H), 2.41 (m, 3H), 2.83 (m, 1H), 4.17 (q, 2H, J = 7.1), 5.13 (s, 2H), 5.66 (m, 4H), 7.36 (m, 5H);

31

P NMR (101 MHz,

CDCl3): 29.46; ESI MS: 599.3 ([M + Na]+); HR ESI MS: calcd for C25H39O13P 577.20445; found 577.20445. 4-((Bis((pivaloyloxy)methoxy)phosphoryl)methyl)-5-methoxy-5-oxopentanoic acid (15a). A solution of benzyl ester 14a (1 mmol) in THF (30 mL) and the mixture was hydrogenated in the presence of 10 % Pd/C (90 mg) at room temperature and atmospheric pressure for 15 h. The catalyst was removed by filtration through a pad of Celite. The crude filtrate was finally purified by additional filtration through a Whatman nylon membrane filter. The filtrate was evaporated to give a desired monoester as a colorless syrup in 90%yield. The reaction course was monitored by TLC in ethyl acetate:acetone:ethanol:water (18:3:2:2), detection was performed by spraying with bromocresol green solution and heating (white spot of the product). 1H NMR (CDCl3):



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δ 1.24, 1.24 (2 × s, 2 × 9H), 1.95 – 2.05 (m, 3H), 2.30 – 2.44 (m, 3H), 2.84 (m, 1H), 3.72 (s, 3H), 5.63 – 5.68 (m, 4H);

13

C NMR (CDCl3): 26.80 (6C), 27.82 (d, J = 12.8), 28.60 (d, J = 142.9),

30.90, 38.56 (d, J = 3.6), 38.71 (2C), 52.23, 81.39, 81.45, 173.86 (d, J = 9.7), 176.60, 176.84, 176.85; 31P NMR (CDCl3): 29.95; ESI MS: 467 ([M - H]-); HR ESI MS: calcd for C19H32O11P 467.16877; found 467.16788. 4-({Bis[(pivaloyloxy)methoxy]phosphoryl}methyl)-5-ethoxy-5-oxopentanoic acid (15b). The title compound was synthesized in the same manner as 15a outlined above in 95% yield. 1H NMR (CDCl3): δ 1.24 (s, 18H), 1.28 (t, 3H, J = 7.1), 1.94 - 2.06 (m, 3H), 2.30 - 2.45 (m, 3H), 2.82 (m, 1H), 4.14 - 4.20 (m, 2H), 5.64 - 5.68 (m, 4H);

13

C NMR (CDCl3): 14.10, 26.80 (6C),

27.76 (d, J = 12.2), 28.50 (d, J = 142.7), 30.98, 38.63 (d, J = 3.4), 38.71 (2C), 61.24, 81.46 (d, J = 6.2), 81.49 (d, J = 6.2), 173.38 (d, J = 10.4), 176.84, 176.85, 177.03;

31

P NMR (CDCl3):

30.67; ESI MS: 505 ([M + Na]+); HR ESI MS: calcd for C20H35O11PNa 505.18092; found 505.18109. 4-((Bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)-5-ethoxy-5-oxopentanoic acid (15c). The title compound was synthesized in the same manner as 15a outlined above in 94% yield. 1H NMR (CDCl3): δ 1.30 (m, 15H), 2.03 (m, 3H), 2.40 (m, 3H), 2.85 (m, 1H), 4.18 (q, 2H, J = 7.1), 4.94 (dq, 2H, J = 12.4 and 6.2), 5.61-5.75 (m, 4H); 13C NMR (CDCl3): 14.08, 21.59 (4C), 27.78 (d, J = 12.5), 28.30 (d, J = 143.5), 30.93, 38.56 (d, J = 3.3), 61.23, 73.32 (2C), 83.99 (d, J = 6.2), 84.02 (d, J = 6.2), 153.12, 153.14, 173.33 (d, J = 10.1), 176.68;

31

P NMR

(CDCl3): 30.07; ESI MS: 509 ([M + Na]+).HR ESI MS: calcd for C18H31O13PNa 509.13945; found 509.13962.


Page 19 of 44

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5-Benzyl 1-(tert-butyl) 2-methylenepentanedioate (17). A dry Schlenk flask was charged with compound 16 (6.62g, 21.62 mmol), N,N-Dimethylmethyleneiminium iodide (10 g, 54.05 mmol, 2.5 equiv.) and then it was flushed with argon. Absolute t-BuOH (265 mL) was added to the flask and the mixture was stirred at 65 °C for 48 h. The organic solvent was evaporated in vacuo. The residue was filtered through pad of silica gel (hexane:ethyl acetate 5:1) to afford the desired product (5 g, 80 %) as an oil. 1H NMR (CDCl3): δ 1.48 (9H, s), 2.54 – 2.64 (4H, m), 5.11 (2H, s), 5.48 (1H, m), 6.07 (1H, d, J = 1.2), 7.30 – 7.40 (5H, m); 13C NMR (CDCl3): 27.59, 28.19, 33.40, 66.39, 80.90, 124.87, 128.33, 128.35, 128.66, 136.10, 140.58, 166.01, 172.75; ESI MS: 313 ([M + Na]+); HR ESI MS: calcd for C17H22O4Na 313.14103; found 313.14103. 5-(Benzyloxy)-2-((diethoxyphosphoryl)methyl)-5-oxopentanoic acid (18). Diethyl phosphite (8.5 mL, 66.1 mmol, 1 equiv.) was dissolved in absolute dichloromethane (57 mL) under argon and cooled to 0 °C. A solution of trimethyl aluminium (2 M in hexanes, 33 mL, 66.1 mmol, 1 equiv.) was added dropwise and the solution was stirred at 0 °C for 30 min. Solution of compound 17 (19.2 g, 66.1 mmol, 1 equiv) in dichloromethane (171 mL) was added and the cooling bath was removed. The reaction mixture was then stirred at room temperature overnight. The reaction was quenched with 2 N hydrochloric acid (40 mL). Then it was extracted with diethyl ether (3 x 40 mL), the combined organic layers were washed with water (40mL), brine (40mL), and dried over anhydrous MgSO4. The evaporation of the solvents afforded an oil, which was filtered through pad of silica gel (hexane-ethyl acetate 3:1 to 1:1) to afford the desired product (28.3 g, 94 %) as an oil. 1H NMR (400 MHz, CDCl3): δ 1.24 – 1.28 (6H, m), 1.40 (9H, s), 1.69 – 1.79 (1H, m), 1.85 – 2.02 (2H, m), 2.11 – 2.24 (1H, m), 2.29 – 2.43 (2H, m), 2.61 – 2.71 (1H, m), 4.00 – 4.07 (4H, m), 5.07 (2H, s), 7.25 – 7.34 (5H); 13C NMR (CDCl3): 16.41 (d, JC,P = 5.9), 27.74 (d, JC,P = 158.8), 27.99, 28.39 (d, JC,P = 4.4), 31.44, 39.93 (d, JC,P = 3.5), 60.92,



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Page 20 of 44

61.67 (d, JC,P = 4.5), 61.73 (d, JC,P = 4.5), 66.32, 81.14, 128.19, 128.22, 128.54, 135.86, 172.40, 173.06 (d, JC,P = 9.4); 31P NMR (CDCl3): 31.76; ESI MS: 451 ([M + Na]+); HR ESI MS: calcd for C21H33O7PNa 451.18561; found 451.18564. The intermediate phosphonate was dissolved in dichloromethane (100 mL) and trifluoroacetic acid (100 mL) was slowly added. The reaction mixture was stirred at room temperature overnight. Then the solvents were removed in vacuo. The residue was filtered through a short pad of silica gel (chloroform-methanol 10 : 1) to furnish the desired product (19.7 g, 88 %) as an oil. 1H NMR (CDCl3): δ 1.27 – 1.31 (6H, m), 1.86 – 1.96 (1H, m), 1.99 – 2.04 (2H, m), 2.24 – 2.34 (1H, m), 2.39 – 2.52 (2H, m), 2.74 – 2.88 (1H, m), 4.03 – 4.17 (4H, m), 5.11 (2H, s), 7.29 – 7.39 (5H, m); 13C NMR (CDCl3): 16.28 (d, JC,P = 5.9), 27.51 (d, JC,P = 132.7), 28.28, 31.49, 39.04, 62.31 (d, JC,P = 6.5), 66.33, 128.18, 128.21, 128.54, 135.89, 172.49, 176.35 (d, JC,P = 8.1);

31

P NMR (CDCl3): 29.20; ESI MS: 395 ([M +

Na]+); HR ESI MS: calcd for C17H25O7NaP 395.12301; found 395.12337. 5-Benzyl 1-((pivaloyloxy)methyl) 2-((diethoxyphosphoryl)methyl) pentanedioate (19a). A dry flask was charged with phosphonate 18 (250 mg, 0.67 mmol), NaI (201 mg, 1.34 mmol, 2 equiv.), K2CO3 (185 mg, 1.34 mmol, 2 equiv.). Dry acetonitrile (5 mL) was added and reaction mixture was stirred at room temperature for 15 min. and then chloromethyl pivalate (0.2 mL, 1.34 mmol, 2 equiv.) was added. Reaction mixture was stirred at 50 °C overnight. The solvent was removed under reduced pressure and the residue was chromatographed on silica gel (hexane - ethyl acetate 1:2) to afford the desired product (230 mg, 70 %) as an oil. 1H NMR (CDCl3): δ 1.16 (9H, s), 1.25 – 1.29 (6H, m), 1.79 – 1.88 (1H, m), 1.91 – 2.08 (2H, m), 2.14 – 2.26 (1H, m), 2.30 – 2.44 (2H, m), 2.78 – 2.88 (1H, m), 3.99 – 4.12 (4H, m), 5.08 (2H, s), 5.69 (1H, d, J = 5.5), 5.79 (1H, d, J = 5.4), 7.27 – 7.35 (5H, m); 13C NMR (101 MHz, CDCl3): 16.40 (d, JC,P = 2.2), 16.46 (d, JC,P = 2.2), 26.88, 27.53 (d, JC,P = 143.1), 28.13 (d, JC,P = 12.0), 31.23, 38.78, 39.12 (d,


Page 21 of 44

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JC,P = 3.7), 61.91 (d, JC,P = 5.5), 61.98 (d, JC,P = 5.4), 66.48, 79.77, 128.31, 128.33, 128.61, 135.81, 172.14, 176.73 (d, JC,P = 9.1), 177.02;

31

P NMR (101 MHz, CDCl3): 30.60; ESI MS:

509 ([M + Na]+); HR ESI MS: calcd for C23H35O9NaP 509.19109; found 509.19102. 5-Benzyl-1-(((isopropoxycarbonyl)oxy)methyl)-2-((diethoxyphosphoryl) methyl)pentanedioate (19b). The same method of preparation was used as outlined for compound 19a to produce the title compound in 72% yield as an oil. 1H NMR (400 MHz, CDCl3): δ 1.27 – 1.31 (12H, m), 1.82 – 1.92 (1H, m), 1.96 – 2.10 (2H, m), 2.19 – 2.30 (1H, m), 2.30 – 2.47 (2H, m), 2.81 – 2.91 (1H, m), 4.00 – 4.14 (4H, m), 4.89 (1H. hept, J = 6.3), 5.10 (2H, s), 5.72 (1H, d, J = 5.7), 5.77 (1H, d, J = 5.6), 7.29 – 7.38 (5H, m); 13C NMR (CDCl3): 16.38 (d, JC,P = 2.8), 16.44 (d, JC,P = 2.8), 21.66, 27.56 (d, JC,P = 143.0), 28.08 (d, JC,P = 12.1), 31.22, 39.14 (d, JC,P = 3.8), 61.92 (d, JC,P = 6.6), 62.02 (d, JC,P = 6.4), 66.50, 73.18, 82.10, 128.31, 128.33, 128.62, 135.83, 153.34, 172.20, 176.62 (d, JC,P = 8.7);

31

P NMR (CDCl3): 30.54; ESI

MS: 511 ([M + Na]+); HR ESI MS: calcd for C22H34O10P 489.18841; found 489.18838. 5-Benzyl-1-((pivaloyloxy)methyl)-2((bis((pivaloyloxy)methoxy)phosphoryl)methyl)pentanedioate (20a). Compound 19a (230 mg, 0.473 mmol) was dissolved in absolute dichloromethane (3 mL) under argon and cooled to 0 °C. Bromotrimethylsilane (0.25 mL, 1.89 mmol, 4 equiv.) was added dropwise and the solution was stirred at 0 °C overnight. The volatiles were removed in vacuo and the residue was diluted with a mixture of acetonitrile and water (5 mL, 4:1) and evaporated. The residue was dissolved in absolute acetonitrile. To the crude reaction mixture was added chloromethyl pivalate (0.68 mL, 4.73 mmol, 10 equiv.) and Ag2CO3 (339 mg, 1.23 mmol, 2.6 equiv.) under argon and reaction mixture was stirred at 80 °C overnight. The solvent was removed under reduced pressure and the residue was chromatographed on silica gel (toluene:acetone 10:1) to afford the



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Page 22 of 44

desired product (255 mg, 82 %) as an oil. 1H NMR (CDCl3): δ 1.15 (9H, s), 1.21 (18H, s), 1.90 – 2.06 (3H, m), 2.23 – 2.38 (3H, m), 2.77 – 2.88 (1H, m), 2.49 – 2.63 (2H, m), 5.07 (2H, s), 5.59 – 5.68 (4H, m), 5.67 (1H, d, J = 5.6), 5.80 (1H, d, J = 5.6), 7.27 – 7.37 (5H, m);

13

C NMR

(CDCl3): 26.82, 26.83 (2C), 28.33 (d, JC,P = 143.2), 27.98 (d, JC,P = 12.8), 30.99 (3C), 38.65 (d, JC,P = 3.6), 38.72, 66.44, 79.87, 81.41 (d, JC,P = 6.2), 81.50 (d, JC,P = 6.2), 128.26 (2C), 128.54, 135.73, 171.92, 172.17 (d, JC,P = 9.5), 176.78, 176.81, 176.90;

31

P NMR (CDCl3): 31.38; ESI

MS: 681 ([M + Na]+); HR ESI MS: calcd for C31H47O13NaP 651.26465; found 651.26430. 5-Benzyl-1-(((isopropoxycarbonyl)oxy)methyl)-2((bis(((isopropoxycarbonyl)oxy)methoxy)phosphoryl)methyl)pentanedioate (20b). The same method of preparation was used as described above for 20a. The title phosphonate was isolated as an oil in 92% yield. 1H NMR (CDCl3): δ 1.26 – 1.28 (6H, m), 1.30 – 1.32 (12H, m), 1.83 – 1.93 (1H, m), 1.98 – 2.08 (3H, m), 2.33 – 2.44 (3H, m), 2.83 – 2.94 (1H, m), 4.83 – 4.97 (3H, m), 5.10 (2H, s), 5.59 – 5.68 (4H, m), 5.70 (1H, d, J = 5.7), 5.79 (1H, d, J = 5.7) 7.29 – 7.39 (5H, m);

13

C NMR (CDCl3): 21.70 (2C), 21.74 (4C), 28.33 (d, JC,P = 143.7), 28.02 (d, JC,P =

13.2), 31.15, 38.72 (d, JC,P = 3.6), 66.59, 73.28, 73.48 (2C), 82.41, 84.14 (d, JC,P = 4.7), 84.20 (d, JC,P = 4.8), 128.38, 128.39, 128.67, 135.87, 153.23, 153.26, 153.37, 172.14 (d, JC,P = 9.0), 172.14; 31P NMR (CDCl3): 31.18; ESI MS: 687 ([M + Na]+); HPLC retention time: 25.84 min (>99%). 4-((Bis((pivaloyloxy)methoxy)phosphoryl)methyl)-5-oxo-5((pivaloyloxy)methoxy)pentanoic acid (21a). Compound 20a (48 mg, 72.9 mmol) was dissolved in dry THF (3 mL). Palladium on carbon (10%, 5 mg) was added and reaction mixture was bubbled with hydrogen for 10 min. Reaction mixture was stirred at room temperature overnight under hydrogen atmosphere. Palladium was filtered through cotton and the volatiles


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were removed in vacuo to afford desired product (40 mg, 98 %) as an oil. 1H NMR (CDCl3): δ 1.14 (9H, s), 1.16 (18H, s), 1.84 – 1.99 (3H, m), 2.20 – 2.34 (3H, m), 2.74 – 2.84 (1H, m), 5.58 (2H, d, J = 13.1), 5.58 (2H, d, J = 12.9), 5.63 (1H, d, J = 5.5), 5.77 (1H, d, J = 5.5); 13C NMR (101 MHz, CDCl3): 26.80 (9C), 27.85 (d, JC,P = 12.8), 28.25 (d, JC,P = 143.2), 30.68, 38.58 (d, JC,P = 3.6), 79.88, 81.49 (d, JC,P = 6.3), 81.49 (d, JC,P = 6.3), 81.56 (d, JC,P = 6.4), 172.15 (d, JC,P = 9.5), 175.75, 176.76, 176.79, 176.91; 31P NMR (101 MHz, CDCl3): 31.51; ESI MS: 591 ([M + Na]+); HR ESI MS: calcd for C24H41O13NaP 591.21770; found 591.21724. 4-((Bis(((isopropoxycarbonyl)oxy)methoxy)phosphoryl)methyl)-5(((isopropoxycarbonyl)oxy)methoxy)-5-oxopentanoic acid (21b). The same method of preparation was used as described for 21a with the product being isolated in 98% yield as an oil. 1

H NMR (CDCl3): δ 1.28 – 1.31 (18H, m), 1.93 – 2.1 (3H, m), 2.31 – 2.44 (1H, m), 2.83 – 2.94

(1H, m), 4.86 – 4.94 (3H, m), 5.59 – 5.68 (4H, m), 5.70 (1H, d, J = 5.7), 5.80 (1H, d, J = 5.7); 13

C NMR (CDCl3): 21.70 (6C), 27.73 (d, JC,P = 12.9), 28.18 (d, JC,P = 144.00), 30.79, 38.56 (d,

JC,P = 3.5), 73.33, 73.53 (2C), 82.39, 84.21 (d, JC,P = 3.7), 84.27 (d, JC,P = 3.9), 153.20, 153.23, 153.37, 172.06 (d, JC,P = 9.4), 176.84; 31P NMR (CDCl3): 31.28; ESI MS: 597 ([M + Na]+); HR ESI MS: calcd for C21H35O16NaP 597.15549; found 597.15553; HPLC retention time: 21.72 min (>99%) Metabolic Stability Studies. For metabolic stability, plasma and liver microsomes (mouse and human) were used. For stability, prodrugs (10 µM) were spiked in each matrix and incubated in an orbital shaker at 37 °C. At predetermined times (0, 30 and 60 min), 100 µL aliquots of the mixture in triplicate were removed and the reaction quenched by addition of three times the volume of ice cold acetonitrile spiked with the internal standard (losartan 5µM). The samples were vortexed for 30 s and centrifuged at 12000 g for 10 min. Then, 50 µL of the supernatant



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

was diluted with 50 µL water and transferred to a 250 µL polypropylene vial sealed with a Teflon cap. Prodrug disappearance was monitored over time using a liquid chromatography and tandem mass spectrometry (LC/MS/MS) method as described below. In vivo Pharmacokinetics of Prodrugs in mice. All of the mice pharmacokinetic studies were performed as per protocols approved by the Institutional Animal Care and Use Committee (Protocol# RA13) at Johns Hopkins University. Prodrugs (30 mg/kg p.o. equivalent 1) were dosed in mice at a dosing volume of 10 mL/kg. For single time point studies blood was collected at 30 min (N=3) following dosing. For full pharmacokinetics of 21b in mice, prodrug was dosed and blood was obtained via cardiac puncture at 0 min, 15 min, 30 min, 1h, 2h, and 4h post dose (n=3 per time point). Plasma was harvested from blood by centrifugation and stored at -80 deg C until analysis by LC/MS/MS. In vivo Pharmacokinetics of 21b in dog. The dog pharmacokinetic study was conducted in accordance with the guidelines recommended in Guide for the Care and Use of Laboratory Animals and was approved by the Absorption Systems (Exton, PA) Institutional Animal Care and Use Committee. A single beagle dog was dosed with 21b (10 mg/kg p.o. equivalent 1). Blood samples were collected from the jugular vein (~1 mL) via direct venipuncture at 0 min, 30 min, 1h, 2h, 4h, 6h, and 8h post dose, placed into potassium oxalate with sodium fluoride tubes, and maintained on wet ice until processing. Blood samples were centrifuged at a temperature of 4°C, at 3000 x g, for 5 minutes. Plasma was collected in tubes and flash frozen. Samples were stored in a freezer set to maintain -60°C to -80°C until bioanalysis.


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Bioanalysis of prodrugs, compound 1 and metabolites in plasma. Prodrugs and their metabolites, including Compound 1, was quantified in plasma by two different methods, as described previously.35,36 Briefly, 2-PMPA was extracted from plasma by protein precipitation with 6X methanol containing 2-(phosphonomethyl) succinic acid (2-PMSA; 1µM) as an internal standard, followed by vortexing for 30s and then centrifugation at 12000 x g for 10 min. Supernatant was transferred and evaporated to dryness at 40°C under a gentle stream of nitrogen. 1 quantified by two different methods for determination of total exposures and the specific release of 1. Method #1 involved use of a strong derivatizing reagent, n-butanol with 3N HCl, which converted the prodrug and all metabolites into butyl esters of 1.36 For derivatization, the residue following extraction was reconstituted with 100 μL of n-butanol with 3N HCl and samples were heated at 60°C in a shaking water bath for 30 min. At the end of 30 min the derivatized samples were dried under a gentle stream of nitrogen. The residue was reconstituted in 100 μL of 30% acetonitrile and analysed via LC/MS/MS. In brief, separation of the analyte from potentially interfering material was achieved using Agilent Eclipse Plus column (100 x 2.1mm i.d.) packed with a 1.8 µm C18 stationary phase. The mobile phase was composed of 0.1% formic acid in acetonitrile and 0.1% formic acid in H2O with gradient elution. Chromatographic analysis was performed using an Accela™ ultra high-performance system consisting of an analytical pump, and an autosampler coupled with TSQ Vantage mass spectrometer (Thermo Fisher Scientific Inc., Waltham MA). The [M+H]+ ion transitions of derivatized 1 at m/z 339.537>191.354, 149.308 and that of the internal standard at m/z 325.522 >121.296,195.345 were monitored. Method #2 involved the use of a milder derivatization procedure to selectively evaluate the release of 1 as previously described.35 Supernatant was dried under a gentle stream of nitrogen at



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45° C and the residue reconstituted with 75 µL of acetonitrile and vortexed.

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25 µL of

derivatizing agent N-tert-Butyldimethysilyl-N-methyltrifluoro-acetamide (MTBSTFA) was added to microcentrifuge tubes, vortexed, and heated at ~ 60 °C for 40 min. At the end of 40 min, the derivatized samples ~75 µL were transferred to a 250 µL polypropylene vials and were analyzed via LC/MS/MS. In brief, separation of the analyte from potentially interfering material was achieved using Waters X-terraR, RP18, 3.5 µm, and (2.1 x 50 mm). The mobile phase used was composed of 0.1% Formic Acid in Acetonitrile and 0.1% Formic Acid in H2O with gradient elution. Chromatographic analysis was performed on Accela UPLC and TSQ vantage mass spectrometer. The [M+H]+ ion transitions of derivatized 1 at m/z 683.0 >551.4 and that of the internal standard at m/z 669.0 >537.2 were monitored. Pharmacokinetic Analysis: Mean concentration-time data was used for pharmacokinetic analysis. Non-compartmental-analysis module in WinNonlin® (version 5.3) was used to assess pharmacokinetic parameters. Peak plasma concentrations (Cmax) and time to Cmax (Tmax) were the observed values. Area under the curve (AUC) was calculated by log-linear (p.o.) trapezoidal rule to the end of sample collection (AUClast).


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AUTHOR INFORMATION Corresponding Authors *Phone, (410) 614-0662; fax, (410) 614-0659; E-mail, [email protected]. *Phone, (410) 614-1906; fax, (410) 614-0659; E-mail, [email protected]. Notes The authors declare no competing financial interest. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENTS



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This research was supported by NIH Grant RO1CA161056 (to BSS) and the Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic v.v.i.

ABBREVIATIONS Glutamate

Carboxypeptidase

phosphonomethyl

II

pentanedioic

(GCPII), acid

N-acetyl-aspartyl-glutamate

(2-PMPA),

PivaloylOxyMethyl

(NAAG),

2-

(POM)

and

isoPropylOxyCarbonyloxymethyl (POC), 2-(3-MercaptoPropyl)Pentane-dioic Acid (2-MPPA)


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Table 1. Structures and in vitro metabolic stability studies of prodrugs of 1 in mouse and human plasma and liver microsomes.

O

R2 O

O

POC =

O

P O R1

O

O R1

O R3

O

POM =

Microsomal Stability (% remaining at 1h) Mouse Human H H H 100 91 1 H Me Me 101 103 5b H Et Et 83 88 5c * POC H H 7 POM Me Me 0 0 9 POC Me Me 0.1 0 10 POM Et Et 0 0 11 POM Me H 0 54 15a POM Et H 0 26 15b POC Et H 0 32 15c POM POM H 0 0 21a POC POC H 0 0 21b * Compound 7 was chemically too unstable for biological testing Compound

R1

O

O

R2

R3


O

Plasma Stability (% remaining at 1h) Mouse Human 85 83 86 96 59 73 0 29 0.1 0 0 18 0 82 3 61 0 80 0 79 0 48


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Page 36 of 44

Scheme 1. Synthesis of compound 7, the bis-POC phosphonate prodrug of 1a

O

O OBn 5a

O

O a

O HO P HO

a

OBn O

O O O

O O P O

O

OBn

O

O b

O OBn

6

O O O O

O O P O

O

OH

O OH

7

Reagents and conditions: (a) POC-Cl, DBU, dioxane, 80°C, 39%; (b) Pd(OH)2, H2, THF r.t.,

97%.


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Scheme 2. Synthesis of the bis-POM and bis-POC dialkyl esters 9-11a O O EtO P EtO

O

OR1 a O OR1

8a (R1 = Me) 8b (R1 = Et)

O HO P HO

O

OR1 b O OR1

5b (R1 = Me) 5c (R1 = Et)

O O P R2 O

R2

OR1

O OR1

9 (R1 = Me, R2 = POM) 10 (R1 = Me, R2 = POC) 11 (R1 = Et, R2 = POM)

a

Reagents and conditions: (a) TMS-Br, DCM, 0°C, 73% for 5b and 60% for 5c; (b) R2-X, DBU, dioxane, 80°C, 40% for 9, 37% for 10, 84% for 11.



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Page 38 of 44

Scheme 3. Synthesis of the bis-POM and bis-POC alkyl esters 15a-ca

O

O

OBn

OBn

a O OR1 12a (R1 = Me) 12b (R1 = Et)

a

O EtO P EtO

O b, c

O OR1

13a (R1 = Me) 13b (R1 = Et)

O O P R2 O

R2

OBn

O d

O OR1

14a (R1 = Me, R2 = POM) 14b (R1 = Et, R2 = POM) 14c (R1 = Et, R2 = POC)

O O P R2 O

R2

OH

O

OR1 , = a e 15 (R1 M R2 = POM) 15b (R1 = Et, R2 = POM) 15c (R1 = Et, R2 = POC)

Reagents and conditions: (a) HPO(OEt)2, AlMe3, DCM, 0°C to r.t., 94% for 13a, 94% for 13b;

(b) TMS-Br, DCM, 0°C; (c) R1-X, DBU, dioxane, 80°C, 40% for 14a, 32% for 14b, 38% for 14c; (d) Pd(OH)2, H2, r.t., 90% for 15a, 95% for 15b, 94% for 15c.


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Scheme 4. Synthesis of tris-POM and tris-POC prodrugs 21a and 21b.a

OBn

O

b, c

a O

O

O O

O d

O EtO P EtO

O OH

O

O

OBn

O

OBn

O

18 17

16

O e

O RO P RO

OBn f O OR

20a (R = POM) 20b (R = POC)

a

O O RO P RO

O EtO P EtO

OBn

O

OR = a 19 (R POM) 19b (R = POC)

OH

O OR

21a (R = POM) 21b (R = POC)

Reagents and conditions: (a) Eschenmosher’s salt, t-BuOH, 65°C, 80%; (b) HPO(OEt)2, AlMe3,

DCM, 0°C to r.t., 94%; (c) TFA, r.t., 88%; (d) POC-Cl or POM-Cl, K2CO3, acetonitrile, 50°C, 70% for 19a and 72% for 19b; (e) TMS-Br, DCM, 0°C, POM-Cl or POC-Cl, AgCO3 82% for 20a and 92% for 20b; (f) Pd(OH)2, H2, r.t., 98% for 21a and 98% for 21b.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

HO

O

HO

HO

O

γ O HO P HO

α

S OH

O , 1 (2 PMPA)

HS

OH O 2, (2 MPPA)

HO O

Page 40 of 44

HO

O

O N N H H O , 3 (DCMC)

OH

HO

H N

OH O

O 4

Figure 1. Representative examples of GCPII inhibitors from 4 distinct chemical classes


O

Page 41 of 44

80

M e ta b o lite s ( m M )

P r o d r u g + A ll R e s p e c t iv e

A

60

40

20

25

20

15

10

5

b 2

1

a 1 2

1

5

c

b 5 1

1

5

a

1 1

0 1

9

c 5

b 5

1

0


b 2

Com pound #

B

Com pound #

1

a 1 2

1

1

5

c

b 5

5

a

1 1

1

1

0

9

c 5

b 5

1

0

2 -P M P A ( m M )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 44

Figure 2. Single time point pharmacokinetics of orally administered prodrugs in mice. Total exposure of the prodrugs and all respective metabolites (A) or specifically released 2-PMPA (compound 1) (B) at 30 minutes following 30 mg/kg p.o. prodrug (equivalent dose of compound 1) in mice. Compared to oral administration of 1 which provided less than 1µM, the prodrugs afforded 5-40µM concentrations, likely due to enhanced permeability through the intestinal mucosa. Although good oral exposure of the prodrugs and metabolites was observed, most did not release significant levels of compound 1, with the exception of 21a and 21b that provided >15mM plasma levels.


MICE

DOG

Page 43 of 44

1 21b

Cmax

Tmax

AUC(0-t)

Cmax

Tmax

AUC(0-t)

(µM)

(h)

(µMol/h/mL)

(µM)

(h)

(µMol/h/L)

0.86 15.1

1 0.5

1.94 35.9

51.1

1

126

B

25

2 1 b p .o . 1 p .o .

20

15

10

5

0 0

1

2

3

4

5

2 - P M P A P la s m a C o n c . ( m M )

A 2 - P M P A P la s m a C o n c . ( m M )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60

40

20

0 0

2

4

6

8

10

T im e ( h r )

T im e ( h r )

Figure 3. In vivo pharmacokinetics of compound 1 following oral administration of 1 and 21b in mice and dog. (A) 1 and 21b were dosed in mice at 30 mg/kg via oral gavage and plasma concentration of compound 1 were evaluated via LC/MS. While oral administration of compound 1 provided very low plasma levels (50mM.



Table of Contents Graphic

O O

O O

O

P OH

20

OH

O

HO

OH

O

O

OH O

2-PMPA (1)

O

P O

O O

O

O O

O O

Tris-POC-2-PMPA (21b)

oral dosing in mice

[2 -P M P A ] (m M )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 44

2 - P M P A fr o m 2 1 b

15

10

5

2 P M P A fr o m 1

0 0

1

2

3

T im e ( h r )


4

5

Discovery of Orally Available Prodrugs of the Glutamate

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