The diverse pharmacology and medicinal chemistry of phosphoramidates – a review

Abstract

The phosphoramidates consist of compounds which possess at least one amino group bound directly to the phosphorus atom and are, therefore, phosphoramide acid derivatives. The inherent chemical properties of the element phosphorus include polarizability, low to medium electronegativity and derivatives exhibit low coordination numbers thereby allowing synthesis of a diverse range of compounds. In line with their physicochemical properties, phosphorus compounds have widespread industrial applications and also demonstrate a diverse range of biological activities. In the last two decades, notably, phosphoramidates have been evaluated for both their antitumor and antiviral efficacy. This brief review describes the most promising examples of this class which possess antiviral, antitumor, antibacterial, antimalarial and anti-protozoal activity, as well as urease, acetyl and butyrylcholinesterase enzyme inhibitor activity.

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Fabrício M. Oliveira

Fabrício Marques de Oliveira was born in 1983. In 2008 obtained a Magister Scienciae Degree in Agrochemistry from Universidade Federal de Viçosa (UFV). He's currently working on his PhD thesis under supervision of Dr Luiz Cláudio de Almeida Barbosa at UFV. Recently he was admitted as Professor at Instituto Federal de Educação, Ciência e Tecnologia de Minas Gerais, in Brazil. His research interests include: synthesis of compounds with potential biological activity, synthesis of phosphorous compounds, and use of theoretical calculations by density functional theory (DFT).

Luiz C. A. Barbosa

Luiz C. A. Barbosa, received a BSc (1981) and MSc (1986) from Federal University of Viçosa (UFV – Brazil). In 1991 he completed his Ph.D. in Organic Synthesis at the University of Reading (UK) and spent a sabbatical year (2008–2009) in the group of Professor Timothy J. Donohoe at the University of Oxford. He started his academic career as an auxiliary professor at UFV (1983) rising to Professor in 1997. Since 2012 he has been a Full Professor at Federal University of Minas Gerais. His research interests include the synthesis of biologically active compounds, analogues of natural products. More details at http://www.luizclaudiobarbosa.com.br

Fyaz M. D. Ismail

Fyaz M. D. Ismail completed his Ph.D. in Physical Organic Chemistry in 1989 (Salford University with Professor Roger Bisby) and went onto “green” the highly polluting Kelly process (by converting it from batch to flow; Monsanto Research Fellow at Reading University with Dr Neal Stuart Isaacs: High Pressure Chemistry Unit). After a decade as senior lecturer in Spectroscopy and Medicinal Chemistry (University of Hertfordshire), he became subject leader at the School of Pharmacy (LJMU). His research interests include modulating free radical processes in disease by perfecting weakly active substance (inspired by natural products) into clinically useful medicines using rational drug design.

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Introduction

The element phosphorus was discovered in 1669 by Henning Brandt and since then, significant discoveries have been made regarding its biological function.¹

The advent of suitable synthetic methodology coupled with advances in analytical science has accelerated research into both isolating and designing both organic and organometallic compounds with ever more diverse applications. Structural incorporation of a phosphorus atom within biomolecules essential for life, notably DNA and RNA, are especially abundant in cells. This element plays a pivotal role within ATP, which is used by most living organisms for storing and translocating energy (Fig. 1). Phosphorus compounds also serve as cofactors of multiple enzyme systems whereas others act as essential buffering agents within the cellular mileau.^1,2

Fig. 1 Fragment of an RNA molecule and the structural formula of adenosine triphosphate (ATP).

The inherent properties of the element phosphorus include: (a) polarizability, (b) low to medium electronegativity and (c) low coordination number, thereby, allowing the synthesis of a diverse range of compounds. Biological and medicinal activities of organophosphorus compounds are modulated, in part, by the nature of the element bound to phosphorus and variations in its own oxidation state. In the last few decades, the phosphoramidates in particular have attracted the attention of many investigators. This class is characterized by having at least one –NR¹R² group (where R¹ and R² = alkyl, aryl, heteroaryl) bound to phosphorus, itself derived from phosphoramidic acid (Fig. 2).²

Fig. 2 (a) Tetrahedral structure of phosphoramidic acid; (b) planar structure of carbamic acid.

Amongst the large array of activities and properties reported for phosphoramidates, the following are particularly noteworthy: biological activities and potential agricultural applications,^3–5 flame retardant properties,^6,7 their use as antirust additives within lubricating oils⁸ and, notably, pharmaceuticals used for treating and controlling various diseases.^1,9–31 Considering the increasing interest in this class of compound, especially within the pharmaceutical arena, this review discusses the most significant examples of phosphoramidates imbued with antiviral, antitumor, antibacterial, anti-malarial, anti-protozoal activities, as well as examples of selected compounds that act as inhibitors of urease, butyryl and acetyl cholinesterase. Hopefully, this review will encourage further attention upon this class of compounds, especially that involving the discovery of new drugs for the treatment of various diseases.

The use of vaccines during the last decades has assisted humankind in the fight against diseases caused by viruses such as rubella, measles, mumps and polio. However, for some viral pathogens such as the human papilloma virus (HPV) and human herpes virus (HHV), amongst others, currently, there is neither any small molecule (denoted here as drug) nor vaccine effective for managing such infections. For other diseases, such as herpes simplex virus (HSV) and Varicella–Zoster virus (VZV), although some effective drugs are available, in many cases off target-side effects in some patients can pose problems, and in such cases, it is imperative to discover new, more efficacious drugs.^28,32 For instance, two-drugs Telaprevir and Boceprevir were approved in 2011 for treating hepatitis C virus infection (HCV) in genotype 1 patients. However, these drugs do not exert effect on genotypes 2–6.²⁸ In these cases, the treatment also requires regular injections of pegalated α-interferon (PEG-IFN)† with daily oral administration of ribavirin (a nucleoside analogue which possesses useful and broad antiviral activity). However, daily administration places additional burden upon the patient arising from management of uncontrolled side effects (Fig. 3). Amongst the large array of possible scaffolds for antiviral drugs development, the phosphoramidates promise considerable potential for development into putative drugs.²⁸

Fig. 3 Structure of Telaprevir, Boceprevir and Ribavirin, drugs approved for treating HCV infection in genotype 1 patients.

Nucleoside analogs are considered an important class of inhibitors of the viral polymerase of several therapeutic targets especially HCMV, HSV, HIV and HBV.³³ Recently some investigators have evaluated phosphoramidate groups covalently linked to nucleosides, a strategy known as ‘phosphoramidate protide’.^{12–14,16,19,20,24,26,31,34–38} This approach was introduced by McGuigan et al. (1992) as a means of improving the therapeutic potential of a prototype drug.²³ The introduction of this group favors the phosphorylation process in vivo, by converting the nucleosides into their active forms as triphosphates derivatives, commonly known as nucleotides. These phosphorylated analogues have significantly advanced both the chemotherapy of neoplasms and antiviral treatments.^23,39 McGuigan et al.¹⁹ have synthesized several phosphorylated nucleoside analogs that proved to be efficient phosphate releasing agents. The phosphoramidate derivatives of 4′-azidocytidine (R1479) were effective in inhibiting hepatitis C virus (HCV). From such compounds, pro-nucleoside derivatives were evolved that were up to 3.4 times more potent (1; EC₅₀ = 0.38 μM) than the standard compounds (R1479; EC₅₀ = 1.28 μM) (Fig. 4). Sofosbuvir (Fig. 4) is a drug for the treatment of HCV approved by FDA in December of 2013. This compound suppresses various HCV genotypes in patients exposed to the drug for up to 12 weeks without causing any serious side effects. Clinical trials revealed a cure rate of 90% when sofosbuvir (Fig. 4) is combined with peginterferon and ribavirin. Notably, with conventional treatment that utilizes only peginterferon and ribavirin, the cure rate is less than 50%.⁴⁰

Fig. 4 Structural formulas of compounds with anti-HCV activity.

Acyclovir^® (also commercially available as Zovirax^®) and a series of derivatives were synthesized by Derudas et al.³⁴ Acyclovir (ACV), a synthetic purine nucleoside analogue, possesses inhibitory activity in vitro and in vivo against human herpes viruses including herpes simplex virus (HSV) types 1 and 2, Varicella–Zoster virus (VZV), Epstein–Barr virus (EBV) and cytomegalovirus (CMV).⁴¹ ACV is considered inactive against virus HSV thymidine kinase deficient (HSV-1 TK⁻) (EC₅₀ = 50 μM), whereas in the case of nucleoside, pro-activity was increased 35 fold (EC₅₀ = 1.4 μM) (Fig. 5). As for activities against HSV-1 and HSV-2, the synthetic compounds proved slightly less active than the standard product ACV employed as a control. Against HSV-1, ACV revealed an EC₅₀ = 0.4 μM, whilst for compounds 6–9 the activities were as follows: compound 6, EC₅₀ = 0.9 μM; 7, 1.4 μM; 8, 0.8 μM; 9, 1.1 μM. For HSV-2, ACV had an EC₅₀ = 0.2 μM and for the synthetic phosphorous compounds the EC₅₀ values were: compound 6, 0.5 μM; 7, 1.6 μM; 8, 0.7 μM; 9, 1.1 μM. In the same study, the investigators found that pro-nucleoside derivatives 10–16, incorporating the amino acid alanine, showed high activity (EC₅₀ values lay between 0.8 and 42 μM) on three cultures of HIV (HIV-1 CEM, HIV-2 CEM and MT-4) (Fig. 5), whilst ACV proved relatively inactive (EC₅₀ > 250 for all cultures). The pro-nucleosides 6–9 were also evaluated against HIV cultures, but failed to demonstrate any useful activity against these infections.³⁴

Fig. 5 Structure of Acyclovir® and synthetic pro-nucleosides actives against HSV-1 TK⁻ and HIV viruses (Derudas et al. 2009).³⁴

Phosphoramidate 2′-C-methyl guanosine monophosphate pro-drugs 18a–i, were active against HCV (Fig. 6). Notably, the addition of a phosphorous group to nucleosides (17a–i) enhanced biological activity over 100 fold. Amongst the compounds synthesized, the insertion of the amino acid alanine produced the most favorable modification, with EC₅₀ lying within the nanomolar range (Fig. 6).²⁰ The phosphoramidate derivatives (20–30) of 4′-azidouridine (AZU, 19, Fig. 7), synthesized by Perrone et al.,²⁶ were up to 450 times more active against HCV when compared with their corresponding, non-phosphorylated, precursors. Data are presented as EC₅₀ values (representing the concentration of compounds reducing HCV replication by 50%) and CC₅₀ values (representing the concentration of compounds reducing cell viability by 50%) as determined using the WST assay. All phosphoramidates tested (20–30) were non-toxic towards HCV in the replication assay (CC₅₀ > 100 μM). In this series, the most active compound was 20 (EC₅₀ = 0.22 μM), with the phosphorus atom in either the R or S configuration. In general, it was observed that compounds bearing naphthyl group (20–22) were more active when compared with their corresponding analogues (23–30) bearing a phenyl group (Fig. 7).

Fig. 6 HCV Replicon activity of 6-substituted 2′-C-methylguanosine nucleosides (17a–i) and ProTides (18a–i).

Fig. 7 HCV Replicon activity of 4′-azidouridine nucleosides derivatives (20–30).

In an attempt to increase the concentration of the bioactive ribavirin monophosphate in the cells, some phosphorylated derivatives of ribavirin (Fig. 8) were prepared.¹² Subsequent pharmacological evaluation revealed that compound (31) was active against vesicular stomatitis virus (EC₅₀ = 20 μM), vaccinia virus (EC₅₀ = 100 μM) and influenza B (EC₅₀ = 14 μM); compound (32) was active against Punta Toro virus (EC₅₀ = 100 μM) and (33) showed a better effect against vesicular stomatitis virus (EC₅₀ = 20 μM), vaccinia virus (EC₅₀ = 20 μM) and Punta Toro virus (EC₅₀ = 58 μM). These values are comparable to the activity of ribavirin (vesicular stomatitis virus, EC₅₀ = 22 μM; vaccinia virus, EC₅₀ = 22 μM; influenza B, EC₅₀ = 9 μM; Punta Toro virus, EC₅₀ = 183 μM), clearly indicating that phosphorylation, in this case, did not improve the desired bioactivity. Studies using cell lysate coupled with molecular modeling investigations revealed a weak activation of ProTide in comparison with the free monophosphate, which may provide one explanation for the observed biological activity.

Fig. 8 Phosphorylated derivatives of ribavirine.

Stavudine (d4T), an analogue of thymidine, is the fourth antiretroviral drug to achieve world-wide commercialization.⁴¹ In 2011, Román et al. synthesized several diasteroisomericaly pure derivatives of d4T phosphoramidates (34–37). Antiviral activities against HIV-1 and HIV-2 were evaluated and the corresponding EC₅₀ against type HIV-1 (IIIB) are shown in Fig. 9. Since the phosphorous center is chiral, all compounds can exist in the S_P and R_P forms. All isomers were examined and, in general, compounds with S_P stereochemistry proved superior to the corresponding R_P analogues, compound 36 being a notable exception.³⁵

Fig. 9 Structural formulas of diasteroisomericaly pure derivatives of d4T phosphoramidates actives HIV-1 (IIIB).

Some reports have demonstrated that phosphoramidate derivatives of dioxolane sugar nucleosides (DOA) possessing pyrimidines improved the antiviral potency in vitro.^42,43 The phosphoramidates 38 and 39, synthesized by Bondada et al.⁴⁴ had evaluated their activities against HIV-1 and HBV. The compound 38 displayed up to a 1070-fold greater potency versus HIV-1 compared to their corresponding parent nucleoside (40), and, notably, were up to 12-fold more potent versus HBV (Fig. 10). The improved and significant dual antiviral activity of these novel phosphoramidate nucleosides was partially explained by the increased intracellular formation of the adenosine dioxolane triphosphate. Cytotoxicity was absent for compound 38 when tested at concentrations of 100 μM in human PBM cells, Vero cells (kidney epithelial cells from the African green monkey) and CEM cells (a human-T-cell-derived cell line), whereas compound 39 showed a value of CC₅₀ = 65 μM for Huh7 cells (Human Hepatoma Cell).

Fig. 10 Adenosine dioxolane nucleoside phosphoramidates as antiviral agents for inhibiting Human Immunodeficiency and Hepatitis B Viruses.

Recently, Cho et al.⁹ have formulated a structure–activity relationship (SAR) of C-nucleosides containing the 2′-C-Me as putative NS5B inhibitors. In search of compounds with a better selectivity compared to their precursors, structural changes were introduced within both the heteroaromatic portion base and sugar moiety, coupled with a monophosphate prodrug approach. Judicious modification of pharmacokinetic parameters led to the discovery of phosphoramidate GS-6620 (Fig. 11), the first C-nucleoside clinical candidate. During phase I clinical trials GS-6620 showed potent anti-HCV activity in HCV-infected patients, but with an unexpectedly high variability in PK/PD investigations.¹⁰

Fig. 11 Phosphoramidate prodrug possessing HCV Replicon activity.

In a study aimed at improving efficacy against HCV, several phosphoramidate derivatives 2′-methycytidine were prepared (Fig. 12). Although valopicitabine 1 (NM283) proved highly efficacious, phase II clinical trials revealed some undesirable side effects. In the case of derivative 41, a thirty fold increase in activity was observed in the replication test, whilst compound 42 proved to be only ten times more active. However, measurements of triphosphate levels in mouse liver after oral administration, showed low levels of nucleoside triphosphate (NTP) for both compounds. In contrast, compound 41, after six hours of subcutaneous administration,¹³ exhibited high levels of NTP.

Fig. 12 Structure of NM283 and 5′-phosphoramidate prodrugs of 2′-C-methylcytidine inhibitors of NS5B.

The anti-influenza activity of 6-modified 2′-fluoro-2′-phosphoramidate deoxyriboside ProTides was evaluated in vitro (Fig. 13). Compounds 43–46 showed EC₉₉ value ranging from 12 μM to 15 μM. Xenobiotic metabolism experiments with carboxypeptidase Y and whole-cell lysates demonstrated that the 2′-fluoro-2′-deoxyguanosine ProTides are readily cleaved by cellular enzymes to release the 6-modified 2′-fluoro-2′-deoxyguanosine 5′-monophosphate. Further conversion into 2′-fluoro-2′-deoxyguanosine 5′-triphosphate, the active metabolite that inhibits the viral polymerase then occurs. Antiviral results were supported by drug metabolism studies and calculations suggest that the c log P value (in the range of 2–3) may be favourable. These compounds proved inactive against HSV-1, HSV-2 TK⁻-deficient HSV-1, as well as vaccinia and vesicular stomatitis viruses.²⁵

Fig. 13 Phosphoramidate ProTides with anti-influenza virus activity.

The synthesis of numerous phosphoramidates with antiviral activities has been described in the literature. Recently, Sofia et al.²⁸ published a review on inhibitors of hepatitis C virus NS5B RNA-dependent RNA-polymerase highlighting in particular the phosphoramidates. Several other research groups are also engaged in the search for promising bioactive phosphorylated molecules but have only achieved sporadic success.^{13,14,16,25,29,31,36–38,43,45–47}

Antitumoral activity

Cancer is a disease characterized by abnormal cell division and migration, accounting for approximately 13% of all human deaths worldwide. In 2008, 12.4 million cases and 7.6 million deaths have been reported. The forecast is that by 2030 there will be 27 million cancer patients.^48,49 Consequently, there is an urgent need to develop new, cost effective and safe drugs (chemotherapy agents) which may be used in combination with surgery and radiation therapy to reduce overall mortality.

Notably, “phosphoramidate protide” has also been used as a strategy for the development of compounds possessing antitumor activity. This strategy combines, generally, the advantage of high water solubility in many cases, with low toxicity (to normal cells) of such compounds.⁵⁰ For instance, brivudine (BVdU) (Fig. 14), a potent inhibitor of herpes simplex virus type 1 (HSV-1) and Varicella–Zoster virus (VZV), has served as a model for the development of newer, active, prototypes drugs.^51,52 The incorporation of a phosphoramidate group within this scaffold led to the discovery of thymectacin (NB1011, 47a) by the company New Biotics. This compound selectively acts on tumor cells that express high levels of thymidylate synthase.^53,54 Subsequently, the replacement of the methyl by a benzyl group within thymectacin (47a) increased the potency against colon cancer HT115 by 175 fold (47b, Fig. 14).²⁴ Congiatu et al.¹¹ have synthesized a number BVdU phosphoramidate derivatives (48–54) (Fig. 14) possessing high cytotoxic activity against the cell lines MDAMB231 (breast cancer) and PC-3 (prostate cancer) cell lines. Compounds 48–50 and 53, tested as diastereisomer mixtures, were the most active on MDAMB231 cells, with EC₅₀ ranging from 0.32 μM to 2.7 μM whilst compounds 49–54 showed EC₅₀ values in the range of 1.4–6.9 μM upon PC-3. These compounds were more active than the standard control NB1011 (EC₅₀ = 79 and 155 μM on MDAMB231 and PC-3, respectively). For compound 53, the mixture was separated by reversed phase chromatography and each diastereoisomer was tested separately against the MDAMB231 (breast cancer) cell line. The rapidly eluting diastereoisomer (R stereochemistry on P center) was less active than the mixture with an EC₅₀ = 7.4 μM, whilst the slow eluting diastereoisomer (S stereochemistry on the P center) was 10 times more potent than the mixture, with an EC₅₀ = 0.5 μM highlighting the importance of investigating pharmacology of individual stereoisomers.

Fig. 14 Structure of brivudine (BVdU) and various pro-nucleoside derivatives.

Some fluorinated pyrimidine nucleosides⁵⁵ have been used as chemotherapeutic agents since 1957 in the treatment of ovarian, breast and gastrointestinal tumors.^56,57 The fluorinated pyrimidine 5-fluorouracil-2′-deoxynucleoside (FUDR) is active against liver metastases, and is beneficially metabolized within the host.⁵⁸ In recent studies, McGuigan et al.²² reported the synthesis of 39 FUDR phosphate pro-drugs derivatives (Fig. 15) and measured their relative cytotoxic activity within tumor cell lines L1210/0, L1210/TK⁻, Cem/0, Cem/TK⁻, HeLa, HeLa/TK⁻. The effect on L1210/0 infected with Mycoplasma hyorhinis (L1210.Hyor) was also reported. In general, the compounds showed ability to release the nucleoside monophosphate within intact cells whilst maintaining activity in tumor cell cultures infected with mycoplasma. The ProTide's stability in both acidic and neutral pH, as well as host plasma, is of great importance, especially considering that some compounds have proven cytotoxicity within the nanomolar range.

Fig. 15 General structure of FUDR prodrugs described of McGuigan et al.²²

In 2013, Lewandowska et al.⁵⁹ reported the synthesis of 4-chlorophenyl N-alkyl phosphoramidate diesters of 3′-azido-2′,3′-dideoxy-5-fluorouridine (AddFU) (Fig. 16) and evaluation of their cytotoxic activity in three human cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7). The most active compound for all cell lines was compound 56, which displayed IC₅₀ = 1.64 μM, IC₅₀ = 1.64 μM and IC₅₀ = 1.88 μM for HeLa, KB and MCF-7 cells, respectively. This compound was more potent than the parent AddFU (IC₅₀ = 11.06 μM, IC₅₀ = 8.11 μM and IC₅₀ = 10.32 μM, respectively). Phosphoramidate 56 also exhibited relatively high activity for these cell lines, displaying IC₅₀ = 4.21 μM for HeLa, IC₅₀ = 3.81 μM for KB and IC₅₀ = 5.41 μM for MCF-7. The authors suggested that the mechanism of inhibition of these compounds differs from that described by McGuigan et al.,²² when devoid of the ester motif, which initiates the hydrolysis process to release the nucleoside analogue 5′-monophosphate.

Fig. 16 3′-Azido-2′,3′-dideoxy-5-fluorouridine phosphoramidates with anticancer activity.

Other notable examples of compounds demonstrating useful antineoplastic activities include triethylenethiophosphoramides and cyclophosphamides (Fig. 17). For instance, Thio-TEPA^®, which has been previously used against a variety of neoplasms, demonstrated consistent efficacy against breast and ovarian adenocarcinomas as well as superficial papillary bladder carcinoma. The pharmacological effect is considered similar to mechlorethamine hydrochloride (57), the first systemic chemotherapeutic agent to be approved in the United States for the treatment of Hodgkin's disease, lymphosarcoma, chronic myelocytic or lymphocytic leukemia, polycythemia vera, bronchogenic carcinoma and mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma (CTCL).^1,60 The cyclophosphamide (2H-1,3,2-oxazaphosphorin-2-amine, N,N-bis(2-chloroethyl)tetrahydro-2-oxide) is the active principle of Endoxan^®. This genotoxic nitrogen mustard is used in the treatment of many cancers and is also endowed with marked immunosuppressant properties.^1,61 In an attempt to alleviate carcinogenic potential and find more selective agents, it has served as a template for the synthesis of TH-302 (Fig. 17). This nitro-1H-imidazole has proven effective in the control of pancreatic tumor cells MIA PaCa-2, whilst also surviving microsomal metabolism. In addition, when combined with Gemcitabine^®, it has proven particularly effective when tested in mice. Such attractive preclinical properties of TH-302 have prompted phase 1 clinical trials for the treatment of solid tumors.⁶² The presence of the nitro group may engender activity in hypoxic conditions found in these neoplasms. Another series of compounds derived from cyclophosphamide, namely trofosfamide (58), mafosfamide (59), and ifosfamide (60) (Fig. 17) represent some of the most widely used anticancer chemotherapeutic agents.¹

Fig. 17 Chemical structures of some highly effective anticancer agents.

Wang et al.³⁰ prepared phosphoramidate derivatives of haematoporphine dihydrochloride (HPD), a drug with anti-tumor properties marketed under the name Apiabasilon®. For instance, compound (61), shows selectivity against the liver tumor cancer cell line (BEL-7402), possessing activity in the micromolar range in vitro (IC₅₀ = 8.41 μM; Fig. 18).

Fig. 18 Structure of the monomeric photosensitiser found within the mixture denoted PdD⁶³ and its phosporamide analogue of chlorin (61). The IC₅₀ reported are against the liver tumor cell line BEL-7402.

Some highly potent anticancer heterocyclic phosphoramidates were synthesized by Kiran et al.¹⁵ Tests in vitro, involving three human tumor cell lines NCI-H460 (large cell lung), MCF-7 (breast adenocarcinoma) and SF-268 (central nervous system glioblastoma), cyclic compounds like 62 (IC₅₀ = 2.8–4.5 μM) showed greater activity when compared to acyclic products such as 63 (IC₅₀ = 71.3–90.4 μM) (Fig. 19). The investigators did not offer an explanation as to why such cyclic compounds were highly active, and considering the limited number of analogues prepared, further investigations, especially those involving substitutions on the aromatic rings may produce compounds with superior activity.

Fig. 19 Anticancer phosphoramidates reported by Kiran et al. (2008).¹⁵

Ghosh et al.⁶⁴ reported that 7-benzylguanosine monophosphate (GMP-7BN) (64) was a potent antagonist of eukaryotic initiation factor 4E (eIF4E) (K_d = 0.8 μM). However, this compound possesses a negative charge at physiological pH preventing penetration across the cell membrane.⁵⁰ Consequently, phosphoramidate derivatives of 64 were prepared and the pro-drug 65 was also active against eIF4E (Fig. 20). Furthermore, it was found that the zwitterion 65 was very efficient in crossing the membrane of MDA-231 breast cancer and lung cancer (H460, H383 and H2009) cell lines. Hydrolysis of the indolyl side chain once inside the cells provided the active anion form 64 which was then entrapped within the cells.⁶⁵

Fig. 20 Structures of 7Bn-GMP and 7Bn-GMP pro-drug.

Antibacterial activity

The incidence of clinically multi-resistant antibiotics bacterial infections caused by bacteria has led to an unacceptable increase in mortality rates. An absence of effective treatments makes it vitally important that both health professionals and the public are sufficiently informed to take necessary precautions so as to maximize the effectiveness and lifetime of currently available antibiotics. Meanwhile, both the pharmaceutical sector and academic investigators continue to search for both novel targets and mechanisms of drug action rational drug design thereby facilitating development of new and effective antibacterial agents.^66–68 In this context, amongst the several classes of compounds with putative antibacterial activity, phosphoramidates possess considerable potential for further development.

In 2011, Gholivand and Dorosti⁶⁹ reported the synthesis of two new N-phosphinyl ureas (Fig. 21) and evaluated their antibacterial activities against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella typhi using the disc diffusion method and also obtained the minimum inhibitory concentration (MIC) for each compound. Compound 66 exhibited high activity against all of the microorganisms under test (MIC = 1.17–18.8 μg cm⁻³ comparable with MIC of genatamycin used as a positive control) whilst compound 67 was virtually inactive (MIC > 385 μg cm⁻³).

Fig. 21 Two novel N-phosphinyl ureas with antibacterial activity.

Prasad et al.²⁷ have synthesized a series of heterocyclic phosphoramidates (68–76), which showed moderate activity against two Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium and Klebsiella pneumoniae). Their antibacterial activity was evaluated at concentrations of 20 μg mL⁻¹ and 40 μg mL⁻¹ with Ciprofloxacin^® a commercial quinolone antibiotic, as a comparator (Fig. 22). Compounds 71–74 inhibited bacterial growth at a concentration of 20 μg mL⁻¹ for both Gram-positive and Gram-negative bacteria. Compounds 73 and 74 exhibited the highest activity. Other diphenylphosphoramidates (77–78), have been prepared by Madhava and coworkers.⁷⁰ When tested against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, they were shown to be twice as effective as Cyprofloxacin^® (Fig. 22).

Fig. 22 Structural formulas of Cyprofloxacin^® and some synthetic phosphoramidates with antibacterial activity.

Antiurease activity

Urease is a nickel-dependent enzyme present in various organisms, such as plants, fungi and bacteria, especially Proteus mirabilis, Klebsiella aerogenes and Helicobacter pylori and, consequently, is of considerable biological significance considering its ubiquity in organisms causing diseases of the gastrointestinal and urinary tracts. By neutralizing gastric acid through ammonia production from urea hydrolysis, H. pylori, can maintain 50% to 60% of the metabolic activity at pH = 2.5 (ref. 71–74) and is considered the leading cause of stomach ulcers. It has been estimated that around half of the world population is infected with this microorganism, and in some developing countries this can affect up to 85% of the adult population.⁷² During the search for novel enzyme inhibitors of urease phosphoramidates showed excellent activity. Amongst putative urease inhibitors, N-(but-1-yl)thiophosphoric triamide (NBPT, Fig. 23) excels in inhibiting the enzyme.^71,74 NBPT is a pro-drug since it has to be oxidized to unmask the active form: N-(but-1-yl)phosphoric triamide (NBPTO, Fig. 23).⁷¹ In an in silico investigation of the active site of urease from H. pylori, a molecular modelling study of phenylphosphorodiamidate (PPD, Fig. 23) demonstrated that this compound interacts with His221 through a hydrogen bond involving its oxygen atoms. A hydrophobic interaction between the benzene group with residues of Ala168, Ala169 and Ala365 (ref. 75) is also involved. A deeper understanding of such non-covalent interactions may well lead to the design of superior inhibitors.

Fig. 23 Chemical structures of phosphoramidates efficient as urease inhibitors.

During 2008, Domínguez et al.⁷⁶ described the synthesis of various phosphoramidates all highly active in the nanomolar range against Jack bean urease in vitro (Fig. 24). The reference compounds NBPT and PPD used, showed IC₅₀ equal to 100 and 3 nM, respectively. Although no clear structure–activity relationship was found, this series of compounds represents a useful template for further investigation as these are currently the most potent urease inhibitors reported so far.

Fig. 24 Aromatic phosphoramidates with high urease inhibitory activity.⁷⁷

Miscellaneous

As this survey has shown, the phosphoramidates encompass a diverse range of biological activities. These have, therefore, led several investigators to explore the potential application of this class of compounds against tropical diseases especially malaria and leishmaniasis. Recent investigations include synthesis of nitrobenzyl phosphoramide mustards (compounds 79–90, Fig. 25) that proved active against Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. These protozoan parasites cause major health problems in many countries of the developing world spread across three continents.^77–79 The highest biological activity was associated with compounds 84 and 85, which showed IC₅₀ values in the nanomolar range against T. brucei.⁸⁰ Compound 86 revealed selectivity towards T. brucei⁸¹ and served as a prototype for the development of novel phosphonates with EC₅₀ lying between 1.7 and 4.2 μM (structures not shown).⁸² Disappointingly, these compounds showed low bioavailability due to their inability to cross cell membranes, therefore, only reaching the site of drug action in sub-therapeutic concentrations in vivo. Thus, Ruda et al.⁸³ prepared a series of phosphoramidate derivatives 86 with better pharmacokinetic properties. Among these compounds 87–90 showed EC₅₀ values equal to 90, 40, 50 and 8 nM, respectively, against T. brucei.

Fig. 25 Compounds with activity against T. brucei.

Malaria is a parasitic disease caused by the protozoan of the Plasmodium genus, with the most virulent species been Plasmodium falciparum. This disease is responsible for about 800 000 deaths per year, many of them including children and pregnant women, and most of them in sub Saharan Africa.⁸⁴ Amongst a number of compounds described by Mara et al.,¹⁸ 91–94 showed good efficacy against P. falciparum (IC₅₀ = 39–50 μM) (Fig. 26). The bio-isosteric replacement of isopropylamino group by other alkylamino substituents resulted in new more pharmacologically potent phosphoramidates 95–104 (Fig. 26). Amongst such compounds, 95, 97, 99 and 104 (IC₅₀ = 17–32 μM) proved most active.¹⁸ Other examples of phosphoramidates (106–109) with antimalarial activity were published recently which employed diphosphonic acid 105 as the lead structure.⁸⁵ Compound 108 (IC₅₀ = 3.4 μM) was more potent than 106 (IC₅₀ = 6.6 μM), 107 (IC₅₀ = 9.7 μM) and 109 (IC₅₀ = 42 μM) cells on D6 (wild-type P. falciparum strain proved most sensitive to these drugs). In cell lines W2 (chloroquine-and pyrimethamine-resistant P. falciparum strain), compound 108 showed IC₅₀ value of 4.0 μM as 106, 107 and 109 had values equal to 7.7, 7.1 and 48 μM, respectively. Compound 109 has little or no effect since it was cytostatic (CC₅₀ > 300 μM) on the cell lines A549 (human lung carcinoma cells), C32 (human melonoma cells) and C32TG (and thioguanine-resistant mutant of HGPRT-deficient cell line C32). The pro-drug 107 shows low levels of cytotoxicity against C32 line cell (CC₅₀ = 130 μM) and C32TG (CC₅₀ = 109 μM). According to the authors, these values are not considered cytotoxic, since these pro-drugs fail to enter mammalian cells. Compound 105 (Fig. 26) represents a promising prototype for the development of new phosphoramidates as pro-drugs which may well possess useful antimalarial activity.⁸⁵

Fig. 26 Chemical structure of compounds with activity against P. falciparum.

The major health problems caused by parasites are currently confined to the developing world but climate change may well shift these zones of infection to the more developed areas. Sadly this area of world health has attracted less attention from the pharma sector than desired. In contrast, within several regions of the world, Alzheimer's disease has attracted considerable attention from both pharmaceuticals companies and academic institutions since increasing longevity has led to the emergence of such conditions. Currently, cholinesterase inhibitors (ChEI) are the main drugs commercially available for the treatment of Alzheimer's disease, which is a leading cause of dementia worldwide, with approximately 24 million cases reported annually.^86,87 The rationale behind the use of these inhibitors is based on the assumption that a cholinergic deficit is associated with the disease, and aims at increasing the availability of synaptic acetylcholine by inhibiting its main catalytic enzymes, acetyl (AChE) and butyrylcholinesterase (BChE).^86,88 In 2010, Gholivand et al.⁸⁹ synthesized and evaluated the effect of compounds 110–114 (Fig. 27) on the aforementioned enzymes. As a result, these compounds revealed a mixed type mechanism of action with moderate activity against BChE (IC₅₀ ranging from 0.43 to 2.45 mM) and AChE (IC₅₀ ranging from 1.95 to 18.62 mM).

Fig. 27 Chemical structure of compounds with activity against AChE and BChE.

Rational drug design using phosporamidates

The previous sections demonstrate that the incorporation of a phosphoramidate at a critical point within the agonist or antagonist is essential for success. During a drug design campaign, bioisosteric substitution should be carried out at a known xenobiotic transformation point inferred from studies in vitro using the relevant receptor, usually, but not exclusively an enzyme, which can now be achieved using recombinant methods. For instance, since folate hydrolase I and glutamate carboxypeptidase II, PSMA possess proteolytic activities toward γ-glutamyl folic acid derivatives and the neuropeptide N-acetylaspartylglutamate.⁹⁰ Studies involving PSMA substrate specificity revealed that acidic residues at the P1 and P19 positions are suitable targets for modification, and several folate-like and N-acetylaspartylglutamate-like dipeptides with modest hydrolytic efficiency were reported.⁹¹ Later, by adapting the dipeptide motif for PSMA targeting, Mallari et al. developed a library of tetrahedral phosphoramidates for PSMA inhibition (Fig. 28).⁹²

Fig. 28 Rational approaches towards the design of inhibitors active in vivo.

Through adopting a seco-analogue approach (molecular pruning), Wu et al. systematically identified several potent inhibitors⁹³ (Fig. 29 & 30) and proposed a pharmacophore model involving the zinc binding site which pseudo-irreversibly bind to PSMA.⁹⁴

Fig. 29 Computational docking of phosphoramidate inhibitors into the active site of PSMA binding with zinc represented by two spheres: (see panel a, inhibitor 1a: (((S)-2-benzamido-2-carboxylatoethoxy)oxidophosphoryl)-L-glutamate; yellow; re-rendered using PyMol from ref. 93; see original paper for details of interactions with relevant amino acids).

Fig. 30 Computational docking of phosphoramidate inhibitors into the active site of PSMA binding with zinc represented by two spheres: (see panel c, inhibitor 1j, phosphonato-D-glutamate, yellow; re-rendered using PyMol from ref. 93; see original paper for details of interactions with relevant amino acids).

An ¹⁸F labelled analogue of phosphoramidate (117, R⁴ = H) showed their suitability in PSMA-targeted delivery for prostate cancer using positron emission tomography⁹⁵ allowing them to track its biodistribution data in murine xenografts in vivo.⁹⁶ The uptake of ¹⁸F-fluorobenzamido-phosphoramidate in vivo 119 in both PSMA+ and PSMA− tumor models by PET and ex vivo biodistribution study revealed that phosphoramidate probe 119 rapidly localized at the LNCaP PSMA+ tumor, whereas there was significantly less activity within the PC-3 PSMA− tumor model (Fig. 31).

Fig. 31 PET transaxial images of male nude mice bearing subcutaneous LNCaP (A and C) and PC-3 (B) tumor xenografts at 2 h after injection of ¹⁸F-fluorobenzamido-phosphoramidate (3). Arrows indicate tumor placement. Blocked LNCaP is shown in C. Reproduced with permission from ref. 96.

As an imaging agent, the ¹⁸F-labeled phosphoramidate, had high uptake in the PSMA-expressing tumor and low background and some of the accumulation seen in the kidney may be specific to the mouse. Such findings clearly demonstrate that the leading phosphoramidate is a PSMA-specific targeting ligand in vivo and uptake was specific to PSMA. Consequently, these findings will allow further development of this class of compounds and also provide a sensitive and specific imaging agent for prostate cancer. Knowledge of drug action through crystallography is the ideal guide for rational synthesis. For instance, the mode of binding to thermolysin of the unsubstituted phosphoramidate inhibitor N-phosphoryl-L-leucinamide was studied by single crystal X-ray diffraction methods (Fig. 32) and these were considered transition state analogues.⁹⁷

Fig. 32 Binding to thermolysin of the unsubstituted phosphoramidate inhibitor N-phosphoryl-L-leucinamide.

In general, the bond that is known to be cleaved is usually modified and, in our experience, often tends to be the weakest bond possessing dimensions that are normally longer than those averages culled from crystal structure databases (F. M. D. Ismail & M. G. B. Drew, unpublished study).

Conclusion

In this review, we briefly reported promising pharmacological activities displayed by the phosphoramidate class of compounds. Although various applications have been discussed in the literature, the biological activity of this class deserves further investigation since they reveal considerable potential for improving drug action especially with respect to antitumor and antiviral activities. Rational drug design will be improved as the structure of the target receptors and underlying biochemistry and genetics becomes available. By a careful consideration of ADMET approaches, more robust series of compounds can be developed especially using 3D-QSARs.

Abbreviations

7Bn-GMP	7-Benzyl guanosine monophosphate
A549 cell	Human lung carcinoma cells
AChE	Acetylcholinesterase
ACV	Acyclovir
AddFU	3′-Azido-2′,3′-dideoxy-5-fluorouridine
ADMET	Absorption, distribution, metabolism, and excretion – toxicity in pharmacokinetics
Ala	Alanine
APM	Amiprophos methyl
ATP	Adenosine triphosphate
AZU	4′-Azidouridine
BChE	Butyrylcholinesterase
BEL-7402 cell	Liver tumor cancer cell line
BVdU	Brivudine
C32 cell	Human melonoma cells
C32TG	Thioguanine-resistant mutant of HGPRT-deficient cell line C32
CEM cell	Human T cell lymphoblast-like cell line
ChEI	Cholinesterase inhibitors
d4T	Stavudine
DNA	Deoxyribonucleic acid
DOA	Dioxolane sugar nucleosides
EBV	Epstein–Barr virus
eIF4E	Eukaryotic initiation factor 4E
MIC	Minimum inhibitory concentration
FUDR	5-Fluorouracil 2′-deoxynucleoside
Gly	Glycine
H460	H383 and H2009 cells, human lung cancer cell lines
HBV	Hepatitis B virus
HCMV	Human cytomegalovirus
HCV	Hepatitis C virus
HeLA cell	Human cervical cancer cell lines
HHV	Human herpes virus
HIV	Human immunodeficiency virus
HPD	Haematoporphine dihydrochloride
HPV	Human papilloma virus
HSV	Herpes simplex virus
HSV-1 TK^−	HSV-deficient thymidine kinase
KB cell	Human oral cancer cell lines
MCF-7 cell	Human breast cancer cell lines
MDA-231 cell	Human membrane of breast cancer cell lines
MDAMB231 cell	Human breast cancer cell lines
MIC	Minimum inhibitory concentration
MT-4 cell	Human T cell line MT4
NBPT	N-(n-Butyl)thiophosphoric triamide
NBPTO	N-(n-Butyl)phosphoric triamide
NCI-H460 cell	Human large cell lung cancer cell lines
NS5B	Non-structural protein 5B
NTP	Nucleoside triphosphate
PC-3 cell	Human prostate cancer cell lines
PEG-IFN	Pegalated α-interferon
Phe	Phenylalanine
PK/PD	Pharmacokinetic/pharmacodynamics
PSMA	Prostate-specific membrane antigen
PPD	Phenylphosphorodiamidate
RNA	Ribonucleic acid
SF-268 cell	Human central nervous system glioblastoma cell lines
VZV	Varicella–Zoster virus.

Acknowledgements

We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for financial support and research fellowships (LCAB and FMO). FMDI thanks LJMU for a research allocation. We sincerely thank both Professors C. E. Berkman (Washington State University) and M. O. Anderson (San Francisco State University) for useful correspondence and generously providing re-rendered figures originally published in 2007.⁹³

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Footnote

† Interferons (IFNs) are proteins made and released by host cells in response to the presence of pathogens such as viruses, bacteria, parasites or tumor cells. They facilitate communication between cells to trigger the protective defences of the immune system allowing eradicate of either pathogens or tumors. The pegylated form of interferon-alpha, with greater stability and in vivo activity, has substantially improved sustained virological response rates when compared with unmodified interferon-alpha.

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