Next Article in Journal
Inhibition of Human Cytochrome P450 Enzymes by Bacopa monnieri Standardized Extract and Constituents
Next Article in Special Issue
Structure-Activity Relationship Study of Sesquiterpene Lactones and Their Semi-Synthetic Amino Derivatives as Potential Antitrypanosomal Products
Previous Article in Journal
Synthesis, Spectroscopic and Theoretical Studies of New Quasi-Podands from Bile Acid Derivatives Linked by 1,2,3-Triazole Rings
Previous Article in Special Issue
Antileishmanial Lead Structures from Nature: Analysis of Structure-Activity Relationships of a Compound Library Derived from Caffeic Acid Bornyl Ester

Molecules 2014, 19(2), 2571-2587; doi:10.3390/molecules19022571

Synthetic Fosmidomycin Analogues with Altered Chelating Moieties Do Not Inhibit 1-Deoxy-d-xylulose 5-phosphate Reductoisomerase or Plasmodium falciparum Growth In Vitro
René Chofor 1, Martijn D.P. Risseeuw 1, Jenny Pouyez 2, Chinchu Johny 3, Johan Wouters 2, Cynthia S. Dowd 4, Robin D. Couch 3 and Serge Van Calenbergh 1,*
Laboratory for Medicinal Chemistry, Ghent University, Harelbekestraat 72, Ghent B-9000, Belgium
Department of Chemistry, University of Namur, UNamur, Rue de Bruxelles 61, Namur B-5000, Belgium
Department of Chemistry and Biochemistry, George Mason University, Manassas, VA 20110, USA
Department of Chemistry, George Washington University, Washington, DC 20052, USA
Author to whom correspondence should be addressed; Tel.: +32-926-481-24; Fax: +32-926-481-46.
Received: 6 February 2014; in revised form: 18 February 2014 / Accepted: 19 February 2014 / Published: 24 February 2014


: Fourteen new fosmidomycin analogues with altered metal chelating groups were prepared and evaluated for inhibition of E. coli Dxr, M. tuberculosis Dxr and the growth of P. falciparum K1 in human erythrocytes. None of the synthesized compounds showed activity against either enzyme or the Plasmodia. This study further underlines the importance of the hydroxamate functionality and illustrates that identifying effective alternative bidentate ligands for this target enzyme is challenging.
fosmidomycin; DOXP reductoisomerase; non-mevalonate pathway; isoprenoid biosynthesis; coordination chemistry

1. Introduction

Yearly, up to 5 million clinical cases and a million fatalities result from malaria, an infectious disease caused by protozoa of the Plasmodium species, with P. falciparum being responsible for the most severe cases [1]. The heaviest caseload is suffered by pregnant women and children in sub-Saharan Africa [2]. Unlike Plasmodia which are endemic in the tropics, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis afflicts one-third of the world’s population annually, leading to about 2–3 million deaths [3]. With resistance emerging to virtually all currently used drugs for the treatment of both diseases, new, safe, effective and low cost antimalarial and antitubercular therapeutics are highly awaited.

The discovery that fosmidomycin (1, Figure 1) and its acetyl congener FR900098 (2), both natural products extracted from Streptomyces species inhibit 1-deoxy-d-xylulose-5-phosphate reducto-isomerase (Dxr), opened interesting opportunities for therapeutics [4,5]. Dxr is the second enzyme in the non-mevalonate pathway (NMP) for isoprenoid biosynthesis, which is absent in humans, but present in most Gram-negative and some Gram-positive bacteria (including Mtb), as well as in apicomplexan parasites (including Plasmodia) [6,7]. Fosmidomycin inhibits the Dxr-catalyzed conversion of 1-deoxy-d-xylulose-5-phosphate (DOXP) to 2C-methyl-d-erythritol-4-phosphate (MEP), by mimicking the binding mode of DOXP to this enzyme [8,9]. SAR studies have indicated the importance of fosmidomycin’s hydroxamate moiety for chelation of a divalent metal cation (M: Mn2+ or Mg2+) present in the enzyme’s active site.

Figure 1. Analogy between DOXP and Fosmidomycin/FR900098.
Figure 1. Analogy between DOXP and Fosmidomycin/FR900098.
Molecules 19 02571 g001 1024

Due to its promising antimalarial activity, fosmidomycin received considerable attention and a combination therapy with clindamycin confirmed its potential as an antimalarial drug, following clinical trials conducted in Gabon and Thailand [10,11]. However, the moderate bioavailability and short serum half-life of fosmidomycin prevented the drug combination from reaching the market. Fosmidomycin’s phosphonate group is highly ionized at physiological pH, which is the main reason for its low bioavailability. While this does not preclude efficient uptake in P. falciparum, other organisms like Mtb, are not sensitive to fosmidomycin because they lack a glycerol-3-phosphate transporter (G1pT) that is known to actively transport fosmidomycin across hydrophobic cell membranes [12,13].

Although the chelating ability of hydroxamates often makes them potent metalloenzyme inhibitors, most hydroxamic acids suffer from poor oral bioavailability and significant binding to other metals (e.g., Zn2+, Cu2+, etc.) besides Mn2+ and Mg2+ [14,15]. In addition, hydroxamic acids may be rapidly degraded in vivo by hydrolysis, glucuronidation and sulfation and may suffer from poor pharmacokinetic and toxicological profiles [16]. In order to circumvent the limitations associated with the phosphonate and hydroxamate moiety of fosmidomycin, two strategies have been widely exploited in the design of potent analogues: masking of the polar phosphonate group as prodrugs and/or substituting the hydroxamate of fosmidomycin with an alternative Mn2+ and Mg2+ binding group. The former strategy has been relatively well investigated [17], while the latter has been studied with less rigor.

Giessmann et al. synthesized a series of amidopropylphosphonates 3 (Figure 2), but none of these showed detectable E. coli Dxr inhibition when tested up to 30 µM, indicating the importance of the N-OH group for Dxr inhibition [18]. This was further proven by Woo et al. following the evaluation of compounds 4 wherein the N-OH was replaced with N-CH3 [19]. During the synthesis of α-substituted fosmidomycin analogues, our group observed that benzyl removal from the retrohydroxamate moiety by catalytic hydrogenation typically resulted in the formation of the desired compound, but also significant amounts of the corresponding deoxygenated derivative, i.e., the amide, due to the competitive side reaction of “full” reduction [20]. Deprotection of the phosphonate moiety of the latter afforded analogues such as 5, which were moderately potent in inhibiting E. coli Dxr and capable of inhibiting the growth of a Dd2 P. falciparum strain at submicromolar concentrations (unpublished results). The Rohmer group demonstrated that the reverse hydroxamate counterparts of fosmidomycin or FR900098 (6) elicit comparable inhibitory activity against E. coli Dxr as the natural products [21]. This observation was further confirmed by other groups which obtained sub-micromolar IC50 values following evaluation of fosmidomycin analogues comprising a reverse hydroxamate moiety [22,23,24]. Nakamura and co-workers showed that a cis arrangement of the two oxygen atoms of the hydroxamate group is required for effective metal chelation. Furthermore, they suggested that alternative functional groups containing cis oxygen atoms might have comparable metal coordination ability [8]. Catechols 7a and 7b showed IC50 values of 24.8 µM and 4.5 µM, respectively, when tested for inhibition of E. coli Dxr, indicating a preference for the 1,3,4-orientation (7b) of the catechol over the 1,2,3-orientation (7a) [25].

Figure 2. Hydroxamate-modified analogs of fosmidomycin.
Figure 2. Hydroxamate-modified analogs of fosmidomycin.
Molecules 19 02571 g002 1024

In search for lipophilic fosmidomycin analogues, Andaloussi et al. resynthesized 7b alongside other hydroxamate-modified compounds with a bulky heteroaryl moiety such as 7c. Tests conducted with these compounds revealed that steric constraint in the vicinity of the Dxr active site was deleterious to inhibitory potency [26]. Other attempts to substitute the hydroxamate group of fosmidomycin with similar sterically demanding alternatives led to the conclusion that the Dxr active site is very narrow around the metal cation [27,28]. Nevertheless, the Dowd group recently observed a more efficient coordination of the metal cation by amide- versus O-linked substituents on the retrohydroxamate of fosmidomycin [29]. They highlighted the importance of having an aromatic group in the inhibitor while also suggesting that an alkyl chain between the retrohydroxamate and the aryl group may be preferable for accessing an alternate binding location.

This paper aims to more systematically investigate the possibilities of replacing the retrohydroxamate group of fosmidomycin with effective alternative bidentate ligands. Amide derivatives represented by the general structure 8 were prepared and evaluated. We envisaged a contribution to chelation by ortho-substituents on the amide-linked aromatic ring. Compounds with a NH moiety between carbonyl and sulfonyl groups are very acidic (pKa ~ 2). At physiological pH, the presence of a negative charge at this position would be expected to improve the interaction with the active-site metal ion [30]. Therefore, we included one analogue with a methylsulfonyl group in the ortho position of the phenyl ring (compound 8h), as well as a (non-aromatic) sulfamate (compound 8m). In order to ascertain the influence of electronic factors on chelation, aromatic substituents with various electronic properties were selected.

2. Results and Discussion

2.1. Synthesis

The synthesis of the amide derivatives 8ai, mq is outlined in Scheme 1. Carboxylic acid 9 was readily prepared starting from commercially available ethyl 4-bromo-butyrate and dibenzyl phosphite as previously described by Kuntz et al. [21]. Anticipation that the cyano substituent on aniline 11q would be susceptible to hydrogenation later in the synthesis necessitated the use of the diethyl protected phosphonate 10, obtained from saponification of commercially available triethyl 4-phosphonobutyrate, for reaction with this aniline. With the exception of anilines 11i and 11l, all other anilines used were commercially available. Synthesis of 11i (Scheme 2) started from 2-nitro-aniline which was easily converted to the NH-Boc protected form as described by McNeil and Kelly [31]. Subsequent N, N-dimethylation, followed by Boc removal afforded the aniline. Compound 11l was prepared from 2,6-dihydroxyaniline according to a literature procedure [32].

Anilines are often poor nucleophiles, thus carboxylic acids 9 and 10 were first converted to their respective acid chlorides by treatment with oxalyl chloride before subsequent nucleophilic substitution of 11am, 11q to generate a small library of the protected amides 12am, and 13q in moderate yields. The 1H-NMR spectrum of 12c displays two peaks at 2.17 ppm and 2.21 ppm for the 2,6-dimethyl protons corresponding to the E and Z amide rotamers in a 5/1 ratio. Hydrolysis of the tertiary butyl ester group of 12j with TFA (20% in dichloromethane) further converted this intermediate to 12n. Using benzyl protection for both the phosphonate and the aryl substituent (12k and 12l) allowed a mild single deprotection by catalytic hydrogenolysis in the presence of palladium over activated charcoal at room temperature to access targets 8ai, mp. TMSBr mediated deprotection of 13q and basic workup yielded 8q as the bisammonium salt.

Scheme 1. General synthesis of amide derivatives 8ai, mq.
Scheme 1. General synthesis of amide derivatives 8ai, mq.
Molecules 19 02571 g004 1024

Reagents and conditions: (i) oxalyl chloride, DMF, CH2Cl2, 45 °C, 2–3 h, 40%–75%; (ii) TFA/CH2Cl2 (for 12j); (iii) H2, Pd/C, MeOH, NaOHaq., 25 °C, 10–15 min, quant.; (iv) TMSBr, CH2Cl2, H2O, NH4OHaq., quant.

Scheme 2. Preparation of aniline 11i.
Scheme 2. Preparation of aniline 11i.
Molecules 19 02571 g005 1024

Reagents and conditions: (i) formaldehyde, H2, Pd/C, MeOH, 90%; (ii) acetyl chloride, MeOH.

2.2. Antiplasmodial and Antitubercular Evaluation

The ability of the final compounds to inhibit the E. coli Dxr and M. tuberculosis Dxr was investigated using a spectrophotometric assay monitoring the substrate dependent oxidation of NADPH, essentially as described in detail elsewhere [33]. As shown in Figure 3, at a concentration of 100 µM, all compounds failed to significantly inhibit the E. coli or Mtb Dxr. Likewise all compounds were found essentially inactive against P. falciparum K1 in human erythrocytes (IC50 > 64 µM).

Figure 3. Relative activity of 8ai, mq on purified E. coli (dark grey) and Mtb Dxr (light-grey).
Figure 3. Relative activity of 8ai, mq on purified E. coli (dark grey) and Mtb Dxr (light-grey).
Molecules 19 02571 g003 1024

Similar to fosmidomycin, we expected that the phosphonate group of these analogs would be accommodated in the phosphate binding pocket of Dxr. With the three-carbon spacer unaltered, the introduced modification of the hydroxamate group is determining the lack of Dxr inhibitory activity. Monodentate ligands include virtually all anions and simple Lewis bases. While anticipating that the bivalent metal cation would be more readily bound by electron rich substituents on the aromatic ring, we expected that the analogs with 2,6-disubstituted aromatic rings would elicit better enzyme inhibition than their monosubstituted counterparts, since possible rotation of the amide bond would still assure a favorable conformation (cis) with respect to the carbonyl oxygen. Even though the hard metal ion character of Mg2+ favors the formation of stable complexes with dioxygen based hard ligands, O-linked substituents on the ring did not improve the inhibitory ability of these analogues. Carboxylate is a known chelating group [34] but in the assay conditions, the group was possibly protonated thereby reducing the chelating potency of the carboxylate oxygen of 8n with the Mg2+ ion. Obviously, the presence of an aromatic ring improved the lipophilicity of these analogs. However, limited flexibility around the amide bond seems detrimental for inhibitory activity. Maybe, the introduction of methylene groups between the NH and the (substituted)phenyl ring could increase the likelihood of adopting of a better conformation for occupation of ‘alternative’ binding pockets or a better fitting of the compound into the active site. In the course of our work, Bodill et al. reported similar modifications of the reytrohydroxamate moiety of fosmidomycin [35]. Out of a series of phosphonated N-(hetero)arylcarboxamide analogues with one, two, three or four methylene groups linking the phosphonate to the carboxamide group, they found that increasing the number of methylene groups in the spacer (particularly to three or four methylene groups) decreases the Dxr inhibitory activity dramatically. The authors noted that while receptor-cavity size constraints is an important determinant of binding, allosteric and reverse-orientation ligand binding modes cannot be excluded.

3. Experimental

3.1. General Methods and Materials

1H-, 13C-, 19F- and 31P-NMR spectra were recorded in CDCl3, or D2O on a Mercury 300 spectrometer (Varian, Palo Alto, CA, USA). Chemical shifts are given in parts per million (ppm) (δ relative to TMS for H and C and to external D3PO4 for 31P. High resolution mass spectroscopy spectra for all compounds were also recorded on a LCT Premier XE orthogonal time-of flight spectrometer with API-ES source (Waters, Alliance 2695XE-LCT Premier XETM, Zellik, Belgium). Silica gel (60 Å, 0.063–0.200 mm) was purchased from Biosolve (Valkenswaard, The Netherlands). All solvents and chemicals were used as purchased unless otherwise stated.

3.2. General Procedure for the Synthesis of Protected Amides

To a 0.5 M solution of the acid 9/10 in dichloromethane under nitrogen atmosphere, was added oxalyl chloride (2 eq.) and a few drops of DMF at room temperature. After effervescence subsided, the mixture was heated to reflux at 45 °C for 2 h. It was then cooled to room temperature, concentrated in vacuo, co-evaporated three times with toluene and then re-dissolved in dichloromethane. The aniline (2 eq.) was then added at 0 °C, followed by DIPEA (3 eq.) and the mixture stirred overnight at room temperature. The reaction was quenched by addition of NaHCO3 and the aqueous layer was extracted three times with dichloromethane. The combined organic layer was washed once with brine, dried over Na2SO4 and concentrated in vacuo. Purification by silica gel chromatography using a toluene/acetone or dichloromethane/methanol solvent system gave access to the pure protected amides (30%–75% yields).

Dibenzyl 3-(phenylcarbamoyl)propylphosphonate (12a). 1H-NMR (300 MHz, CDCl3) δH ppm 1.55–2.09 (m, 4H, P-CH2-CH2), 2.47 (t, J = 6.82 Hz, 2H, CH2-CONHPh), 4.89–5.11 (m, 4H, CH2-Ph), 6.99–7.55 (m, 15H, Ar-H), 8.27 (br. s, 1H, NH). 13C-NMR (75 MHz, CDCl3) δC ppm 18.93 (d, 2JP-C = 6.32 Hz, C2), 24.42 (d, 1JP-C = 139.32 Hz, C1), 36.82 (d, 3JP-C = 8.85 Hz), 67.39 (d, 2JP-C = 6.63 Hz, PhCH2, C3), 119.77 (Ar-C), 119.87 (Ar-C), 124.23 (Ar-C), 128.22 (Ar-C), 128.80 (Ar-C), 136.12 (3JP-C = 5.53 Hz, Cipso-PhCH2), 136.31, (Ar-C) 138.39 (Ar-C), 170.71 (CO). 31P-NMR (121.5 MHz, CDCl3): δPppm = 34.00. HRMS (ESI): calculated for C24H27NO4P [(M+H)+], 424.1672; found 424.1698.

Dibenzyl 3-(o-tolylcarbamoyl)propylphosphonate (12b). 1H-NMR (300 MHz, CDCl3) δH ppm 1.70–2.12 (m, 4H, P-CH2-CH2), 2.24 (s, Ph-CH3), 2.51 (t, J = 6.74 Hz, 2H, CH2-CONHPh), 4.87–5.12 (m, 4H, CH2-Ph), 7.00–7.23 (m, 3H), 7.28–7.38 (m, 10H, Ar-H), 7.55 (br. s, 1H, NH), 7.78 (d, J = 7.91 Hz, 1H, Ar-H). 13C-NMR (75 MHz, CDCl3) δC ppm 18.18 (PhCH3), 19.16 (d, 2JP-C = 6.32 Hz, C2), 24.75 (d, 1JP-C = 140.21 Hz, C1), 36.87 (d, 3JP-C = 9.34 Hz, C3), 67.52 (2JP-C = 6.54 Hz, PhCH2), 123.35 (Ar-C), 125.32 (Ar-C), 126.85 (Ar-C), 128.18 (Ar-C), 128.74 (Ar-C), 128.86 (Ar-C), 130.70 (Ar-C), 136.00 (Ar-C), 136.43 (d, 3JP-C = 5.93 Hz, Cipso-PhCH2), 170.70 (CO). 31P-NMR (121.5 MHz, CDCl3): δP ppm = 33.77. HRMS (ESI): calculated for C25H29NO4P [(M+H)+], 438.1829; found 438.1831.

Dibenzyl 3-(2,6-dimethylphenylcarbamoyl)propylphosphonate (12c). 1H-NMR (300 MHz, CDCl3) δH ppm 1.69–2.12 (m, 4H, P-CH2-CH2), 2.17 (5/6 of 6H, s, Ph-CH3), 2.17 (1/6 of 6H, s, Ph-CH3), 2.49 (t, J = 7.16 Hz, 2H, CH2-CONHPh), 4.86–5.14 (m, 4H, CH2-Ph), 7.02–7.14 (m, 3H, Ar-H), 7.29–7.38 (m, 10H, Ar-H). 13C-NMR (75 MHz, CDCl3) δC ppm 18.79 (Ph-CH3), 19.22 (d, 2JP-C = 5.21 Hz, C2), 25.20 (d, 1JP-C = 140.50 Hz, C1), 36.24 (d, 3JP-C = 10.92 Hz, C3), 67.49 (d, 2JP-C = 6.69 Hz, PhCH2), 127.47 (Ar-C), 128.28 (Ar-C), 128.38 (Ar-C), 128.74 (Ar-C), 128.87 (Ar-C), 134.14 (Ar-C), 135.53 (Ar-C), 136.48 (d, 3JP-C = 5.85 Hz, Cipso-PhCH2), 170.57 (CO). 31P-NMR (121.5 MHz, CDCl3): δP ppm = 33.63. HRMS (ESI): calculated for C26H31NO4P [(M+H)+], 452.1985; found 452.1990.

Dibenzyl 3-(2-methoxyphenylcarbamoyl)propylphosphonate (12d). 1H-NMR (300 MHz, CDCl3) δH ppm 1.74–2.12 (m, 4H, P-CH2-CH2), 2.47 (t, J = 7.04 Hz, 2H, CH2-CONHPh), 3.83 (s, 3H, NHPh-O-CH3), 4.91–5.10 (m, 4H, CH2-Ph), 6.86 (dd, J = 1.17 Hz, 7.91 Hz, 1H, Ar-H), 6.94 (td, J = 1.46 Hz, 7.61 Hz, 1H, Ar-H), 7.03 (td, J = 1.76 Hz, 7.62 Hz), 7.26–7.40 (m, 10H, Ar-H), 7.82 (br. s, 1H, NH), 8.33 (dd, J = 1.17 Hz, 7.91 Hz, 1H, Ar-H). 13C-NMR (75 MHz, CDCl3) δC ppm 18.61 (d, 2JP-C = 4.98 Hz, C2), 25.09 (d, 1JP-C = 140.42 Hz, C1), 37.48 (d, 3JP-C = 13.27 Hz, C3), 55.67 (Ph-O-CH3) 67.23 (2JP-C = 6.64 Hz, PhCH2), 109.97 (Ar-C), 119.98 (Ar-C), 121.08 (Ar-C), 123.76 (Ar-C), 127.61 (Ar-C), 127.99 (Ar-C), 128.46 (Ar-C), 128.65 (Ar-C), 136.41 (d, 3JP-C = 6.08 Hz, Cipso-PhCH2), 147.889 (Ar-C), 170.03 (CO). 31P-NMR (121.5 MHz, CDCl3): δP ppm = 33.52. HRMS (ESI): calculated for C25H29NO5P [(M+H)+], 454.1778; found 454.1791.

Dibenzyl 3-(2,6-dimethoxyphenylcarbamoyl)propylphosphonate (12e). 1H-NMR (300 MHz, CDCl3) δH ppm 1.85–2.11 (m, 4H, P-CH2-CH2), 2.33–2.59 (m, 2H, CH2-CONHPh), 3.75 (br. s, 6H, OCH3) 4.86–5.12 (m, 4H, CH2-Ph), 6.55 (d, J = 8.51 Hz, 2H, Ar-H), 7.17 (t, J = 8.52 Hz, 1H, Ar-H) 7.27–7.36 (m, 10H, Ar-H). 13C-NMR (75 MHz, CDCl3) δC ppm 18.12 (Ph-CH3), 22.26 (d, 2JP-C = 5.35 Hz, C2), 25.28 (d, 1JP-C = 139.10 Hz, C1), 36.21 (d, 3JP-C = 9.83 Hz, C3), 67.38 (d, 2JP-C = 6.58 Hz, PhCH2), 127.39 (Ar-C), 128.20 (Ar-C), 128.32 (Ar-C), 128.45 (Ar-C), 128.92 (Ar-C), 129.13 (Ar-C), 135.51 (Ar-C), 136.97 (d, 3JP-C = 6.08 Hz, Cipso-PhCH2), 165.22 (CO). 31P-NMR (121.5 MHz, CDCl3): δP ppm = 33.07. HRMS (ESI): calculated for C26H31NO6P [(M+H)+], 484.1884 ; found 484.0402.

Dibenzyl 3-(2-fluorophenylcarbamoyl)propylphosphonate (12f). 1H-NMR (300 MHz, CDCl3) δH ppm 1.76–2.01 (m, 4H, P-CH2-CH2), 2.50 (t, J = 7.06 Hz, 2H, CH2-CONHPh), 4.88–5.15 (m, 4H, CH2-Ph), 6.96–7.16 (m, 3H, Ar-H), 7.28–7.39 (m, 10H, Ar-H), 7.84 (br. s, 1H, NH), 8.25 (t, J = 8.18 Hz, 1H, Ar-H). 13C-NMR (75 MHz, CDCl3) δC ppm 18.64 (d, 2JP-C = 5.24 Hz, C2), 24.71 (d, 1JP-C = 140.37 Hz, C1), 36.85 (d, 3JP-C = 10.64 Hz, C3), 67.29 (d, 2JP-C = 6.59 Hz, PhCH2), 114.86 (d, 2JF-C = 19.38 Hz, F-Ph), 122.02 (Ar-C), 124.36 (d, 2JF-C = 7.58 Hz, F-Ph), 124.49 (d, 3JF-C = 3.79 Hz, F-Ph), 128.00 (Ar-C), 128.49 (Ar-C), 128.62 (Ar-C), 136.26 (d, 3JP-C = 5.71 Hz, Cipso-PhCH2), 152.42 (d, 1JF-C = 243.71 Hz, F-Ph), 170.43 (CO). 31P-NMR (121.5 MHz, CDCl3): δP ppm = 33.60. HRMS (ESI): calculated for C24H26FNO4P [(M+H)+], 442.1578; found 442.1586.

Dibenzyl 3-(2-acetylphenylcarbamoyl)propylphosphonate (12g). 1H-NMR (300 MHz, CDCl3) δH ppm 1.80–2.12 (m, 4H, P-CH2-CH2), 2.49 (t, J = 7.11 Hz, 2H, CH2-CONHPh), 2.65 (s, 3H, OCCH3), 4.93–5.11 (m, 4H, CH2-Ph), 7.11 (dd, 1H, J = 1.17 Hz, 8.23 Hz, Ar-H), 7.29–7.38 (m, 10H, Ar-H), 7.54 (dd, J = 1.68 Hz, 8.52 Hz, 1H, Ar-H), 7.88 (dd, J = 1.50 Hz, 7.86 Hz, 1H, Ar-H), 8.72 (dd, J = 1.10 Hz, 8.52, 1H Ar-H), 11.70 (br. s, 1H, NH). 13C-NMR (75 MHz, CDCl3) δC ppm 18.59 (d, 2JP-C = 4.42 Hz, C2), 25.45 (d, 1JP-C = 140.98 Hz, C1), 28.69 (PhCOCH3), 38.45 (d, 3JP-C = 16.03 Hz, C3), 67.25 (d, 2JP-C = 6.63 Hz, PhCH2), 120.82 (Ar-C), 121.90 (Ar-C), 122.45 (Ar-C), 128.01 (Ar-C), 128.45 (Ar-C), 128.70 (Ar-C), 131.79 (Ar-C), 135.30 (Ar-C), 136.45 (d, 3JP-C = 6.08 Hz, Cipso-PhCH2), 141.07 (Ar-C), 174.24 (CO), 202.91 (PhCOCH3). 31P-NMR (121.5 MHz, CDCl3): δP ppm = 33.43. HRMS (ESI): calculated for C26H29NO5P [(M+H)+], 466.1778; found 466.1779.

Dibenzyl 3-(2-(methylsulfonyl)phenylcarbamoyl)propylphosphonate (12h). 1H-NMR (300 MHz, CDCl3) δH ppm 1.74–2.12 (m, 4H, P-CH2-CH2), 2.50 (t, J = 7.10 Hz, 2H, CH2-CONHPh), 2.99 (br. s, 3H, SO2-CH3), 4.92–5.11 (m, 4H, CH2-Ph), 7.21–7.29 (m, 2H, Ar-H), 7.30–7.37 (m, 10H, Ar-H), 7.62 (td, J = 1.62 Hz, 7.07 Hz, 1H, Ar-H), 7.90 (dd, J = 1.62 Hz, 7.98 Hz), 8.45 (dd, J = 1.27 Hz, 8.01 Hz, 1H, Ar-H). 13C-NMR (75 MHz, CDCl3) δC ppm 18.56 (d, 2JP-C = 5.07 Hz, C2), 25.42 (d, 1JP-C = 141.36 Hz, C1), 37.92 (d, 3JP-C = 14.76 Hz, C3), 44.41 (-PhSO2CH3), 67.47 (d, 2JP-C = 6.82 Hz, PhCH2), 123.06 (Ar-C), 124.40 (Ar-C), 127.28 (Ar-C), 128.21 (Ar-C), 128.68 (Ar-C),128.85 (Ar-C), 129.54 (Ar-C), 135.54 (Ar-C), 136.53 (d, 3JP-C = 5.81 Hz, Cipso-PhCH2), 137.11 (Ar-C), 170.66 (CO). 31P-NMR (121.5 MHz, CDCl3): δP ppm = 33.10. HRMS (ESI): calculated for C25H29NO6PS [(M+H)+], 502.1448; found 502.1470.

Dibenzyl 3-(2-(dimethylamino)phenylcarbamoyl)propylphosphonate (12i). 1H-NMR (300 MHz, CDCl3) δH ppm 1.75–2.12 (m, 4H, P-CH2-CH2), 2.49 (t, J = 7.07 Hz, 2H, CH2-CONHPh), 2.60 (br. s, 6H, N-(CH3)2), 4.92–5.10 (m, 4H, CH2-Ph), 7.00–7.18 (m, 3H, Ar-H), 7.27–7.38 (m, 10H, Ar-H), 8.33 (d, 1H, J = 7.78, Ar-H), 8.43 (br. s, 1H, NH). 13C-NMR (75 MHz, CDCl3) δC ppm 18.80 (d, 2JP-C = 4.81 Hz, C2), 25.51 (d, 1JP-C = 140.87 Hz, C1), 37.89 (d, 3JP-C = 14.19 Hz, C3), 45.00 (N-CH3), 67.41 (d, 2JP-C = 6.60 Hz, PhCH2), 119.72 (Ar-C), 120.12 (Ar-C), 123.92 (Ar-C), 125.26 (Ar-C), 128.15 (Ar-C), 128.63 (Ar-C), 128.82 (Ar-C), 133.53 (Ar-C), 136.58 (d, 3JP-C = 6.02 Hz, Cipso-PhCH2), 142.87 (Ar-C), 170.16 (CO). 31P-NMR (121.5 MHz, CDCl3): δP ppm = 33.53. HRMS (ESI): calculated for C26H32N2O4P [(M+H)+], 467.2094; found 467.2330.

Dibenzyl 3-(2-(tert-butoxycarbonyl)phenylcarbamoyl)propylphosphonate (12j). 1H-NMR (300 MHz, CDCl3) δH ppm 1.59 (br. s, 9H, O-tBu), 1.77–2.13 (m, 4H, P-CH2-CH2), 2.50 (t, J = 7.21 Hz, 2H, CH2-CONHPh), 4.93–5.11 (m, 4H, CH2-Ph), 7.05 (td, J = 1.10 Hz, 7.38, 1H, Ar-H), 7.25–7.38 (m, 10H, Ar-H), 7.49 (td, J = 1.75 Hz, 7.38 Hz, 1H, Ar-H), 7.97 (dd, J = 1.75 Hz, 8.32 Hz 1H, Ar-H), 8.67 (dd, J = 1.06 Hz, 8.51 Hz 1H, Ar-H), 11.20 (br. s, 1H, NH). 13C-NMR (75 MHz, CDCl3) δC ppm 18.66 (d, 2JP-C = 4.94 Hz, C2), 25.54 (d, 1JP-C = 140.62 Hz, C1), 28.41 (PhCOOCCH3), 38.55 (d, 3JP-C = 15.82 Hz, C3), 67.38 (d, 2JP-C = 6.35 Hz, PhCH2), 82.66 (Ar-C), 116.62 (Ar-C), 120.47 (Ar-C), 122.48 (Ar-C), 128.15 (Ar-C), 128.57 (Ar-C), 128.79 (Ar-C), 131.24 (Ar-C), 134.31 (Ar-C), 136.63 (d, 3JP-C = 5.92 Hz, Cipso-PhCH2), 141.71 (Ar-C), 167.91 (COOtBu), 170.96 (C0). 31P-NMR (121.5 MHz, CDCl3): δP ppm = 33.41.

Dibenzyl 3-(2-(benzyloxy)phenylcarbamoyl)propylphosphonate (12k). 1H-NMR (300 MHz, CDCl3) δH ppm 1.74–2.10 (m, 4H, P-CH2-CH2), 2.40 (t, J = 7.21 Hz, 2H, CH2-CONHPh), 4.86–5.16 (m, 4H, CH2-Ph), 5.10 (br. s, 2H, NH-Ph-O-CH2-Ph), 6.88–7.05 (m, 3H, Ar-H), 7.24–7.43 (m, 15H, Ar-H), 7.79 (br. s, 1H, NH), 8.35 (td, J = 2.47 Hz, 7.84 Hz, 1H, Ar-H). 13C-NMR (75 MHz, CDCl3) δC ppm 19.25 (d, 2JP-C = 4.98 Hz, C2), 25.66 (d, 1JP-C = 140.43 Hz, C1), 38.00 (d, 3JP-C = 14.37 Hz, C), 67.54 (2JP-C = 6.6.63 Hz, PhCH2), 71.50 (NH-Ph-O-CH2-Ph), 112.35 (Ar-C), 120.72 (Ar-C), 122.04 (Ar-C), 124.28 (Ar-C), 128.09(Ar-C), 128.49(Ar-C), 128.90 (Ar-C), 128.95 (Ar-C), 129.14 (Ar-C), 129.35 (Ar-C), 136.91 (d, 3JP-C = 6.09 Hz, Cipso-PhCH2), 136.97 (Ar-C), 147.66 (Ar-C), 170.49 (CO). 31P-NMR (121.5 MHz, CDCl3): δP ppm = 33.55. HRMS (ESI): calculated for C31H33NO5P [(M+H)+], 530.2091; found 530.2122.

Dibenzyl 3-(2,6-bis(benzyloxy)phenylcarbamoyl)propylphosphonate (12l). 1H-NMR (300 MHz, CDCl3) δH ppm 1.69–1.98 (m, 4H, P-CH2-CH2), 2.38 (app. s, 2H, CH2-CONHPh), 5.82–5.01 (m, 4H, P-O-CH2-Ph), 5.06 (s, 4H, N-Ph-O-CH2-Ph), 6.62 (d, J = 8.57 Hz, 2H, Ar-H), 7.12 (t, J = 8.39 Hz, 1H, Ar-H), 7.21–7.45 (m, 20 H, Ar-H). 13C-NMR (75 MHz, CDCl3) δC ppm 18.19 (C2), 24.49 (d, 1JP-C = 138.22 Hz, C1), 36.07 (C3), 67.02 (d, 2JP-C = 6.59 Hz, PhCH2OP), 70.71 (NH-PhOCH2Ph), 106.08 (Ar-C), 115.21 (Ar-C), 127.33 (Ar-C), 127.89 (Ar-C), 128.31 (Ar-C), 128.53 (Ar-C), 136.39, (d, 3JP-C = 5.53 Hz, Cipso-PhCH2), 138.81 (Ar-C), 154.92 (CO). 31P-NMR (121.5 MHz, CDCl3): δPppm = 34.16.

Dibenzyl (4-(methylsulfonamido)-4-oxobutyl)phosphonate (12m). 1H-NMR (300 MHz, CDCl3) δH ppm 1.75–2.04 (m, 4H, P-CH2-CH2), 2.47 (t, J = 7.03 Hz, 2H, CH2-CONHPh), 3.21 (s, 3H, SO2NHCH3), 4.89–5.11 (m, 4H, CH2-Ph), 7.28–7.40 (m, 10H, Ar-H), 10.63 (br.s, 1H, NH). 13C-NMR (75 MHz, CDCl3) δC ppm 17.79 (d, 2JP-C = 5.93 Hz, C2), 24.46 (d, 1JP-C = 140.57 Hz, C1), 35.84 (d, 3JP-C = 10.02 Hz, C3), 41.61 (SO2NHCH3), 67.93 (d, 2JP-C = 6.47 Hz, PhCH2), 128.14 (Ar-C), 128.88 (Ar-C), 128.93 (Ar-C), 136.18 (d, 3JP-C = 5.81 Hz, Cipso-PhCH2), 172.17 (CO). 31P-NMR (121.5 MHz, CDCl3): δP ppm = 33.41. HRMS (ESI): calculated for C19H25NO6PS [(M+H)+], 426.1140; found 426.1162.

2-(4-(Bis(benzyloxy)phosphoryl)butanamido)benzoic acid (12n). Compound 12j (0.416 g) was dissolved in a dichloromethane/TFA mixture (5/1, 8 mL) at 0 °C. After stirring for an hour, TLC analysis showed a completed reaction. Toluene (15 mL) was then added to the reaction mixture before concentration in vacuo. Column chromatography (97.5% CH2Cl2/2% MeOH/0.5% CH3COOH) yielded 272 mg of 12n as an oil (73% yield). 1H-NMR (300 MHz, CDCl3) δH ppm 1.94–2.17 (m, 4H, P-CH2-CH2), 2.52 (t, J = 6.26 Hz, 2H, CH2-CONHPh), 4.87–5.14 (m, 4H, CH2-Ph), 7.06 (td, J = 1.08 Hz, 8.10 Hz, 1H, Ar-H), 7.27–7.35 (m, 10H, Ar-H), 7.51 (td, J = 1.08 Hz, 8.28 Hz, 1H, Ar-H), 8.10 (dd, J = 1.68 Hz, 8.10 Hz, 1H, Ar-H), 8.66 (td, J = 1.00 Hz, 8.39 Hz, 1H, Ar-H), 11.44 (br.s 1H, NH). 13C-NMR (75 MHz, CDCl3) δC ppm 18.25 (d, 2JP-C = 5.04 Hz, C2), 25.22 (d, 1JP-C = 140.04 Hz, C1), 38.52 (d, 3JP-C = 17.22 Hz, C3), 67.96 (d, 2JP-C = 6.52 Hz, PhCH2), 115.55 (Ar-C), 120.23 (Ar-C), 122.72 (Ar-C), 128.21 (Ar-C), 128.77 (Ar-C), 128.86 (Ar-C), 131.81 (Ar-C), 134.71 (Ar-C), 136.14 (d, 3JP-C = 5.92 Hz, Cipso-PhCH2), 141.89 (Ar-C), 170.90 (CO), 170.98 (CO). 31P-NMR (121.5 MHz, CDCl3): δP ppm = 26.02.

Diethyl 3-(2-cyanophenylcarbamoyl)propylphosphonate (13q). 1H-NMR (300 MHz, CDCl3) δH ppm 1.34 (t, J = 7.11 Hz, 6H, P-O-CH2CH3), 1.79–2.17 (m, 4H, P-CH2-CH2), 2.64 (t, J = 7.11 Hz, 2H, CH2-CONHPh), 4.01–4.23 (m, 4H, -O-CH2-CH3), 7.19 (dd, J = 1.05 Hz, 7.64 Hz, 1H, Ar-H), 7.51–7.66 (m, 2H, Ar-H), 8.14 (br. s, 1H, NH), 8.28 (dd, J = 1.10 Hz, 8.96 Hz, 1H Ar-H). 13C-NMR (75 MHz, CDCl3) δC ppm 16.49 (d, 3JP-C = 6.32 Hz, P-O-CH2-CH3), 18.58 (d, 2JP-C = 6.32 Hz, C2), 24.43 (d, 1JP-C = 141.21 Hz, C1), 37.02 (d, 3JP-C = 12.19 Hz, C3), 61.75 (d, 2JP-C = 6.06 Hz, P-O-CH2-CH3), 102.97 (Ar-C), 116.46 (CN), 122.10 (Ar-C), 124.39 (Ar-C), 132.46 (Ar-C), 134.04 (Ar-C), 140.37 (Ar-C), 170.86 (CO). 31P-NMR (121.5 MHz, CDCl3): δP ppm = 32.14. HRMS (ESI): calculated for C25H22N2O4P [(M+H)+], 325.1317; found 325.1317.

3.3. General Procedure for Amide Deprotection Yielding Targets 8ai, mp

The amide (100–150 mg) was dissolved in MeOH (10 mL) and Pd/C (10%) was added under inert atmosphere. The resulting mixture was then stirred under hydrogen atmosphere for 10 min and the progress monitored by mass spectrometry. At completion, the reaction mixture was filtered and neutralized with 1 eq. of a NaOH. The mixture was concentrated in vacuo, re-dissolved in a mixture of water and ter-butanol, frozen and lyophilized to afford the desired targets compounds 8ai, mp as a white powder in quantitative yield.

Sodium hydrogen 3-(phenylcarbamoyl)propylphosphonate (8a). 1H-NMR (300 MHz, D2O) δH ppm 1.40–1.56 (m, 2H, -CH2-), 1.78–1.93 (m, 2H, P-CH2-), 2.46 (t, J = 7.47 Hz, 2H, CH2-CONHPh), 7.24 (dt, J = 5.78, 2.82 Hz, 1H, Ar-H), 7.34–7.46 (m, 4 H, Ar-H). 13C-NMR (75 MHz, D2O) δC ppm 21.13 (d, 2JP-C = 3.71 Hz, C2), 28.55 (d, 1JP-C = 131.25 Hz, C1), 38.10 (d, 3JP-C = 16.61 Hz, C3), 122.45 (Ar-C), 125.79 (Ar-C), 129.33 (Ar-C), 136.92 (Ar-C), 176.00 (CO). 31P-NMR (121.5 MHz, D2O): δP ppm = 22.06. HRMS (ESI): calculated for C10H13NO4P [(M−H)], 242.0588; found 242.0061.

Sodium hydrogen 3-(o-tolylcarbamoyl)propylphosphonate (8b). 1H-NMR (300 MHz, D2O) δH ppm 1.47–1.62 (m, 2H, -CH2-), 1.70–1.90 (m, 2H, P-CH2-), 2.10 (s, Ph-CH3), 2.40 (t, J = 7.44 Hz, 2H, CH2-CONHPh), 7.18–7.35 (m, 4H, Ar-H), 8.42 (br. s, 1H, NH). 13C-NMR (75 MHz, D2O) δC ppm 17.14 (Ph-CH3), 21.06 (d, 2JP-C = 3.65 Hz, C2), 28.42 (d, 1JP-C = 131.87 Hz, C1), 37.68 (d, 3JP-C = 16.61 Hz, C3), 126.76 (Ar-C), 127.21 (Ar-C), 127.81 (Ar-C), 130.89 (Ar-C), 134.56 (Ar-C), 137.97 (Ar-C), 176.47 (CO). 31P-NMR (121.5 MHz, D2O): δP ppm = 23.60. HRMS (ESI): calculated for C11H15NO4P [(M−H)], 256.0744; found 256.0322.

Sodium hydrogen 3-(2,6-dimethylphenylcarbamoyl)propylphosphonate (8c). 1H-NMR (300 MHz, D2O) δH ppm 1.47–1.65 (m, 2H, -CH2-), 1.80–2.20 (m, 2H, P-CH2-), 2.17 (s, 6H, Ph-CH3), 2.54 (t, J = 7.47 Hz, 2H, CH2-CONHPh), 7.07–7.25 (m, 3H, Ar-H). 13C-NMR (75 MHz, D2O) δC ppm 17.44 (Ph-CH3), 21.11 (d, 2JP-C = 3.44 Hz, C2), 28.68 (d, 1JP-C = 131.75 Hz, C1), 37.24 (d, 3JP-C = 17.28 Hz, C3), 128.21 (Ar-C), 133.48 (Ar-C), 136.31 (Ar-C), 176.37 (CO). 31P-NMR (121.5 MHz, D2O): δP ppm = 22.47. HRMS (ESI): calculated for C12H17NO4P [(M−H)], 270.0901; found 270.0319.

Sodium hydrogen 3-(2-methoxyphenylcarbamoyl)propylphosphonate (8d). 1H-NMR (300 MHz, D2O) δH ppm 1.37–1.52 (m, 2H, -CH2-), 1.77–1.92 (m, 2H, P-CH2-), 2.47 (t, J = 7.52 Hz, 2H, CH2-CONHPh), 3.83 (s, 3H, Ph-O-CH3), 7.00 (td, J = 7.65 Hz, 1.33 Hz, 1H, Ar-H), 7.09 (dd, J = 8.31 Hz, 1.24 Hz, 1H, Ar-H), 7.20–7.32 (m, 1H, Ar-H), 7.52 (dd, J= 7.87 Hz, 1.68 Hz, 1H, Ar-H). 13C-NMR (75 MHz, D2O) δC ppm 21.40 (d, 2JP-C = 3.36 Hz, C2), 28.94 (d, 1JP-C = 130.12 Hz, C1), 38.04 (d, 3JP-C = 16.84 Hz, C3), 56.01 (-Ph-O-CH3), 112.30 (Ar-C), 121.07 (Ar-C), 125.20 (Ar-C), 125.53 (Ar-C), 127.65 (Ar-C), 152.22 (Ar-C), 176.45 (CO). 31P-NMR (121.5 MHz, D2O): δP ppm = 21.28. HRMS (ESI): calculated for C11H15NO5P [(M−H)], 272.0693; found 272.0129.

Sodium hydrogen 3-(2,6-dimethoxyphenylcarbamoyl)propylphosphonate (8e). 1H-NMR (300 MHz, D2O) δH ppm 1.42–1.57 (m, 2H, -CH2-), 1.69–1.88 (m, 2H, P-CH2-), 2.40 (t, J = 7.39 Hz, 2H, CH2-CONHPh), 3.71 (s, 6H, Ph-O-CH3), 6.66 (d, J = 8.47 Hz, 2H, Ar-H), 7.33 (t, J = 8.47 Hz, 1H, Ar-H). 13C-NMR (75 MHz, D2O) δC ppm 20.82 (d, 2JP-C = 3.64 Hz, C2), 28.05 (d, 1JP-C = 132.26 Hz, C1), 37.12 (d, 3JP-C = 17.22 Hz, C3), 56.34 (PhOCH3), 105.38 (Ar-C), 113.06 (Ar-C), 129.44 (Ar-C), 155.33 (Ar-C), 176.72 (CO). 31P-NMR (121.5 MHz, D2O): δP ppm = 24.34. HRMS (ESI): calculated for C12H17NO6P [(M+H)+], 302.0799; found 302.0074.

Sodium hydrogen 3-(2-fluorophenylcarbamoyl)propylphosphonate (8f). 1H-NMR (300 MHz, D2O) δH ppm 1.44–1.61 (m, 2H, -CH2-), 1.80–1.94 (m, 2H, P-CH2-), 2.51 (t, J = 7.32 Hz, 2H, CH2-CONHPh), 7.13–7.33 (m, 3H, Ar-H), 7.53 (td, J = 1.74 Hz, 7.63 Hz, 1H, Ar-H). 13C-NMR (75 MHz, D2O) δC ppm 20.89 (d, 2JP-C = 3.54 Hz, C2), 28.30 (d, 1JP-C = 131.37 Hz, C1), 37.50 (d, 3JP-C = 17.13 Hz, C3), 116.05 (d, JF-C = 19.91 Hz, Ar-C), 124.03 (d, JF-C = 3.36 Hz, Ar-C), 124.71 (d, JF-C = 12.74 Hz, Ar-C), 126.62 (Ar-C), 128.08 (d, JF-C = 7.95 Hz, Ar-C), 157.44 (Ar-C), 176.42 (CO). 31P-NMR (121.5 MHz, D2O): δP ppm = 22.63. HRMS (ESI): calculated for C10H12FNO4P [(M−H)], 260.0494; found 260.0001.

Sodium hydrogen 3-(2-acetylphenylcarbamoyl)propylphosphonate (8g). 1H-NMR (300 MHz, D2O) δH ppm 1.24 (s, 3H, PhCOCH3), 1.40–1.58 (m, 2H, -CH2-), 1.84–1.99 (m, 2H, P-CH2-), 2.51 (t, J = 7.13 Hz, 2H, CH2-CONHPh), 7.20–7.43 (m, 4H, Ar-H), 13C-NMR (75 MHz, D2O) δC ppm 21.43 (d, 2JP-C = 3.87 Hz, C2), 29.10 (d, 1JP-C = 129.92 Hz, C1), 29.71 (PhCOCH3), 37.69 (d, 3JP-C = 16.58 Hz, C3), 126.83 (Ar-C), 127.98 (Ar-C), 128.23 (Ar-C), 129.40 (Ar-C), 133.94 (Ar-C), 141.189 (Ar-C), 177.12 (-CO-), 177.20 (-COCH3). 31P-NMR (121.5 MHz, D2O): δP ppm = 22.19. HRMS (ESI): calculated for C12H15NO5P [(M−H)], 284.0693; found 284.0693.

Sodium hydrogen 3-(2-(methylsulfonyl)phenylcarbamoyl)propylphosphonate (8h). 1H-NMR (300 MHz, D2O) δH ppm 1.40–1.58 (m, 2H, -CH2-), 1.81–1.98 (m, 2H, P-CH2-), 2.57 (t, J = 7.66 Hz, 2H, CH2-CONHPh), 3.23 (s, 3H, -Ph-SO2CH3), 7.57 (td, J = 1.36 Hz, 7.73 Hz, 1H, Ar-H), 7.65 (dd, J = 1.36 Hz, 8.13 Hz, 1H, Ar-H), 7.79 (td, J = 1.49 Hz, 7.73 Hz, 1H, Ar-H), 8.01 (dd, J = 8.00 Hz, 1.53 Hz, 1H, Ar-H). 13C-NMR (75 MHz, D2O) δC ppm 20.97 (d, 2JP-C = 3.37 Hz, C2), 28.89 (d, 1JP-C = 130.92 Hz, C1), 37.91 (d, 3JP-C = 17.13 Hz, C3), 43.14 (-Ph-SO2CH3), 128.31 (Ar-C), 129.60 (Ar-C), 129.83 (Ar-C), 133.81 (Ar-C), 134.67 (Ar-C), 135.82 (Ar-C), 177.16 (CO). 31P-NMR (121.5 MHz, D2O): δP ppm = 22.51. HRMS (ESI): calculated for C11H15NO6PS [(M−H)], 320.0363; found 319.9703.

Sodium hydrogen 3-(2-(dimethylamino)phenylcarbamoyl)propylphosphonate (8i). 1H-NMR (300 MHz, D2O) δH ppm 1.37–1.56 (m, 2H, -CH2-), 1.79–1.96 (m, 2H, P-CH2-), 2.51 (t, J = 7.52 Hz, 2H, CH2-CONHPh), 2.62 (s, 6H, Ph-N-CH3), 7.07–7.15 (m, 1H, Ar-H), 7.22–7.29 (m, 2H, Ar-H), 7.45 (app. d, J = 7.65 Hz, 1H, Ar-H). 13C-NMR (75 MHz, D2O) δC ppm 21.31 (d, 2JP-C = 3.69 Hz, C2), 29.11 (d, 1JP-C = 130.27 Hz, C1), 38.17 (d, 3JP-C = 16.91 Hz, C3), 43.71 (Ph-N-CH3), 120.11 (Ar-C), 123.95 (Ar-C), 126.90 (Ar-C), 127.79 (Ar-C), 129.99 (Ar-C), 147.99 (Ar-C), 176.57 (CO). 31P-NMR (121.5 MHz, D2O): δP ppm = 24.24. HRMS (ESI): calculated for C12H18N2O4P [(M−H)], 285.1010; found 285.0459.

Sodium hydrogen (4-(methylsulfonamido)-4-oxobutyl)phosphonate (8m). 1H-NMR (300 MHz, D2O) δH ppm 1.55–1.69 (m, 2H, -CH2-), 1.78–1.92 (m, 2H, P-CH2-), 2.40 (t, J = 7.27 Hz, 2H, CH2-CONHPh), 2.39 (s, 3H, -N-SO2CH3). 13C-NMR (75 MHz, D2O) δC ppm 19.78 (d, 2JP-C = 3.87 Hz, C2), 27.41 (d, 1JP-C = 133.24 Hz, C1), 38.62 (d, 3JP-C = 17.14 Hz, C3), 40.10 (-N-SO2CH3), 180.33 (CO). 31P-NMR (121.5 MHz, D2O): δP ppm = 25.22. HRMS (ESI): calculated for C5H11NO6PS [(M−H)], 244.0050; found 244.0611.

Sodium hydrogen 3-(2-carboxyphenylcarbamoyl)propylphosphonate (8n). 1H-NMR (300 MHz, D2O) δH ppm 1.55–1.70 (m, 2H, -CH2-), 1.81–1.98 (m, 2H, P-CH2-), 2.51 (t, J = 7.31 Hz, 2H, CH2-CONHPh), 7.22 (td, J = 1.03 Hz, 7.64 Hz, 1H, Ar-H), 7.50 (td, J = 1.65 Hz, 7.64 Hz, 1H, Ar-H), 7.85 (dd, J = 7.83, 1.60 Hz, 1H, Ar-H), 8.01 (app. d, 1H, Ar-H). 13C-NMR (75 MHz, D2O) δC ppm 19.97 (d, 2JP-C = 3.95 Hz, C2), 27.37 (d, 1JP-C = 133.39 Hz, C1), 38.45 (d, 3JP-C = 17.66 Hz, C3), 121.99 (Ar-C), 124.67 (Ar-C), 125.10 (Ar-C), 130.71 (Ar-C), 132.22 (Ar-C), 137.17 (Ar-C), 173.83 (CO, PhCOOH), 174.88 (CO, -CH2-CO-NH-). 31P-NMR (121.5 MHz, D2O): δP ppm = 24.98. HRMS (ESI): calculated for C11H13NO6P [(M−H)], 286.0486; found 286.0268.

Sodium hydrogen 3-(2-hydroxyphenylcarbamoyl)propylphosphonate (8o). 1H-NMR (300 MHz, D2O) δH ppm 1.46–1.61 (m, 2H, -CH2-), 1.79–1.96 (m, 2H, P-CH2-), 2.51 (t, J = 7.44 Hz, 2H, CH2-CONHPh), 6.88–7.03 (m, 2H, Ar-H), 7.18 (td, J = 1.79 Hz, 7.45 Hz, 1H, Ar-H), 7.35 (dd, J = 7.83 Hz, 1.60 Hz, 1H, Ar-H). 13C-NMR (75 MHz, D2O) δC ppm 20.85 (d, 2JP-C = 3.84 Hz, C2), 28.25 (d, 1JP-C = 131.65 Hz, C1), 37.47 (d, 3JP-C = 17.18 Hz, C3), 116.89 (Ar-C), 120.73 (Ar-C), 124.10 (Ar-C), 126.37 (Ar-C), 128.15 (Ar-C), 149.83 (Ar-C), 176.39 (CO). 31P-NMR (121.5 MHz, D2O): δP ppm = 22.85. HRMS (ESI): calculated for C10H13NO5P [(M+H)+], 258.0537; found 258.0058.

Sodium hydrogen 3-(2,6-dihydroxyphenylcarbamoyl)propylphosphonate (8p). 1H-NMR (300 MHz, D2O) δH ppm 1.48–1.66 (m, 2H, -CH2-), 1.78–1.99 (m, 2H, P-CH2-), 2.54 (t, J = 7.43 Hz, 2H, CH2-CONHPh), 6.52 (d, J = 8.33 Hz, 2H, Ar-H), 7.07 (t, J = 8.22 Hz, 1H, Ar-H). 13C-NMR (75 MHz, D2O) δC ppm 20.63 (d, 2JP-C = 3.95 Hz, C2), 28.12 (d, 1JP-C = 131.80 Hz, C1), 37.02 (d, 3JP-C = 16.52 Hz, C3), 108.25 (Ar-C), 111.93 (Ar-C), 129.19 (Ar-C), 152.84 (Ar-C), 177.01 (CO). 31P-NMR (121.5 MHz, D2O): δP ppm = 22.22. HRMS (ESI): calculated for C10H13NO6P [(M−H)], 274.0486; found 273.9962.

Bisammomium 3-(2-cyanophenylcarbamoyl)propylphosphonate (8q). Intermediate 13q (150 mg, 0.334 mmol) was dissolved in dry dichloromethane (6 mL) under inert atmosphere and cooled to 0 °C. TMSBr (0.5 mL, 3.3 mmol) was added dropwise while stirring. The icebath was removed after 10 min and the reaction stirred at room temperature for 24 h. 31P-NMR confirmed that the starting phosphonate was completely deprotected (shift from δ = 32–25 ppm). The volatiles were removed in vacuo, the crude material was dissolved in 5% aqueous ammonia and washed with diethyl ether. Lyophilisation of the ammonia solution yielded the product as a brown solid in quantitative yield. 1H-NMR (300 MHz, D2O) δH ppm 1.50–1.65 (m, 2H, -CH2-), 1.85–2.20 (m, 2H, P-CH2-), 2.68 (t, J = 7.58 Hz, 2H, CH2-CONHPh), 7.45 (td, J = 0.99 Hz, 7.96 Hz, 1H, Ar-H), 7.60 (d, J = 8.05 Hz, 1H, Ar-H), 7.77 (td, J = 1.51 Hz, 7.20 Hz, 1H, Ar-H), 8.08 (dd, J = 1.33 Hz, 7.96 Hz, 1H, Ar-H). 13C-NMR (75 MHz, D2O) δC ppm 21.55 (d, 2JP-C = 3.87 Hz, C2), 27.80 (d, 1JP-C = 136.00 Hz, C1), 35.65 (d, 3JP-C = 16.59 Hz, C3), 121.59 (Ph-CN), 126.40 (Ar-C), 126.65 (Ar-C), 127.45 (Ar-C), 134.95 (Ar-C), 149.45 (Ar-C), 157.68 (Ar-C), 162.45 (CO). 31P-NMR (121.5 MHz, D2O): δP ppm = 25.00. HRMS (ESI): calculated for C11H13N2O4P [(M−H)], 267.0540; found 267.0823.

3.4. Synthesis of o-(Dimethylamino)aniline (11i)

To a solution of 14 (0.5 g; 2 mmol) in MeOH (100 mL) was added formalin (14 mL), Pd/C 10% (160 mg) and formic acid (1 mL). The resulting mixture was allowed to stir under a hydrogen atmosphere for 3 h, after which, the mixture was filtered over a celite path and the filtrate concentrated to about 25 mL. The mixture was then basified by adding NaHCO3 and the water layer was extracted three times with EtOAc (3 × 50 mL). The combined organic phase was washed once with brine and dried over Na2SO4. Column chromatography (hexane/EtOAc 95:5) yielded 15 (0.450 g, 90%) as a colorless oil. Subsequent treatment of 15 with 30% TFA in dichloromethane at 0 °C afforded 11i which was used for the next step without further purification.

tert-Butyl 2-(dimethylamino)phenylcarbamate (15). 1H-NMR (300 MHz, CDCl3) δH ppm 1.54 (br. s, 9H, tert-Bu), 2.62 (s, 6H, N-CH3), 6.96 (td, J = 1.16 Hz, 7.57 Hz, 1H, Ar-H), 7.05–7.16 (m, 2H, Ar-H), 7.70 (br. s, 1H, NH), 8.07 (d, J = 8.17). 13C-NMR (75 MHz, CDCl3) δC ppm 28.93 (CH3 of tert-Bu), 44.83 (N-CH3), 80.27 (Cq of tert-Bu), 117.97 (Ar-C), 120.16 (Ar-C), 122.51 (Ar-C), 125.22 (Ar-C), 134.13 (Ar-C), 142.35 (Ar-C), 153.29 (CO). HRMS (ESI): calculated for C13H21N2O2 [(M+H)+], 237.1598; found 237.1602.

4. Conclusions

In conclusion, amide derivatives of fosmidomycin were synthesized from simple starting materials. These analogues were inactive against E. coli Dxr, Mtb Dxr and P. falciparum K1 possibly due their inability to adopt a favorable conformation necessary for the Dxr active site metal chelation. Replacing the hydroxamate group of fosmidomycin with an alternative and efficient bidentate metal binding group in Dxr inhibitors, remains a daunting challenge as previously noted [36].


RDC acknowledges Amanda Haymond and Jessica Bases for their assistance with recombinant protein purification and Prof. Cynthia Dowd for the Mtb DXR expression construct. RDC is supported by the U.S. Army Medical Research and Materiel Command W23RYX1291N601.

Author Contributions

R.C. synthesized the target phosphonates under daily supervision of M.R. J.P. performed the E. coli Dxr experiments under the supervision of J.W., while C.J. performed the M. tuberculosis Dxr experiments under supervision of R.D.C. R.C. and S.V.C. wrote the manuscript. C.D. contributed with valuable discussions and revised the manuscript. S.V.C. coordinated this study.

Conflicts of Interest

The authors declare no conflict of interest.


  1. Snow, R.W.; Guerra, C.A.; Noor, A.M.; Myint, H.Y.; Hay, S.I. The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature 2005, 434, 214–217. [Google Scholar] [CrossRef]
  2. World Health Organization. World Malaria Report 2011. WHO Press: Geneva, Switzerland, 2011. [Google Scholar]
  3. World Health Organization. Global Tuberculosis Control 2009: Epidemiology, Strategy, Financing; WHO Press: Geneva, Switzerland, 2009. [Google Scholar]
  4. Kuzuyama, T.; Shimizu, T.; Takahashi, S.; Seto, H. Fosmidomycin, a specific inhibitor of 1-deoxy-d-xylulose 5-phosphate reductoisomerase in the nonmevalonate pathway for terpenoid biosynthesis. Tetrahedron Lett. 1998, 39, 7913–7916. [Google Scholar] [CrossRef]
  5. Zeidler, J.; Schwender, J.; Muller, C.; Wiesner, J.; Weidemeyer, C.; Beck, E.; Jomaa, H.Z.; Lichtenthaler, H.K. Inhibition of the non-mevalonate 1-deoxy-d-xylulose-5-phosphate pathway of plant isoprenoid biosynthesis by fosmidomycin. Z. Naturforsch. 1998, 53c, 980–986. [Google Scholar]
  6. Rohmer, M.; Knani, M.; Simonin, P.; Sutter, B.; Sahm, H. Isoprenoid biosynthesis in bacteria: A novel pathway for the early steps leading to isopentenyl diphosphate. Biochem. J. 1993, 295 (Pt 2), 517–524. [Google Scholar]
  7. Jomaa, H.; Wiesner, J.; Sanderbrand, S.; Altincicek, B.; Weidemeyer, C.; Hintz, M.; Turbachova, I.; Eberl, M.; Zeidler, J.; Lichtenthaler, H.K.; et al. Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science 1999, 285, 1573–1575. [Google Scholar] [CrossRef]
  8. Umeda, T.; Tanaka, N.; Kusakabe, Y.; Nakanishi, M.; Kitade, Y.; Nakamura, K.T. Molecular basis of fosmidomycin’s action on the human malaria parasite Plasmodium falciparum. Sci. Rep. 2011, 1, 1–8. [Google Scholar]
  9. Steinbacher, S.; Kaiser, J.; Eisenreich, W.; Huber, R.; Bacher, A.; Rohdich, F. Structural basis of fosmidomycin action revealed by the complex with 2-C-methyl-D-erythritol 4-phosphate synthase (IspC). Implications for the catalytic mechanism and anti-malaria drug development. J. Biol. Chem. 2003, 278, 18401–18407. [Google Scholar]
  10. Borrmann, S.; Issifou, S.; Esser, G.; Adegnika, A.A.; Ramharter, M.; Matsiegui, P.B.; Oyakhirome, S.; Mawili-Mboumba, D.P.; Missinou, M.A.; Kun, J.F.; et al. Fosmidomycin-clindamycin for the treatment of Plasmodium falciparum malaria. J. Infect. Dis. 2004, 190, 1534–1540. [Google Scholar] [CrossRef]
  11. Borrmann, S.; Adegnika, A.A.; Matsiegui, P.B.; Issifou, S.; Schindler, A.; Mawili-Mboumba, D.P.; Baranek, T.; Wiesner, J.; Jomaa, H.; Kremsner, P.G. Fosmidomycin-clindamycin for Plasmodium falciparum Infections in African Children. J. Infect. Dis. 2004, 189, 901–908. [Google Scholar] [CrossRef]
  12. Dhiman, R.K.; Schaeffer, M.L.; Bailey, A.M.; Testa, C.A.; Scherman, H.; Crick, D.C. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) from Mycobacterium tuberculosis: Towards understanding mycobacterial resistance to fosmidomycin. J. Bacteriol. 2005, 187, 8395–8402. [Google Scholar] [CrossRef]
  13. Sakamoto, Y.; Furukawa, S.; Ogihara, H.; Yamasaki, M. Fosmidomycin resistance in adenylate cyclase deficient (cya) mutants of Escherichia coli. Biosci. Biotechnol. Biochem. 2003, 67, 2030–2033. [Google Scholar] [CrossRef]
  14. Lou, B.; Yang, K. Molecular diversity of hydroxamic acids. Part II. Potential therapeutic applications. Mini Rev. Med. Chem. 2003, 3, 609–620. [Google Scholar] [CrossRef]
  15. O’Brien, E.C.; Farkas, E.; Gil, M.J.; Fitzgerald, D.; Castineras, A.; Nolan, K.B. Metal complexes of salicylhydroxamic acid (H2Sha), anthranilic hydroxamic acid and benzohydroxamic acid. Crystal and molecular structure of [Cu(phen)2(Cl)]ClPH2Sha, a model for a peroxidaseinhibitor complex. J. Inorg. Biochem. 2000, 79, 47–51. [Google Scholar] [CrossRef]
  16. Sanderson, L.; Taylor, G.W.; Aboagye, E.O.; Alao, J.P.; Latigo, J.R.; Coombes, R.C.; Vigushin, D.M. Plasma pharmacokinetics and metabolism of the histone deacetylase inhibitor trichostatin a after intraperitoneal administration to mice. Drug Metab. Dispos. 2004, 32, 1132–1138. [Google Scholar] [CrossRef]
  17. Jackson, E.R.; Dowd, C.S. Inhibition of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr): A review of the synthesis and biological evaluation of recent inhibitors. Curr. Top. Med. Chem. 2012, 12, 706–728. [Google Scholar] [CrossRef]
  18. Giessmann, D.; Heidler, P.; Haemers, T.; van Calenbergh, S.; Reichenberg, A.; Jomaa, H.; Weidemeyerd, C.; Sanderbrand, S.; Wiesner, J.; Link, A. Towards new antimalarial drugs: Synthesis of non-hydrolyzable phosphate mimics as feed for a predictive QSAR study on 1-deoxy-d-xylulose-5-phosphate reductoisomerase inhibitors. Chem. Biodivers. 2008, 5, 643–656. [Google Scholar] [CrossRef]
  19. Woo, Y.; Fernandes, R.; Proteau, P. Evaluation of fosmidomycin analogs as inhibitors of the Synechocystis sp PCC6803 1-deoxy-d-xylulose 5-phosphate reductoisomerase. Bioorg. Med. Chem. 2006, 14, 2375–2385. [Google Scholar]
  20. Haemers, T.; Wiesner, J.; Busson, R.; Jomaa, H.; van Calenbergh, S. Synthesis of α-aryl-substituted and conformationally restricted fosmidomycin analogues as promising antimalarials. Eur. J. Org. Chem. 2006, 17, 3856–3863. [Google Scholar]
  21. Kuntz, L.; Tritsch, D.; Grosdemange-Billiard, C.; Hemmerlin, A.; Willem, A.; Bacht, T.; Rohmer, M. Isoprenoid biosynthesis as a target for antibacterial and antiparasitic drugs: Phosphonohydroxamic acids as inhibitors of deoxyxylulose phosphate reducto-isomerase. Biochem. J. 2005, 386, 127–135. [Google Scholar] [CrossRef]
  22. Behrendt, C.T.; Kunfermann, A.; Illarionova, V.; Matheeussen, A.; Pein, M.K.; Gräwert, T.; Kaiser, J.; Bacher, A.; Eisenreich, W.; Illarionov, B.; et al. Reverse fosmidomycin derivatives against the antimalarial drug target IspC (Dxr). J. Med. Chem. 2011, 54, 6796–6802. [Google Scholar] [CrossRef]
  23. Brücher, K.; Illarionov, B.; Held, J.; Tschan, S.; Kunfermann, A.; Pein, M.K.; Bachar, A.; Gräwert, T.; Maes, L.; Mordmüller, B.; et al. α-ubstituted β-Oxa isosteres of fosmidomycin: Synthesis and biological evaluation. J. Med. Chem. 2012, 55, 6566–6575. [Google Scholar] [CrossRef]
  24. Kunfermann, A.; Lienau, C.; Illarionov, B.; Held, J.; Gräwert, T.; Behrendt, C.T.; Werner, P.; Hähn, S.; Eisenreich, W.; Riederer, U.; et al. IspC as target for antiinfective drug discovery: Synthesis, enantiomeric separation and structural biology of fosmidomycin thia isosters. J. Med. Chem. 2013, 56, 8151–8162. [Google Scholar] [CrossRef]
  25. Deng, L.; Sundriyal, S.; Rubio, V.; Shi, Z.Z.; Song, Y. Coordination chemistry based approach to lipophilic inhibitors of 1-deoxy-d-xylulose-5-phosphate reductoisomerase. J. Med. Chem. 2009, 52, 6539–6542. [Google Scholar] [CrossRef]
  26. Andaloussi, M.; Lindh, M.; Bjorkelid, C.; Suresh, S.; Wieckowska, A.; Iyer, H.; Karlen, A.; Larhed, M. Substitution of the phosphonic acid and hydroxamic acid functionalities of the Dxr inhibitor FR900098: An attempt to improve the activity against Mycobacterium tuberculosis. Bioorg. Med. Chem. Lett. 2011, 21, 5403–5407. [Google Scholar] [CrossRef]
  27. Williams, S.L.; de Oliveira, C.A.F.; Vazquez, H.; McCammon, J.A. From Zn to Mn: The Study of novel manganese-binding groups in the search for new drugs against tuberculosis. Chem. Biol. Drug Des. 2011, 77, 117–123. [Google Scholar] [CrossRef]
  28. Bodill, T.; Conibear, A.C.; Blatch, G.L.; Lobb, K.A.; Kaye, P.T. Synthesis and evaluation of phosphonated N-heteroarylcarboxamides as DOXP-reductoisomerase (Dxr) inhibitors. Bioorg. Med. Chem. 2011, 19, 1321–1327. [Google Scholar] [CrossRef]
  29. San Jose, G.; Jackson, E.R.; Uh, E.; Johny, C.; Haymond, A.; Lundberg, L.; Pinkham, C.; Kehn-Hall, K.; Boshoff, H.I.; Couch, R.D.; et al. Design of potential bisubstrate inhibitors against Mycobacterium tuberculosis (Mtb) 1-deoxy-d-xylulose 5-phosphate reductoisomerase (Dxr)—evidence of a novel binding mode. Med. Chem. Commun. 2013, 4, 1099–1104. [Google Scholar] [CrossRef]
  30. Gavalda, S.; Braga, R.; Dax, C.; Vigroux, A. N-Sulfonyl hydroxamate derivatives as inhibitors of class II fructose-1,6-diphosphate aldolase. Bioorg. Med. Chem. Lett. 2005, 15, 5375–5377. [Google Scholar] [CrossRef]
  31. McNeil, D.W.; Kelly, T.A. Asimple method for the protection of aryl amines as their t-butylcarbamoyl (Boc) derivatives. Tetrahedron Lett. 1994, 35, 9003–9006. [Google Scholar] [CrossRef]
  32. Sumandeep, K.G.; Hao, X.; Kirchoff, P.D.; Cierpicki, T.; Turbiak, A.J.; Wan, B.; Zhang, N.; Peng, K.W.; Franzblau, S.G.; Garcia, G.A.; et al. Structure-based design of novel benzoxazinorifamycins with potent binding affinity to wild-type and rifampin-resistant mutant Mycobacterium tuberculosis RNA polymerases. J. Med. Chem. 2012, 55, 3814–3826. [Google Scholar] [CrossRef]
  33. Jawaid, S.; Seidle, H.; Zhou, W.; Abdirahman, H.; Abadeer, M.; Hix, J.H.; van Hoek, M.L.; Couch, R.D. Kinetic characterization and phosphoregulation of the Francisella tularensis 1-deoxy-d-xylulose 5-phosphate reductoisomerase (MEP Synthase). PLoS One 2009, 4, e8288. [Google Scholar] [CrossRef]
  34. Larsen, T.M.; Wedekind, J.E.; Rayment, I.; Reed, G.H. A Carboxylate oxygen of the substrate bridges the magnesium ions at the active Site of enolase: Structure of the yeast enzyme complexed with the equilibrium mixture of 2-phosphoglycerate and phosphoenolpyruvate at 1.8 Å Resolution. Biochemistry 1996, 35, 4351–4358. [Google Scholar]
  35. Bodill, T.; Conibear, A.C.; Mutorwa, M.K.M.; Goble, J.L.; Blatch, G.L.; Lobb, K.A.; Klein, R.; Kaye, P.T. Exploring DOXP-reductoisomerase binding limits using phosphonated N-aryl and N-heteroarylcarboxamides as DXR inhibitors. Bioorg. Med. Chem. 2013, 21, 4332–4341. [Google Scholar] [CrossRef]
  36. Zinglé, C.; Kuntz, L.; Tritsch, D.; Grosdemange, C.B.; Rohmer, M. Modifications around the hydroxamic acid chelating group of fosmidomycin, an inhibitor of the metalloenzyme 1-deoxyxylulose 5-phosphate reductoisomerase (DXR). Bioorg. Med. Chem. Lett. 2012, 22, 6563–6567. [Google Scholar] [CrossRef]
  • Sample Availability: Not available.
Molecules EISSN 1420-3049 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert