α- and β-Substituted phosphonate analogs of LPA as autotaxin inhibitors

Abstract

Autotaxin (ATX) is an attractive pharmacological target due to its lysophospholipase D activity which leads to the production of lysophosphatidic acid (LPA). Blockage of ATX produced LPA by small molecules could be a potential anticancer chemotherapy. In our previous study, we have identified the two β-hydroxy phosphonate analogs of LPA (compounds f17 and f18) as ATX inhibitors. With this work, we investigated α- and β-substituted phosphonate analogs of LPA and evaluated them for ATX inhibitory activity. The stereochemistry of β-hydroxy phosphonates was also studied.

1. Introduction

Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphatase and phosphodiesterase (NPP) family of enzymes which are able to hydrolyze phosphoester bonds in nucleotides.^1,2 It can be biosynthesized by a variety of cells and tissues and highly expressed in various malignancies.^3–8 ATX is a multifunctional enzyme which potently stimulates cancer cell proliferation and tumor cell motility, augments the tumorigenicity, and induces angiogenic response.^3,9–14 In 2002, two independent laboratories discovered that ATX is identical to plasma lysophospholipase D (lysoPLD) and acts by hydrolyzing lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA).^15,16 LPA is a lipid mediator that signals cell proliferation, migration, and survival by activating its specific extracellular and intracellular receptors.^17–27 The generation of LPA by ATX can fully account for the biological effects of ATX in cell culture. Considering the fact that ATX is the major enzyme in biosynthesis of LPA in plasma, the development of ATX inhibitors could lead to novel anticancer chemotherapies.

There are a limited number of ATX inhibitors which have been reported.^28–32 In our laboratory, we have developed a series of β-keto and β-hydroxy phosphonate derivatives and tested the ATX inhibitory activity in a choline detection assay.³³ Among them, two β-hydroxy phosphonates originated from L-tyrosine were identified as the lead compounds and they inhibited approximately 70% of ATX activity at 1 μM. The pK^a of the second phosphonate proton (~7.6) can be influenced by the substitutions on the α- and β-positions of the phosphonate. ³⁴ This electronic property is believed to affect enzyme–substrate interaction.³⁵ In this report, we synthesized both α- and β-substituted phosphonate analogs based on the lead compounds and investigated their activities on ATX inhibition.

2. Results and discussion

2.1. Chemistry

Synthesis of α-fluoro phosphonates (Scheme 1) began with esterification of the free acid of N-tert-butyl carbamate, O-benzyl protected L-tyrosine 5, with trimethylsilyl diazomethane. The methyl ester 6 was reduced to the corresponding alcohol with sodium borohydride in the presence of calcium chloride and then oxidized to the desired aldehyde 8 under the Swern oxidative condition. The tetraethyl monofluoromethylene diphosphonate 2 was formed by the fluorination of the tetraethyl diphosphonate anion with Selectfluor.³⁶ Next, treatment of the monofluorodiphosphonate 2 with n-butyl lithium at −78 °C generated the lithiated carbanion, which condensed with the aldehyde to afford α-fluorovinylphosphonate 9. The protecting N-Boc group was removed by treatment of trifluoroacetic acid and the remaining ammonium trifluoroacetic acid salt was acylated with palmitoyl chloride to generate amide 11. After dual hydrogenation/hydrogenolysis of the olefin and benzyl group, respectively, the resulting phenol 12, which was a mixture of two diastereomers, was coupled with the mesylate 4 under basic conditions to give the ether mixture 13. The α-fluoro phosphonate 14 was obtained by trimethylsilyl bromide mediated deprotection of diethyl esters.³⁷

Scheme 1.

Synthesis of 14. Reagents and conditions: (a) NaH, THF, 0 °C; Selectfluor, rt, overnight, 56%; (b) NEt₃, CH₂Cl₂, 0 °C, then MsCl, 5 h, 90%; (c) TMS–CHN₂, 0 °C, 10 min, anhydrous MeOH, 100%; (d) NaBH₄, CaCl₂, THF, EtOH, 3 h, 0 °C—rt, 94%; (e) oxalyl chloride, DMSO, CH₂Cl₂, −78 °C, NEt₃, 2 h, 83%; (f) 2, n-BuLi, THF, −78 °C, 2 h, 70%; (g) TFA, CH₂Cl₂, rt, 5 h; (h) palmitoyl chloride, TEA, 0 °C, 3 h; (i) Pd(OH)₂, H₂, EtOH, rt, overnight, 85%, three steps; (j) 4, K₂CO₃, acetone, 18-crown-6, reflux, overnight, 90%; (k) TMS–Br, then MeOH and H₂O, rt, overnight, 82%.

Synthesis of α-hydroxy phosphonates (Scheme 2) commenced with the Arndt–Eistert reaction. Treatment of N-tert-butyl carbamate, O-benzyl ether protected L-tyrosine 5 with isobutyl chloroformate gave the mixed anhydride. The diazoketone 16 was prepared by adding the pre-cooled freshly distilled diazomethane to the mixed anhydride. Next, a carbene mediated Wolff rearrangement furnished the transformation from a diazoketone to a ketene, which underwent a nucleophilic addition by methanol to afford the desired homologated methyl ester 17. The aldehyde 19 was obtained by a two-step reduction/oxidation process. Then the sodium anion of dimethyl phosphite was generated and reacted with the aldehyde to give α-hydroxy phosphonates 20. Next, the N-Boc protecting group was removed and the resulting ammonium trifluoroacetate salt was acylated with palmitoyl chloride to generate amides 21a and b. The two diastereomers in terms of α-hydroxy group were separated by column chromatography at this step. The exact stereochemistry for each diastereomer was determined (unpublished results). The benzyl ether was cleaved by treatment with palladium hydroxide on carbon and the resulting phenols 22a and b were reacted with 4-methoxy-3,5-dimethyl-2-pyridine-methanol under Mitsunobu condition to give 23a and b. Finally, the fully functionalized α-hydroxy phosphonates were obtained by the same method mentioned above.

Scheme 2.

Synthesis of 24a and b. Reagents and conditions: (a) KOH, 65 °C, isobutyl chloroformate, THF, NEt₃, 0 °C; (b) CH₂N₂, overnight, 51%; (c) silver benzoate, NEt₃, MeOH, THF, −78–0 °C, overnight, 73%; (d) NaBH₄, CaCl₂, THF, EtOH, 0 °C—rt, overnight, 97%; (e) oxalyl chloride, DMSO, CH₂Cl₂, −78 °C, NEt₃, 2 h, 94%; (f) dimethyl phosphite NaH, THF, 0 °C, 1 h, 60%; (g) TFA, CH₂Cl₂, rt, 5 h, then palmitoyl chloride, NEt₃, CH₂Cl₂, 0 °C, 3 h, 82%; (h) Pd(OH)₂, H₂, EtOH, rt, overnight, 58–73%; (i) 4-methoxy-3,5-dimethyl-2-pyridine-methanol, DIAD, PPh₃, NEt₃, CH₂Cl₂, 0 °C—rt, overnight, 80%; (j) TMS–Br, then MeOH and H₂O rt, overnight, 90%.

The transformation of the β-hydroxy phosphonates (49a and b)³³ to β-fluoro phosphonates (25a and b) was accomplished by using diethylaminosulfur trifluoride (DAST) (Scheme 3). The product was then formed with the above-mentioned deprotection of the phosphonate group using TMS–Br.

Scheme 3.

Synthesis of 25a and b. Reagents and conditions: (a) DAST, CH₂Cl₂, −78 °C, 1 h, 50%; (b) TMS–Br, CH₂Cl₂, rt, 4 h, then H₂O and MeOH, overnight, 62%.

2.2. Biological evaluation

The ATX activity was measured in the presence of the compounds under different concentrations (100 μM, 10 μM, and 1 μM). The ATX activity without compounds was used as the standard (100% activity). The data are shown in Table 1. α-Hydroxy (24a and b) and α-fluoro (14a and b) phosphonates did not show any substantial ATX inhibition. The β-fluoro phosphonate demonstrated the same levels of activity as the lead compounds (f17 and f18)³³ at higher concentrations (10 μM and 100 μM); however it lost some potency at the lowest concentration (1 μM). Further optimization was attempted on β-hydroxy and β-keto phosphonates. We investigated a variety of natural amino acid backbones and the compounds and data are shown in Table 2. Most of the compounds could inhibit 50–80% ATX activity at the highest concentration and lost the potency when lower concentrations were applied. The β-keto phosphonate derived from L-proline 38 has proved to be the most potent β-keto phosphonate which could inhibit 80% of ATX activity at medium concentration (10 μM). Tryptophan was chosen to compare with the linker region in tyrosine. The compounds were not as potent as the lead compounds and the β-hydroxy phosphonate with 4-methoxy-3,5-dimethyl-pyridyl moiety 44 did show higher potency than the one with benzyl group 45.

Table 1.

ATX inhibitory evaluation of compounds 14, 24, and 25

Compounds	X	α	Y	β	ATX activity (%)
					1 μM	10 μM	100 μM
24a	OH	S	H		71	70	53
24b	OH	R	H		73	68	87
14	Fa	RS	H		74	68	25
f18	H		OH	bb	63	21	8
f17	H		OH	a	27	13	6
25	H		Fa	RS	59	22	11

^aRacemic products were tested.

^ba refers to the diastereomer that elutes first, b refers to the diastereomer that elutes second from normal phase silica gel based column chromatography.

Table 2.

ATX inhibitory evaluation of compounds 26–46

Compounds	X	R	ATX activity (%)
			1 μM	10 μM	100 μM
26	=O	–H	79	95	76
27	–OHa	–H	88	88	56
28	=O	–CH3	60	67	72
29	–OHa	–CH3	50	14	25
30	=O	–CH(CH3)2	109	77	24
31	–OH (a)b	–CH(CH3)2	115	57	13
32	–OH (b)b	–CH(CH3)2	72	78	23
33	=O	–CH2CH(CH3)2	89	48	25
34	–OH (a)	–CH2CH(CH3)2	68	53	23
35	–OH (b)	–CH2CH(CH3)2	93	64	52
36	–OH (a)	–CH2Ph	88	70	70
37	–OH (b)	–CH2Ph	92	99	67
38	=O	–CH2CH2CH2–c	49	18	9
39	–OHa	–CH2CH2CH2–c	83	43	16
40	=O		91	70	28
41	–OH (a)		88	51	25
42	–OH (b)		93	75	26
43	–OHa		80	35	28
44	–OHa		64	28	13
45	–OH (a)		83	41	19
46	–OH (b)		78	46	61

^aRacemic products were tested.

^ba refers to the diastereomer that elutes first, b refers to the diastereomer that elutes second from normal phase silica gel based column chromatography.

^cThe compounds were synthesized from protected L-proline.

2.3. Structure determination of lead compounds

The lead compounds f17 and f18 are diastereomers originating from reduction of β-keto phosphonate. To further study this reaction and determine the absolute stereochemistry of β-hydroxy phosphonates, a variety of reductive agents and reaction conditions were applied (Table 3). Sodium borohydride gave diastereoselectivity in 1:2.5 ratio favoring the more polar isomer. Lewis acid mediated reduction gave higher reaction yields but lost the diastereoselectivity. Application of bulky hydride reducing reagents such as lithium triethylborohydride (Super-Hydride) and lithium tris[(3-ethyl-3-pentyl) oxy]aluminohydride resulted in lower reaction yields but significantly improved the selectivity.

Table 3.

Reduction of β-keto phosphonate

Entry	Hydride	Conditions	Yield (%)	Ratio (a:b)a
1	NaBH4	EtOH, 0 °C	75	1:2.5
2	NaBH4, CeCl3	EtOH, −78 °C	82	1:1
3	NaBH4, CeCl3	EtOH, 0 °C	85	1:1
4	NaBH4, MgCl2	EtOH, 0 °C	60	1:1
5	Li(C2H5)3BH	THF, 0 °C	33	1:9
6	LiAl[OC(C2H5)3]3H	THF, 0 °C	40	1:11
7	Catecholborane	THF, 0 °C	NRb	NR

^aa refers to the diastereomer that elutes first, b refers to the diastereomer that elutes second.

^bNR, no reaction.

The relationship between the dihedral angle and the vicinal coupling constant ³J was given theoretically by the Karplus relationship.³⁸ Due to the single bond rotation the coupling constants are revealed as an average value contributed from relatively stable rotational isomers. It is expected that the ³J difference between syn and anti isomers could be enlarged if the hydroxyl group and amide in the β-hydroxy phosphonate substrate are fixed in a ring form which prevents a free rotation of carbon bond. Oxazolidines 47 and 48 were prepared (Scheme 4) from β-hydroxy phosphonates 49a and 49b (49a was the less polar isomer and 49b was the more polar isomer). The results of the decoupling study show that the J values between geminal benzylic protons H₃ and H₄ are approximately 14 Hz in both oxazolidines (Fig. 1). These two protons couple with H₂ to give J values corresponding to 6 Hz and 9 Hz, respectively. The ³J values between H₁ and H₂ are close to 0 Hz in 47 and 5 Hz in 48. According to the Karplus relationship, 47 has the anti configuration and the less polar isomer 49a corresponds to the R alcohol; 48 has the syn configuration and the more polar isomer 49b corresponds to the S alcohol. This result is consistent with the reported ³J values of oxazolidone derivatives of α-amino-β-hydroxy acids.^39,40 Taken into account the outcome of diastereoselectivity, the reaction is likely governed by Felkin–Ahn model (Fig. 2).

Scheme 4.

Synthesis of 47 and 48. Reagents and conditions: (a) 2-methoxypropene, CSA, CH₂Cl₂, 0 °C, 30–35%.

Figure 1.

¹H homonuclear decoupling study.

Figure 2.

Modified Felkin–Ahn model of reductive reaction.

3. Conclusion

We have synthesized a series of α-/β-substituted phosphonate analogs of LPA and evaluated them for ATX inhibitory activity. The β-substituted analogs showed higher potency than the α-substituted analogs. Further structural optimization was attempted on β-keto and β-hydroxy phosphonates. We investigated a variety of amino acid backbones. Some analogs showed comparable potency with the lead compounds (f17 and f18) at high concentrations (10 μM and 100 μM). However, at the lowest concentration (1 μM), these newer analogs showed reduced potency compared to the lead compounds. The stereochemistry of the β-hydroxy phosphonates was also determined by ¹H homonuclear decoupling study. The most potent compound (f17) was proven to be a β-hydroxy phosphonate with R-hydroxy moiety. Additional synthetic efforts are needed to fully elucidate the SAR of the phosphonate analogs and toward the goal of developing potent ATX inhibitors as the potential anticancer chemotherapy.

4. Experimental

4.1. Materials

Chemicals for syntheses were purchased from Aldrich Chemical Company (Milwaukee, WI), Acros Chemical Company (Morris Plains, NJ), Novabiochem Chemical Company (Laufelfingen, Switzerland), and Fluka Chemical Company (Milwaukee, WI), and were used without further purification. All reactions were carried out under an inert atmosphere of nitrogen unless otherwise stated. All solvents were filtered through alumina (activity 1) immediately prior to use in reactions. In certain case, THF was filtered through alumina and subsequently distilled over a mixture of sodium and benzophenone immediately prior to use. Merck silica gel F-254 pre-coated, aluminum backed plates and Merck Silica Gel 60 (230–400 mesh) or Silicycle Ultra pure silica gel (230–400 mesh) were used in thin layer chromatography and flash column chromatography, respectively. Anal-tech (Newark, NJ) alumina GF Preparative TLC plates (500 μm, 1000 μm, and 2000 μm 20 × 20 cm) were used for preparative TLC separation. All nuclear magnetic resonance (NMR) spectra were collected using a Varian UnityInova 300 NMR spectrometer at 300 MHz and chemical shifts are reported in ppm.

4.2. General procedures

4.2.1. General procedure A: acylation of amines

To a solution of the amine (1 equiv) to be acylated and TEA (3 equiv) in CH₂Cl₂ at 0 °C was added the appropriate acyl chloride dropwise by syringe. The reaction mixture was then allowed to stir for several hours. After completion of the reaction as determined by TLC, it was quenched with 10% aqueous HCl and further extracted with 10% aqueous HCl. The aqueous layer was then extracted once with CH₂Cl₂ followed by a brine extraction of the organic layer. The organic layer was then dried using magnesium sulfate, filtered, and concentrated under reduced pressure. Then the product was purified using the appropriate purification techniques.

4.2.2. General procedure B: palladium hydroxide on carbon mediated hydrogenolysis of benzyl ethers

To a solution of the benzyl ether to be cleaved in ethanol was added a catalytic amount of 20% palladium hydroxide on carbon; the reaction was then subjected to a hydrogen atmosphere (typically 1ATM) and was allowed to stir overnight. The following day the reaction mixture was filtered through a pad of Celite to remove all palladium using methanol as the eluent followed by removal of all solvent under reduced pressure. Then any purification that was deemed necessary was undertaken.

4.2.3. General procedure C: sodium borohydride reduction of esters

NaBH₄ (2 equiv) and CaCl₂ (2 equiv) are dissolved in ethanol so that a solution of milky appearance is formed. This solution is cooled to 0 °C and the ester to be reduced is dissolved in THF, so that a 2:1 ethanol/THF mixture will be the resulting solution ratio. Then the ester in THF is added dropwise to the reaction mixture via canula. The reaction mixture is allowed to slowly warm to rt and then it is stirred overnight. The following day the reaction is quenched with a saturated solution of aqueous ammonium chloride, then extracted with EtOAc, brine, dried with sodium sulfate, and concentrated under reduced pressure. The product is then either purified via flash column chromatography or used unpurified.

4.2.4. General procedure D: Swern oxidation of alcohols

To dry dichloromethane in a flame-dried RBF was added oxalyl chloride (1.5 equiv, 2 M in CH₂Cl₂), the mixture was then cooled to −78 °C before a solution of dimethylsulfoxide (3 equiv) in CH₂Cl₂ was added dropwise via syringe. The resulting mixture was stirred at this temperature for 30 min before a solution of the alcohol (1 equiv) to be oxidized in CH₂Cl₂ was added dropwise via canula. The mixture was stirred for 30 min before triethyl amine was added via syringe; the resulting mixture was stirred for an additional 15 min before it was warmed to rt. Then a 10% HCl solution in water was added to the reaction mixture until it turned acidic as indicated by pH paper. Then the organic layer was extracted with another small portion of 10% HCl, then the aqueous layer was twice back extracted with CH₂Cl₂. The organic layer was further extracted with brine, dried using magnesium sulfate, filtered, and then concentrated under reduced pressure. The resulting aldehyde was either purified using flash column chromatography or used unpurified.

4.2.5. General procedure E: mesylate coupling

To a stirring solution of phenol (1 equiv), potassium carbonate (4 equiv), and appropriate mesylate (or alkyl halide) (1.2 equiv) in acetone was added a catalytic amount of 18-crown-6 and the reaction mixture was heated to reflux. The reaction was maintained at this temperature overnight and the next morning allowed too cool to room temperature. After sufficient cooling the reaction mixture was concentrated under reduced pressure and then the resulting mixture was solvated with ethyl acetate and extracted with ammonium chloride, then the aqueous layer was further extracted with ethyl acetate, the organic layer was washed with brine, dried with sodium sulfate, filtered, and then concentrated under reduced pressure. The product was then purified using flash column chromatography.

4.2.6. General procedure F: synthesis of β-keto phosphonates

To a solution of diethylmethylphosphonate (1 equiv) in THF at −78 °C was added n-BuLi (1.1 equiv, 2.5 M) dropwise via syringe. The reaction mixture was allowed to stir at this temperature for 1 h before a solution of the ester (1.05 equiv) in THF was added. The reaction mixture was stirred for an additional hour before a saturated aqueous solution of ammonium chloride was added and the reaction mixture was allowed to slowly warm to room temperature. Then the reaction mixture was extracted with ethyl acetate, followed by brine, dried over sodium sulfate and concentrated under reduced pressure. The product was then purified using flash column chromatography.

4.2.7. General procedure G: sodium borohydride reduction of ketones

NaBH₄ (2 equiv) is dissolved in ethanol so that a solution of milky appearance is formed. This solution is cooled to 0 °C and the ketone to be reduced is dissolved in THF, so that a 2:1 ethanol/THF mixture will be the resulting solution ratio. Then the ester in THF is added dropwise to the reaction mixture via canula. The reaction mixture is allowed to slowly warm to rt and then it is stirred overnight. The following day the reaction is quenched with a saturated solution of aqueous ammonium chloride, then extracted with EtOAc, brine, dried with sodium sulfate, and concentrated under reduced pressure. The product was then purified using flash column chromatography.

4.2.8. General procedure H: bromotrimethylsilane mediated deprotection of phosphate and phosphonate esters

To a stirring solution of the ester to be deprotected is added bromotrimethylsilane (10 equiv). The reaction mixture is then allowed to stir for about 4 h and progress is monitored by TLC; after completion of the reaction the solvent is removed under reduced pressure and then the resulting oil is solvated with 95% methanol 5% water and the reaction mixture is allowed to stir overnight. The following day the solvent is removed under reduced pressure and the appropriate purification technique is employed.

4.2.9. General procedure I: mitsunobu coupling

To a solution of the alcohol (2 equiv), the coupling phenol or acid (1 equiv), diisopropylethyl amine (2 equiv), and polymer bound triphenylphine (2 equiv) in CH₂Cl₂ at 0 °C was added diisopropylazodicarboxylate (2 equiv). The reaction mixture was then allowed to slowly warm to room temperature and to stir overnight; the following day the reaction mixture is diluted with methanol and filtered through a pad of Celite using methanol as the eluent. The organic layer is then concentrated under reduced pressure and the product is purified using flash column chromatography.

4.3. Synthesis of compounds 2–25, 47, and 48

4.3.1. [(Diethoxy-phosphoryl)-fluoro-methyl]-phosphonic acid diethyl ester (2)

To a solution of sodium hydride (700 mg, 60%, 17.5 mmol) in THF at 0 °C was added a solution of (diethoxy-phosphorylmethyl)-phosphonic acid diethyl ester (5 g, 17.3 mmol) in THF. The reaction mixture was allowed to stir at this temperature for 1 h before solid Selectfluor (6.146 g, 17.3 mmol) was added to the mixture. The reaction mixture was stirred at room temperature overnight and then the solid was filtered off through a pad of Celite using methanol as the eluent, concentrated under reduced pressure, and purified using flash column chromatography (4% MeOH:EtOAc, stained with Henessian stain) to yield the monofluorinated product in 3.0 g (56%). ¹H NMR (300 MHz, CDCl₃) δ 1.37 (t, 12 H, J = 7.31 Hz), 4.28 (p, 8H, J = 8.05 Hz), 5.50 (m, 1H).

4.3.2. Methanesulfonic acid 4-methoxy-3,5-dimethyl-pyridin-2-ylmethyl ester (4)

To a stirring solution of (4-methoxy-3,5-dimethyl-pyridin-2-yl)-methanol (500 mg, 3.0 mmol) and triethylamine (0.63 ml, 4.52 mmol) in CH₂Cl₂ at 0 °C was slowly added methane sulfonylchloride (0.28 ml, 3.62 mmol) via syringe. The reaction mixture was slowly warmed to room temperature and stirred for an additional 4 h at which time the reaction was stopped. It was stopped prematurely and some starting material was retained. The solvent was removed under reduced pressure and then the resulting deep red oil was placed directly onto a flash column and purified via flash column chromatography (1:1 EtOAc/hexanes) to give 660 mg product (90%). ¹H NMR (300 MHz, CDCl₃) 2.15 (s, 3H), 2.24 (s, 3H), 3.67 (s, 3H), 4.57 (s, 2H), 8.10 (s, 1H); ¹³C NMR δ 11.11, 13.54, 46.02, 60.12, 126.04, 126.79, 149.60, 154.73, 164.53.

4.3.3. 3-(4-Benzyloxy-phenyl)-2-tert-butoxycarbonylamino-propionic acid methyl ester (6)

To a solution of N-Boc O-Bnz L-tyrosine (2.5 g, 6.74 mmol) in anhydrous methanol was added trimethylsilyl diazomethane (2 M in ether, 5.1 ml, 10.1 mmol) dropwise to the stirring mixture at 0 °C until no more nitrogen gas was evolved and a bright yellow color persisted (about 20 min). The mixture was then concentrated under reduced pressure and purified by flash column chromatography (20% EtOAc:hexanes) to yield the crude product 2.8 g. ¹H NMR (300 MHz, CDCl₃) δ 1.43 (s, 9H), 3.03 (m, 2H), 3.69 (s, 3H), 4.57 (m, 1H), 5.02 (s, 2H), 5.08 (m, 1H), 6.91 (d, 2H, J = 8.42 Hz), 7.05 (d, 2H, J = 8.42 Hz), 7.31–7.44 (m, 5H). ¹³C NMR δ 28.58, 37.72, 52.41, 54.86, 70.22, 115.20, 127.75, 128.23, 128.63, 128.85, 130.61, 137.31, 158.17, 172.70.

4.3.4. [1-(4-Benzyloxy-benzyl)-2-hydroxy-ethyl]-carbamic acid tert-butyl ester (7)

This compound was synthesized according to general procedure C from 6 (2.8 g, 7.27 mmol) to yield the resulting alcohol in 2.26 g (94%). ¹H NMR (300 MHz, CDCl₃) δ 1.42 (s, 9H), 2.78 (d, 2H, J = 7.32 Hz), 3.60 (m, 2H), 3.82 (b, 1H), 5.04 (s, 2H), 6.92 (d, 2H, J = 8.78 Hz), 7.13 (d, 2H, J = 8.42 Hz), 7.31–7.45 (m, 5H).

4.3.5. [1-(4-Benzyloxy-benzyl)-2-oxo-ethyl]-carbamic acid tert-butyl ester (8)

This compound was synthesized according to general procedure D from 7 (2.26 g, 8.79 mmol) to yield the resulting aldehyde in 1.87 g (83%).¹H NMR (300 MHz, CDCl₃) δ 1.45 (s, 9H), 3.06 (d, 2H, J = 6.58 Hz), 4.38 (d, 1H, J = 6.59 Hz), 5.04 (s, 2H), 5.11 (m, 1H), 6.92 (d, 2H, J = 8.42 Hz), 7.09 (d, 2H, J = 8.42 Hz), 7.31–7.44 (m, 5H), 9.62 (s, 1H). ¹³C NMR δ 28.54, 34.78, 61.14, 70.26, 80.37, 115.34, 127.72, 128.25, 128.85, 128.91, 130.63, 137.18, 158.14, 199.91.

4.3.6. [4-(4-Benzyloxy-phenyl)-3-tert-butoxycarbonylamino-1-fluoro-but-1-enyl]-phosphonic acid diethyl ester (9)

To a solution of 2 (500 mg, 1.63 mol) in THF at −78 °C was added a solution of n-butyl lithium (0.67 ml, 2.5 M in THF). The reaction mixture was allowed to stir for 1 h and then a solution of 8 (775 mg, 2.18 mmol) in THF was added and the reaction mixture was allowed to slowly warm to room temperature. The reaction mixture was allowed to stir overnight and the next day the reaction was quenched with ammonium chloride and then the aqueous layer was extracted with EtOAc, then the organic layer was extracted with brine, the organic was then dried with sodium sulfate, filtered, and then concentrated under reduced pressure. The product was purified via flash column chromatography (1:1 EtOAc/hexanes) to afford the product in 600 mg (70%) yield. ¹H NMR (300 MHz, CDCl₃) δ 1.33 (m, 6H); 1.46 (m, 9H); 2.04 (s, 1H), 2.78 (m, 2H), 4.07 (m, 4H), 4.88 (m, 1H), 5.02 (s, 2H), 5.15 (m, 1H), 5.95 (m, 1H), 6.92 (d, 2H, J = 8.45 Hz), 7.14 (d, 2H, J = 8.46 Hz), 7.44 (m, 5H).

4.3.7. [4-(4-Benzyloxy-phenyl)-1-fluoro-3-hexadecanoylamino-but-1-enyl]-phosphonic acid diethyl ester (11)

A solution of 9 (600 mg, 1.18 mmol) in dichloromethane and trifluoroacetic acid (2:1) was stirred for several hours and progress was monitored by TLC. After the reaction had proceeded to completion the solvent was removed under reduced pressure and the remaining TFA was removed after repeated azeotropic distillations with both diethyl ether and methanol. The product was prepared from palmitoyl chloride according to the general procedure A. The crude products were collected without purification in a yield of 860 mg.

4.3.8. [1-Fluoro-3-hexadecanoylamino-4-(4-hydroxy-phenyl)-butyl]-phosphonic acid diethyl ester (12)

This compound was synthesized according to the general procedure B from 11 (860 mg) in a yield of 570 mg (85%, three steps). ¹H NMR (300 MHz, CDCl₃) δ 0.84 (t, 3H, J = 5.12 Hz), 1.21 (s, 24H), 1.30 (m, 6H), 1.52 (m, 2H), 2.09 (t, 3H, J = 7.31 Hz), 2.72 (m, 2H), 3.61(s, 1H), 4.14 (m, 4H), 4.39 (b, 1H), 4.82 (m, 1H), 6.11 (t, 1H, J = 8.79 Hz), 6.72 (d, 2H, J = 8.05 Hz), 6.95 (d, 2H, J = 7.32 Hz). ¹³C NMR δ 14.32, 16.52, 16.60, 16.65, 22.89, 25.90, 25.99, 29.47, 29.49, 29.57, 29.73, 29.86, 29.89, 29.91, 32.12, 36.99, 37.01, 63.54, 64.02, 70.71, 115.73, 127.25, 128.00, 128.09, 130.48, 130.50, 156.02, 173.87, 174.13.

4.3.9. {1-Fluoro-3-hexadecanoylamino-4-[4-(4-methoxy-3,5-dimethyl-pyridin-2-ylmethoxy)-phenyl]-butyl}-phosphonic acid dimethyl ester (13)

This compound was synthesized according to the general procedure E from 12 (1.098 g, 1.97 mmol) and 4 (531 mg, 2.17 mmol) in a yield of 1.151 g (83%). The product was then purified by using flash column chromatography (5% MeOH:CH₂Cl₂). ¹H NMR (300 MHz, CDCl₃) δ 0.84 (t, 3H, J = 6.5 Hz), 1.21 (m, 26H), 1.29 (m, 8H), 1.52 (p, 2H, J = 6.5 Hz), 2.08 (m, 3H), 2.23 (s, 3H), 2.29 (s, 3H), 2.77 (m, 2H), 3.76 (s, 3H), 4.135 (q, 4H, J = 8.1 Hz), 4.36 (m, 1H), 5.09 (s, 2H), 5.69–5.76 (m, 1H), 6.92 (d, 2H, J = 8,8 Hz), 7.06 (d, 2H, J = 8.5 Hz), 8.20 (s, 1H).

4.3.10. {1-Fluoro-3-hexadecanoylamino-4-[4-(4-methoxy-3,5-dimethyl-pyridin-2-ylmethoxy)-phenyl]-butyl}-phosphonic acid (14)

This product was formed by using general procedure H from 13 (70 mg, 0.099 mmol) to afford 41 mg yield (63%). ¹H NMR (300 MHz, CD₃OD) δ 0.90 (t, J = 6.8 Hz, 3H); 1.27 (m, 26H); 1.52 (m, 2H); 1.53 (m, 2H); 2.15 (m, 4H); 2.39 (s, 3H); 2.46 (s, 3H); 2.76 (m, 2H); 4.10 (s, 3H); 4.30 (m, 1H); 5.35 (s, 2h); 7.05 (d, J = 8.4 Hz, 2H); 7.23 (d, J = 7.9 Hz, 2H); 8.42 (s, 1H); elemental analysis CHN calcd % C = 64.59%, H = 8.67%, N = 4.30%; found C = 62.51%, H = 8.61%, N = 4.22%.

4.3.11. [1-(4-Benzyloxy-benzyl)-3-diazo-2-oxo-propyl]-carbamic acid tert-butyl ester (16)

To N-Boc, O-benzyl L-tyrosine acid (4 g, 10.8 mmol) in THF at 0 °C in a erlenmeyer flask free of internal scratches is added isobutyl chloroformate (2.206 g, 16.2 mmol). The reaction mixture is allowed to stir for 30 min before a solution of ethereal diazomethane (approximately 2.3 g, 54 mmol) is added via canula. Reaction mixture is allowed to slowly warm to room temperature and then allowed to stir overnight. The following day the reaction mixture was then stirred open to air in order to allow for unreacted diazomethane to dissipate. After a few hours the reaction mixture is extracted (sodium bicarbonate, then brine), dried (sodium sulfate), and then concentrated under reduced pressure. The reaction mixture is purified via flash chromatography (50% EtOAc in hexanes) to yield the product (2.16 g, 51%). ¹H NMR (300 MHz, CDCl₃) δ 1.42 (s, 9H), 2.96 (d, 2H, J = 6.77 Hz), 4.36 (b, 1H), 5.05 (s, 2H), 5.16 (b, 1H), 5.23 (b, 1H), 6.91 (d, 2H, J = 8.71 Hz), 7.10 (d, 2H, J = 8.71 Hz), 7.30–7.44 (m, 5H); ¹³C NMR δ 28.58, 37.98, 54.73, 58.90, 70.27, 115.28, 127.78, 128.82, 128.87, 130.69, 137.23, 158.09.

4.3.12. 4-(4-Benzyloxy-phenyl)-3-tert-butoxycarbonylamino-butyric acid methyl ester (17)

To a solution of the α-diazoketone 16 (2.16 g, 5.47 mmol), TEA (2.3 ml, 16.4 mmol) in 25 ml anhydrous methanol, and a small amount of THF (added to help solubilize the diazoketone) in a flask wrapped with tin foil, to protect reaction from light, and cooled to −78 °C was added a catalytic amount of silver benzoate. Silver benzoate was dissolved in a small amount of TEA before it is added to reaction mixture. Reaction mixture was allowed to stir at −78 °C for several hours before a second portion of silver benzoate (catalytic amount, dissolved in TEA) was added. Then reaction mixture was allowed to slowly warm to room temperature and then allowed to stir overnight. The next day the reaction mixture is filtered through silica gel by using EtOAc as eluent to remove any silver salts that are left over from the reaction. The mixture is then concentrated under reduced pressure and purified via flash column chromatography (20% EtOAc in hexanes) to yield the product (1.63 g, 73%). ¹H NMR (300 MHz, CDCl₃) δ 1.42 (s, 9H), 2.47 (m, 2H), 2.75 (m, 2H), 3.66 (s, 3H), 4.12 (b, 1H), 5.02 (s, 2H), 5.11 (b, 1H), 6.90 (d, 2H, J = 8.61 Hz), 7.10 (d, 2H, J = 8.42 Hz), 7.30–7.44 (m, 5H); ¹³C NMR δ 28.66, 37.88, 39.80, 49.25, 51.92, 70.22, 79.59, 115.14, 127.75, 128.20, 128.85, 130.34, 130.66, 137.39, 155.45, 157.85, 172.41.

4.3.13. [1-(4-Benzyloxy-benzyl)-3-hydroxy-propyl]-carbamic acid tert-butyl ester (18)

This product was synthesized according to general procedure C using 17 as the starting material (1.63 g, 3.95 mmol). The product was purified by flash column chromatography (EtOAc) and the yield was 1.41 g (97%). ¹H NMR (300 MHz, CDCl₃) δ 1.44 (s, 9H), 1.81 (b, 2H), 2.75 (d, 2H, J = 4.95 Hz), 3.65 (b, 2H), 4.02 (b, 1H), 4.78 (d, 1H, J = 8.98 Hz), 5.03 (s, 2H), 5.06 (b, 1H), 6.92 (d, 2H, J = 8.72 Hz), 7.11 (d, 2H, J = 8.73 Hz), 7.30–7.46 (m, 5H); ¹³C NMR δ 28.69, 37.85, 40.81, 48.69, 59.19, 70.30, 79.94, 115.14, 127.78, 128.26, 128.90, 130.53, 130.63, 137.39, 157.10, 157.77.

4.3.14. [1-(4-Benzyloxy-benzyl)-3-oxo-propyl]-carbamic acid tert-butyl ester (19)

This compound was prepared from 18 (1.41 g, 3.81 mmol) by using general procedure D. The product was purified by flash column chromatography (20% EtOAc in hexanes) in a yield of 1.32 g (94%). ¹H NMR (300 MHz, CDCl₃) δ 1.42 (s, 9H), 2.55 (m, 2H), 2.77 (m, 2H), 4.22 (b, 1H), 4.85 (d, 1H, J = 8.06 Hz), 5.03 (s, 2H), 5.06 (b, 1H), 6.94 (d, 2H, J = 8.60 Hz), 7.11 (d, 2H, J = 8.43 Hz), 7.32–7.46 (m, 5H), 9.68 (s, 1H); ¹³C NMR δ 28.63, 40.15, 47.78, 48.05, 70.27, 79.86, 115.28, 127.78, 128.26, 128.87, 130.02, 130.63, 137.31, 157.98, 201.44.

4.3.15. [4-(4-Benzyloxy-phenyl)-3-tert-butoxycarbonylamino-1-hydroxy-butyl]-phosphonic acid dimethyl ester (20)

To a solution of 60% NaH in mineral oil (215 mg, 5.38 mmol) in THF at 0 °C was added dimethyl phosphite (0.5 ml, 5.38 mmol) dropwise via syringe. The resulting mixture was allowed to stir at this temperature for 1 h before a solution of aldehyde 19 (1.32 g, 3.58 mmol) was added. The reaction mixture is then allowed to stir for 15 min before it was quenched with saturated ammonium chloride in water. The organic layer is then extracted with EtOAc, followed by brine, dried with sodium sulfate, and then concentrated under reduced pressure. The product was purified by flash column chromatography (EtOAc) in a yield of 1.02 g (60%). ¹H NMR (300 MHz, CDCl₃) δ 1.37 (s, 9H), 1.66 (m, 1H), 1.89 (m, 1H), 2.75 (d, 2H, J = 6.41 Hz), 3.75 (m, 6H), 4.05 (m, 2H), 4.80 (d, 1H, J = 8.97 Hz), 4.99 (s, 2H), 6.88 (d, 2H, J = 7.33 Hz), 7.07 (d, 2H, J = 7.33 Hz), 7.25–7.40 (m, 5H); ¹³C NMR δ 28.55, 37.02, 40.47, 48.08, 48.29, 53.61, 63.28, 65.52, 70.22, 80.26, 115.01, 115.20, 127.70, 128.18, 128.82, 130.07, 130.50, 130.80, 157.40, 157.82.

4.3.16. [4-(4-Benzyloxy-phenyl)-3-hexadecanoylamino-1-hydroxy-butyl]-phosphonic acid dimethyl ester (21a and b)

This compound was prepared from 20. The N-Boc protecting group was removed with the same method in preparation of 11 and then the TFA salt was reacted with palmitoyl chloride according to the general procedure A. The products were purified by column chromatography (2% MeOH in CH₂Cl₂) in a yield of 1.1 g (82% two steps). Two diastereomers were separated in the ratio of 1:4.

Compound 21a: ¹H NMR (300 MHz, CDCl₃) δ 0.87 (t, 3H, J = 5.85 Hz), 1.25 (s, 24H), 1.48 (p, 2H, J = 6.96 Hz), 1.69 (m, 1H), 2.00 (m, 1H), 2.12 (dt, 2H, J = 2.56, 7.30 Hz), 2.79 (dq, 2H, J = 5.49, 15.37 Hz), 3.81 (dd, 6H, J = 2.92, 10.61 Hz), 3.92 (m, 1H), 4.33 (m, 1H), 5.01 (s, 2H), 6.18 (d, 1H, J = 8.79 Hz), 6.89 (d, 2H, J = 8.05 Hz), 7.08 (d, 2H, J = 8.42 Hz), 7.25– 7.41 (m, 5H); ¹³C NMR δ 14.40, 22.95, 26.07, 29.41, 29.62, 29.76, 29.97, 32.19, 36.75, 37.50, 40.23, 47.09, 47.28, 53.71, 53.79, 63.20, 65.47, 70.25, 115.25, 127.67, 128.20, 128.82, 129.81, 129.94, 130.31, 137.23, 157.93, 165.76, 175.72.

Compound 21b: ¹H NMR (300 MHz, CDCl₃) δ 0.87 (t, 3H, J = 6.22 Hz), 1.23 (s, 24H), 1.51 (p, 2H, J = 6.95 Hz), 1.85 (m, 1H), 1.99 (m, 1H), 2.12 (t, 2H, J = 8.42 Hz), 2.80 (d, 2H, J = 6.59 Hz), 3.74 (t, 6H, J = 9.88 Hz), 4.04 (m, 1H), 4.25 (m, 1H), 5.00 (s, 2H), 6.18 (d, 1H, J = 7.69 Hz), 6.88 (d, 2H, J = 8.42 Hz), 7.09 (d, 2H, J = 8.79 Hz), 7.25–7.42 (m, 5H); ¹³C NMR δ 14.40, 22.95, 25.94, 29.57, 29.62, 29.68, 29.81, 29.92, 29.97, 32.19, 35.90, 37.13, 40.17, 49.49, 49.71, 53.45, 53.53, 53.82, 65.47, 67.66, 70.25, 115.06, 127.70, 128.20, 128.82, 130.25, 130.66, 157.82, 173.96.

4.3.17. [3-Hexadecanoylamino-1-hydroxy-4-(4-hydroxy-phenyl)-butyl]-phosphonic acid dimethyl ester (22a and b)

The two diastereomers were synthesized according to general procedure B from 21a (290 mg, 0.46 mmol) and 21 b (190 mg, 3.01 mmol) in the yields of 95 mg (58%) and 180 mg (73%).

Compound 22a: ¹H NMR (300 MHz, CDCl₃) δ 0.86 (t, 3H, J = 6.59 Hz), 1.23 (s, 24H), 1.50 (p, 2H, J = 5.85 Hz), 1.68 (m, 1H), 1.95 (m, 1H), 2.13 (t, 2H, J = 8.05 Hz), 2.74 (m, 2H), 3.79 (dd, 6H, J = 4.76, 10.24 Hz), 3.89 (m, 1H), 4.34 (m, 1H), 6.14 (d, 1H, J = 8.78 Hz), 6.73 (d, 2H, J = 8.42 Hz), 6.96 (d, 2H, J = 8.42 Hz); ¹³C NMR δ 14.34, 22.90, 26.02, 29.44, 29.54, 29.58, 29.72, 29.89, 29.94, 32.14, 36.74, 36.87, 40.14, 53.67, 53.74, 53.96, 54.06, 115.88, 128.08, 130.31, 155.94, 175.84.

Compound 22b: ¹H NMR (300 MHz, CDCl₃) δ 0.86 (t, 3H, J = 6.95 Hz), 1.23 (s, 24H), 1.51 (p, 2H, J = 6.90 Hz), 1.82 (m, 1H), 1.99 (m, 1H), 2.12 (t, 2H, J = 7.68 Hz), 2.72 (d, 2H, J = 5.49 Hz), 3.73 (t, 6H, J = 9.88 Hz), 4.00 (t, 1H, J = 7.69 Hz), 4.24 (m, 1H), 6.38 (m, 1H), 6.74 (d, 2H, J = 8.41 Hz), 6.95 (d, 2H, J = 8.42 Hz); ¹³C NMR δ 14.34, 22.92, 25.92, 29.54, 29.59, 29.79, 29.95, 32.14, 37.04, 53.49, 53.58, 53.76, 54.06, 115.78, 128.43, 130.58, 155.80, 174.36.

4.3.18. {3-Hexadecanoylamino-1-hydroxy-4-[4-(4-methoxy-3,5-dimethyl-pyridin-2-ylmethoxy)-phenyl]-butyl}-phosphonic acid dimethyl ester (23a and b)

The two diastereomers were synthesized according to general procedure I from 22a (250 mg, 0.474 mmol) and 22b (106 mg, 0.201 mmol) in the yields of 216 mg (67%) and 86 mg (63%).

Compound 23a: ¹H NMR (300 MHz, CDCl₃) δ 0.87 (t, 3H, J = 7.0 Hz), 1.24 (m, 26H); 1.49 (p, 2H, J = 7.0 Hz), 1.68 (m, 1H), 2.02 (m, 1H), 2.12 (m, 2H), 2.27 (s, 3H), 2.32 (s, 3H), 2.79 (dq, 2H, J = 7.9, 14.9 Hz), 3.78 (s, 3H), 3.81 (dd, 6H, J = 4.9, 10.3 Hz), 3.87 (m, 1H), 4.36 (m, 1H), 5.12 (s, 2H), 5.72 (d, 1H, J = 8.6 Hz), 6.95 (d, 2H, J = 8.8 Hz), 7.07 (d, 2H, J = 8.8 Hz), 8.23 (s, 1H).

Compound 23b: ¹H NMR (300 MHz, CDCl₃) δ 0.86 (t, 3H, J = 6.9 Hz), 1.23 (m, 26H), 1.53 (m, 2H), 1.83 (m, 1H), 2.03 (m, 1H), 2.09 (t, 2H, J = 8.5 Hz), 2.26 (s, 3H), 2.31 (s, 3H), 2.79 (d, 2H, J = 6.2 Hz), 3.75 (m, 9H), 4.01 (m, 1H), 4.12 (q, 1H, J = 6.5 Hz), 4.21 (m, 1H), 5.11 (s, 2H), 5.99 (d, 1H, J = 7.7 Hz), 6.93 (d, 2H, J = 8.8 Hz), 7.07 (d, 2H, J = 8.8 Hz), 8.23 (s, 1H).

4.3.19. {3-Hexadecanoylamino-1-hydroxy-4-[4-(4-methoxy-3,5-dimethyl-pyridin-2-ylmethoxy)-phenyl]-butyl}-phosphonic acid (24a and b)

The two diastereomers were synthesized according to general procedure H from 23a (56 mg, 0.0825 mmol) and 23b (86 mg, 0.127 mmol) in the yields of 43 mg (80%) and 70 mg (86%).

Compound 24a: ¹H NMR (300 MHz, CD₃OD) δ 0.89 (t, 3H, J = 6.8 Hz, 3H), 1.26 (m, 26H), 1.51 (m, 2H), 1.83 (m, 2H), 2.13 (t, 2H, J = 6.6 Hz), 2.35 (s, 3H), 2.39 (s, 3H), 2.75 (d, 2H, J = 6.6 Hz), 3.38 (m, 1H), 3.99 (s, 2H), 4.32 (m, 1H), 4.94 (s, 2H), 5.25 (m, 1H), 6.99 (d, 2H, J = 9.2 Hz), 7.19 (d, 2H, J = 8.2 Hz), 8.35 (s, 1H); ¹³C NMR (300 MHz, CD₃OD) δ 14.03, 14.46, 23.75, 27.20, 30.32, 30.48, 30.61, 30.83, 33.10, 37.29, 41.67, 93.43, 98.51, 115.92, 131.68, 133.47, 157.96, 170.14; elemental analysis CHN calcd % C = 64.79%, H = 8.86%, N = 4.32%; found C = 64.06%, H = 9.14%, N = 4.56%.

Compound 24b: ¹H NMR (300 MHz, CD₃OD) δ 0.89 (t, 3H, J = 6.5 Hz), 1.27 (m, 26H), 1.46 (p, 2H, J = 7.3 Hz), 1.84 (m, 1H), 1.99 (m, 1H), 2.06 (t, 2H, J = 6.9 Hz), 2.36 (s, 3H), 2.41 (s, 3H), 2.60 (m, 1H), 2.90 (m, 1H), 3.82 (m, 1H), 4.01 (s, 2H), 4.30 (m, 1H), 5.27 (s, 2H), 6.68 (d, 1H, J = 8.1 Hz), 7.01 (m, 2H), 7.20 (d, 2H, J = 7.0 Hz), 8.37 (s, 1H); ¹³C NMR (300 MHz, CD₃OD) δ 9.95, 12.88, 13.27, 22.57, 25.89, 29.04, 29.07, 29.31, 29.34, 29.41, 29.45, 29.64, 31.90, 36.15, 36.67, 38.41, 60.38, 66.73, 95.54, 114.65, 114.79, 130.22, 130.54, 132.42, 174.37; elemental analysis CHN calcd % C = 64.79%, H = 8.86%, N = 4.32%; found C = 62.91%, H = 8.94%, N = 4.03%.

4.3.20. {2-Fluoro-3-hexadecanoylamino-4-[4-(4-methoxy-3,5-dimethyl-pyridin-2-ylmethoxy)-phenyl]-butyl}-phosphonic acid (25a)

To a solution of 49a (30 mg, 0.04 mmol) in CH₂Cl₂ at −78 °C was added via cannula a pre-cooled solution of DAST (0.015 ml, 0.11 mmol) CH₂Cl₂. The reaction mixture was allowed to warm to room temperature and after 1 h, a saturated solution of sodium bicarbonate was added and the mixture was diluted with EtOAc. The mixture was extracted with EtOAc and combined organic layers were washed with brine and dried over magnesium sulfate and concentrated under vacuum. The residue was purified by column chromatography (4% MeOH:CH₂Cl₂) to give the dimethyl ester product in 14 mg (47%). ¹H NMR (300 MHz, CDCl₃) δ 0.87 (t, 3H, J = 7.57 Hz), 1.25 (s, 24H), 1.61 (p, 2H, J = 7.10 Hz), 2.05 (m, 2H), 2.26 (s, 3H), 2.28 (t, 2H, J = 7.48 Hz), 2.32 (s, 3H), 2.75 (dq, 2H, J = 8.32, 15.05 Hz), 3.75 (m, 6H), 3.77 (s, 3H), 4.38 (q, 1H, J = 7.48 Hz), 4.87 (dq, 1H, J = 3.49, 10.48 Hz), 5.12 (s, 2H), 6.95 (d, 2H, J = 8.73 Hz), 7.12 (d, 2H, J = 8.73 Hz), 8.23 (s, 1H). MS (ESI) m/z 679.6 [M+H]⁺. Compound 25a was prepared according to the general procedure H to give 8 mg (62%). ¹H NMR (300 MHz, CD ₃OD) δ 0.89 (t, 3H, J = 6.35 Hz), 1.28 (s, 24H), 1.64 (p, 2H, J = 6.80 Hz), 2.19 (dd, 2H, J = 7.26, 19.05 Hz), 2.40 (s, 3H), 2.45 (t, 2H, J = 7.71 Hz), 2.50 (s, 3H), 2.63 (dd, 1H, J = 9.53, 14.06 Hz), 3.19 (dd, 1H, J = 5.44, 14.97 Hz), 3.68 (m, 6H), 3.91 (m, 1H), 4.16 (s, 3H), 5.45 (s, 2H), 7.23 (d, 2H, J = 8.62 Hz), 7.38 (d, 2H, J = 8.62 Hz), 8.50 (s, 1H). MS (ESI) m/z 651.2 [M+H]⁺.

4.3.21. {2-Fluoro-3-hexadecanoylamino-4-[4-(4-methoxy-3,5-dimethyl-pyridin-2-ylmethoxy)-phenyl]-butyl}-phosphonic acid (25b)

It was prepared from 49b with the same method as in the preparation of 25a with same reaction yield. ¹H NMR (300 MHz, CD₃OD) δ 0.89 (t, 3H, J = 6.54 Hz), 1.28 (s, 24H), 1.67 (p, 2H, J = 6.94 Hz), 2.15 (m, 2H), 2.40 (s, 3H), 2.47 (t, 2H, J = 6.54 Hz), 2.50 (s, 3H), 2.95 (m, 1H), 3.94 (m, 1H), 4.16 (s, 3H), 5.39 (m, 1H), 5.45 (s, 2H), 7.10 (m, 1H), 7.29 (m, 4H), 8.51 (s, 1H). MS (ESI) m/z 651.8 [M+H]⁺.

4.3.22. {3-Hexadecanoyl-4-[4-(4-methoxy-3,5-dimethyl-pyridin-2-ylmethoxy)-benzyl]-2,2-dimethyl-oxazolidin-5-ylmethyl}-phosphonic acid dimethyl ester (47)

2-Methoxypropene (0.06 ml, 0.63 mmol) was added to a solution of 49a (80 mg, 0.12 mmol) in CH₂Cl₂ at 0 °C, then CSA (5.5 mg, 0.02 mmol) was added and the mixture was allowed to warm to room temperature overnight. The next day the reaction mixture was diluted with CH₂Cl₂ and washed with saturated sodium bicarbonate solution then dried over magnesium sulfate and concentrated under vacuum. The residue was purified by preparative TLC (4% MeOH:CH₂Cl₂) to give 27 mg product (32%). ¹H NMR (300 MHz, CDCl₃) δ 0.86 (t, 3H, J = 6.06 Hz), 1.24 (bs, 24H), 1.60 (m, 2H), 1.63 (s, 3H), 1.66 (s, 3H), 2.00 (m, 2H), 2.25 (s, 3H), 2.30 (s, 3H), 2.85 (m, 2H), 3.50 (m, 6H), 3.77 (s, 3H), 4.13 (dd, 1H, J = 5.30, 9.60 Hz), 4.33 (m, 1H), 5.12 (s, 2H), 6.98 (d, 2H, J = 8.61 Hz), 7.11 (d, 2H, J = 8.61 Hz), 8.24 (s, 1H). ¹³C NMR δ 11.14, 13.62, 14.37, 22.95, 25.43, 28.13, 28.58, 29.62, 29.70, 29.81, 29.94, 31.25, 32.16, 33.07, 35.85, 40.68, 52.46, 52.54, 60.15, 64.61, 64.66, 70.99, 74.89, 115.41, 126.76, 129.57, 130.58, 149.33, 154.33, 154.38, 158.01, 170.59.

4.3.23. {3-Hexadecanoyl-4-[4-(4-methoxy-3,5-dimethyl-pyridin-2-ylmethoxy)-benzyl]-2,2-dimethyl-oxazolidin-5-ylmethyl}-phosphonic acid dimethyl ester (48)

2-Methoxypropene (0.06 ml, 0.63 mmol) was added to a solution of 49b (80 mg, 0.12 mmol) in CH₂Cl₂ at 0 °C, then CSA (5.5 mg, 0.02 mmol) was added and the mixture was allowed to warm to room temperature overnight. The next day the reaction mixture was diluted with CH₂Cl₂ and washed with saturated sodium bicarbonate solution then dried over magnesium sulfate and concentrated under vacuum. The residue was purified by preparative TLC (4% MeOH:CH₂Cl₂) to give 25 mg product (30%). ¹H NMR (300 MHz, CDCl₃) δ 0.87 (t, 3H, J = 6.72 Hz), 1.24 (br s, 24H), 1.60 (s, 3H), 1.65 (m, 2H), 1.77 (s, 3H), 2.10 (m, 2H), 2.26 (s, 3H), 2.30 (s, 3H), 2.65 (m, 1H), 2.95 (dd, 1H, J = 5.60, 4.18 Hz), 3.77 (s, 3H), 3.78 (m, 6H), 4.06 (m, 1H), 4.60 (m, 1H), 5.11 (s, 2H), 6.95 (d, 2H, J = 8.21 Hz), 7.07 (d, 2H, J = 8.21 Hz), 8.25 (s, 1H). ¹³C NMR δ 11.17, 13.65, 14.40, 22.95, 24.25, 25.16, 27.22, 27.65, 29.49, 29.62, 29.73, 29.78, 29.97, 32.19, 35.34, 35.63, 52.19, 52.75, 53.21, 61.38, 61.49, 70.97, 72.17, 101.76, 115.54, 129.89, 130.74, 149.38, 154.35, 158.04, 171.31.

4.4. Synthesis of compounds 26–46

These compounds were synthesized from appropriate amino acids by using General procedures A, E, F, G, and H.²⁴

4.4.1. (3-Hexadecanoylamino-2-oxo-propyl)-phosphonic acid (26)

¹H NMR (300 MHz, CD₃OD) δ 0.90 (t, 3H, J = 7.03 Hz), 1.28 (s, 24H), 1.62 (q, 2H, J = 7.03 Hz), 2.25 (t, 2H, J = 7.03 Hz), 3.15 (m, 2H), 4.20 (s, 2H). MS (ESI) m/z 392.5 [M+H]⁺.

4.4.2. (3-Hexadecanoylamino-2-hydroxy-propyl)-phosphonic acid (27)

¹H NMR (300 MHz, CD₃OD) δ 0.90 (t, 3H, J = 6.93 Hz), 1.28 (s, 24H), 1.62 (q, 2H, J = 6.93 Hz), 1.90 (m, 2H), 2.22 (t, 2H, J = 6.94 Hz), 3.22 (dd, 1H, J = 6.63, 14.47 Hz), 3.38 (dd, 1H, J = 3.38, 14.32 Hz), 4.03 (m, 1H). MS (ESI) m/z 394.3 [M+H]⁺.

4.4.3. (3-Hexadecanoylamino-2-oxo-butyl)-phosphonic acid (28)

¹H NMR (300 MHz, CD₃OD) δ 0.90 (t, 3H, J = 6.93 Hz), 1.28 (s, 24H), 1.33(d, 3H, J = 7.42 Hz), 1.61 (q, 2H, J = 7.28 Hz), 2.23 (t, 2H, J = 7.43 Hz), 3.20 (m,2H), 4.50 (m, 1H). MS (ESI) m/z 406.5 [M+H]⁺.

4.4.4. (3-Hexadecanoylamino-2-hydroxy-butyl)-phosphonic acid (29)

¹H NMR (300 MHz, CD₃OD) δ 0.90 (t, 3H, J = 6.96 Hz), 1.14 (d, 3H, J = 6.50 Hz), 1.29 (s, 24H), 1.61 (q, 2H, J = 6.86 Hz), 1.91 (m, 2H), 2.19 (t, 2H, J = 6.97 Hz), 3.70 (m, 1H), 3.91(m, 2H). MS (ESI) m/z 408.4 [M+H]⁺.

4.4.5. (3-Hexadecanoylamino-4-methyl-2-oxo-pentyl)-phosphonic acid (30)

¹H NMR (300 MHz, CD₃OD) δ 0.90 (m, 9H), 1.28 (s, 24H), 1.61 (m, 2H), 2.13 (m, 1H), 2.26 (t, 2H, J = 7.52 Hz), 4.31 (m, 1H), 8.21 (b, 1H). MS (ESI) m/z 434.6 [M+H]⁺.

4.4.6. (3-Hexadecanoylamino-2-hydroxy-4-methyl-pentyl)-phosphonic acid (31)

¹H NMR (300 MHz, CD₃OD) δ 0.93 (m, 9H), 1.29 (s, 24H), 1.63 (p, 2H, J = 7.09 Hz), 1.92 (m, 2H), 2.28 (m, 1H), 2.40 (t, 2H, J = 7.10 Hz), 3.58 (m, 2H). MS (ESI) m/z 436.1 [M+H]⁺.

4.4.7. (3-Hexadecanoylamino-2-hydroxy-4-methyl-pentyl)-phosphonic acid (32)

¹H NMR (300 MHz, CD₃OD) δ 0.89 (m, 9H), 1.28 (s, 24H), 1.62 (p, 2H, J = 6.62 Hz), 1.95 (m, 2H), 2.28 (m, 1H), 2.25 (t, 2H, J = 7.66 Hz), 3.67 (m, 2H), 3.78 (m, 1H). MS (ESI) m/z 436.1 [M+H]⁺.

4.4.8. (3-Hexadecanoylamino-5-methyl-2-oxo-hexyl)-phosphonic acid (33)

¹H NMR (300 MHz, CD₃OD) δ 0.94 (m, 9H), 1.28 (s, 24H), 1.64 (m, 5H), 2.25 (t, 2H, J = 6.73 Hz), 3.16 (m, 2H), 4.59 (m, 1H). MS (ESI) m/z 448.4 [M+H]⁺.

4.4.9. (3-Hexadecanoylamino-2-hydroxy-5-methyl-hexyl)-phosphonic acid (34)

¹H NMR (300 MHz, CD₃OD) δ 0.92 (m, 9H), 1.28 (s, 24H), 1.62 (m, 5H), 1.90 (m, 2H), 2.25 (t, 2H, J = 7.27 Hz), 3.72 (m, 1H), 3.99 (m, 1H). MS (ESI) m/z 450.4 [M+H]⁺.

4.4.10. (3-Hexadecanoylamino-2-hydroxy-5-methyl-hexyl)-phosphonic acid (35)

¹H NMR (300 MHz, CD₃OD) δ 0.92 (m, 9H), 1.28 (s, 24H), 1.43 (m, 2H), 1.61 (m, 3H), 1.91 (m, 2H), 2.22 (t, 2H, J = 7.41 Hz), 3.72 (m, 1H), 3.84 (m, 1H), 3.95 (b, 1H). MS (ESI) m/z 450.2 [M+H]⁺.

4.4.11. (3-Hexadecanoylamino-2-hydroxy-4-phenyl-butyl)-phosphonic acid (36)

¹H NMR (300 MHz, CD₃OD) δ 0.90 (t, 3H, J = 7.05 Hz), 1.28 (s, 24H), 1.46 (p, 2H, J = 7.05 Hz), 1.92 (m, 2H), 2.13 (t, 2H, J = 7.40 Hz), 2.80 (m, 1H), 2.93 (m, 1H), 4.07(m, 1H), 4.16 (m, 1H), 7.24 (m, 5H). MS (ESI) m/z 484.4 [M+H]⁺.

4.4.12. (3-Hexadecanoylamino-2-hydroxy-4-phenyl-butyl)-phosphonic acid (37)

¹H NMR (300 MHz, CD₃OD) δ 0.90 (t, 3H, J = 6.74 Hz), 1.29 (s, 24H), 1.37 (p, 2H, J = 6.37 Hz), 1.90 (m, 2H), 2.04 (t, 2H, J = 7.11 Hz), 2.60 (m, 1H), 3.15 (m, 1H), 3.95 (m, 1H), 4.12 (m, 1H), 7.22 (m, 5H). MS (ESI) m/z 484.5 [M+H]⁺.

4.4.13. [2-(1-Hexadecanoyl-pyrrolidin-2-yl)-2-oxo-ethyl]-phosphonic acid (38)

¹H NMR (300 MHz, CD₃OD) δ 0.90 (t, 3H, J = 7.16 Hz), 1.29 (s, 24H), 1.59 (q, 2H, J = 7.16 Hz), 2.00 (m, 2H), 2.21 (m, 2H), 2.36 (t, 2H, J = 7.47 Hz), 3.12 (m, 1H), 3.21 (m, 1H), 3.62 (m, 2H), 4.66 (m, 1H). MS (ESI) m/z 432.4 [M+H]⁺.

4.4.14. [2-(1-Hexadecanoyl-pyrrolidin-2-yl)-2-hydroxy-ethyl]-phosphonic acid (39)

¹H NMR (300 MHz, CD₃OD) δ 0.90 (t, 3H, J = 7.51 Hz), 1.24 (s, 24H), 1.61 (q, 2H, J = 7.00 Hz), 1.92 (m, 2H), 2.05 (m, 4H), 2.38 (t, 2H, J = 7.51 Hz), 3.59 (m, 2H), 4.10 (m, 1H), 4.25 (m,1H). MS (ESI) m/z 434.7 [M+H]⁺.

4.4.15. [3-Hexadecanoylamino-4-(1H-indol-3-yl)-2-oxo-butyl]-phosphonic acid (40)

¹H NMR (300 MHz, CD₃OD) δ 0.90 (t, 3H, J = 6.98 Hz), 1.28 (s, 24H), 1.45 (q, 2H, J = 6.98 Hz), 2.13 (t, 2H, J = 7.29 Hz), 3.10 (m, 2H), 3.17 (m, 2H), 7.05 (m, 2H), 7.08 (s, 1H), 7.31 (d, 1H, J = 8.25 Hz), 7.58 (d, 1H, J = 8.25 Hz). MS (ESI) m/z 521.6 [M+H]⁺.

4.4.16. [3-Hexadecanoylamino-2-hydroxy-4-(1H-indol-3-yl)-butyl]-phosphonic acid (41)

¹H NMR (300 MHz, CD₃OD) δ 0.89 (t, 3H, J = 7.09 Hz), 1.27 (s, 24H), 1.46 (m, 2H), 1.93 (m, 2H), 2.16 (t, 2H, J = 7.00 Hz), 3.06 (m, 2H), 4.15 (m, 1H), 4.25 (m, 1H), 7.03 (m, 2H), 7.09 (s, 1H), 7.30 (d, 1H, J = 8.42 Hz), 7.62 (d, 1H, J = 8.42 Hz), 8.25 (s, 1H). MS (ESI) m/z 523.9 [M+H]⁺.

4.4.17. [3-Hexadecanoylamino-2-hydroxy-4-(1H-indol-3-yl)-butyl]-phosphonic acid (42)

¹H NMR (300 MHz, CD₃OD) δ 0.89 (t, 3H, J = 7.38 Hz), 1.28 (br s, 26H), 2.11 (m, 2H), 2.14 (t, 2H, J = 7.98 Hz), 2.93 (m, 1H), 3.21 (m, 1H), 4.09 (m, 1H), 4.37 (m, 1H), 7.01 (m, 2H), 7.08 (s, 1H), 7.30 (d, 1H, J = 8.60 Hz), 7.61 (d, 1H, J = 8.60 Hz). MS (ESI) m/z 523.9 [M+H]⁺.

4.4.18. [4-(1-Benzyl-1H-indol-3-yl)-3-hexadecanoylamino-2-hydroxy-butyl]-phosphonic acid (43)

¹H NMR (300 MHz, CD₃OD) δ 0.89 (t, 3H, J = 6.51 Hz), 1.27 (br s, 26H), 2.03 (t, 2H, J = 6.76 Hz), 3.73 (d, 2H, J = 9.85 Hz), 4.02 (m, 1H), 4.27 (m, 1H), 5.30 (s, 2H), 7.01–7.07 (m, 3H), 7.09 (s, 1H), 7.20–7.33 (m, 5H), 7.62 (d, 1H, J = 7.88 Hz). MS (ESI) m/z 613.3 [M+H]⁺.

4.4.19. {3-Hexadecanoylamino-2-hydroxy-4-[1-(4-methoxy-3,5-dimethyl-pyridin-2-ylmethyl)-1H-indol-3-yl]-butyl}-phosphonic acid (44)

¹H NMR (300 MHz, CD₃OD) δ 0.90 (t, 3H, J = 7.40 Hz), 1.28 (s, 26H), 1.65 (m, 2H), 2.01 (t, 2H, J = 7.93 Hz), 2.40 (s, 3H), 2.46 (s, 3H), 3.24 (m, 2H), 3.34 (s, 3H), 3.37 (m, 2H), 4.15 (s, 2H), 5.73 (d, 1H, J = 7.17 Hz), 7.10 (s, 1H), 7.08–7.18 (m, 2H), 7.24 (d, 1H, J = 6.86 Hz), 7.70 (d, 1H, J = 7.17 Hz), 8.36 (s, 1H). MS (ESI) m/z 672.3 [M+H]⁺.

4.4.20. (4-Benzyloxy-3-hexadecanoylamino-2-hydroxy-butyl)-phosphonic acid (45)

¹H NMR (300 MHz, CD₃OD) δ 0.89 (t, 3H, J = 7.22 Hz), 1.27 (s, 24H), 1.61 (q, 2H, J = 7.23 Hz), 1.88–1.96 (m, 2H), 2.26 (t, 2H, J = 7.22 Hz), 3.60 (m, 2H), 4.16 (m, 1H), 4.25 (m, 1H), 4.53 (s, 2H), 7.25–7.35 (m, 5H). MS (ESI) m/z 514.3 [M+H]⁺.

4.4.21. (4-Benzyloxy-3-hexadecanoylamino-2-hydroxy-butyl)-phosphonic acid (46)

¹H NMR (300 MHz, CD₃OD) δ 0.89 (t, 3H, J = 6.86 Hz), 1.27 (s, 24H), 1.59 (q, 2H, J = 6.86 Hz), 1.89–2.00 (m, 2H), 2.22 (t, 2H, J = 6.86 Hz), 3.86 (m, 2H), 4.07 (m, 1H), 4.52 (s, 2H), 7.27–7.35 (m, 5H). MS (ESI) m/z 514.2 [M+H]⁺.