Synthesis of Polyfluoro Ketones for Selective Inhibition of Human Phospholipase A₂ Enzymes

Abstract

The development of selective inhibitors for individual PLA₂ enzymes is necessary in order to target PLA₂-specific signaling pathways; but it is challenging due to the observed promiscuity of known PLA₂ inhibitors. In the current work, we present the development and application of a variety of synthetic routes to produce pentafluoro, tetrafluoro and trifluoro derivatives of activated carbonyl groups in order to screen for selective inhibitors and characterize the chemical properties that can lead to selective inhibition. Our results demonstrate that the pentafluoroethyl ketone functionality favors selective inhibition of the GVIA iPLA₂, a very important enzyme for which specific, potent reversible inhibitors are needed. We find that 1,1,1,2,2-pentafluoro-7-phenyl-heptan-3-one (FKGK11) is a selective inhibitor of GVIA iPLA₂ (X_I(50) = 0.0073). Furthermore, we conclude that the introduction of an additional fluorine atom at the α′ position of a trifluoromethyl ketone constitutes an important strategy for the development of new potent GVIA iPLA₂ inhibitors.

Introduction

Phospholipase A₂ (PLA₂) enzymes catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids producing free fatty acids and lysophospholipids.^1,2 Both products are precursor signaling molecules that are involved in a plethora of biological functions. The PLA₂ superfamily currently consists of fifteen groups and many subgroups of which a number of enzymes differ in primary sequence, structure and catalytic mechanism.¹ Among the various PLA₂ enzymes, Group IVA cPLA₂ (GIVA cPLA₂) is considered the rate-limiting provider of arachidonic acid and lysophospholipids that can be converted into prostaglandins, leukotrienes and PAF, respectively.^1–3 Another major intracellular PLA₂, the calcium-independent PLA₂ (GVIA iPLA₂) appears to be the primary phospholipase for basal metabolic functions within the cell.^1,2,4,5 Both intracellular enzymes share the same catalytic mechanism of utilizing a serine residue as the nucleophile. The PLA₂ superfamily also includes a type of small, secreted phospholipase (sPLA₂) that is characterized by a catalytic His/Asp dyad as well as a catalytic Ca²⁺.^1,2,6 A well-studied example of this class is the human Group V secreted phospholipase A₂ (GV sPLA₂).⁷ In many cases the activity of sPLA₂ has been shown to be dependent on or linked to the activity of GIVA cPLA₂.^8–10

Various classes of synthetic compounds have been studied as inhibitors of human GIVA cPLA₂, GVIA iPLA₂ and GV sPLA₂; and the results are summarized in recent review articles.^11,12 One of the most potent inhibitors of GIVA cPLA₂ is pyrrophenone (1, Figure 1).¹³ Other recently reported inhibitors include 2-propanone derivatives combined with the indole ring (e.g. 2, Figure 1)^14–16 and a series of indole derivatives^17–19 presented by Wyeth (for example compounds 3a and 3b, Figure 1) of which Efipladib (3b) is currently in phase I clinical trials.¹⁹ Our laboratories have reported on the development of 2-oxoamide inhibitors of GIVA cPLA₂ (e.g. 4a–d, Figure 1). ^20–26

Figure 1.

Some known inhibitors of GIVA cPLA₂.

Historically, the first potent inhibitor of GIVA cPLA₂ was a trifluoromethyl ketone analogue of arachidonic acid (AACOCF₃) in which the carboxyl group was replaced by COCF₃ (5, Figure 2).²⁷ This analogue was shown to be a slow- and tight-binding inhibitor of GIVA cPLA₂ and its mechanism of inhibition has been characterized via ¹⁹F NMR and ¹³C NMR.²⁸ Trifluoromethyl ketone analogues of H-linolenic and linoleic acid as well as the analogue of palmitic acid (6, Figure 2) also inhibit GIVA cPLA₂.^29,30 Furthermore, a variety of trifluoromethyl ketones have been analyzed with phospholipid vesicle-, detergent-phospholipid mixed micelle-, and natural membrane-based assays.³¹

Figure 2.

Trifluoromethyl ketone inhibitors of GIVA cPLA₂ and GVIA iPLA₂.

AACOCF₃ has been used as a tool to study the role of GIVA cPLA₂ inhibition in various animal models. Using this inhibitor, it was demonstrated that GIVA cPLA₂ plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis.³² AACOCF₃ was also used to study possible contributions of central nervous PLA₂ enzymes to the development of allodynia after facial carrageenan injection in mice.³³ Intrathecal administration of AACOCF₃ prevented thermal hyperalgesia induced by intraplantar carrageenan as well as formalin-induced flinching in a dose-dependent manner.³⁴ Intrathecal injection of AACOCF₃, at antihyperalgesic doses, decreased the release of prostaglandin PGE-2 into spinal dialysate-evoked N-methyl-D-aspartate (NMDA).³⁵ Similarly, treatment of prion-infected cell lines indicated a pivotal role for PLA₂ enzymes in prion diseases.³⁵ Even so, the various in vivo activities of AACOCF₃ should be viewed with some caution, since this inhibitor is not selective for GIVA cPLA₂ and has been reported to cause cell lysis.³⁶ Additional trifluoromethyl ketone derivatives are also observed to inhibit GIVA cPLA₂.^37–40 For example, BMS-229724⁴¹ (7, Figure 2) was reported to be a tight-binding inhibitor of GIVA cPLA₂ possessing anti-inflammatory activity in skin inflammation models.⁴¹

Trifluoromethyl ketone analogs of arachidonic and palmitic acids also inhibit GVIA iPLA₂.⁴² Both compounds inhibited macrophage GVIA iPLA₂ in a concentration-dependent manner and, in contrast to GIVA cPLA₂, GIVA iPLA₂ showed a preference for the saturated fatty chain.⁴² Inhibition studies of a variety of trifluoromethyl ketones as inhibitors of GVIA iPLA₂ in mixed-micelle assays found that one trifluoromethyl ketone (8, Figure 2) is a potent inhibitor of GVIA iPLA₂ presenting a X_I(50) value of 0.0043, which is ten-fold more potent than the corresponding value against GIVA cPLA₂.³¹

Continuing our efforts to synthesize selective inhibitors for the various PLA₂ enzyme types, we designed a variety of polyfluoro ketone-based derivatives. In this work, we present routes for the synthesis of polyfluoro ketones and demonstrate their inhibition of the three major human PLA₂ enzymes: GIVA cPLA₂, GVIA iPLA₂ and GV sPLA₂, but with vastly different specificities. Of particular note is the development of specific GVIA iPLA₂ inhibitors.

Design and Synthesis of Polyfluoro Ketones

We designed a variety of polyfluoro ketones and examples of such activated carbonyl functionalities are depicted in Figure 3. The rationale behind our design of polyfluoro ketones was based on: (a) Increase of the carbonyl reactivity by introduction of additional fluorine atoms at the β- or α′-positions. The inductive effect of additional fluorine atoms may increase carbonyl reactivity against nucleophiles, such as the active-site serine hydroxyl group in GIVA cPLA₂ and GVIA iPLA₂; and (b) Increase of the inhibitor binding affinity to the target enzymes. Additional fluorine atoms at the β- or α′-position may contribute to the development of additional interactions, further stabilizing the enzyme-inhibitor complex. Recently, it has become clear that fluorine can enhance binding efficacy and selectivity in pharmaceuticals due to a variety of multipolar C-F^···H-N, C-F^···C=O, and C-F^···H-C_α interactions between a fluorinated ligand and protein binding-site.^43,44 Since the natural substrates of PLA₂ enzymes are long chain phospholipids, we chose to attach the polyfluoro ketone functionality to a long aliphatic chain as well as to short or medium chains carrying a non-substituted or para-alkoxy (or aryloxy) substituted ring.

Figure 3.

Polyfluoro ketone functionalities.

Among the existing methods, the synthesis of trifluoromethyl ketones through conversion of carboxylic acids into chlorides followed by subsequent treatment with trifluoroacetic anhydride and pyridine⁴⁵ has found wide application. We observe that simple carboxylic acids, amino acids and peptides,⁴⁶ and even lipophilic glyceride analogues, as we have demonstrated for the synthesis of potent gastric lipases inhibitors,⁴⁷ are able to produce trifluoromethyl ketones in satisfactory yields. For the synthesis of pentafluoroethyl ketones, carboxylic acids 9a–c were converted to chlorides by treatment with oxalyl chloride and then to the target compounds 10a–c using pentafluoropropionic anhydride and pyridine (Figure 4). For comparison purposes, we prepared pentafluoroethyl ketone 11 corresponding to palmitic acid as well as trifluoromethyl ketones 12a,b corresponding to pentafluoro derivatives 10b,c.

Figure 4.

Reagents and conditions: (a) i) (COCl₂)₂, CH₂Cl₂; ii) (CF₃CF₂CO)₂O, pyridine, CH₂Cl₂.

The synthesis of various trifluoromethyl and pentafluoroethyl ketones is depicted in Figure 5. The hydroxymethyl group of compounds 13a,b was oxidized to an aldehyde by the NaClO/TEMPO method.⁴⁸ Wittig olefination of aldehydes 14a,b and Wadworth-Horner-Emmons reaction led to elongation of the chain by two or four carbon atoms, respectively. After hydrogenation and saponification, carboxylic acids 17a,b and 18a,b were converted to fluoroketones 19a,b, 20a,b and 21 as described above. The trifluoromethyl ketone 23 was prepared from he t known carboxylic acid 22 (Figure 6).

Figure 5.

Reagents and conditions: (a) NaOCl, TEMPO, NaBr, NaHCO₃, toluene/EtOAc, H₂O; (b) Ph₃P=CHCOOCH₃, CH₂Cl₂; (c) C₂H₅OOCH=CHCH₂P(=O)(OC₂H₅), LiOH, THF; (d) i) H₂, 10% Pd, ii) NaOH, CH₃OH; (e) i) (COCl₂)₂, CH₂Cl₂, ii) (CF₃CO)₂O, pyridine, CH₂Cl₂.

Figure 6.

Reagents and conditions: (a) i) (COCl₂)₂, CH₂Cl₂, ii) (CF₃CO)₂O, pyridine, CH₂Cl₂.

Tetrafluoro derivative 26 was synthesized as shown in Figure 7. The replacement of the hydroxyl group of methyl 2-hydroxy-hexadecanoate (24) with fluorine was carried out by treatment with diethylaminosulfur trifluoride (DAST), a well-known fluorinating agent.⁴⁹ Treatment of methyl ester 25 by (trifluoromethyl)trimethylsilane in the presence of a catalytic amount of cesium fluoride, followed by hydrolysis of silyl ether intermediate,⁵⁰ led directly to tetrafluoro derivative 26. It should be noted that a 2-fluorocarboxylic acid cannot transform into a trifluoromethyl ketone by conversion to chloride and treatment with anhydride and pyridine, probably because the intermediate ketene required for such a transformation⁴⁵ cannot be formed.

Figure 7.

Reagents and conditions: (a) Deoxofluor, dry CH₂Cl₂; (b) i) (CH₃)₃SiCF₃, CsF, CH₃OCH₂CH₂OCH₃, ii) conc. HCl.

To synthesize pentafluoro derivative 30, we explored two different routes (Figures 8 and 9). Reaction of diethyl oxalate with Grignard reagent⁵¹ 27 led to 2-oxoester 28 (Figure 8). DAST is an efficient reagent for the conversion of 2-oxoesters to 2,2-difluoroesters;^52,53 therefore, 2-oxoester 28 was fluorinated by treatment with DAST and ethyl ester 29 was converted to trifluoromethyl ketone 30 as described above. Alternatively, compound 30 was prepared starting from aldehyde 31 (Figure 9). Formation of cyanohydrin 32 was followed by methanolysis and finally oxidation to produce 2-oxoester 34. By similar procedures to those described above, the pentafluoro derivative 30 was prepared.

Figure 8.

Reagents and conditions: (a) dry Et₂O, diethyl oxalate; (b) Et₂NSF₃; (c) i) (CH₃)₃SiCF₃, CsF, CH₃OCH₂CH₂OCH₃, ii) conc. HCl.

Figure 9.

Reagents and conditions: (a) NaHSO₃, KCN, CH₂Cl₂; (b) HCl, MeOH; (c) Dess-Martin periodinate, CH₂Cl₂; (d) Et₂NSF₃, CH₂Cl₂; (e) i) (CH₃)₃SiCF₃, CsF, CH₃OCH₂CH₂OCH₃, ii) conc. HCl.

Electrophilic ketones, like fluoroketones, may exist in equilibrium with their corresponding hydrates (gem diols) depending on the environment. Based on the ¹H NMR data, the trifluoromethyl ketones and the pentafluoroethyl ketones synthesized in this work were found to exist solely in their ketone forms in chloroform solution. However, tetrafluoro derivative 26 appears to be a mixture of ketone-hydrate form in a ratio 1:2, whereas pentafluoro derivative 30 is completely hydrated (see NMR data in experimental section).¹⁹F NMR spectroscopic data confirm the existence of the hydrated form in the cases of compounds 26 and 30.

In Vitro Inhibition of GIVA cPLA₂, GVIA iPLA₂ and GV sPLA₂

All synthesized inhibitors were tested for inhibition of human GIVA cPLA₂, GVIA iPLA₂ and GV sPLA₂ using previously described mixed micelle-based assays.^20,21,24,25 The resulting degrees of inhibition are presented in Table 1 as either percent inhibition or X_I(50) values. Initially, the percent of inhibition for each PLA₂ enzyme at 0.091 mole fraction of each inhibitor was determined; and, X_I(50) values were estimated for compounds that displayed greater than 90% inhibition. The X_I(50) is the mole fraction of the inhibitor in the total substrate interface required to inhibit the enzyme by 50%.

Table 1. Inhibition of PLA₂ by fluoroketones.

Average percent inhibition and standard error (n=3) reported for each compound at 0.091 mole fraction. X_I(50) values determined for inhibitors with greater than 90% inhibition. N.D. signifies compounds with less than 25% inhibition (or no detectable inhibition).

No	Structure	GIVA cPLA2		GVIA iPLA2		GV sPLA2
		% Inhibition	XI(50)	% Inhibition	XI(50)	% Inhibition	XI(50)
6		96 ± 2	0.0223 ± 0.0023	92 ± 3	0.0195 ± 0.0053	79 ± 9
11		N.D.		50 ± 13		43 ± 8
8		38 ± 2		96 ± 3	0.0096 ±0.0008	N.D.
10a		N.D.		98 ± 16	0.0073 ± 0.0007	28 ± 1
12a		62 ± 5		96 ± 6	0.0025 ± 0.0003	48 ± 6
10b		56 ± 4		98 ± 5	0.0065 ± 0.001	46 ± 8
12b		68 ± 6		99 ± 10	0.0018 ± 0.0005	53 ± 14
10c		65 ± 12		98 ± 4	0.0065 ± 0.0008	75 ± 10
19a		91 ± 2	0.0199 ± 0.0025	85 ± 4	0.0328 ±0.0035	82 ± 8
20a		92 ± 3	0.0098 ± 0.0006	91 ± 4	0.0169 ±0.0021	86 ± 2
19b		96 ± 2	0.0156 ± 0.0019	94 ± 8	0.0208 ±0.0032	80 ± 6
20b		95 ± 2	0.0116 ± 94 ± 8	94 ± 8	0.0166 ±0.0022	84 ± 7
23		88 ± 1		71 ± 14		49 ± 12
21		73 ± 4		95 ± 5	0.0075 ±0.0011	86 ± 4
30		27 ± 3		49 ± 12		59 ± 12
26		94 ± 2	0.0167 ± 0.0018	93 ± 4	0.0011 ± 0.0002	86 ± 10	0.0236 ± 0.004

In accordance with the literature, the long-chain saturated palmitoyl trifluoromethyl ketone 6 inhibits both intracellular enzymes GIVA cPLA₂ and GVIA iPLA₂ at a similar level. In this work, we show that compound 6 is also a weak inhibitor of GV sPLA₂ (79% inhibition at 0.091 mole fraction). However, compound 8 is considered to be a selective inhibitor of GVIA iPLA₂ with an observed X_I(50) 0.0096, while high mole fraction of the inhibitor causes only 38% inhibition of GIVA cPLA₂ and does not affect GV sPLA₂.

The introduction of a pentafluoroethyl ketone functionality led to adverse effects depending on the nature of the chain. 1,1,1,2,2-Pentafluoro-7-phenyl-heptan-3-one (10a, FKGK11) presents slightly higher inhibitory activity on GVIA iPLA₂ (X_I(50) 0.0073) than the corresponding trifluoromethyl derivative 8. The dose-response curve for the inhibition of GVIA iPLA₂ by pentafluoroethyl ketone 10a is shown in Figure 10. In addition, it demonstrates selective inhibition for GVIA iPLA₂ since high mole fractions (0.091) do not affect GVIA cPLA₂ and caused slight inhibition (28%) of GV sPLA₂. Interestingly, the long-chain saturated pentafluoroethyl ketone 11 abolished the inhibitory potency and selectivity, demonstrating only 50% inhibition of GVIA iPLA₂ and 43% inhibition of GV sPLA₂ at 0.091 mole fraction.

Figure 10.

Inhibition curve for pentafluoro ketone 10a in a mixed-micelle assay with human GVIA iPLA₂. Non-linear regression (hyperbolic) estimated a X_I(50) value of 0.0073 ± 0.0007. Compound 10a inhibited GIVA cPLA₂ less than 25% and GV sPLA₂ approximately 28% at 0.091 mole fraction.

In pentafluoroethyl derivatives, increasing the chain length (from four to five or six carbon atoms) between the activated carbonyl group and the aromatic ring resulted in decreased selectivity for GVIA iPLA₂. Derivatives 10b and 10c (five and six carbon atoms, respectively) inhibit GVIA iPLA₂ at a similar level as inhibitor 10a (X_I(50) 0.0065). However, both 10b and 10c are weak inhibitors of GIVA cPLA₂ (56% and 65%, respectively) and GV sPLA₂ (46% and 75%, respectively). For the trifluoromethyl ketone derivatives 12a and 12b, the inhibitory activity increased as the chain length increased between the carbonyl group and the aromatic ring. Both 12a and 12b are more potent inhibitors of GVIA iPLA₂ (X_I(50) 0.0025 and X_I(50) 0.0018, respectively) than compound 8; however, these compounds also weakly inhibit GIVA cPLA₂ (62% and 68%, respectively) and GV sPLA₂ (48% and 53%, respectively) at 0.091 mole fraction. These results demonstrate that an increase of carbon atoms between the activated carbonyl group and the aromatic ring leads to a loss in selectivity.

Trifluoromethyl ketones 19a, 19b, 20a and 20b containing a medium (hexyloxy) or a long (decyloxy) chain substituent at the para position of the aromatic ring inhibit both GIVA cPLA₂ and GVIA iPLA₂. The dose-response curves for the inhibition of GVIA iPLA₂ and GIVA cPLA₂ by 1,1,1-trifluoro-6-(4-hexyloxy-phenyl)-hexan-2-one (20a, FKGK2) are shown in Figure 11. Comparison of 19a with 20a and 19b with 20b shows that the increase of the chain length between the carbonyl group and the aromatic ring from two to four carbon atoms results in increased inhibitory potency for both GIVA cPLA₂ and GVIA iPLA₂. All of these compounds (19a, 19b, 20a and 20b) also inhibit GV sPLA₂. Thus, trifluoromethyl ketones containing an alkoxy group at the para position of the aromatic group can be considered to be pan inhibitors of the all three enzymes: GIVA cPLA₂, GVIA iPLA₂ and GV sPLA₂. In particular, compound 20a is an inhibitor suitable for applications involving the inhibition of both intracellular and extracellular PLA₂ enzymes. The replacement of the hexyloxy by a benzyloxy group led to derivative 23, which weakly inhibited all the three PLA₂ enzymes. Comparison of inhibitors 8, 20a, 20b and 23 demonstrates that the introduction of an alkoxy or a benzyloxy group in the aromatic ring destroys the selectivity for GVIA iPLA₂.

Figure 11.

Inhibition curves for trifluoromethyl ketone 20a in a mixed-micelle assay with human GIVA cPLA₂ and GVIA iPLA₂. Non-linear regressions (hyperbolic) estimated X_I(50) values of 0.0169 ± 0.0021 and 0.0098 ± 0.0006 for GIVA cPLA₂ and GVIA iPLA₂, respectively. Compound 20a inhibited GV sPLA₂ approximately 86% at 0.091 mole fraction.

Comparison of pentafluoroethyl ketone 21 with the corresponding trifluoromethyl ketone 19a reinforces our observation that pentafluoroethyl ketone functionality favors the inhibition of GVIA iPLA₂ (X_I(50) 0.0075). However, the presence of a hexyloxy substituent leads to loss of selectivity for GVIA iPLA₂, since compound 21 weakly inhibits GIVA cPLA₂ (73%) and GV sPLA₂ (86%) at 0.091 inhibitor mole fraction.

Comparison of compound 26 with 6 shows that the introduction of an additional fluorine atom at the α′ position in a long chain saturated derivative results in a derivative with slightly better activity for GIVA cPLA₂ (X_I(50) 0.0167) than the parent trifluoromethyl ketone 6 (X_I(50) 0.0223). More importantly, tetrafluoro derivative 26 is approximately twenty-fold more potent inhibitor of GVIA iPLA₂ (X_I(50) 0.0011) than the trifluoro derivative 6 (X_I(50) 0.0195). To our knowledge, compound 26 is the most potent inhibitor of GVIA iPLA₂ reported, indicating that introduction of an additional fluorine atom at the α′ position constitutes an important strategy for the development of new potent GVIA iPLA₂ inhibitors. However, the tetrafluoro derivative 26 also inhibits GIVA and GVA PLA₂. Interestingly, the introduction of two fluorine atoms at the α′ position in an aromatic ring containing derivative destroyed the inhibitory potency and the selectivity for GVIA iPLA₂. For example, at 0.091 mole fraction, derivative 30 is a weak inhibitor of GVIA iPLA₂ (49%), GV sPLA₂ (59%), and presents no significant inhibition of GIVA cPLA₂ (27%).

Our data indicates the importance of screening selective inhibitors against multiple enzyme classes within the PLA₂ superfamily. As mentioned above, our work shows that the known inhibitor palmitoyl trifluoromethyl ketone 6, reported to strictly inhibit intracellular GVIA iPLA₂ and GIVA cPLA_2, also weakly inhibits GV sPLA₂. Similarly, some of our synthesized trifluoromethyl, pentafluoroethyl and tetrafluoro derivatives (for example, compounds 20a, 21, 26) were found to inhibit GV sPLA₂. Futhermore, Gelb et al. demonstrated that difluoro ketones similar to 36 (Figure 12) inhibit cobra venom PLA₂.⁵⁴ Therefore activated ketones, such as polyfluoro ketones, are likely to inhibit serine enzymes, GIVA cPLA₂ and GVIA iPLA₂, as well as histidine enzymes like secreted PLA₂.

Figure 12.

Structures of difluoro ketone inhibitor 36 of cobra venom PLA₂ and BEL inhibitor 37.

Bromoenol lactone (BEL) 37 (Figure 12) is considered to be a selective and irreversible GVIA iPLA₂ and has been widely applied to study potential biological roles for GVIA iPLA₂.^55,56 However, Turk et al. have recently reported that BEL inactivates GVIA iPLA₂ by generating a diffusible bromomethyl keto acid that alkylates cysteine thiols, rather than creating an acyl-enzyme intermediate with the active-site serine.⁵⁷ Therefore, it is likely that BEL affects multiple enzymes and should be used with appropriate caution when studying potential roles of GVIA iPLA₂.⁵⁷ These observations lead us to design selective inhibitors of GVIA iPLA₂ such as the pentafluoroethyl ketone 10a.

In conclusion, we developed and applied a variety of synthetic routes to produce various pentafluoro, tetrafluoro and trifluoro derivatives containing activated carbonyl groups. We studied their in-vitro activity on the three major human PLA₂ enzyme classes and demonstrated that the pentafluoroethyl ketone functionality favors GVIA iPLA₂ inhibition. Furthermore, 1,1,1,2,2-pentafluoro-7-phenyl-heptan-3-one (10a) was shown to be a selective inhibitor of GVIA iPLA₂. Additionally, introduction of an additional fluorine atom at the α′ position of a trifluoromethyl ketone constitutes an important strategy for the development of new potent GVIA iPLA₂ inhibitors. The tetrafluoro derivative of palmitic acid 26 is observed to be the most potent inhibitor of GVIA iPLA₂ to date; however, it also inhibits GIVA cPLA₂ and GV sPLA₂. Polyfluoro ketones displaying an array of selectivities for the major PLA₂ enzyme classes will prove to be valuable tools for the in-vivo characterization of the roles of PLA₂ enzymes. Furthermore, we found that these compounds do not show cytotoxcity toward cells in culture and we are currently utilizing these polyfluoro ketone derivatives for the comparison of intracellular versus extracellular PLA₂ enzyme roles in animal models of neurological disorders such as multiple sclerosis, spinal cord injury and peripheral nerve injury.⁵⁸

Experimental Section

Synthesis of fluoroketone inhibitors

Melting points were determined on a Buchi 530 apparatus and are uncorrected. Nuclear magnetic resonance spectra were obtained on a Varian Mercury spectrometer (¹H NMR recorded at 200 MHz, ¹³C NMR recorded at 50 MHz, ¹⁹F NMR recorded at 188 MHz) and are referenced in ppm relative to TMS for ¹NMR and ¹³C NMR, and relative to TFA as an internal standard for ¹⁹F NMR. Thin layer chromatography (TLC) plates (silica gel 60 F₂₅₄) and silica gel 60 (230–400 mesh) for flash column chromatography were purchased from Merck. Visualization of spots was effected with UV light and/or phosphomolybdic acid, in EtOH stain. Tetrahydrofuran (THF), toluene, and Et₂O were dried by standard procedures and stored over molecular sieves or Na. All other solvents and chemicals were reagent grade and used without further purification. All the products gave satisfactory elemental analysis results.

General Procedure for the Synthesis of Pentafluoroethyl Ketones

Oxalyl chloride (0.38 g, 3 mmol) and N,N-dimethylformamide (40 μL) were added to a solution of carboxylic acid (1 mmol) in dry dichloromethane (40 mL). After 3 h stirring at room temperature, the solvent and excess reagent were evaporated under reduced pressure and the residue was dissolved in dry dichloromethane (10 mL). Pyridine (0.64 mL, 8 mmol) and pentafluoropropionic anhydride (0.85 mL, 6 mmol) were added dropwise to this solution at 0 °C consecutively. After stirring at 0 °C for 30 min and at room temperature for 1.5 h, the reaction mixture was cooled again at 0 °C and water (2 mL) was added dropwise. After stirring for 30 min at 0 °C and another 30 min at room temperature, the reaction mixture was diluted with dichloromethane (10 mL). The organic phase was then washed with brine and dried (Na₂SO₄). The solvent was evaporated under reduced pressure and the residual oil was purified by flash column chromatography [EtOAc-petroleum ether (bp 40–60 °C) 1/9].

1,1,1,2,2-Pentafluoro-7-phenyl-heptan-3-one (10a)

Yield 53%; yellowish oil; ¹H NMR (CDCl₃): δ 7.31-7.17 (5H, m, Ph), 2.80 (2H, t, J=6.2 Hz, CH₂), 2.66 (2H, t, J=6.6 Hz, CH₂), 1.73-1.67 (4H, m, 2×CH₂); ¹³C NMR: δ 194.2 (t, J_C-C-F=26 Hz, CO), 141.6 (Ph), 128.4 (Ph), 128.3 (Ph), 125.9 (Ph), 117.8 (qt, J_C-F3=287 Hz, J_C-CF2=34 Hz, CF₃), 106.8 (tq, J_C-F2=267 Hz, J_C-CF3=38 Hz, CF₂), 37.1 (CH₂), 35.5 (CH₂), 30.3 (CH₂), 21.9 (CH₂); ¹⁹F NMR: δ -4.1 (CF₃), -45.5 (CF₂); MS (ESI) m/z (%): 279 (M⁻, 100). Anal. (C₁₃H₁₃F₅O) C, H.

1,1,1,2,2-Pentafluoro-8-phenyl-octan-3-one (10b)

Yield 75%; yellowish oil; ¹H NMR (CDCl₃): δ 7.35-7.21 (5H, m, Ph), 2.79 (2H, t, J=6.8 Hz, CH₂), 2.68 (2H, t, J=7.4 Hz, CH₂), 1.80-1.68 (4H, m, 2×CH₂), 1.48-1.40 (2H, m, CH₂); ¹³C NMR: δ 194.3 (t, J_C-C-F=26 Hz, CO), 142.2 (Ph), 128.3 (Ph), 128.2 (Ph), 125.7 (Ph), 117.8 (qt, J_C-F3=285 Hz, J_C-CF2=34 Hz, CF₃), 106.9 (tq, J_C-F2=265 Hz, J_C-CF3=37 Hz, CF₂), 37.2 (CH₂), 35.6 (CH₂), 31.0 (CH₂), 28.2 (CH₂), 22.1 (CH₂); ¹⁹F NMR: δ -4.2 (CF₃), -45.6 (CF₂); MS (ESI) m/z (%): 293 (M⁻, 100). Anal. (C₁₄H₁₅F₅O) C, H.

1,1,1,2,2-Pentafluoro-9-phenyl-nonan-3-one (10c)

Yield 60%; yellowish oil; ¹H NMR (CDCl₃): δ 7.31-7.18 (5H, m, Ph), 2.76 (2H, t, J=6.8 Hz, CH₂), 2.64 (2H, t, J=8.0 Hz, CH₂), 1.72-1.58 (4H, m, 2×CH₂), 1.44-1.34 (4H, m, 2×CH₂); ¹³C NMR: δ 194.4 (t, J_C-C-F=26 Hz, CO), 142.5 (Ph), 128.4(Ph), 128.3 (Ph), 125.7 (Ph), 117.8 (qt, J_C-F3=285 Hz, J_C-CF2=34 Hz, CF₃), 106.9 (tq, J_C-F2=265 Hz, J_C-CF3 = 37 Hz, CF₂), 37.3 (CH₂), 35.8 (CH₂), 31.1 (CH₂), 28.8 (CH₂), 28.5 (CH₂), 22.2 (CH₂); ¹⁹F NMR: δ -4.2 (CF₃), -45.6 (CF₂); MS (ESI) m/z (%): 307 (M⁻, 100). Anal. (C₁₅H₁₇F₅O) C, H.

1,1,1,2,2-Pentafluoro-octadecan-3-one (11)

Yield 24%; colorless oil; ¹H NMR (CDCl₃): δ 2.75 (2H, t, J=7.4 Hz, CH₂), 1.67 (2H, t, J=7.0 Hz, CH₂), 1.38-1.20 (24H, m, 12×CH₂), 0.88 (3H, t, J=7.0 Hz, CH₃); ¹³C NMR: δ 194.5 (t, J_C-CF2=26 Hz, CO), 117.8 ppm (qt, J_C-F3=285 Hz, J_C-CF2=34 Hz, CF₃), 106.9 ppm (tq, J_C-F2=265 Hz, J_C-CF3 = 38 Hz, CF₂), 37.4 (CH₂), 31.9 (CH₂), 30.3 (CH₂), 29.7 (CH₂), 29.6 (CH₂), 29.5 (CH₂), 29.4 (CH₂), 29.2 (CH₂), 28.7 (CH₂), 22.7 (CH₂), 22.3 (CH₂), 14.1 (CH₃); ¹⁹F NMR: δ -4.2 (CF₃), -45.6 (CF₂); MS (ESI) m/z (%): 357 (M⁻, 93). Anal. (C₁₈H₃₁F₅O) C, H.

1,1,1,2,2-Pentafluoro-5-(4-hexyloxy-phenyl)-pentan-3-one (21)

Yield 76%; yellowish oil; ¹H NMR (CDCl₃): δ 7.10 (2H, d, J=8.6 Hz, Ph), 6.85 (2H, d, J=8.6 Hz, Ph), 3.94 (2H, t, J=6.6 Hz, CH₂O), 3.02 (2H, t, J=7.0 Hz, CH₂), 2.96 (2H, t, J=7.0 Hz, CH₂), 1.86-1.73 (2H, m, CH₂), 1.60-1.25 (6H, m, 3×CH₂), 0.93 (3H, t, J=6.4 Hz, CH₃). ¹³C NMR (CDCl₃): δ 193.5 (t, J_C-C-F=26 Hz, CO), 157.9 (Ph), 130.9 (Ph), 129.2 (Ph), 117.8 (qt, J_C-F3 =286 Hz, J_C-CF2=34 Hz, CF₃), 114.7 (Ph), 106.8 (tq, J_C-F2=265 Hz, J_C-CF3=38 Hz, CF₂), 68.0 (CH₂O), 39.4 (CH₂), 31.6 (CH₂), 29.3 (CH₂), 27.5 (CH₂), 25.7 (CH₂), 22.6 (CH₂), 13.9 (CH₃). ¹⁹F NMR: δ -4.2 (CF₃), -45.6 (CF₂). MS (ESI) m/z (%): 351 (M⁻, 100). Anal. (C₁₇H₂₁F₅O₂) C, H.

Synthesis of Trifluoromethyl Ketones

The synthesis of trifluoromethyl ketones was carried out following the procedure described above for pentafluoromethyl ketones, except that trifluoroacetic anhydride was used instead of pentafluoropropionic anhydride. The products were purified by flash column chromatography [EtOAc-petroleum ether (bp 40–60 °C) 3/7].

1,1,1-Trifluoro-7-phenylheptan-2-one (12a).⁵⁹

Yield 45%; yellowish oil; ¹H NMR (CDCl₃): δ 7.34-7.19 (5H, m, Ph), 2.76-2.62 (4H, m, 2×CH₂), 1.77-1.66 (4H, m, 2×CH₂), 1.46-1.39 (2H, m, CH₂); ¹³C NMR: δ 191.8 (q, J_C-C-F=35 Hz, COCF₃), 142.2 (Ph), 128.3 (Ph), 128.2 (Ph), 125.7 (Ph), 115.5 (q, J_C-F=290 Hz, CF₃), 36.2 (CH₂), 35.7 (CH₂), 30.9 (CH₂), 28.2 (CH₂), 22.2 (CH₂); ¹⁹F NMR: δ -1.5 (CF₃); MS (ESI) m/z (%): 243 (M⁻, 100).

1,1,1-Trifluoro-8-phenyloctan-2-one (12b).⁵⁹

Yield 42%; yellowish oil; ¹H NMR (CDCl₃): δ 7.28-7.17 (5H, m, Ph), 2.72-2.60 (4H, m, 2×CH₂), 1.70-1.61 (4H, m, 2×CH₂), 1.42-1.24 (4H, m, 2×CH₂); ¹³C NMR: δ 191.4 (q, J_C-C-F=35 Hz, COCF₃), 142.5 (Ph), 128.3 (Ph), 128.2 (Ph), 125.7 (Ph), 115.6 (q, J_C-F=291 Hz, CF₃), 36.3 (CH₂), 35.8 (CH₂), 31.1 (CH₂), 28.7 (CH₂), 28.6 (CH₂), 22.3 (CH₂); ¹⁹F NMR: δ -1.5 (CF₃); MS (ESI) m/z (%): 257 (M⁻, 100).

1,1,1-Trifluoro-4-(4-hexyloxy-phenyl)-butan-2-one (19a)

Yield 53%; yellowish oil; ¹H NMR (CDCl₃): δ 7.10 (2H, d, J=8.6 Hz, Ph), 6.84 (2H, d, J=8.6 Hz, Ph), 3.93 (2H, t, J=6.2 Hz, CH₂O), 3.10-2.92 (4H, m, 2×CH₂), 1.82-1.62 (2H, m, CH₂), 1.55-1.22 ( 6H, m, 3×CH₂), 0.91 (3H, t, J=6.6 Hz, CH₃); ¹³C NMR: δ 190.7 (q, J_C-C-F = 35 Hz, COCF₃), 157.9 (Ph), 131.0 (Ph), 129.2 (Ph), 115.5 (q, J_C-F=292 Hz, CF₃), 114.7 (Ph), 68.0 (OCH₂), 38.3 (CH₂), 31.6 (CH₂), 29.2 (CH₂), 27.5 (CH₂), 25.7 (CH₂), 22.6 (CH₂), 13.9 (CH₃); ¹⁹F NMR: δ -1.5 (CF₃); MS (ESI) m/z (%): 301 (M⁻, 100). Anal. (C₁₆H₂₁F₃O₂) C, H.

4-(4-Decyloxy-phenyl)-1,1,1-trifluoro-butan-2-one (19b)

Yield 46%; yellowish oil; ¹H NMR (CDCl₃): δ 7.12 (2H, d, J=8.6 Hz, Ph), 6.85 (2H, d, J=8.6 Hz, Ph), 3.95 (2H, t, J=6.6 Hz, CH₂O), 3.05-2.85 (4H, m, 2×CH₂), 1.81-1.62 (2H, m, CH₂), 1.56-1.22 (14H, m, 7×CH₂), 0.92 (3H, t, J=6.8 Hz, CH₃); ¹³C NMR: δ 190.5 (q, J_C-C-F=35 Hz, COCF₃), 157.9 (Ph), 131.0 (Ph), 129.2 (Ph), 115.5 (q, J_C-F=292 Hz, CF₃), 114.6 (Ph), 68.0 (CH₂O), 38.3 (CH₂), 31.8 (CH₂), 29.6 (CH₂), 29.3 (CH₂), 29.2 (CH₂), 27.4 (CH₂), 26.0 (CH₂), 22.7 (CH₂), 14.0 (CH₃); ¹⁹F NMR: δ -1.5 (CF₃); MS (FAB) m/z (%): 358 (M⁺, 85). Anal. (C₂₀H₂₉F₃O₂) C, H.

1,1,1-Trifluoro-6-(4-hexyloxy-phenyl)-hexan-2-one (20a)

Yield 45%; yellowish oil; ¹H NMR (CDCl₃): δ 7.09 (2H, d, J=8.0 Hz, Ph), 6.85 (2H, d, J=8.0 Hz, Ph), 3.95 (2H, t, J=6.6 Hz, CH₂), 2.74 (2H, t, J=6.6 Hz, CH₂), 2.60 (2H, t, J=6.2 Hz, CH₂), 1.82-1.62 (6H, m, 3×CH₂), 1.46-1.25 (6H, m, 3×CH₂), 0.94 (3H, t, J=6.8 Hz, CH₃); ¹³C NMR: δ 191.4 (q, J_C-C-F=34 Hz, COCF₃), 157.9 (Ph), 133.4 (Ph), 129.1 (Ph), 115.4 (q, J_C-F=290 Hz, CF₃), 114.4 (Ph), 67.9 (CH₂O), 36.1 (CH₂), 34.5 (CH₂), 31.6 (CH₂), 30.6 (CH₂), 29.3 (CH₂), 25.7 (CH₂), 22.6 (CH₂), 21.8 (CH₂), 13.9 (CH₃); ¹⁹F NMR: δ -1.6 (CF₃); MS (FAB) m/z (%): 330 (M⁺, 23). Anal. (C₁₈H₂₅F₃O₂) C, H.

6-(4-Decyloxy-phenyl)-1,1,1-trifluoro-hexan-2-one (20b)

Yield 46%; yellowish oil; ¹H NMR (CDCl₃): δ 7.08 (2H, d, J=8.6 Hz, Ph), 6.84 (2H, d, J=8.6 Hz, Ph), 3.94 (2H, t, J=6.6 Hz, CH₂O), 2.73 (2H, t, J=6.6 Hz, CH₂), 2.59 (2H, t, J=7.0 Hz, CH₂), 1.82-1.62 (6H, m, 3×CH₂), 1.45-1.22 (14H, m, 7×CH₂), 0.90 (3H, t, J=6.8 Hz, CH₃); ¹³C NMR: δ 191.6 (q, J_C-C-F=35 Hz, COCF₃), 157.7 (Ph), 133.7 (Ph), 129.4 (Ph), 115.8 (q, J_C-F = 292 Hz, CF₃), 114.6 (Ph), 68.2 (CH₂O), 36.4 (CH₂), 34.8 (CH₂), 32.1 (CH₂), 30.9 (CH₂), 29.8 (CH₂), 29.7 (CH₂), 29.6 (CH₂), 29.5 (CH₂), 26.6 (CH₂), 26.3 (CH₂), 22.9 (CH₂), 22.1 (CH₂), 14.32 (CH₃); ¹⁹F NMR: δ -1.5 (CF₃); MS (FAB) m/z (%): 386 (M⁺, 100). Anal. (C₂₂H₃₃F₃O₂) C, H.

4-(4-Benzyloxy-phenyl)-1,1,1-trifluoro-butan-2-one (23)

Yield 43%; yellowish solid; mp 71–72 °C; ¹H NMR (CDCl₃): δ 7.46-7.35 (5H, m, Ph), 7.15 (2H, d, J=8.4 Hz, Ph), 6.95 (2H, d, J=8.4 Hz, Ph), 5.07 (2H, s, PhCH₂), 3.12-2.85 (4H, m, 2×CH₂); ¹³C NMR: δ 190.7 (q, J_C-C-F = 35 Hz, COCF₃), 157.4 (Ph), 136.9 (Ph), 131.5 (Ph), 129.2 (Ph), 128.5 (Ph), 127.9 (Ph), 127.4 (Ph), 115.4 (q, J_C-F=290 Hz, CF₃), 114.9 (Ph), 70.0 (CH₂O), 38.3 (CH₂), 27.4 (CH₂); ¹⁹F NMR: δ -1.4 (CF₃); MS (ESI) m/z (%): 307 (M⁻, 100). Anal. (C₁₇H₁₅F₃O₂) C, H.

Intermediate compounds 14a,b and 22 were prepared by known methods and their spectroscopic data were in accordance with those in the literature.^60,61

Horner-Wadsworth-Emmons Olefination

A suspension of aldehyde 14a or 14b (1 mmol), triethyl 4-phosphonocrotonate (0.37 g, 1.5 mmol), lithium hydroxide (0.036 g, 1.5 mmol) and molecular sieves (beads, 4–8 mesh, 1.5 g/mmol aldehyde) in dry tetrahydrofuran (10 mL) was refluxed under argon for 24 h. The reaction mixture was then cooled to room temperature, filtered through a thin pad of celite and the solvent evaporated under reduced pressure. The residual oil was purified by chromatography on silica gel eluting with ether-petroleum ether (bp 40–60 °C) 1/9.

Ethyl (2E,4E)-5-(4-Hexyloxy-phenyl)-penta-2,4-dienoate (16a)

Yield 71%; white solid; mp 68–69 °C; ¹H NMR (CDCl₃): δ 7.48-7.20 (3H, m, CH, Ph), 6.90-6.75 (3H, m, CH, Ph), 6.71 (1H, d, J=15.4 Hz, CH), 5.94 (1H, d, J=15.4 Hz, CHCOO), 4.23 (2H, q, J=7.4 Hz, OCH₂CH₃), 3.97 (2H, t, J=6.2 Hz, CH₂O), 1.85-1.62 (2H, m, CH₂CH₂O), 1.45-1.02 (9H, m, 3×CH₂, CH₃), 0.92 (3H, t, J=6.8 Hz, CH₃); ¹³C NMR: δ 167.2 (COO), 160.0 (Ph), 145.0 (CH), 140.2 (CH), 131.9 (Ph), 128.6 (Ph), 123.9 (CH), 119.9 (CH), 114.7 (Ph), 68.0 (CH₂O), 60.2 (OCH₂CH₃), 31.6 (CH₂), 29.1 (CH₂), 25.7 (CH₂), 22.6 (CH₂), 14.3 (CH₃), 14.0 (CH₃). Anal. (C₁₉H₂₆O₃) C, H.

Ethyl (2E,4E)-5-(4-Decyloxy-phenyl)-penta-2,4-dienoate (16b)

Yield 65%; white solid; mp 80–81 °C; ¹H NMR (CDCl₃): δ 7.45-7.38 (3H, m, CH, Ph), 6.88-6.80 (3H, m, CH, Ph), 6.78 (1H, d, J=12 Hz, CH), 5.94 (1H, d, J=15.4 Hz, CHCOO), 4.23 (2H, q, J=7.4 Hz, OCH₂CH₃), 3.97 (2H, t, J=6.6 Hz, CH₂O), 1.81-1.75 (2H, m, CH₂CH₂O), 1.50-1.14 (17H, m, 7×CH₂, CH₃), 0.89 (3H, t, J=6.8 Hz, CH₃); ¹³C NMR: d167.3 (COO), 160.0 (Ph), 145.1 (CH), 140.2 (CH), 131.9 (Ph), 128.6 (Ph), 124.0 (CH), 119.9 (CH), 114.8 (Ph), 68.1 (CH₂O), 60.2 (CH₂), 31.9 (CH₂), 29.5 (CH₂), 29.4 (CH₂), 29.3 (CH₂), 29.2 (CH₂), 26.0 (CH₂), 22.7 (CH₂), 14.3 (CH₃), 14.1 (CH₃). Anal. (C₂₃H₃₄O₃) C, H.

Wittig Olefination

A solution of aldehyde 14a or 14b (1 mmol) and methyl (triphenylphosphanylidene)acetate (0.334 g, 1 mmol) in dry dichloromethane (3 mL) was refluxed under argon for 24 h. The reaction mixture was then cooled to room temperature and the solvent evaporated under reduced pressure. The residual oil was purified by flash column chromatography on silica gel eluting with EtOAc-petroleum ether (bp 40–60 °C) 1/9.

Methyl (E)-3-(4-Hexyloxy-phenyl)-acrylate (15a)

Yield 93%; white solid; mp 84–85 °C; ¹H NMR (CDCl₃): δ 7.63 (1H, d, J=15.8 Hz, CH=CHCO), 7.43 (2H, d, J=8.8 Hz, Ph), 6.87 (2H, d, J=8.8 Hz, Ph), 6.28 (1H, d, J=15.8 Hz, CHCOO), 3.95 (2H, t, J=6.4 Hz, CH₂O), 3.77 (3H, s, OCH₃), 1.76 (2H, m, CH₂CH₂O), 1.46-1.21 (6H, m, 3×CH₂), 0.89 (3H, t, J=6.8 Hz, CH₃); ¹³C NMR: δ 167.7 (COO), 161.0 (Ph), 144.6 (CH), 129.6 (Ph), 126.8 (Ph), 115.0 (CH), 114.7 (Ph), 68.1 (CH₂O), 51.5 (OCH₃), 31.5 (CH₂), 29.0 (CH₂), 25.6 (CH₂), 22.5 (CH₂), 13.9 (CH₃). Anal. (C₁₆H₂₂O₃) C, H.

Methyl (E)-3-(4-Decyloxy-phenyl)-acrylate (15b)

Yield 92%; white solid; mp 75–76 °C; ¹H NMR (CDCl₃): δ 7.63 (1H, d, J=15.8 Hz, CH=CHCOO), 7.37 (2H, d, J=8.8 Hz, Ph), 6.85 (2H, d, J=8.8 Hz, Ph), 6.23 (1H, d, J=15.8 Hz, CHCOO), 3.87 (2H, t, J=6.6 Hz, CH₂O), 3.71 (3H, s, OCH₃), 1.78-1.62 (2H, m, CH₂CH₂O), 1.40-1.22 (14H, m, 7×CH₂), 0.84 (3H, t, J=7 Hz, CH₃); ¹³C NMR: δ 167.4 (COO), 160.8 (Ph), 144.3 (CH), 129.4 (Ph), 126.6 (Ph), 114.8 (CH), 114.5 (Ph), 67.8 (CH₂O), 51.2 (OCH₃), 31.7 (CH₂), 29.4 (CH₂), 29.2 (CH₂), 29.1 (CH₂), 29.0 (CH₂), 25.8 (CH₂), 22.5 (CH₂), 13.9 (CH₃). Anal. (C₂₀H₃₀O₃) C, H.

Hydrogenation and Saponification of Unsaturated Esters

A mixture of the unsaturated ester (0.7 mmol) in dry 1,4-dioxane (7 mL) and 10% palladium on activated carbon (0.07 g) was hydrogenated for 12 h under atmospheric conditions. After filtration through a pad of celite, the solvent was removed in vacuo to give the saturated compound.

The solution of the saturated ester in methanol (1.4 mL) was treated with sodium hydroxide 1N (1 mL, 1 mmol). The mixture was stirred at room temperature for 12 h, acidified with 1N HCl and extracted with EtOAc (3 × 10 mL). The solvent was removed in vacum to afford the saturated acid as a white solid.

3-(4-Hexyloxy-phenyl)-propanoic acid (17a)

Yield 90%; white solid; mp 70–72 °C; ¹H NMR (CDCl₃): δ 7.14 (2H, d, J=8.2 Hz, Ph), 6.86 (2H, d, J=8.2 Hz, Ph), 3.96 (2H, t, J=6.6 Hz, CH₂O), 2.93 (2H, t, J=7.6 Hz, CH₂), 2.67 (2H, t, J=7.6 Hz, CH₂), 1.76-1.60 (2H, m, CH₂), 1.41-1.30 (6H, m, 3×CH₂), 0.92 (3H, t, J=6.7 Hz, CH₃); ¹³C NMR: δ 179.0 (COO), 157.6 (Ph), 132.0 (Ph), 129.1 (Ph), 114.5(Ph), 67.9 (CH₂O), 35.9 (CH₂), 31.5 (CH₂), 29.7 (CH₂), 29.2 (CH₂), 25.7 (CH₂), 22.5 (CH₂), 14.0 (CH₃). Anal. (C₁₅H₂₂O₃) C, H.

3-(4-Decyloxy-phenyl)-propanoic acid (17b)

Yield 96%; white solid; mp 74–76 °C; ¹H NMR (CDCl₃): δ 7.14 (2H, d, J=8.2 Hz, Ph), 6.86 (2H, d, J=8.2 Hz, Ph), 3.95 (2H, t, J=6.5 Hz, CH₂O), 2.93 (2H, t, J=7.7 Hz, CH₂CH₂COO), 2.67 (2H, t, J=7.7 Hz, CH₂COO), 1.85-1.68 (2H, m, CH₂CH₂O), 1.50-1.21 (14H, br s, 7×CH₂), 0.92 (3H, t, J=6.2 Hz, CH₃); ¹³C NMR: δ 179.3 (COO), 157.6 (Ph), 132.0 (Ph), 129.1 (Ph), 114.5 (Ph), 67.9 (CH₂O), 35.9 (CH₂), 31.9 (CH₂), 29.7 (CH₂), 29.5 (CH₂), 29.3 (CH₂), 26.0 (CH₂), 22.6 (CH₂), 14.1 (CH₃). Anal. (C₁₉H₃₀O₃) C, H.

5-(4-Hexyloxy-phenyl)-pentanoic acid (18a)

Yield 96%; white solid; mp 90–91 °C; ¹H NMR (CDCl₃): δ 7.03 (2H, d, J=8.4 Hz, Ph), 6.77 (2H, d, J=8.4 Hz, Ph), 3.88 (2H, t, J=6.2 Hz, CH₂O), 2.52 (2H, t, J=6.8 Hz, CH₂), 2.32 (2H, t, J=6.7 Hz, CH₂COO), 1.80-1.60 (6H, m, 3×CH₂), 1.60-1.21 (6H, m, 3×CH₂), 0.89 (3H, t, J=6.7 Hz, CH₃); ¹³C NMR: δ 180.1 (COO), 157.3 (Ph), 133.9 (Ph), 129.2 (Ph), 114.4 (Ph), 68.0 (CH₂O), 34.6 (CH₂), 33.9 (CH₂), 31.6 (CH₂), 31.0 (CH₂), 29.3 (CH₂), 25.7 (CH₂), 24.2 (CH₂), 22.6 (CH₂), 14.0 (CH₃). Anal. (C₁₇H₂₆O₃) C, H.

5-(4-Decyloxy-phenyl)-pentanoic acid (18b)

Yield 94%; white solid; mp 101–102 °C; ¹H NMR (CDCl₃): δ 7.08 (2H, d, J=8.4 Hz, Ph), 6.82 (2H, d, J=8.4 Hz, Ph), 3.93 (2H, t, J=6.2 Hz, CH₂O), 2.57 (2H, t, J=6.8 Hz, PhCH₂), 2.37 (2H, t, J=7 Hz, CH₂COOH), 1.80-1.60 (6H, m, 3×CH₂), 1.51-1.22 (14H, m, 7×CH₂), 0.89 (3H, t, J=6.6 Hz, CH₃); ¹³C NMR: δ 179.5 (COO), 157.2 (Ph), 133.8 (Ph), 129.1 (Ph), 114.3 (Ph), 67.9 (CH₂O), 34.5 (CH₂), 33.8 (CH₂), 31.8 (CH₂), 30.9 (CH₂), 29.5 (CH₂), 29.4 (CH₂), 29.3 (CH₂), 29.2 (CH₂), 26.0 (CH₂), 24.2 (CH₂), 22.6 (CH₂), 14.0 (CH₃). Anal. (C₂₁H₃₄O₃) C, H.

Methyl 2-Fluoro-hexadecanoate (25)

Compound 24 (1 mmol) was added to a solution of bis(2-methoxyethyl)amino-sulfur-trifluoride, Deoxofluor (0.2 mL, 1 mmol) in dry dichloromethane (0.2 mL) at −78 °C. After stirring for 2 h at −78 °C and another 3 h at room temperature, the reaction mixture quenched with saturated aqueous NaHCO₃ (2.5 mL). The organic phase was then washed with brine and dried (Na₂SO₄). The solvent was evaporated under reduced pressure and the residual oil was purified by flash column chromatography on silica gel eluting with EtOAc-petroleum ether (bp 40–60 °C) 3/7. Yield 64%; yellowish oil; ¹H-NMR (CDCl₃): δ 4.86 (1H, dt, J_H-F=49.2 Hz, J_H-H=6.6 Hz, CH), 3.74 (3H, s, OCH₃), 2.00-1.72 (2H, m, CH₂), 1.45-1.10 (24H, br, 12×CH₂), 0.83 (3H, t, J=6.2 Hz, CH₃); ¹³C NMR: δ 170.4 (d, J_C-C-F=24 Hz, COO), 88.9 (d, J_C-F=183 Hz, CF), 52.0 (OCH₃), 32.3 (d, J_C-C-F=21, CH₂), 31.9 (CH₂), 29.6 (CH₂), 29.5 (CH₂), 29.4 (CH₂), 29.3 (CH₂), 29.2 (CH₂), 29.0 (CH₂), 24.6 (CH₂), 24.4 (CH₂), 24.3 (CH₂), 22.7 (CH₂), 14.0 (CH₃); ¹⁹F NMR: δ -120.8 (m, CF). Anal. (C₁₇H₃₃FO₂) C, H.

1,1,1,3-Tetrafluoro-heptadecan-2-one (in equilibrium with 1,1,1,3-Tetrafluoro-heptadecane-2,2-diol) (26)

A solution of compound 25 (173 mg, 0.6 mmol) and trifluoromethyltrimethylsilane (170 μL, 1.15 mmol) in ethylene glycol dimethyl ether (0.55 mL) at 0 °C was treated with cesium fluoride (3 mg). After stirring for 30 min at 0 °C and another 18 h at 25 °C the reaction mixture was treated with concentrated HCl (1 mL). After stirring for another 18 h at 25 °C, the reaction mixture was diluted with EtOAc (10 mL). The organic phase was then washed with brine and dried (Na₂SO₄). The solvent was evaporated under reduced pressure and the residual oil was purified by flash column chromatography on silica gel eluting with EtOAc-petroleum ether (bp 40–60 °C) 3/7. Yield 58%; white solid; mp 34–35 °C; ¹H-NMR (CDCl₃): δ 5.23 (1/3H, dt, J_H-F=48.2 Hz, J_H-H=6.2 Hz, CH), 4.65 (2/3H, dt, J_H-F=49.4 Hz, J_H-H=6.6 Hz, CH), 3.74 (2/3H, s, OH), 3.49 (2/3H, s, OH), 2.08-1.27 (26H, m, 13×CH₂), 0.89 (3H, t, J=7 Hz, CH₃); ¹³C NMR: δ 122.6 (q, J_C-F3=286 Hz, CF₃), 115.4 (q, J_C-F3=290 Hz, CF₃) 92.9 [C(OH)₂], 92.4 (d, J_C-F=186 Hz, CF), 32.1 (CH₂), 31.6 (d, J_C-C-F=20 Hz, CH₂), 29.9 (CH₂), 29.8 (CH₂), 29.7 (CH₂), 29.6 (CH₂), 29.5 (CH₂), 29.4 (CH₂), 29.1 (CH₂), 28.5 (CH₂), 28.1 (CH₂), 22.7 (CH₂), 22.3 (CH₂), 14.3 (CH₃); ¹⁹F NMR: δ 1.6 (CF₃), -5.3 (CF₃), -121.7 (CF); MS (ESI) m/z (%): 343 (M⁻, 100).

Ethyl 2,2-Difluoro-5-phenyl-pentanoate (29)

To a stirring mixture of magnesium (350 mg, 14.6 mmol) and iodine in dry THF (10 ml), (3-bromo-propyl)-benzene (2.87 g, 14.4 mmol) was added dropwise under N₂ atmosphere. Once the Grignard reagent was formed, the resulting mixture was added dropwise to a cooled (−78 °C) solution of diethyl oxalate (1.6 mL, 11.8 mmol) in dry ether (17.3 mL). The reaction mixture was stirred at −78 °C for 45 min and then was quenched with 1N HCl. The aqueous layer was extracted with ether (3 × 25 mL) and the combined organic layers were washed with brine, dried (Na₂SO₄) and the solvent was evaporated in vacuo. After flash column chromatography, a mixture of methyl 2-oxo-5-phenyl-pentanoate (28) with diethyl oxalate was obtained and treated with DAST (1 eq) at room temperature. After stirring for 4 h at 45 °C, the reaction mixture was quenched with ice water. The reaction mixture was diluted with dichlolomethane and the organic phase was then washed with brine and dried (Na₂SO₄). The solvent was evaporated under reduced pressure and the residual oil was purified by flash column chromatography on silica gel eluting with EtOAc-petroleum ether (bp 40–60 °C) 1/9. Yield 69%; yellowish oil; ¹H NMR (CDCl₃): δ 7.38-7.12 (5H, m, Ph), 4.30 (2H, q, J=6.8 Hz, OCH₂), 2.68 (2H, t, J=7.4 Hz, PhCH₂), 2.21-1.93 (2H, m, CH₂CF₂), 1.90-1.75 (2H, m, CH₂), 1.34 (3H, t, J=6.8 Hz, CH₃); ¹³C NMR: δ 164.2 (t, J_C-C-F=24 Hz, COO), 140.9 (Ph), 128.4 (Ph), 128.3 (Ph), 126.1 (Ph), 116.2 (t, J_C-F=248 Hz, CF₂), 62.7 (OCH₂), 34.9 (CH₂), 33.8 (t, J_C-C-F=23 Hz, CH₂CF₂), 23.0 (t, J_C-C-C-F=4 Hz, CH₂CH₂CF₂), 13.8 (CH₃); ¹⁹F NMR: δ - 28.0 (t, J=17 Hz, CF₂). Anal. (C₁₃H₁₆F₂O₂) C, H.

1,1,1,3,3-Pentafluoro-6-phenyl-hexane-2,2-diol (30)

It was prepared following the method used for the synthesis of compound 26. Yield 35%; yellowish oil; ¹H NMR (CDCl₃): δ 7.41-7.18 (5H, m, Ph), 3.93 (2H, br, 2×OH), 2.69 (2H, t, J=7.6 Hz, PhCH₂), 2.22-1.88 (4H, m, 2×CH₂); ¹³C NMR: δ 141.3 (Ph), 128.4 (Ph), 126.3 (Ph), 126.1 (Ph), 121.5 (q, J=286 Hz, CF₃), 120.7 (t, J=249 Hz, CF₂), 92.3 [C(OH)₂], 35.2 (CH₂), 30.8 (t, J_C-C-F2=23 Hz, CH₂CF₂), 22.5 (t, J_C-C-C-F2=2.4 Hz, CH₂CH₂CF₂); ¹⁹F NMR: δ –3.2 (CF₃), –36.4 (CF₂); Ms (ESI) m/z (%): 283 (M⁻, 65), 213 (100). Anal. (C₁₂H₁₃F₅O₂) C, H.

2-Hydroxy-5-phenyl-pentanenitrile (32).⁶²

A solution of 4-phenylbutanal 31 (0.56 g, 3.78 mmol) and NaHSO₃ (0.59 g in 1 ml H₂O) in dichloromethane was stirred for 30 min at room temperature. After the formation of the white salt, the organic solvent was evaporated and water (3.8 mL) was added. The mixture cooled to 0 °C and an aqueous solution of KCN (0.368 g, 567 mmol in 1 mL H₂O) was added dropwise. The reaction mixture was stirred for another 18 h at room temperature and then CH₂Cl₂ (10 mL) and water (10 mL) were added. The organic phase was washed with brine and dried (Na₂SO₄). The solvent was evaporated under reduced pressure and the residual oil was purified by flash column chromatography on silica gel eluting with EtOAc-petroleum ether (bp 40–60 °C) 2/8 to give 0.653 g (99%) of the title compound as a clear oil; ¹H-NMR (CDCl₃): δ 7.32-7.15 (5H, m, Ph), 4.40 (1H, t, J=8.8 Hz, CH), 2.63 (2H, t, J=6.6 Hz, CH₂), 1.90-1.70 (4H, m, 2×CH₂); ¹³C NMR: 141.5 (Ph), 128.7 (Ph), 128.6 (Ph), 126.3 (Ph), 120.4 (CN), 61.2 (CH), 35.3 (CH₂), 34.7 (CH₂), 26.4 (CH₂). Anal. (C₁₁H₁₃NO) C, H.

Methyl 2-Hydroxy-5-phenyl-pentanoate (33).⁶³

Compound 32 (0.63 g, 3.59 mmol) was treated with HCl (0.6 mL. 6N) in MeOH for 18 h at room temperature. The organic solvent was evaporated and an aqueous solution of K₂CO₃ was added to neutralize the pH of the mixture. After extraction with EtOAc (3 × 15 mL), the combined organic phases were washed with brine and dried (Na₂SO₄). The solvent was evaporated under reduced pressure and the residual oil was purified by flash column chromatography on silica gel eluting with EtOAc-petroleum ether (bp 40–60 °C) 3/7 to give 0.56 g (79%) of the title compound as a clear oil; ¹H-NMR (CDCl₃): δ 7.30-7.12 (5H, m, Ph), 4.22 (1H, t, J=4.0 Hz, CH), 3.74 (3H, s, OCH₃), 3.18 (1H, s, OH), 2.66 (2H, t, J=6.6 Hz, CH₂), 1.85-1.62 (4H, m, 2×CH₂); ¹³C NMR: 175.4 (COO), 141.6 (Ph), 128.1 (Ph), 128.0 (Ph), 125.5 (Ph), 70.1 (CHOH), 52.1 (OCH₃), 35.2 (CH₂), 33.6 (CH₂), 26.3 (CH₂). Anal. (C₁₂H₁₆O₃) C, H.

Methyl 2-Oxo-5-phenyl-pentanoate (34)

Compound 33 (0.20 g, 0.96mmol) was dissolved in CH₂Cl₂ (20 mL) and treated with Dess-Martin periodinane (0. 43 g) under stirring for 40 min. The organic phase was washed with brine and dried (Na₂SO₄). The solvent was evaporated under reduced pressure and the residual oil was purified by flash column chromatography on silica gel eluting with EtOAc-petroleum ether (bp 40–60 °C) 3/7 to give 0.195 g (99%) of the title compound as an yellowish oil; ¹H-NMR (CDCl₃): δ 7.31-7.15 (5H, m, Ph), 3.85 (3H, s, OCH₃), 2.86 (2H, t, J=6.6 Hz, CH₂), 2.63 (t, J=6.6 Hz, 2H, CH₂), 1.71-1.62 (2H, m, CH₂); ¹³C NMR: 194.0 (CO), 161.2 (COO), 141.8 (Ph), 128.3 (Ph), 128.0 (Ph), 125.8 (Ph), 52.8 (OCH₃), 35.5 (CH₂), 30.5 (CH₂), 22.5 (CH₂). Anal. (C₁₂H₁₄O₃) C, H.

Methyl 2,2-Difluoro-5-phenyl-pentanoate (35)

A solution of compound 34 (0.404 g, 1.67 mmol) in CH₂Cl₂ (3.3 mL) was treated dropwise with DAST (0.489 mL, 3.6 mmol) at room temperature. After heating at 55 °C for 5 h, it was poured into H₂O, cautiously neutralized by the addition of solid K₂CO₃, and extracted with CHCl₃ (2 × 15 mL). The organic solvent was dried over Na₂SO₄, filtered and evaporated, and the crude product purified by flash column chromatography on silica gel eluting with EtOAc-petroleum ether (bp 40–60 °C) 1/9 to give 0.202 g (50%) of the title compound as an yellowish oil; ¹H NMR (CDCl₃): δ 7.32-7.12 (5H, m, Ph), 4.10 (3H, s, OCH₃), 2.69 (2H, t, J=7.4 Hz, PhCH₂), 2.21-1.90 (2H, m, CH₂CF₂), 1.80-1.72 (2H, m, CH₂); ¹³C NMR: δ 164.2 (t, J_C-C-F=33 Hz, COO), 140.9 (Ph), 128.4 (Ph), 128.3 (Ph), 126.1 (Ph), 116.2 (t, J_C-F=248 Hz, CF₂), 52.7 (OCH₃), 34.9 (CH₂), 33.8 (t, J_C-C-F=23 Hz, CH₂CF₂), 23.0 (t, J_C-C-C-F=2.4 Hz, CH₂CH₂CF₂); ¹⁹F NMR: δ -28.0 (2F, t, J=17 Hz, CF₂). MS (ESI) m/z (%): 229 (M⁺+1, 100). Anal. (C₁₂H₁₄F₂O₂) C, H.

In-vitro PLA₂ Assays

Phospholipase A₂ activity was determined using the previously described modified Dole assay²⁰ with buffer and substrate conditions optimized for each enzyme as described previously^20,21,24,25: (i) GIVA cPLA₂ substrate mixed-micelles were composed of 400 μM Triton X-100, 97 μM PAPC, 1.8 μM ¹⁴C-labeled PAPC, and 3 μM PIP₂ in buffer containing 100 mM HEPES pH 7.5, 90 μM CaCl₂, 2 mM DTT and 0.1 mg/ml BSA; (ii) GVI iPLA₂ substrate mixed-micelles were composed of 400 μM Triton X-100, 99 μM DPPC, and 1.5 μM ¹⁴C-labeled DPPC in buffer containing 200 mM HEPES pH 7.0, 1 mM ATP, 2 mM DTT and 0.1 mg/ml BSA; and (iii) GV sPLA₂ substrate mixed-micelles were composed of 400 μM Triton X-100, 99 μM DPPC, and 1.5 μM ¹⁴C-labeled DPPC in buffer containing 50 mM Tris pH 8.0 and 5 mM CaCl₂.

In-vitro PLA₂ Inhibition Studies

Initial screening of compounds at 0.091 mole fraction inhibitor in mixed-micelles was carried out. We considered compounds displaying 25% or less inhibition to have no inhibitory affect (designated N.D.). We report average percent inhibition (and standard error, n=3) for compounds displaying more than 25% and less than 90% enzyme inhibition. If percent inhibition was greater than 90%, we determined its X_I(50) by plotting percent inhibition vs. inhibitor molar fraction (7 points; typically 0.005 to 0.091 mole fraction). Inhibition curves were modeled in Graphpad Prism using either a linear (x, y intercept = 0) or non-linear regression (one-site binding model - hyperbola, BMAX = 100) to calculate the reported X_I(50) and associated error values.

Supplementary Material

1_si_001. Supporting Information Available.

Elemental analysis results for the compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

Abbreviations

AACOCF₃

arachidonyl trifluoromethyl ketone

ATP

adenosine triphosphate

BEL

bromoenol lactone, DAST, diethylaminosulfur trifluoride

Deoxofluor

bis(2-methoxyethyl)amino-sulfur-trifluoride

DIBALH

diisobutylaluminium hydride

DPPC

1,2-dipalmitoylphosphotidylcholine

DTT

dithiothreitol

EAE

experimental autoimmune encephalomyelitis

EtOAc

ethyl acetate

GIVA cPLA₂

Group VIA cytosolic phospholipase A₂

GV sPLA₂

Group V secreted phospholipase A₂

GVIA iPLA₂

Group VIA calcium-independent phospholipase A₂

NMDA

N-methyl-D-aspartate

PAF

platelet activating factor

PAPC

1-palmitoyl, 2-arachidonal phosphatidylcholine

PIP₂

phosphatidyl inositol (4,5)-bisphosphate

TBAF

tetra-n-butylammonium fluoride

TEMPO

2,2,6,6-tetramethylpiperidine-1-yloxy free radical

TFA

trifluoroacetic acid

THF

tetrahydrofuran

TLC

thin-layer chromatography

TMS

tetramethylsilane