Synthesis of Mevalonate- and Fluorinated Mevalonate Prodrugs and Their in vitro Human Plasma Stability

Abstract

The mevalonate pathway is essential for the production of many important molecules in lipid biosynthesis. Inhibition of this pathway is the mechanism of statin cholesterol-lowering drugs, as well as the target of drugs to treat osteoporosis, to combat parasites, and to inhibit tumor cell growth. Unlike the human mevalonate pathway, the bacterial pathway appears to be regulated by diphosphomevalonate (DPM). Enzymes in the mevalonate pathway act to produce isopentenyl diphosphate, the product of the DPM decarboxylase reaction, utilize phosphorylated (charged) intermediates, which are poorly bioavailable. It has been shown that fluorinated DPMs (6-fluoro- and 6,6,6-trifluoro-5-diphosphomevalonate) are excellent inhibitors of the bacterial pathway; however, highly charged DPM and analogues are not bioavailable. To increase cellular permeability of mevalonate analogues, we have synthesized various prodrugs of mevalonate and 6-fluoro- and 6,6,6-trifluoromevalonate that can be enzymatically transformed to the corresponding DPM or fluorinated DPM analogues by esterases or amidases. To probe the required stabilities as potentially bioavailable prodrugs, we measured the half-lives of esters, amides, carbonates, acetals, and ketal promoieties of mevalonate and the fluorinated mevalonate analogues in human blood plasma. Stability studies showed that the prodrugs are converted to the mevalonates in human plasma with a wide range of half-lives. These studies provide stability data for a variety of prodrug options having varying stabilities and should be very useful in the design of appropriate prodrugs of mevalonate and fluorinated mevalonates.

Introduction

The mevalonate pathway (Figure 1) is an important cellular metabolic pathway present in all higher eukaryotes and many bacteria. Isopentenyl diphosphate (IPP), an intermediate in this pathway, is an important precursor of isoprenoids, which leads to many biologically active small molecules, including cholesterol, steroid hormones, and vitamin A. ¹ Therefore, it is not surprising that enzymes in the mevalonate pathway are targets for a variety of drug discovery programs. ² The statin cholesterol-lowering drugs target 3-hydroxy-3-methylglutaryl CoA reductase, the enzyme that produces mevalonate;³ the osteoporosis drug alendronate inhibits the synthesis of farnesyl diphosphate from IPP; ⁴ enzymes in the mevalonate pathway are also targeted for cancer⁵ and parasites.⁶ Leyh and co-workers discovered that the mevalonate pathway in S. pneumoniae is regulated by 5-diphosphomevalonate (DPM).⁷ They showed that DPM is a feedback inhibitor of mevalonate kinase (MK), and binds tightly to an allosteric site⁸ of the pneumococcal MK. However, human MK is not inhibited by DPM at concentrations that effectively inhibit the S. pneumoniae system.⁹ Therefore, DPM can be a lead compound for the development of new anti-pneumococcal antibiotics that do not perturb human metabolism.

Figure 1.

The bacterial mevalonate pathway. Conversion of mevalonic acid to isopentenyl diphosphate occurs in three ATP-dependent steps. DPM is a feedback inhibitor of MK: MK, mevalonate kinase; PMK, phosphomevalonate kinase; DPM-DC, diphosphomevalonate decarboxylase.

In early studies of DPM analogues, ¹⁰ it was found that 6,6,6-trifluoromevalonate was converted into the corresponding diphosphate enzymatically, which inhibited DPM decarboxylase (DPM-DC) and led to the accumulation of DPM in rat liver homogenates.¹¹ Moreover, 6-fluoromevalonate causes the accumulation of phosphorylated mevalonates and completely blocks the related bioactivity of mevalonate at 200 μM concentration.¹² However, whereas a functional mevalonate pathway is essential for the survival of bacteria, suppression of this pathway in humans results in minimal side effects, as evidenced by the common use of statin drugs, which block cholesterol biosynthesis at a step prior to DPM-DC, and by antiproliferative drugs, such as bisphosphonates, which block farnesyltransferase.¹³ Furthermore, antibacterial treatment is short in duration, which should not have a serious effect on the products of this pathway. Nonetheless, diphosphate compounds are generally not suitable for use as drugs; because of their highly charged structure (4-), they are not expected to penetrate the negatively charged bacterial cell membrane.¹⁴ Also phosphatases can degrade the diphosphate group easily.

Because of the importance of mevalonate and phosphorylated metabolites to drug discovery, neutral and less polar prodrugs, chemically modified molecules of the pharmacologically active moiety that are transformed into the active form in vivo,¹⁵ were designed to avoid these potential bioavailability problems. The charged carboxylic acid was protected as an ester, lactone, or amide to make it neutral. To explore the influence of polarity of the prodrug on human absorption and bacterial cell permeation, the two hydroxyl groups of the mevalonate were converted to carbonate, acetal, and ketal prodrugs. These esters, ¹⁶ lactones, ¹⁷ amides, ¹⁸ carbonates,¹⁹ acetals,^20,21 and ketals,²² having half-lives ranging from a couple of minutes to several days in human plasma, were chosen as the promoieties of the carboxyl and hydroxyl functionalities of mevalonate. These analogues should be enzymatically hydrolyzed to their original mevalonate or fluorinated mevalonates in both humans and bacteria, and then the fluorinated mevalonates can be enzymatically converted to the phosphorylated forms.²³ The stabilities of diverse analogues (Figure 2) in human blood plasma were studied to develop an armamentarium of promoieties for further in vitro and in vivo studies.

Figure 2.

Cyclic carbonate, cyclic acetal/ketal, ester, and lactone prodrugs of mevalonate

Results and Discussion

Cyclic carbonate prodrug 4 was prepared from mevalonolactone (1) using the synthetic route described in Scheme 1. The hydrolysis of 1 with aqueous KOH afforded a solution of 2, which was neutralized to pH 7–8 with aqueous HCl and lyophilized to remove water. If neutralization was not carried out, starting material 1 was regenerated during lyophilization. The crude carboxylic acid (2) was converted to the corresponding benzyl ester (3) via treatment with benzyl bromide and tetrabutylammonium bromide. Although a portion of ester 3 was converted to the starting material (1) during column chromatography with silica gel, 3 was isolated as the major product (69% yield). Ester 3 was easily converted to the cyclic carbonate (4) via treatment with triphosgene. An alternative route to the benzyl ester (3) is also shown in Scheme 1; TBS protection of the hydroxyl group of 4-hydroxy-2-butanone (5), followed by an aldol reaction with benzyl acetate using LDA, afforded 7 in excellent yields. Deprotection of the TBS group in compound 7, with tetrabutylammonium fluoride and two equivalents of acetic acid at 0 °C, afforded desired alcohol 3. The reaction of crude product 3 with triphosgene yielded cyclic carbonate 4; lactone 1 was still generated gradually before 3 disappeared completely.

Scheme 1. Synthesis of compound 4^a.

^aReagents and Conditions: (i) aq. KOH, 40 °C; aq. HCl, (ii) BnBr, TBAB, THF, 50 °C (69% for 2 steps), (iii) triphosgene, pyridine, 0 °C (88%); (iv) TBSCl, imidazole, DMF (90%), (v) benzyl acetate, LDA, THF, −78 °C (93%), (vi) TBAF, AcOH, THF, 0 °C (72%).

Cyclic carbonate analogues 9a,b and 10a–c were synthesized from benzyl ester 4 after removal of the benzyl group via palladium-catalyzed hydrogenolysis (Scheme 2). The coupling reaction of carboxylic acid 8 with various phenols and amines using EDCI or HBTU provided the desired esters (9a,b) and amides (10a–c) in moderate to good yields (Scheme 2). The obtained benzyl amide derivatives were expected to be more stable than the ester derivatives in plasma. To confirm the relative stability of other less stable amides (due to the electron withdrawing effect), a phenyl amide (10a) and a 4-fluorobenzyl amide (10c) were prepared.

Scheme 2. Synthesis of 9a–b and 10a–c^a.

^aReagents and Conditions: (i) Pd/C, H₂, quantitative; (ii) EDCI, DMAP, CH₂Cl₂, ROH R = Ph (9a, 54%), 4-F-Ph (9b, 65%); (iii) R-NH₂, HBTU, DIEA, DMF, R = Ph (10a, 55%), Bn (10b, 61%), 4-F-Ph (10c, 38%)

The synthetic route for the 6-fluoromethyl cyclic carbonate analog is shown in Scheme 3. The addition of allylmagnesiun bromide (1.95 equiv) to ethyl fluoroacetate 11 at 0 °C for 30 min afforded diolefin 12. This reaction was sensitive to the duration and the equivalents of Grignard reagent; an addition of excess allymagnesium bromide or prolonged reaction times resulted in undesired side product generation. Ozonolysis of crude product 12, followed by oxidation with H₂O₂ gave dicarboxylic acid 13. The benzylation of crude product 13 was conducted to give diester 14 in more than 50% yield for four steps. Partial reduction of 14 with DIBAL-H (3–4 equiv) at 0 °C in THF afforded the desired compound (15). Because 15 easily underwent intramolecular lactone formation on silica gel, the crude mixture was allowed to react with triphosgene without further column purification to obtain cyclic carbonate 16 in moderate yields. The benzyl deprotection of 16, followed by esterification with 4-fluorobenzyl bromide, or 2,4-difluorobenzyl bromide, provided the corresponding esters (18a–b), respectively.

Scheme 3. Synthesis of Compounds 18a–b^a.

^aReagents and Conditions: (i) allylmagnesium bromide (1.95 equiv), Et₂O, 0 °C, < 30 min. (ii) (1) ozone, CH₂Cl₂, −78 °C (2) aq. H₂O₂, AcOH, H₂SO₄, reflux, (iii) BnBr, K₂CO₃, DMF, 54% for 4 steps (69% for 2 steps), (iv) DIBAL-H, THF, 0 °C, (v) (Cl₃CO)₂CO, pyridine, CH₂Cl₂, 0 °C (37% for 2 steps), (vi) H₂, Pd/C, AcOEt (99%), (vii) NaHCO₃, DMF, R-X; R= 4-F-BnBr (18a, 77%), 2,4-diF-BnBr (18b, 72%).

4-Trifluoromethyl carbonate analogues were synthesized from commercially available 4-ethoxy-1,1,1-trifluoro-3-buten-2-one (Scheme 4). The aldol reaction of benzyl acetate and enone 19 with LDA gave 20. The produced enol ether was hydrolyzed to the corresponding aldehyde (21) under acidic conditions, which was reduced to alcohol 22 by sodium triacetoxyborohydride. When sodium borohydride was used, the benzyl ester group was gradually reduced to the hydroxyl group; perhaps the reactivity of the ester group was increased by the electron withdrawing effect of the trifluoromethyl group. Cyclic carbonate 23 was obtained by treatment of the crude diol (22) with triphosgene. The benzyl group of 23 was removed by palladium-catalyzed hydrogenolysis in ethyl acetate under a hydrogen atmosphere. When MeOH was used as a solvent in this reaction, the carbonate group decomposed. Carboxylic acid 24 was esterified with 4-fluorobenzyl bromide, or 2,4-difluorobenzyl bromide using sodium bicarbonate as a base, to give 25a–b. When potassium carbonate was used for the esterification of 24, the cyclic carbonate group decomposed.

Scheme 4. Synthesis of 25a–b^a.

^aReagents and Conditions: (i) benzyl acetate, LDA, THF, −78 °C (88%), (ii) aq. HCl/acetone, 0 °C (80%), (iii) NaBH(OAc)₃, benzene, (iv) triphosgene, pyridine, CH₂Cl₂, 0 °C (68% for 2 steps), (v) H₂, Pd/C, AcOEt (98%), (vi) NaHCO₃, DMF, R-X = 4-F-BnBr (25a, 88%), 2,4-diF-BnBr (25b, 85%).

Stability of the cyclic carbonate prodrugs in PBS buffer

The stabilities of several cyclic carbonate analogues in PBS buffer (pH 7.4) were tested to determine baseline level of drug decomposition by the medium. The cyclic carbonate analogues (4, 16, 18a–b, 23, 25a–b) were UV active, which allowed us to determine the amount of drug in the culture media by HPLC with UV detection. After the mixture of the compounds and PBS buffer were incubated at 37 °C, the degradation of the compounds over time was monitored. The half-life (t_1/2) of 4, which is a mevalonate prodrug, was more than 48 h (Table 1). The t_1/2 of the 4-monofluoromethyl analogues (16, 18a–b) were around 25 h, and the t_1/2 of the 4-trifluoromethyl analogues (23, 25a–b) were around 5 h. As the 4-substituent became increasingly more electron deficient, the t_1/2 decreased dramatically. However, electron-withdrawing groups on the aromatic ring of the ester (benzyl, 4-fluorobenzyl, or 2,4-difluorobenzyl) did not impact the half-lives of the molecules, as long as the R₂- substituent was constant (CH₃, CH₂F, or CF₃).

Table 1.

Half-lives of the diverse carbonates in PBS buffer at 37 °C.

#	R1	R2	t1/2 (h)
4	Bn	CH3	>48
16	Bn	CH2F	25
18a	4-F-Bn	CH2F	26
18b	2,4-di-F-Bn	CH2F	22
23	Bn	CF3	5
25a	4-F-Bn	CF3	6
25b	2,4-di-F-Bn	CF3	5

HPLC and ¹H NMR analysis of trifluoromethyl analogue 23 during incubation with PBS buffer was carried out to delineate its decomposition pathway and related intermediates (Figure 3). As the peak for 23 decreased, the peak for benzyl alcohol increased, but the peak for 22, which results from decomposition of the carbonate moiety, was not observed by HPLC. ¹H NMR analysis of the incubation mixture after lyophilization showed carboxylic acid 24 was only a minor component; desired final product 27 was the major product. Further stability tests of prospective intermediates were carried out. By monitoring the ¹H NMR spectrum of synthesized 24 in PBS buffer it was found that the t_1/2 of 24 was about 3 h, shorter than the t_1/2 of 23 (5 h). The existence of about 8% of lactone 26 was also observed. Pure 22 was incubated with PBS buffer to confirm the decomposition pathway of 23 via 22. Interestingly, the obtained t_1/2 for 22 was very short (< 1 min), so even through little 22 was formed during the decomposition of 23, whatever small amount was formed would quickly undergo further degradation to 26 or 27. A similar experiment using lactone 26 was carried out. The spectra showed that lactone 26 was converted to carboxylic acid 27 rapidly (t_1/2 in PBS buffer was about 1 min). This indicates that the decomposition through 22 and 26 is also possible even though 22 was not detected.

Figure 3.

Pathways for the decomposition of trifluoromethyl benzyl ester carbonate 23

Stability of the cyclic carbonate prodrugs in human plasma

Human plasma stabilities were evaluated,²⁴ for the cyclic carbonate analogues (4, 9a–b, 10a–c, 16, 18a–b, 23, 25a–b), and reported as their corresponding half-lives (Table 2). In all cases, quantification was performed in at least duplicate (<25% error) using HPLC in the presence of the plasma-stable internal standard haloperidol.²⁵ The mixture of test compounds and human plasma was incubated at 37 °C, and the degradation of the compounds was monitored by HPLC. Slight differences in the t_1/2 were observed for the various ester groups, but the t_1/2 for 4 was 4 min and the t_1/2 for the remaining esters (9a–b, 16, 18a–b, 23, 25a–b) were less than 3 min. While the benzyl and phenyl ester moieties were unstable, the cyclic carbonate moiety was relatively more stable, and the peaks of the corresponding diols (3, 15, or 22), which correspond to the products when the carbonate moiety is hydrolyzed faster than the ester moiety, were not observed. The t_1/2 of these benzyl and phenyl ester analogues was very short. The t_1/2 for N-phenyl amide 10a was also less than 3 min. Interestingly, the t_1/2 values for benzyl amide analogues 10b and 10c were 8 min, resulting from the hydrolysis of the carbonate moiety rather than the amide moiety. No benzylamine peak was observed by HPLC through 12 h incubation, illustrating the high stability of the benzyl amide moiety in human plasma. These results demonstrate that the cyclic carbonate moiety of these analogues is more stable than the benzyl ester moiety but less stable than the benzyl amide moiety.

Table 2.

Various half-lives of the diverse carbonate analogues in human plasma at 37 °C.

#	R1	R2	t1/2 (min)
4	BnO	CH3	4
9a	PhO	CH3	< 3
9b	4-F-PhO	CH3	< 3
10a	PhNH	CH3	< 3
10b	BnNH	CH3	8a
10c	4-F-BnNH	CH3	8a
16	BnO	CH2F	2
18a	4-F-BnO	CH2F	2
18b	2,4-di-F-BnO	CH2F	1
23	BnO	CF3	< 1
25a	4-F-BnO	CF3	< 1
25b	2,4-di-F-BnO	CF3	<1

^aHydrolysis occurred only at carbonate promoieties

The enantiomeric ratio of the synthesized mevalonate derivatives was monitored by chiral chromatography and LC/MS during incubation with human plasma to determine the influence of stereochemistry on the hydrolysis rate.²⁶ At t_1/4 and t_1/2, a portion of the incubation mixtures of 4 and 10b were quenched with acetonitrile and injected into a Chiral Cel OD-H column attached to a HPLC system. The observed integration ratio of each enantiomer of 4 was 35/65 at 2 min, and 41/59 at 4 min. In the case of compound 10b, the enantiomeric ratio was 48/52 at 4 min and 46/54 at 8 min. Within the margin of error, hydrolysis of the carbonate was not significantly affected by the stereochemistry. Although a stereospecific hydrolysis was observed during incubation of a racemic mevalonate ester with human plasma, the difference did not seriously affect the overall hydrolysis rate.

Syntheses of acetal/ketal prodrugs

The synthesis of the acetal/ketal analogues shown in Scheme 5 was initiated by the ring-opening of 1 and 26 with benzylamine. The introduction of the MOM group, followed by treatment with BF₃•Et₂O, gave methylene acetals 30a–b. Reaction of 29a with 2,2-dimethoxypropane and a catalytic amount of camphorsulfonic acid (CSA) gave acetonides 31. Treatment of 29a,b with benzaldehyde dimethoxy acetal and catalytic CSA in CH₂Cl₂ gave diastereomeric mixtures 32a–b. Diol 29b was converted to the p-methoxybenzylidene acetal 32c–d by treatment with anisaldehyde dimethyl acetal. Because electron-rich benzylidene acetals are known to undergo hydrolysis more quickly, 4-methoxybenzylidene derivative 32c and 2,4-dimethoxybenzylidene derivative 32d were expected to decompose more easily than 32b. Compounds 29a–b were converted to Boc-protected 33-b by treatment with an excess amount of Boc₂O in anhydrous acetonitrile.

Scheme 5. Synthesis of Compounds 30–33^a.

^aReactions and Conditions: (i) BnNH₂, DMF, 80 °C (80–88%), (ii) (a) MOMCl, CH₂Cl₂, 0 °C to room temperature, (b) BF₃OEt₂, CH₂Cl₂, 0 °C to room temperature, 77% yield for 30a, 69% yield for 30b, (iii) 2,2-dimethoxypropane, CSA, CH₂Cl₂, 27% yield, (iv) benzaldehyde dimethylacetal, CSA, CH₂Cl₂, 34% yield for 32a, 42% yield for 32b, (v) anisaldehyde dimethyl acetal, CSA, CH₂Cl₂, reflux, 60% yield for 32c; 2,4-dimethoxybenzaldehyde, CSA, MS 4Å, benzene, reflux, 6% yield for 32d, (vi) Boc₂O, DMAP, acetonitrile, reflux, 88% yield for 33a, 68% yield for 33b

Stability of the acetal/ketal amide prodrugs in human plasma

Prodrugs with the acetal, ketal, and carbonate promoieties were further investigated for their stability in human plasma (Table 3). The t_1/2 values for the acetal, ketal, and carbonate derivatives (30a–b, 31, 32a–d, 33a–b) in human plasma were much greater than 48 h. An electron-withdrawing group (trifluoromethyl) at C4 did not affect the stability of the acetal groups. These results show that the methylene, isopropylidene, and benzylidene acetal groups of the tested compounds may be useful if the mevalonate prodrug requires long-term stability.

Table 3.

Stability of the acetal/ketal amides in human plasma at 37 °C

#	R1	R2	R3	t1/2 (h)
30a	CH3	H	H	> 48
31	CH3	CH3	CH3	> 48
32a	CH3	Ph	H	> 48
30b	CF3	H	H	> 48
32b	CF3	Ph	H	> 48
32c	CF3	4-MeO-Ph	H	> 48
32d	CF3	2,4-(MeO)2-Ph	H	> 48
33a	CH3			> 48
33b	CF3			> 48

Syntheses of ester prodrugs

Syntheses of diverse ester and amide analogues are shown in Scheme 6 and Scheme 7. The ring opening of 1 was carried with various electron deficient amines, and the introduction of the acetyl group was performed to give diverse acetyl esters (35, 36). The reaction of 34 with excess acetic anhydride in pyridine gave both mono- and di-acetylated products (35, 36). The 4-monofluoromethyl analogues (38a–c) were prepared from dibenzyl ester 14 (Scheme 7). The dibenzyl ester was reduced to the triol using LiBH₄, and addition of one equivalent of acetic anhydride in pyridine yielded mono- acetylated intermediate 37. That intermediate underwent oxidation by pyridinium dichromate (PDC), and was then coupled with various alcohols and an amine to obtain the corresponding products in moderate yields (38a–c).

Scheme 6^a.

^aReagents and Conditions: (i) 2,3,4,5,6-pentafluorobenzylamine, DMF, 80 °C, 53% yield, (ii) Ac₂O, pyridine, 41% yield for 35, 20% yield for 36

Scheme 7^a.

^a Reagents and Conditions: (i) LiBH₄ (3 eq.), THF, 0 °C (70%) (ii) Ac₂O, DMAP, pyridine (45%), (iii) PDC, DMF, 24 h, room temperature (iv) RNH₂,EDCI, K₂CO₃, or R-X, K₂CO₃, DMF, two steps 11~25%.

Stability of the ester promoiety in PBS buffer

The stability of ethyl ester analogue 38c in deuterated PBS buffer (pH 7.4) at 37 °C was monitored by ¹H NMR spectroscopy as a control to determine the decomposition of the ester prodrug by the medium. The half-life (t_1/2) of 38c in PBS was > 48 h.

Stability of the ester promoiety in human plasma

The stabilities of the electron-deficient benzyl amide and ester analogues (34–36, 38a–c) in human plasma were determined (Table 4). The half-life of the 2,3,4,5,6-pentafluorobenzyl amide (34) in human plasma was greater than 2 h; therefore, the amide of the parent compounds is expected to be useful if the prodrug requires long-term stability. The half-life of the acetyl groups on 35 and 38a was 20 – 30 min, suggesting that this ester is a promising promoiety for the alcohols if the prodrug needs moderate stability in human plasma. The half-lives of the monofluoromethyl alkyl esters containing an acetyl moiety were around 10 min, suggesting that the alkyl ester group is a viable promoiety for the carboxylic acid and for moderate stability.

Table 4.

Stability of the benzyl amide analogues in human plasma at 37 °C

#	R1	R2	R3	R4	t1/2 (min)
34	C6F5CH2NH	CH3	H	H	> 120
35	C6F5CH2NH	CH3	H	Ac	20a
36	C6F5CH2NH	CH3	Ac	Ac	12
38a	BnNH	CH2F	H	Ac	30a
38b	MeO	CH2F	H	Ac	10
38c	EtO	CH2F	H	Ac	11

^aHydrolysis occurred only at the acetyl promoiety

Inhibition activity of diverse prodrugs against Streptococcus pneumoniae

Diverse prodrugs of fluoromevalonates were evaluated for their in vitro antibacterial activity against S. pneumoniae (Table 5) in THB medium using a well-established technique,²⁷ and the minimum inhibitory concentration values (MICs) were determined. Carbonate prodrugs, which undergo relatively fast hydrolysis (1–4 min), were only marginally active (25a) or generally inactive (16, 18a). Moderately stable (10–20 min) ester prodrugs (38a–c) and a quite stable acetal prodrug (32b) were found to lack activity. Lactones (26 and 28²⁸), which were determined to undergo the fastest hydrolysis, were the most active compounds among these diverse prodrugs against S. pneumoniae in THB media; 6-fluoromevalolactone (28) is 4-fold more potent (MIC = 200 μM) than 6,6,6-trifluoromevalolactone (26, MIC = 800 μM). S. pneumoniae active compounds 25a, 26, and 28 were further evaluated against Streptococcus pyogenes (GAS 5448), Streptococcus agalactiae (GBS COH1), Staphylococcus aureus (TCH1516), vancomycin resistant Enterococcus (VRE), and E. coli K12. Only 26 displayed activity, albeit low, against Streptococcus pyogenes (MIC = 200 μM) and Streptococcus agalactiae (MIC = 800 μM); the others exhibited MIC values >1.6 mM concentration. For all of these additional organisms, vancomycin and penicillin G exhibited MIC values <0.0125 mM. No evidence of general cytotoxicity was observed for 25a, 26, and 28 against RAW murine macrophages and HaCaT human keratinocytes at concentrations up to 1.6 mM for 24 h as measured by lactate dehydrogenase (LDH) release vs media alone. Although these experiments do not consider clinical parameters of host bioavailability such as oral absorption, these results suggest that unstable prodrugs of fluoromevalonates are more beneficial than stable prodrugs to penetrate the bacterial cell and interact with enzymes of the mevalonate pathway. It is possible that the other relatively stable prodrugs, which probably have a better opportunity to penetrate the bacterial cell, are not hydrolyzed efficiently in the bacteria to reach their minimum effective concentration for a desired pharmacological effect in the bacteria.

Table 5.

Antibacterial activity of diverse prodrugs toward S. pneumoniae

name	MICa (mM)
16	>1.6
18a	>1.6
25a	1.6
26	0.8
28	0.2
32b	>1.6
38a	>1.6
38b	>1.6
38c	>1.6
Vancomycin

^aMinimum inhibitory concentration (MIC): lowest drug concentration that reduced growth by 80% or more.

Conclusion

Stability studies of diverse analogues of mevalonate and fluorinated mevalonates using human blood plasma and PBS buffer show that they are converted to mevalonate or fluorinated mevalonates via hydrolysis, mediated by human plasma or solvent. We also found that decomposition of the cyclic carbonates and esters is faster than that of the amides and cyclic acetals/ketals in the tested analogues. In general, an aliphatic ester promoiety is converted to the desired carboxylic acid with a t_1/2 of 10–20 min in human plasma. Although the cyclic carbonate decomposes relatively rapidly (4–8 min), and cyclic ketal, acetal, and amide moieties decompose relatively slowly in human plasma, this study shows that the t_1/2 in human plasma for each functional group is controllable by modifying the electron density of the promoiety. These plasma stability studies demonstrate that ester, amide, carbonate, acetal, or ketal promoieties have the potential to enhance biopermeability. We have observed that only rapidly hydrolyzed prodrugs of fluorinated mevalonates possess intracellular antibacterial activity against S. pneumoniae. Slowly hydrolyzed prodrugs may not have the appropriate pharmacokinetics with S. pneumoniae. Other prodrug analogues may be more effective with different bacteria, or for use against human enzymes.

Experimental Section

General Chemistry Procedures

All reagents were obtained from commercial suppliers and used without further purification. All solvents were distilled and stored under argon or a nitrogen atmosphere before use. All reactions were performed under an argon atmosphere unless otherwise noted. Analytical thin layer chromatography was visualized by ultraviolet light or by using phosphomolybdic acid as a universal stain. Flash chromatography was carried out under a positive pressure of nitrogen. ¹H NMR and ¹³C NMR spectra were obtained with a Bruker AVANCE III 500 (500 MHz) spectrometer; chemical shift (δ) values were referenced to tetramethylsilane as internal standard and reported as follows: (δ) shift (multiplicity, coupling constants, proton count). Coupling constants were taken directly from spectra and are uncorrected. ¹³C NMR spectra were recorded at 125 MHz, and all chemical shifts are reported in ppm on the δ scale with an internal reference of δ 77.16 or 49.0 for CDCl₃ or CD₃OD, respectively. High-resolution mass spectra (HRMS) were measured with an Agilent 6210 LC-TOF (ESI, APCI, APPI) mass spectrometer. The purity of the synthesized final compounds was determined by HPLC analysis to be ≥95%. The column used was a Phenomenex Luna 5 μm 200 Å, 4.6 × 250 mm.

Synthesis and characterization of compounds

Benzyl 3,5-dihydroxy-3-methylpentanoate (3)

(±)-Mevalonolactone 1 (268 mg, 2.0 mmol) was added to a solution of KOH (123 mg, 2.2 mmol) in H₂O (4 mL). The solution was stirred at 40 °C for 1 h, adjusted to pH 7–8 with aqueous HCl (0.1 M), and lyophilized. The residue was mixed with benzyl bromide (363 μL, 3.0 mmol) and tetrabutylammonium bromide (967 mg, 3.0 mmol) in THF (8 mL), and stirred at 50 °C for 4 h. After the mixture was diluted with AcOEt and brine, the organic layer was partitioned, dried (Na₂SO₄), and evaporated. The residue was purified by silica gel column chromatography (50% AcOEt in hexane) to give 3 (329 mg, 69%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 7.33–7.38 (m, 5H, aromatic), 5.17 (s, 2H, benzyl-CH₂), 4.04 (br s, 1H, OH), 3.88 (m, 1H, CH₂CH₂O), 3.81 (m, 1H, CH₂CH₂O), 2.90 (br s, 1H, OH), 2.69 (d, 1H, OC(O)CH₂, J = 15.5 Hz), 2.52 (d, 1H, OC(O)CH₂, J = 15.5 Hz), 1.80 (m, 1H, CH₂CH₂O), 1.74 (m, 1H, CH₂CH₂O), 1.32 (s, 3H, CH₃); ¹³C NMR (125 MHz, CDCl₃) δ 172.7, 135.3, 128.7, 128.5, 128.4, 72.2, 66.7, 59.4, 45.1, 42.1, 26.9; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₃H₁₈NaO₄: 261.1103, found: 261.1105.

Benzyl 2-(4-methyl-2-oxo-1,3-dioxan-4-yl)acetate (4)

To a solution of 3 (436 mg, 1.83 mmol) in CH₂Cl₂ (15 mL) was added pyridine (224 μL, 2.75 mmol), and the mixture was stirred at 0 °C for 15 min. A solution of triphosgene (98%, 665 mg, 2.20 mmol) in CH₂Cl₂ (5 mL) was added to the mixture, and the resulting mixture was stirred at 0 °C for 30 min. The reaction was quenched with saturated aqueous NH₄Cl and extracted with AcOEt. The organic layer was washed with brine, dried (Na₂SO₄), and evaporated. The residue was purified by silica gel column chromatography (50% AcOEt in hexane) to give 4 (425 mg, 88%) as a light yellow oil.

Synthesis from 7

To a solution of 7 (324 mg, 0.919 mmol) in THF (8 mL) was added tetrabutylammonium fluoride (1.0 M solution in THF, 1.10 mL, 1.10 mmol) and AcOH (1.0 M solution in THF, 2.20 mL, 2.2 mmol) at 0 °C, and the mixture was stirred at 0 °C for 24 h. The mixture was diluted with AcOEt and washed with aqueous NaHCO₃. The organic layer was washed with brine, dried with Na₂SO₄, and evaporated. The residue was purified quickly by silica gel column chromatography (50% to 80% AcOEt in hexane) to give compound 6. After the residue was dissolved in CH₂Cl₂ (8 mL), pyridine (163 μL, 2.0 mmol) was added to the solution, and the mixture was cooled at 0 °C. To the mixture was added a solution of triphosgene (98%, 306 mg, 1.0 mmol) in CH₂Cl₂ (1 mL), and the resulting mixture was stirred at 0 °C for 30 min. The reaction was quenched with addition of saturated aqueous NH₄Cl and extracted with AcOEt. The organic layer was washed with brine, dried with Na₂SO₄, and evaporated. The residue was purified by silica gel column chromatography (33% to 50% AcOEt in hexane) to give 4 (155 mg, 64% for 2 steps) as a light yellow oil, and 1 was recovered (39 mg, 12%). ¹H NMR (500 MHz, CDCl₃) δ 7.34–7.40 (m, 5H, aromatic), 5.14 (s, 2H, benzyl-CH₂), 4.42 (m, 2H, CH₂CH₂O), 2.83 (s, 2H, OC(O)CH₂), 2.36 (m, 1H, CH₂CH₂O), 2.08 (m, 1H, CH₂CH₂O), 1.57 (s, 3H, CH₃); ¹³C NMR (125 MHz, CDCl₃) δ 168.7, 148.5, 135.1, 128.6, 128.5, 128.4, 81.0, 66.9, 64.5, 44.8, 30.4, 25.8; HRMS (pos. ion ESI) m/z calcd for [M+Na]⁺ C₁₄H₁₆NaO₅: 287.0890, found: 287.0899.

4-((tert-butyldimethylsilyl)oxy)butan-2-one (6)

To a solution of 4-hydryoxy-2-butanone (273 μL, 3.0 mmol) in DMF (20 mL) was added tert-butylchlorodimethylsilane (559 mg, 3.6 mmol) and imidazole (490 mg, 7.2 mmol) at 0 °C, and the mixture was stirred at room temperature for 12 h. After the addition of MeOH, the mixture was diluted with Et₂O and washed with H₂O (x 3). The organic layer was washed with brine, dried with Na₂SO₄, and evaporated. The residue was purified by silica gel column chromatography (5% AcOEt in hexane) to give 6 (548 mg, 90%) as a colorless liquid. ¹H NMR (500 MHz, CDCl₃) δ 3.88 (t, 2H, CH₂CH₂O, J = 6.3 Hz), 2.62 (t, 2H, CH₂CH₂O, J = 6.3 Hz), 2.19 (s, 3H, CH₃), 0.88 (s, 9H, C(CH₃)₃), 0.05 (s, 6H, Si(CH₃)₂); ¹³C NMR (125 MHz, CDCl₃) δ 208.2, 58.8, 46.5, 30.9, 25.8, 18.2, −5.5; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₀H₂₂NaO₂Si: 225.1287, found: 225.1280.

Benzyl 5-((tert-butyldimethylsilyl)oxy)-3-hydroxy-3-methylpentanoate (7)

To a solution of lithium diisopropylamide (1.8 M solution in heptane/THF/ethyl benzene, 7.93 mL, 14.3 mmol) in THF (105 mL) was added benzyl acetate (2.04 mL, 14.3 mmol) at −78 °C, and the mixture was stirred at −78 °C for 30 min. A solution of 6 (2.41 g, 11.9 mmol) in THF (5 mL) was added to the mixture via cannula at −78 °C, and the resulting mixture was stirred at −78 °C for 1 h. After addition of sat. aq. NH₄Cl, the mixture was warmed to room temperature and evaporated. The residue was partitioned between AcOEt and sat. aq. NH₄Cl. The organic layer was washed with brine, dried (Na₂SO₄), and evaporated. The residue was purified by silica gel column chromatography (8% AcOEt in hexane) to give 7 (3.92 mg, 93%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 7.31–7.35 (m, 5H, aromatic), 5.17 (d, 1H, benzyl-CH₂, J = 7.5 Hz), 5.12 (d, 1H, benzyl-CH₂, J = 7.5 Hz), 4.20 (s, 1H, OH), 3.86 (t, 2H, CH₂CH₂O, J = 3.6 Hz), 2.66 (d, 1H, OC(O)CH₂, J = 15.6 Hz), 2.60 (d, 1H, OC(O)CH₂, J = 15.6 Hz), 1.82 (m, 2H, CH₂CH₂O), 1.30 (s, 3H, CH₃), 0.89 (s, 9H, C(CH₃)₃), 0.07 (s, 6H, Si(CH₃)₂); HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₉H₃₂NaO₄Si: 375.1962, found: 375.1965.

2-(4-Methyl-2-oxo-1,3-dioxan-4-yl)acetic acid (8)

To a solution of 4 (340 mg, 1.29 mmol) in AcOEt (15 mL) was added Pd/C (10%, 33 mg), and the mixture was stirred under H₂ gas at room temperature for 30 min. The resulting mixture was filtered through Celite with acetone, and the filtrate was evaporated to give 8 (224 mg, quant.) as a colorless solid; mp. 83–85 °C; ¹H NMR (500 MHz, CD₃OD) δ 4.48 (m, 2H, CH₂CH₂O), 2.82 (d, 1H, OC(O)CH₂, J = 15.5 Hz), 2.78 (d, 1H, OC(O)CH₂, J = 15.5 Hz), 2.48 (m, 1H, CH₂CH₂O), 2.12 (m, 1H, CH₂CH₂O), 1.57 (s, 3H, CH₃); ¹³C NMR (125 MHz, CD₃OD) δ 172.5, 151.9, 83.3, 66.2, 45.2, 31.3, 26.1; HRMS (ESI) m/z calcd for [M+Na]⁺ C₇H₁₀NaO₅: 197.0420, found: 197.0426.

Phenyl 2-(4-methyl-2-oxo-1,3-dioxan-4-yl)acetate (9a)

To a solution of 8 (35 mg, 0.20 mmol) in CH₃CN/CH₂Cl₂ (1/1, 1.5 mL) was added phenol (85 mg, 0.90 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (38 mg, 0.20 mmol), and DMAP (20 mg, 0.16 mmol) at 0 °C, and the mixture was stirred at room temperature for 1 h. The mixture was diluted with AcOEt and partitioned between AcOEt and aq. HCl (0.5 M). The organic layer was washed with sat. aq. NaHCO₃, brine, dried with Na₂SO₄, and evaporated. The residue was purified by silica gel column chromatography (33% to 50% AcOEt in hexane) to give 9a (27 mg, 54%) as a white solid; mp. 61–63 °C; ¹H NMR (500 MHz, CDCl₃) δ 7.40 (t, 2H, aromatic, J = 7.9, 8.0 Hz), 7.26 (t, 1H, aromatic, J = 8.0 Hz), 7.09 (d, 2H, aromatic, J = 7.9 Hz), 4.49 (t, 2H, CH₂CH₂O, J = 5.1 Hz), 3.05 (s, 2H, OC(O)CH₂), 2.47 (m, 1H, CH₂CH₂O), 2.19 (m, 1H, CH₂CH₂O), 1.68 (s, 3H, CH₃); ¹³C NMR (125 MHz, CDCl₃) δ 167.6, 150.0, 148.4, 129.6, 126.3, 121.3, 81.0, 64.5, 44.9, 30.6, 25.9; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₃H₁₄NaO₅: 273.0733, found: 273.0731.

4-Fluorophenyl 2-(4-methyl-2-oxo-1,3-dioxan-4-yl)acetate (9b)

9b (35 mg, 65%, white solid) was prepared from 8 (35 mg, 0.20 mmol) as described for the preparation of 9a using 4-fluorophenol instead of phenol; mp. 68–69 °C; ¹H NMR (500 MHz, CDCl₃) δ 7.06–7.08 (m, 4H, aromatic), 4.48 (m, 2H, CH₂CH₂O), 3.03 (s, 2H, OC(O)CH₂), 2.48 (m, 1H, CH₂CH₂O), 2.17 (m, 1H, CH₂CH₂O), 1.67 (s, 3H, CH₃); ¹³C NMR (125 MHz, CDCl₃) δ 167.6, 160.4(d), 148.4, 145.8, 122.8(d), 116.3(d), 80.9, 64.5, 44.9, 30.6, 25.9; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₃H₁₃FNaO₅: 291.0639, found: 291.0648.

2-(4-Methyl-2-oxo-1,3-dioxan-4-yl)-N-phenylacetamide (10a)

To a solution of 8 (29 mg, 0.17 mmol) in DMF (1 mL) was added aniline (45 μL, 0.50 mmol), HBTU (64 mg, 0.17 mmol), and N,N-diisopropylethylamine (59 μL, 0.34 mmol) at 0 °C, and the mixture was stirred at room temperature for 8 h. The mixture was diluted with AcOEt and partitioned between AcOEt and aq. HCl (0.5 M). The organic layer was washed with sat. aq. NaHCO₃, brine, dried with Na₂SO₄, and evaporated. The residue was purified by silica gel column chromatography (50% AcOEt in hexane) to give 10a (23 mg, 55%) as a colorless oil; ¹H NMR (500 MHz, CDCl₃) δ 8.03 (br s, 1H, NH), 7.54 (d, 2H, aromatic, J = 7.8 Hz), 7.33 (t, 2H, aromatic, J = 7.8, 8.2 Hz), 7.13 (t, 1H, aromatic, J = 8.2 Hz), 4.51 (m, 2H, CH₂CH₂O), 2.85 (s, 2H, OC(O)CH₂), 2.52 (m, 1H, CH₂CH₂O), 2.20 (m, 1H, CH₂CH₂O), 1.64 (s, 3H, CH₃); ¹³C NMR (125 MHz, CDCl₃) δ 166.1, 148.9, 137.4, 129.0, 124.7, 120.1, 82.4, 65.0, 48.4, 30.7, 25.6; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₃H₁₅NNaO₄: 272.0893, found: 272.0894.

N-Benzyl-2-(4-methyl-2-oxo-1,3-dioxan-4-yl)acetamide (10b)

10b (27 mg, 61%, colorless oil) was prepared from 8 (29 mg, 0.17 mmol) as described for the preparation of 10a using benzylamine instead of aniline. ¹H NMR (500 MHz, CDCl₃) δ 7.26–7.35 (m, 5H, aromatic), 6.47 (br s, 1H, NH), 4.38–4.46 (m, 4H, CH₂CH₂O and benzyl-CH₂), 2.68 (d, 1H, OC(O)CH₂, J = 14.5 Hz), 2.63 (d, 1H, OC(O)CH₂, J = 14.5 Hz), 2.46 (m, 1H, CH₂CH₂O), 2.09 (m, 1H, CH₂CH₂O), 1.55 (s, 3H, CH₃); ¹³C NMR (126 MHz, CDCl₃) δ 167.7, 148.7, 137.8, 128.8, 127.8, 127.7, 82.0, 64.9, 47.5, 43.8, 30.7, 25.7; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₄H₁₇NNaO₄: 286.1055, found: 286.1052.

N-(4-Fluorobenzyl)-2-(4-methyl-2-oxo-1,3-dioxan-4-yl)acetamide (10c)

10c (19 mg, 38%, colorless oil) was prepared from 8 (29 mg, 0.17 mmol) as described for the preparation of 10a using 4-fluorobenzylamine instead of aniline. ¹H NMR (500 MHz, CDCl₃) δ 7.24–7.27 (m, 2H, aromatic), 6.99–7.02 (m, 2H, aromatic), 6.70 (br s, 1H, NH), 4.37–4.44 (m, 4H, CH₂CH₂O and benzyl-CH₂), 2.66 (s, 2H, OC(O)CH₂), 2.45 (m, 1H, CH₂CH₂O), 2.10 (m, 1H, CH₂CH₂O), 1.54 (s, 3H, CH₃); ¹³C NMR (126 MHz, CDCl₃) δ 170.8, 162.2(d), 147.8, 134.0(d), 127.46, 115.6(d), 81.8, 64.2, 46.5, 43.1, 29.2, 25.0; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₄H₁₆FNNaO₄: 304.0956, found: 304.0960.

Dibenzyl 3-(fluoromethyl)-3-hydroxypentanedioate (14)

To a solution of ethyl fluoroacetate (1.94 mL, 20.0 mmol) in Et₂O (120 mL) was added allylmagnesium bromide (1.0 M solution in Et₂O, 39.0 mL, 39.0 mmol) at 0 °C, and the mixture was stirred at 0 °C for 20 min. After addition of saturated aqueous NH₄Cl, the organic layer was separated, washed with brine, dried with Na₂SO₄, and evaporated. The residue (12) was used in the next reaction without further purification. The crude product 12 (2.60 g) was dissolved in CH₂Cl₂ (40 mL) and cooled to −78 °C. Ozone was bubbled into the solution at −78 °C for 30 min until the color of the solution turned to light purple. Oxygen was bubbled into the solution for 20 min to remove ozone, and the solution was warmed to room temperature. Acetic acid (20 mL) was added to the solution and then the solvent was reduced in vacuo until the amount of the solution was a few milliliters. To the residue was added acetic acid (15 mL), H₂O (15 mL), conc. H₂SO₄ (0.40 mL), and aq. H₂O₂ (30%, 9.0 mL), and the mixture was stirred under reflux for 4 h. After cooling to room temperature, the mixture was neutralized with BaCO₃ (1.5 g) and filtered through Celite with acetone. To the filtrate was added Pd/C (30 mg), and the mixture was stirred at room temperature for 8 h to decompose the H₂O₂. The mixture was filtered through Celite with acetone to remove the Pd/C, and the filtrate was evaporated. The residue was co-evaporated with H₂O (x 2) and toluene (x 3) to give crude dicarboxylic acid 13 as a brown oil. To a solution of crude product 13 in DMF (80 mL) was added benzyl bromide (98%, 4.85 mL, 40 mmol) and K₂CO₃ (5.53 g, 40 mmol), and the mixture was stirred at room temperature for 8 h. After filtration through Celite to remove K₂CO₃, the solvent was evaporated. The residue was partitioned between AcOEt and aq. HCl (0.5 M). The organic layer was washed with sat. aq. NaHCO₃ and brine, dried (Na₂SO₄), and evaporated. The residue was purified by silica gel column chromatography (10% to 20% AcOEt in hexane) to give diester 14 (3.87 g, 54% for 4 steps) as a light yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 7.32–7.38 (m, 10H, aromatic), 5.14 (s, 4H, benzyl-CH₂ x2), 4.42 (d, 2H, CH₂F, J = 47 Hz), 4.25 (s, 1H, OH), 2.78 (m, 4H, C(O)CH₂ x2); ¹³C NMR (125 MHz, CDCl₃) δ 171.0, 135.3, 128.6, 128.5, 128.3, 86.4(d), 71.1, 66.8, 39.9; HRMS (ESI) m/z calcd for [M+Na]⁺ C₂₀H₂₁FNaO₅: 383.1265, found: 383.1276.

Benzyl 2-(4-(fluoromethyl)-2-oxo-1,3-dioxan-4-yl)acetate (16)

To a solution of 14 (280 mg, 0.777 mmol) in THF (8 mL) was added DIBAL-H (1.0 M solution in THF, 0.932 mL, 0.932 mmol) at 0 °C, and the mixture was stirred at 0 °C for 10 min. DIBAL-H (1.0 M solution in THF, 1.86 mL, 1.86 mmol) was added to the mixture at 0 °C, and the resulting mixture was stirred at 0 °C for 15 min. The reaction was quenched with addition of aq. HCl (1.5 M) that was saturated with NaCl and extracted with AcOEt. The organic layer was washed with aq. HCl (1.5 M), which was saturated with NaCl, sat. aq. NaHCO₃, and brine, and then dried (Na₂SO₄) and evaporated. After the residue (15) was dissolved in CH₂Cl₂ (60 mL), pyridine (285 μL, 3.50 mmol) was added to the solution and the mixture was cooled at 0 °C. To the mixture was added a solution of triphosgene (706 mg, 2.33 mmol) in CH₂Cl₂ (4 mL), and the resulting mixture was stirred at 0 °C for 30 min. The reaction was quenched with addition of sat. aq. NH₄Cl and extracted with AcOEt. The organic layer was washed with brine, dried with Na₂SO₄, and evaporated. The residue was purified by silica gel column chromatography (33% to 50% AcOEt in hexane) to give 16 (81 mg, 37% for 2 steps) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 7.33–7.40 (m, 5H, aromatic), 5.15 (s, 2H, benzyl-CH₂), 4.62 (dd, 1H, CH₂F, J = 10.0, 12.6 Hz), 4.54 (dd, 1H, CH₂F, J = 10.0, 12.2 Hz), 4.40 (m, 2H, CH₂CH₂O), 2.86 (m, 2H, C(O)CH₂), 2.37 (m, 1H, CH₂CH₂O), 2.30 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, CDCl₃) δ 168.0, 148.1, 134.9, 128.7, 128.7, 128.5, 85.0(d), 81.1, 67.2, 64.1, 39.8, 25.9; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₄H₁₅FNaO₅: 305.0796, found: 305.0800.

2-(4-(Fluoromethyl)-2-oxo-1,3-dioxan-4-yl)acetic acid (17)

17 (249 mg, 99%, colorless oil) was prepared from 16 (368 mg, 1.30 mmol) as described for the preparation of 8. ¹H NMR (500 MHz, acetone-d₆) δ 4.72 (d, 2H, CH₂F, J = 48 Hz), 4.48 (m, 2H, CH₂CH₂O), 2.94 (m, 2H, C(O)CH₂), 2.47 (m, 1H, CH₂CH₂O), 2.32 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, acetone-d₆) δ 170.6, 148.7, 86.4(d), 82.3, 64.9, 39.4, 26.4; HRMS (ESI) m/z calcd for [M+Na]⁺ C₇H₉FNaO₅: 215.0326, found: 215.0330.

4-Fluorobenzyl 2-(4-(fluoromethyl)-2-oxo-1,3-dioxan-4-yl)acetate (18a)

18a (41 mg, 77%, colorless oil) was prepared from 17 (34 mg, 0.18 mmol) as described for the preparation of 9a using 4-fluorobenzyl bromide instead of iodomethane. ¹H NMR (500 MHz, CDCl₃) δ 7.33–7.36 (m, 2H, aromatic), 7.05–7.08 (m, 2H, aromatic), 5.12 (s, 2H, benzyl-CH₂), 4.61 (dd, 1H, CH₂F, J = 10.0, 14.0 Hz), 4.52 (dd, 1H, CH₂F, J = 10.0, 13.6 Hz), 4.42 (m, 2H, CH₂CH₂O), 2.86 (m, 2H, C(O)CH₂), 2.35 (m, 1H, CH₂CH₂O), 2.28 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, CDCl₃) δ 168.0, 162.8(d), 148.0, 130.8(d), 130.7(d), 115.7(d), 85.0(d), 81.0, 66.5, 64.1, 40.0, 26.1; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₄H₁₄F₂NaO₅: 323.0702, found: 323.0707.

2,4-Difluorobenzyl 2-(4-(fluoromethyl)-2-oxo-1,3-dioxan-4-yl)acetate (18b)

18b (36 mg, 72%, colorless oil) was prepared from 17 (30 mg, 0.16 mmol) as described for the preparation of 9a using 2,4-difluorobenzyl bromide instead of iodomethane. ¹H NMR (500 MHz, CDCl₃) δ 7.38 (m, 1H, aromatic), 6.83–6.92 (m, 2H, aromatic), 5.17 (s, 2H, benzyl-CH₂), 4.62 (dd, 1H, CH₂F, J = 10.0, 13.1 Hz), 4.52 (dd, 1H, CH₂F, J = 10.0, 12.8 Hz), 4.42 (m, 2H, CH₂CH₂O), 2.85 (m, 2H, C(O)CH₂), 2.37 (m, 1H, CH₂CH₂O), 2.29 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, CDCl₃) δ 167.9, 163.4(d), 161.4(d), 148.0, 132.2(m), 118.2(d), 111.7(m), 104.2(t), 85.0(d), 81.0, 64.2, 60.6, 39.9, 26.0; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₄H₁₃F₃NaO₅: 341.0607, found: 341.0612.

Benzyl 5-ethoxy-3-hydroxy-3-(trifluoromethyl)pent-4-enoate (20)

To a solution of lithium diisopropylamide (1.8 M solution in heptane/THF/ethyl benzene, 3.05 mL, 5.50 mmol) in THF (55 mL) was added benzyl acetate (784 μL, 5.50 mmol) at −78 °C, and the mixture was stirred at −78 °C for 30 min. A solution of 4-ethoxy-1,1,1,- trifluoro-3-buten-2-one (19, 712 μL, 5.00 mmol) in THF (5 mL) was added to the mixture via cannula at −78 °C, and the resulting mixture was stirred at −78 °C for 30 min. After the addition of sat. aq. NH₄Cl, the mixture was warmed to room temperature, and evaporated. The residue was partitioned between AcOEt and sat. aq. NH₄Cl. The organic layer was washed with brine, dried with Na₂SO₄, and evaporated. The residue was purified by silica gel column chromatography (6% AcOEt in hexane) to give 20 (1.36 g, 86%) as a light yellow oil; ¹H NMR (500 MHz, CDCl₃) δ 7.33–7.40 (m, 5H, aromatic), 6.74 (d, 1H, CH=CHOEt, J = 12.6 Hz), 5.20 (d, 1H, benzyl-CH₂, J = 14.1 Hz), 5.16 (d, 1H, benzyl-CH₂, J = 14.1 Hz), 4.76 (s, 1H, OH), 4.71 (d, 1H, CH=CHOEt, J = 12.6 Hz), 3.70 (q, 2H, CH₂CH₃), 2.86 (d, 1H, C(O)CH₂, J = 15.6 Hz), 2.71 (d, 1H, C(O)CH₂, J = 15.6 Hz), 1.27 (t, 3H, CH₂CH₃, J = 7.0 Hz); ¹³C NMR (125 MHz, CDCl₃) δ 171.2, 151.8, 134.8, 128.7, 128.5, 128.3, 124.6(q), 99.4, 73.3(q), 67.5, 65.5, 38.9, 14.7; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₅H₁₇F₃NaO₄: 341.0977, found: 341.0978.

Benzyl 4,4,4-trifluoro-3-formyl-3-hydroxybutanoate (21)

To a solution of 20 (334 mg, 1.05 mmol) in acetone (16 mL) was added aq. HCl (12 M, 4 mL) at 0 °C, and the mixture was stirred vigorously at 0 °C for 8 min. To the mixture was added sat. aq. NaHCO₃ to neutralize and extracted with AcOEt. The organic layer was washed with sat. aq. NaHCO₃ and brine, dried (Na₂SO₄), and evaporated. The residue was purified by silica gel column chromatography (20% AcOEt in hexane) to give 21 (243 mg, 80%) as a light yellow oil; ¹H NMR (500 MHz, CDCl₃) δ 9.85 (s, 1H, CHO), 7.34–7.40 (m, 5H, aromatic), 5.35 (s, 1H, OH), 5.19 (s, 2H, benzyl-CH₂), 2.75–2.93 (m, 4H, CH₂CHO and C(O)CH₂); ¹³C NMR (125 MHz, CDCl₃) δ 199.0, 170.8, 134.5, 128.8, 128.7, 128.5, 124.8(q), 73.3(q), 67.7, 46.1, 36.9; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₃H₁₃F₃NaO₄: 313.0658, found: 313.0660.

Benzyl 2-(2-oxo-4-(trifluoromethyl)-1,3-dioxan-4-yl)acetate (23)

To a solution of 21 (320 mg, 1.10 mmol) in benzene (10 mL) was added sodium triacetoxyborohydride (95%, 701 mg, 3.31 mmol) at 0 °C, and the mixture was stirred at room temperature for 2 h. The mixture was quenched with addition of sat. aq. NaHCO₃ and extracted with AcOEt. The organic layer was washed with sat. aq. NaHCO₃, brine, dried (Na₂SO₄), and evaporated. After the residue was dissolved in CH₂Cl₂ (10 mL), pyridine (125 μL, 1.54 mmol) was added to the solution, and the mixture was cooled at 0 °C. To the mixture was added a solution of triphosgene (98%, 400 mg, 1.32 mmol) in CH₂Cl₂ (2 mL) at 0 °C, and the resulting mixture was stirred at 0 °C for 30 min. The reaction was quenched with addition of sat. aq. NH₄Cl, and extracted with AcOEt. The organic layer was washed with brine, dried with Na₂SO₄, and evaporated. The residue was purified by silica gel column chromatography (33% AcOEt in hexane) to give 23 (281 mg, 80% for 2 steps) as a colorless solid; mp. 87–88 °C; ¹H NMR (500 MHz, CDCl₃) δ 7.34–7.41 (m, 5H, aromatic), 5.18 (s, 2H, benzyl-CH₂), 4.43 (m, 1H, CH₂CH₂O), 4.37 (m, 1H, CH₂CH₂O), 3.10 (d, 1H, C(O)CH₂, J = 16.5 Hz), 2.87 (d, 1H, C(O)CH₂, J = 16.5 Hz), 2.74 (m, 1H, CH₂CH₂O), 2.33 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, CDCl₃) δ 167.2, 146.8, 134.8, 128.8, 128.7, 128.5, 123.6(q), 80.2(q), 67.3, 64.1, 37.9, 24.2; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₄H₁₃F₃NaO₅: 341.0607, found: 341.0613.

2-(2-Oxo-4-(trifluoromethyl)-1,3-dioxan-4-yl)acetic acid (24)

24 (176 mg, 98%, colorless oil) was prepared from 23 (250 mg, 0.786 mmol) as described for the preparation of 8. ¹H NMR (500 MHz, D₂O) δ 4.61 (m, 2H, CH₂CH₂O), 3.22 (d, 1H, C(O)CH₂, J = 16.5 Hz), 3.12 (d, 1H, C(O)CH₂, J = 16.5 Hz), 2.76 (m, 1H, CH₂CH₂O), 2.59 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, D₂O) δ 171.4, 150.4, 123.4(q), 81.1(q), 65.3, 36.8, 23.5;HRMS (ESI) m/z calcd for [M+Na]⁺ C₇H₇F₃NaO₅: 251.0138, found: 251.0140.

4-Fluorobenzyl 2-(2-oxo-4-(trifluoromethyl)-1,3-dioxan-4-yl)acetate (25a)

Ester 25a (48 mg, 88%, white solid) was prepared from 24 (39 mg, 0.17 mmol) as described for the preparation of 18a; mp. 84–85 °C; ¹H NMR (500 MHz, CDCl₃) δ 7.35 (m, 2H, aromatic), 7.07 (m, 2H, aromatic), 5.14 (s, 2H, benzyl-CH₂), 4.37–4.47 (m, 2H, CH₂CH₂O), 3.09 (d, 1H, C(O)CH₂, J = 16.4 Hz), 2.86 (d, 1H, C(O)CH₂, J = 16.4 Hz), 2.73 (m, 1H, CH₂CH₂O), 2.33 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, CDCl₃) δ 167.1, 162.9(d), 146.7, 130.8(d), 130.7, 123.6(q), 115.6(d), 80.1(q), 66.8, 64.0, 37.7, 24.1; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₄H₁₂F₄NaO₅: 359.0519, found: 359.0518.

2,4-Difluorobenzyl 2-(2-oxo-4-(trifluoromethyl)-1,3-dioxan-4-yl)acetate (25b)

Ester 25b (45 mg, 85%, colorless oil) was prepared from 24 (36 mg, 0.16 mmol) as described for the preparation of 18b. ¹H NMR (500 MHz, CDCl₃) δ 7.38 (m, 1H, aromatic), 6.88 (m, 2H, aromatic), 5.21 (d, 1H, benzyl-CH₂, J = 12.2 Hz), 5.17 (d, 1H, benzyl-CH₂, J = 12.2 Hz), 4.45 (m, 2H, CH₂CH₂O), 3.09 (d, 1H, C(O)CH₂, J = 16.4 Hz), 2.87 (d, 1H, C(O)CH₂, J = 16.4 Hz), 2.75 (m, 1H, CH₂CH₂O), 2.35 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, CDCl₃) δ 167.0, 163.4(d), 161.4(d), 146.6, 132.3(m), 123.5(q), 118.0(d), 111.6(d), 104.2(t), 80.0(q), 64.1, 60.9, 37.6, 24.1; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₄H₁₁F₅NaO₅: 377.0419, found: 377.0427.

4-Hydroxy-4-(trifluoromethyl)tetrahydro-2H-pyran-2-one (26)

To a solution of 23 (267 mg, 0.919 mmol) in benzene (10 mL) was added sodium triacetoxyborohydride (95%, 615 mg, 2.76 mmol) at 0 °C, and the mixture was stirred at room temperature for 2 h. The mixture was quenched with addition of sat. aq. NaHCO₃ and extracted with AcOEt. The organic layer was washed with sat. aq. NaHCO₃, brine, dried (Na₂SO₄), and evaporated. After the residue was dissolved in CH₂Cl₂ (10 mL), trifluoroacetic acid (1 mL) was added to the solution at 0 °C, and the mixture was stirred at room temperature for 2 h. The mixture was diluted with AcOEt and evaporated. The residue was purified by silica gel column chromatography (33% AcOEt in hexane) to give 26 (135 mg, 80% for 2 steps) as a yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 4.61 (m, 1H, CH₂CH₂O), 4.45 (m, 1H, CH₂CH₂O), 2.86 (d, 1H, CH₂C(O), J = 14.7 Hz), 2.84 (br s, 1H, OH), 2.78 (d, 1H, CH₂C(O), J = 14.7 Hz), 2.24 (m, 1H, CH₂CH₂O), 2.07 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, CDCl₃) δ 167.5, 124.7(q), 71.5(q), 64.4, 36.9, 28.6; HRMS (ESI) m/z calcd for [M+Na]⁺ C₆H₇F₃NaO₃: 207.0239, found: 207.0233.

3,5-Dihydroxy-3-(trifluoromethyl)pentanoic acid (27)

To a solution of 26 (10 mg, 0.054 mmol) in H₂O (1 mL) was added KOH (>90%, 3.4 mg, 0.052 mmol) at room temperature, and the mixture was stirred at 40 °C for 2 h. The pH of the solution was lowered to about pH 7–8 (detected by pH indicator paper) with aq. HCl (0.1 M). The solvent was evaporated and lyophilized to give 27 (15 mg) as a white powder, including KCl. ¹H NMR (500 MHz, D₂O) δ 3.79 (m, 2H, CH₂CH₂O), 2.60 (d, 1H, C(O)CH₂, J = 15.5 Hz), 2.52 (d, 1H, C(O)CH₂, J = 15.5 Hz), 2.04 (m, 1H, CH₂CH₂O), 1.96 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, D₂O) δ 178.4, 125.9(q), 73.2(q), 56.4, 38.3, 35.9; LRMS (ESI) m/z = 225 [M+Na]⁺.

N-Benzyl-3,5-dihydroxy-3-methylpentanamide (29a)

To a solution of (±)-mevalonolactone (1, 97%, 134 mg, 1.00 mmol) in DMF (1 mL) was added benzyl amine (131 μL, 1.20 mmol) at room temperature, and the mixture was stirred at 80 °C for 12 h. After being evaporated, the residue was partitioned between AcOEt and H₂O. The organic layer was washed with brine, dried (Na₂SO₄), and evaporated. The residue (29a, light yellow oil) was used without further purification. ¹H NMR (500 MHz, CDCl₃) δ 7.36 – 7.18 (m, 5H, aromatic), 6.68 (br s, 1H, NH), 4.39 (d, 2H, PhCH₂N, J = 5.8 Hz), 3.82 (ddd, 1H, CH₂CH₂O, J = 11.5, 7.4, 4.3 Hz), 3.74 (ddd, 1H, CH₂CH₂O, J = 11.1, 6.7, 4.4 Hz), 2.48 (d, 1H, NHC(O)CH₂, J = 14.6 Hz), 2.26 (d, 1H, NHC(O)CH_2, J = 14.6 Hz), 1.74 (ddd, 1H, CH₂CH₂O, J = 14.5, 7.4, 4.4 Hz, 1H), 1.66 (ddd, 1H, CH₂CH₂O, J = 14.6, 6.7, 4.3 Hz), 1.25 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 172.4, 138.0, 128.7, 127.6, 127.5, 72.3, 59.2, 46.6, 43.3, 42.0, 26.9; LRMS (ESI) m/z = 237.1 [M+H]⁺.

N-Benzyl-2-(4-methyl-1,3-dioxan-4-yl)acetamide (30a)

To a solution of the crude product 29a (26 mg, ≤0.11 mmol) in CH₂Cl₂ was added chloromethyl methyl ether (84 μL, 1.1 mmol), N,N-diisopropylethylamine (383 μL, 2.2 mmol), and DMAP (1.2 mg) at 0 °C, and the mixture was stirred at room temperature for 2 h. After dilution with AcOEt, the mixture was partitioned between AcOEt and 0.5 M aq. HCl. The organic layer was washed with sat. aq. NaHCO₃, brine, dried (Na₂SO₄), and evaporated. The residue was dissolved in CH₂Cl₂ (17 mL), and BF₃•Et₂O (30 μL, 0.24 mmol) was added at 0 °C. The mixture was stirred at room temperature for 8 h. After addition of sat. aq. NaHCO₃, the mixture was extracted with AcOEt. The organic layer was washed with brine, dried (Na₂SO₄), and evaporated. The residue was purified by silica gel column chromatography (50% AcOEt in hexane) to give 30a (21 mg, 77% for 3 steps) as a white solid; mp. 82–84 °C; ¹H NMR (500 MHz, CDCl₃) δ 7.25–7.35 (m, 5H, aromatic), 6.80 (br s, 1H, NH), 4.92 (d, 1H, OCH₂O, J = 6.5 Hz), 4.85 (d, 1H, OCH₂O, J = 6.5 Hz), 4.48 (d, 2H, benzyl-CH₂, J = 5.5 Hz), 3.95 (m, 1H, CH₂CH₂O), 3.89 (m, 1H, CH₂CH₂O), 2.60 (d, 1H, NHC(O)CH₂, J = 14.5 Hz), 2.47 (d, 1H, NHC(O)CH₂, J = 14.5 Hz), 2.02 (m, 1H, CH₂CH₂O), 1.48 (m, 1H, CH₂CH₂O), 1.40 (s, 3H, CH₃); ¹³C NMR (125 MHz, CDCl₃) δ 169.8, 138.5, 128.6, 127.5, 127.3, 87.8, 72.1, 62.8, 49.0, 43.3, 34.7, 20.9; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₄H₁₉NNaO₃: 272.1257, found: 272.1259.

N-Benzyl-2-(2,2,4-trimethyl-1,3-dioxan-4-yl)acetamide (31)

To a solution of the crude product 29a (55 mg, 0.23 mmol) in CH₂Cl₂ (2 mL) was added 2,2-dimethoxypropane (112 μL, 0.91 mmol) and camphor sulfonic acid (4 mg, 0.02 mmol) at 0 °C, and the mixture was stirred at room temperature for 16 h. After addition of sat. aq. NaHCO₃, the mixture was extracted with AcOEt. The organic layer was washed with brine, dried (Na₂SO₄), and evaporated. The residue was purified by silica gel column chromatography (50% AcOEt in hexane) to give 31 (17 mg, 27% for 2 steps) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 7.27–7.35 (m, 5H, aromatic), 6.97 (br s, 1H, NH), 4.53 (dd, 1H, benzyl-CH₂, J = 6.0, 14.8 Hz), 4.39 (dd, 1H, benzyl-CH₂, J = 5.3, 14.8 Hz), 4.02 (m, 1H, CH₂CH₂O), 3.83 (m, 1H, CH₂CH₂O), 2.49 (d, 1H, NHC(O)CH₂, J = 14.4 Hz), 2.44 (d, 1H, NHC(O)CH₂, J = 14.4 Hz), 1.91 (m, 1H, CH₂CH₂O), 1.51 (m, 1H, CH₂CH₂O), 1.41 (s, 6H, C(CH₃)₂), 1.23 (s, 3H, CH₃); ¹³C NMR (125 MHz, CDCl₃) δ 170.3, 138.4, 128.6, 127.7, 127.4, 98.5, 72.0, 56.5, 51.1, 43.4, 33.3, 29.9, 26.7, 25.7; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₆H₂₃NNaO₃: 300.1570, found: 300.1586.

N-Benzyl-2-(4-methyl-2-phenyl-1,3-dioxan-4-yl)acetamide (32a)

To a solution of the crude product 29a (54 mg, 0.23 mmol) in CH₂Cl₂ (2 mL) was added benzaldehyde dimethyl acetal (51 μL, 0.34 mmol) and camphor sulfonic acid (4 mg, 0.02 mmol) at 0 °C, and the mixture was stirred at room temperature for 16 h. After addition of sat. aq. NaHCO₃, the mixture was extracted with AcOEt. The organic layer was washed with brine, dried (Na₂SO₄), and evaporated. The residue was purified by silica gel column chromatography (33–50% AcOEt in hexane) to give 32a (25 mg, 34% for 2 steps) as a white solid; mp. 108–110 °C; ¹H NMR (500 MHz, CDCl₃) δ 7.20–7.32 (m, 10H, aromatic), 6.83 (br s, 1H, NH), 5.72 (s, 1H, benzyl-CH), 4.47 (dd, 1H, benzyl-CH₂, J = 5.8, 14.6 Hz), 4.38 (dd, 1H, benzyl-CH₂, J = 5.3, 14.6 Hz), 4.14 (m, 2H, CH₂CH₂O), 2.55 (s, 2H, NHC(O)CH₂), 2.16 (m, 1H, CH₂CH₂O), 1.53 (s, 3H, CH₃), 1.46 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, CDCl₃) δ 169.7, 138.3, 138.1, 128.9, 128.7, 128.4, 127.9, 127.4, 125.8, 95.3, 73.4, 63.2, 50.8, 43.6, 33.6, 20.3; HRMS (ESI) m/z calcd for [M+Na]⁺ C₂₀H₂₃NNaO₃: 348.1570, found: 348.1569.

5-(Benzylamino)-3-hydroxy-3-methyl-5-oxopentyl tert-butyl carbonate (33a)

To a solution of crude product 29a (24 mg, ≤0.10 mmol) in acetonitrile (1 mL) was added (Boc)₂O (99 mg, 0.45 mmol) and DMAP (1.2 mg, 0.01 mmol) at room temperature, and the mixture was stirred under reflux conditions for 16 h. After evaporated, the residue was purified by silica gel column chromatography (50% AcOEt in hexane) to give 32a (30 mg, 88% for 2 steps) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 7.27–7.36 (m, 5H, aromatic), 6.23 (br s, 1H, NH), 4.65 (s, 1H, OH), 4.46 (m, 2H, benzyl-CH₂), 4.22 (m, 2H, CH₂CH₂O), 2.45 (d, 1H, NHC(O)CH₂, J = 14.5 Hz), 2.34 (d, 1H, NHC(O)CH₂, J = 14.5 Hz), 1.90 (t, 2H, CH₂CH₂O, J = 6.8 Hz), 1.45 (s, 9H, C(CH₃)₃), 1.28 (s, 3H, CH₃); ¹³C NMR (126 MHz, CDCl₃) δ 172.0, 153.4, 137.8, 128.8, 127.8, 127.7, 82.2, 70.5, 63.5, 46.4, 43.5, 40.2, 27.8, 27.1; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₈H₂₇NNaO₅: 360.1781, found: 360.1785.

N-Benzyl-3,5-dihydroxy-3-(trifluoromethyl)pentanamide (29b)

To a solution of 26 (120 mg, 0.652 mmol) in DMF was added benzyl amine (142 μL, 1.30 mmol), and the mixture was stirred at 80 °C for 12 h. After the solvent was evaporated, the residue was purified by silica gel column chromatography (50–100% AcOEt in hexane) to give 29b (189 mg, 99%) as a colorless oil: ¹H NMR (500 MHz, CDCl₃) δ 7.34–7.37 (m, 2H, aromatic), 7.26–7.32 (m, 3H, aromatic), 6.62 (s, 1H, OH), 6.29 (br s, 1H, NH), 4.49 (dd, 1H, benzyl-CH₂, J = 6.0, 14.5 Hz), 4.43 (dd, 1H, benzyl-CH₂, J = 5.5, 14.5 Hz), 3.95 (m, 1H, CH₂CH₂O), 3.89 (m, 1H, CH₂CH₂O), 2.73 (d, 1H, NHC(O)CH₂, J = 14.7 Hz), 2.51 (m, 1H, OH), 2.49 (d, 1H, NHC(O)CH₂, J = 14.7 Hz), 2.09 (m, 1H, CH₂CH₂O), 1.84 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, CDCl₃) δ 170.6, 137.2, 128.9, 128.7, 127.9, 125.7(q), 75.0(q), 58.4, 43.7, 38.0, 35.3; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₃H₁₆F₃NNaO₃: 314.0974, found: 314.0991.

N-Benzyl-2-(4-(trifluoromethyl)-1,3-dioxan-4-yl)acetamide (30b)

30b (26 mg, 69% for 2 steps, colorless oil) was prepared from 29b (36 mg, 0.12 mmol) as described for the preparation of 30a. ¹H NMR (500 MHz, CDCl₃) δ 7.27–7.36 (m, 5H, aromatic), 6.43 (br s, 1H, NH), 5.06 (d, 1H, OCH₂O, J = 6.3 Hz), 4.94 (d, 1H, OCH₂O, J = 6.3 Hz), 4.55 (dd, 1H, benzyl-CH₂, J = 6.1, 14.9 Hz), 4.42 (dd, 1H, benzyl-CH₂, J = 6.0, 14.9 Hz), 3.99 (m, 2H, CH₂CH₂O), 2.76 (d, 1H, NHC(O)CH₂, J = 14.4 Hz), 2.56 (d, 1H, NHC(O)CH₂, J = 14.4 Hz), 2.37 (m, 1H, CH₂CH₂O), 1.98 (dt, 1H, CH₂CH₂O, J = 3.7, 3.7, 14.7 Hz); ¹³C NMR (125 MHz, CDCl₃) δ 167.4, 137.9, 129.1, 128.7, 128.6, 128.0, 127.5, 125.7(q), 89.9, 73.4(q), 62.5, 43.8, 41.2, 25.7; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₄H₁₆F₃NNaO₃: 326.0974, found: 326.0970.

N-Benzyl-2-(2-phenyl-4-(trifluoromethyl)-1,3-dioxan-4-yl)acetamide (32b)

To a solution of 29b (60 mg, 0.20 mmol) in CH₂Cl₂ (2 mL) was added benzaldehyde dimethyl acetal (151 μL, 1.00 mmol) and camphor sulfonic acid (4.7 mg, 0.02 mmol) at room temperature, and the mixture was stirred under reflux for 12 h. After addition of sat. aq. NaHCO₃, the mixture was extracted with AcOEt. The organic layer was washed with brine, dried (Na₂SO₄), and evaporated. The residue was purified by silica gel column chromatography (10–25% AcOEt in hexane) to give major diastereomer-32b (25 mg, 33%) as a colorless oil or minor diastereomer-32b (7 mg, 9%) as a white solid. major-32b: ¹H NMR (500 MHz, CDCl₃) δ 7.25–7.35 (m, 8H, aromatic), 7.16–7.18 (m, 2H, aromatic), 6.46 (br s, 1H, NH), 5.92 (s, 1H, benzylidene-CH), 4.41 (d, 2H, benzyl-CH₂, J = 5.6 Hz), 4.18 (m, 2H, CH₂CH₂O), 2.73 (d, 1H, NHC(O)CH₂, J = 14.3 Hz), 2.57 (d, 1H, NHC(O)CH₂, J = 14.3 Hz), 2.50 (m, 1H, CH₂CH₂O), 1.97 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, CDCl₃) δ 167.3, 137.5, 129.4, 128.7, 128.5, 128.0, 127.5, 125.5(q), 98.0, 74.7(q), 63.2, 43.9, 42.8, 25.0; HRMS (pos. ion ESI) m/z calcd for (M+Na)⁺ C₂₀H₂₀F₃NNaO₃: 402.1293. Found: 402.1285. minor-32b: ¹H NMR (500 MHz, CDCl₃) δ 7.27–7.38 (m, 8H, aromatic), 7.21–7.23 (m, 2H, aromatic), 6.29 (br s, 1H, NH), 5.78 (s, 1H, benzylidene-CH), 4.49 (dd, 1H, benzyl-CH₂, J = 5.9, 14.6 Hz), 4.36 (dd, 1H, benzyl-CH₂, J = 5.3, 14.6 Hz), 4.27 (m, 1H, CH₂CH₂O), 4.19 (m, 1H, CH₂CH₂O), 3.01 (d, 1H, NHC(O)CH₂, J = 15.2 Hz), 2.94 (d, 1H, NHC(O)CH₂, J = 15.2 Hz), 2.36 (m, 1H, CH₂CH₂O), 2.02 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, CDCl₃) δ 167.2, 137.5, 137.0, 129.4, 128.8, 128.4, 127.9, 127.7, 125.9(q), 96.4, 76.0(q), 62.5, 44.0, 36.6, 25.5; HRMS (ESI) m/z calcd for [M+Na]⁺ C₂₀H₂₀F₃NNaO₃: 402.1287, found: 402.1283.

N-Benzyl-2-(2-(4-methoxyphenyl)-4-(trifluoromethyl)-1,3-dioxan-4-yl)acetamide (32c)

major diasteromer 32c (30 mg, 35%, white solid) and minor diasteromer 32c (22 mg, 25%, colorless oil,) were prepared from 29b (62 mg, 0.21 mmol) as described for the preparation of 32b using anisaldehyde dimethyl acetal instead of benzaldehyde demethyl acetal: major-32c ¹H NMR (500 MHz, CDCl₃) δ 7.27–7.31 (m, 3H, aromatic), 7.17–7.20 (m, 4H, aromatic), 6.75 (d, 2H, aromatic, J = 8.8 Hz), 6.50 (br s, 1H, NH), 5.86 (s, 1H, benzylidene-CH), 4.42 (d, 2H, benzyl-CH₂, J = 5.6 Hz), 4.15 (m, 2H, CH₂CH₂O), 3.79 (s, 3H, OCH₃), 2.73 (d, 1H, NHC(O)CH₂, J = 14.4 Hz), 2.56 (d, 1H, NHC(O)CH₂, J = 14.4 Hz), 2.46 (m, 1H, CH₂CH₂O), 1.96 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, CDCl₃) δ 167.4, 160.2, 137.6, 129.8, 128.7, 128.1, 127.5, 125.5(q), 113.8, 97.9, 74.7(q), 63.2, 55.3, 43.9, 42.8, 25.0; HRMS (ESI) m/z calcd for (M+Na)⁺ C₂₁H₂₂F₃NNaO₄: 432.1399. Found: 432.1408. minor-32c ¹H NMR (500 MHz, CDCl₃) δ 7.28–7.30 (m, 5H, aromatic), 7.22 (m, 2H, aromatic), 6.84 (d, 2H, aromatic, J = 8.8 Hz), 6.30 (br s, 1H, NH), 5.73 (s, 1H, benzylidene-CH), 4.51 (dd, 1H, benzyl-CH₂, J = 6.0, 14.6 Hz), 4.35 (dd, 1H, benzyl-CH₂, J = 5.2, 14.6 Hz), 4.25 (m, 1H, CH₂CH₂O), 4.17 (m, 1H, CH₂CH₂O), 3.80 (s, 3H, OCH₃), 2.99 (d, 1H, NHC(O)CH₂, J = 15.3 Hz), 2.95 (d, 1H, NHC(O)CH₂, J = 15.3 Hz), 2.34 (m, 1H, CH₂CH₂O), 1.98 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, CDCl₃) δ 167.2, 160.3, 137.5, 129.4, 128.8, 127.9, 127.6, 124.9(q), 113.8, 96.3, 76.1(q), 62.5, 55.3, 44.0, 36.5, 25.5; HRMS (ESI) m/z calcd for [M+Na]⁺ C₂₁H₂₂F₃NNaO₄: 432.1393, found: 432.1389.

N-Benzyl-2-(2-(2,4-dimethoxyphenyl)-4-(trifluoromethyl)-1,3-dioxan-4-yl)acetamide (32d)

To a solution of 29b (74 mg, 0.25 mmol) in benzene (3 mL) was added 2,4-dimethoxybenzaldehyde (98%, 52 mg, 0.31 mmol), camphor sulfonic acid (4.7 mg, 0.02 mmol), and 4Å molecular sieves (powder, 18 mg) at room temperature, and the mixture was stirred under reflux for 36 h. After addition of sat. aq. NaHCO₃, the mixture was extracted with AcOEt. The organic layer was washed with brine, dried (Na₂SO₄), and evaporated. The residue was purified by preparative TLC (50% AcOEt in hexane x 2) to give 31d (7 mg, 6%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 7.52 (d, 1H, aromatic, J = 8.5 Hz), 7.24–7.26 (m, 3H, aromatic), 7.17–7.19 (m, 2H, aromatic), 6.57 (br s, 1H, NH), 6.52 (d, 1H, aromatic, J = 8.5 Hz), 6.32 (s, 1H, aromatic), 6.10 (s, 1H, benzylidene-CH), 4.44 (d, 2H, benzyl-CH₂, J = 5.4 Hz), 4.24 (m, 1H, CH₂CH₂O), 4.14 (m, 1H, CH₂CH₂O), 3.80 (s, 3H, OCH₃), 3.57 (s, 3H, OCH₃), 3.12 (d, 1H, NHC(O)CH₂, J = 15.3 Hz), 2.90 (d, 1H, NHC(O)CH₂, J = 15.3 Hz), 2.28 (m, 1H, CH₂CH₂O), 1.88 (m, 1H, CH₂CH₂O); ¹³C NMR (125 MHz, CDCl₃) δ 167.7, 161.7, 157.3, 137.7, 129.4, 128.6, 128.4, 127.5, 127.3, 124.6(q), 117.9, 104.9, 98.2, 91.1, 76.1(q), 62.8, 55.4, 43.9, 36.6, 26.0; HRMS (ESI) m/z calcd for [M+Na]⁺ C₂₂H₂₄F₃NNaO₅: 462.1499, found: 462.1503.

5-(Benzylamino)-3-hydroxy-5-oxo-3-(trifluoromethyl)pentyl tert-butyl carbonate (33b)

33b (50 mg, 68%, colorless oil) was prepared from 30b (55 mg, 0.19 mmol) as described for the preparation of 33a. ¹H NMR (500 MHz, CDCl₃) δ 7.26–7.38 (m, 5H, aromatic), 6.55 (s, 1H, OH), 6.05 (br s, 1H, NH), 4.53 (dd, 1H, benzyl-CH₂, J = 6.0, 14.7 Hz), 4.42 (dd, 1H, benzyl-CH₂, J = 5.5, 14.7 Hz), 4.29 (m, 2H, CH₂CH₂O), 2.69 (d, 1H, J = 15.3 Hz, NHC(O)CH₂), 2.50 (d, 1H, J = 15.3 Hz, NHC(O)CH₂), 2.15 (m, 1H, CH₂CH₂O), 1.99 (m, 1H, CH₂CH₂O), 1.47 (s, 9H, C(CH₃)₃); ¹³C NMR (125 MHz, CDCl₃) δ 170.8, 153.1, 137.0, 128.9, 127.9, 127.6, 125.6(q), 82.5, 73.7(q), 61.8, 43.8, 37.1, 33.6, 27.7; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₈H₂₄F₃NNaO₅: 414.1504, found: 414.1500.

3,5-Dihydroxy-3-methyl-N-((perfluorophenyl)methyl)pentanamide (34)

34 (173 mg, 53%, colorless oil) was prepared from (±)-mevalonolactone and 2,3,4,5,6-pentafluorobenzylamine as described for the preparation of 29a. ¹H NMR (500 MHz, CDCl₃) δ 7.02 (bs, 1H, OH), 4.57 (s, 2H, benzyl-CH₂), 4.01 – 3.80 (m, 2H, CH₂CH₂OH), 2.54 (d, J = 14.9 Hz, 1H, NHC(O)CH₂), 2.30 (dd, J = 14.9, 1H, NHC(O)CH₂), 1.86 – 1.62 (m, 2H, CH₂CH₂OH), 1.29 (s, 3H, CH₃); ¹³C NMR (126 MHz, CDCl₃) δ 172.07, 146.28, 144.30, 139.92, 138.50, 136.49, 111.44, 72.33, 59.55, 46.84, 41.66, 30.96, 26.72; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₃H₁₄F₅NNaO₃: 350.0786, found: 350.0788.

3-Hydroxy-3-methyl-5-oxo-5-(((perfluorophenyl)methyl)amino)pentyl acetate (35) and 3-Methyl-5-oxo-5-(((perfluorophenyl)methyl)amino)pentane-1,3-diyl diacetate (36)

To a solution of 34 (163 mg, 0.5 mmol) in CH₂Cl₂ (5 mL) was added acetyl chloride (142 μL, 2.0 mmol) and pyridine (242 μL, 3 mmol) at 0 °C, and the mixture was stirred at room temperature for 48 h. After dilution with CH₂Cl₂ (15 mL), the mixture was partitioned between CH₂Cl₂ and 0.1 M aq. HCl (20 mL). The organic layer was washed with sat. aq. NaHCO₃, brine, dried (MgSO₄), and evaporated. The residue was purified by silica gel column chromatography (50% to 100% AcOEt in hexane) to give 35 (76 mg, 41%, colorless oil) and 36 (41 mg, 20%, colorless oil). 35: ¹H NMR (500 MHz, CDCl₃) δ 7.22 (s, 1H, OH), 4.73 – 4.39 (m, 2H, benzyl-CH₂), 4.29 – 4.03 (m, 2H, CH₂CH₂OH), 2.49 – 2.28 (m, 2H, NHC(O)CH₂), 2.01 (s, 3H, COCH₃), 1.87 – 1.75 (m, 2H, CH₂CH₂OH), 1.21 (s, 3H, CH₃); ¹³C NMR (126 MHz, CDCl₃) δ 172.04, 171.25, 147.39 – 143.21 (m), 142.41 – 139.45 (m), 139.14 – 135.47 (m), 111.42 (td, J = 17.6, 3.9 Hz), 70.46, 60.80, 46.25, 39.97, 30.89, 26.89, 20.92; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₅H₁₆F₅NNaO₄: 392.0892, found: 392.0896. 36: ¹H NMR (500 MHz, CDCl₃) δ 6.26 (s, 1H, OH), 4.52 (d, J = 5.7 Hz, 2H, benzyl-CH₂), 4.17 (td, J = 7.0, 2.8 Hz, 2H, CH₂CH₂O), 2.98 – 2.70 (m, 2H, NHC(O)CH₂), 2.37 (dt, J = 14.1, 6.7 Hz, 1H, CH₂CH₂O), 2.18 – 2.10 (m, 1H, CH₂CH₂O), 2.04 (s, 3H, CH₃CO), 1.97 (s, 3H, CH₃COOCH₂), 1.55 (s, 3H, CH₃); ¹³C NMR (126 MHz, CDCl₃) δ 171.03, 170.98, 168.76, 146.84 – 143.78 (m), 142.37 – 139.63 (m), 139.01 – 135.86 (m), 111.45 (t, J = 18.1 Hz), 81.24, 60.19, 45.17, 37.14, 31.26, 24.22, 22.14, 20.96; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₇H₁₈F₅NNaO₅: 434.1003, found: 434.1006.

3-(Fluoromethyl)-3,5-dihydroxypentyl acetate (37)

To a solution of 14 (500 mg, 1.39 mmol) in THF (10 mL) was added LiBH₄ (0.067 g, 3.08 mmol) at 0 °C, and the mixture was stirred at 0 °C for 30 min. The reaction was quenched with addition of aq. HCl (1.5 M). The solution was co-evaporated with toluene, the product was extracted from the dry slurry with THF, and the solvent was evaporated to give the crude diol as a colorless oil. To a solution of the crude product (250 mg, 1.64 mmol) in THF (5 mL) was added pyridine (0.132 mL, 1.64 mmol) at room temperature. To the mixture was added acetic anhydride (0.24 mL, 2.46 mmol), and the mixture was stirred for 8 h. The reaction was quenched with aq. HCl (1.5 M) and extracted with AcOEt. The organic layer was washed with aq. HCl (1.5 M), sat. NaHCO₃, and brine, and then dried (MgSO₄) and evaporated. The residue was purified by silica gel column chromatography (50% to 75% AcOEt in hexane) to give 37 (239 mg, 75%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 4.40 (m, 1H, CHH₂F), 4.29 (m, 3H, CHH₂F and CH₂OAc), 3.94 (m, 2H, CH₂OH), 2.06 (s, 3H, CH₃CO), 1.97 (m, 2H, CH₂CH₂OAc), 1.85 (m, 2H, CH₂CH₂OH); ¹³C NMR (125 MHz, CDCl₃) δ 171.0, 87.3, 85.9, 73.2(d), 60.3, 59.3, 36.8, 35.6, 21.1; LRMS (ESI) m/z = 195.10 [M+Na]⁺.

5-(Benzylamino)-3-(fluoromethyl)-3-hydroxy-5-oxopentyl acetate (38a)

To a solution of 6-fluoromevalonate (200 mg, 1.35 mmol) in DMF (5 mL) was added benzylamine (0.18 mL, 1.62 mmol) at room temperature, and the reaction was stirred at 60 °C for 24 h. The reaction was quenched by aq. HCl (1.5 M) and extracted with AcOEt. The organic layer was washed with aq.

HCl (1.5 M) and brine, then dried (MgSO₄) and evaporated. After the residue was dissolved in THF, pyridine (0.11 mL, 1.35 mmol) was added at room temperature. To the solution was added acetic anhydride (0.25 mL, 2.7 mmol), and the mixture was stirred at room temperature for 24 h. The reaction was quenched with aq. HCl (1.5 M) and extracted with AcOEt. The organic layer was washed with aq. HCl (1.5 M) and brine, then dried (MgSO₄) and evaporated. The residue was purified by silica gel column chromatography (15% to 25% AcOEt in hexane) to give 38a as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 7.24 (m, 5H), 6.03 (s, 1H), 4.98 (s, 1H), 4.43 – 4.35 (m, 2H), 4.25 (s, 1H), 4.24 – 4.17 (m, 2H), 4.16 (s, 1H), 2.41 (m, 2H), 1.96 (s, 3H), 1.87 – 1.84 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 171.5, 170.9, 137.4, 128.9, 127.8(δ), 87.1, 85.7, 71.3 (δ) 59.8, 43.6, 40.7, 35.8, 21.0; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₅H₂₀FNNaO₄: 320.1269, found: 320.1268.

Methyl 5-acetoxy-3-(fluoromethyl)-3-hydroxypentanoate (38b)

To a solution of 37 (300 mg, 1.54 mmol) in DMF (8 mL) was added pyridinium dichromate at room temperature, and the mixture was stirred at room temperature for 24 h. The reaction was quenched with aq. HCl (1.5 M) and extracted with AcOEt. The organic layer was washed with aq. HCl (1.5 M) and brine, then dried (MgSO₄) and evaporated. The crude pro duct was dissolved in DMF (5 mL), K₂CO₃ (638 mg, 4.62 mmol) was added to the mixture followed by the addition of methyl iodide (0.21 mL, 3.08 mmol), and the mixture was stirred for 24 h. The reaction was quenched with aq. HCl (1.5 M) and extracted with AcOEt. The organic layer was washed with aq. HCl (1.5 M) and brine, dried (MgSO₄), and evaporated. The residue was purified by silica gel column chromatography (15% to 25% AcOEt in hexane) to give 38b (105 mg, 31%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 4.41(dd, 1H, J = 9.91 Hz) 4.33 – 4.24 (m, 3H), 3.92 (s, 1H), 2.72 – 2.62 (m, 2H), 2.07 (s, 3H), 1.95 (m, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 172.7, 170.9, 87.3, 85.9, 71.2 (d), 59.7, 52.1, 39.5(d), 35.2, 21.0; HRMS (ESI) m/z calcd for [M+Na]⁺ C₉H₁₅FNaO₅: 245.0796, found: 245.0933.

Ethyl 5-acetoxy-3-(fluoromethyl)-3-hydroxypentanoate (38c)

38c (26 mg, 7%, colorless oil) was prepared from 36 as described for the preparation of 38b using ethyl iodide instead of methyl iodide. ¹H NMR (500 MHz, CDCl₃) δ 4.37 (dd, 1H), 4.30 – 4.23 (m, 3H), 4.19 (q, 2H, J = 7.19), 3.99 (s, 1H), 2.62 (dq, 2H), 2.04 (s, 3H), 1.93 (m, 2H), 1.28 (t, 3H, J = 7.19 Hz); ¹³C NMR (125 MHz, CDCl₃) δ 172.4, 170.9, 87.3, 85.9, 71.2(δ), 61.2, 59.8, 39.7(d), 35.2(d), 21.0, 14.1; HRMS (ESI) m/z calcd for [M+Na]⁺ C₁₀H₁₇FNaO₅: 259.0952, found: 259.1080.

Evaluation of stability of the ester promoiety in PBS buffer (pH 7.4)

Deuterated PBS buffer (pH 7.4) was prepared by dissolving NaCl (80 mg, 137 mM), KCl (2.0 mg, 2.7 mM), Na₂HPO₄ (14.4 mg, 10.1 mM), and KH₂PO₄ (2.4 mg, 1.8 mM) in 10 mL of deuterium oxide. Compound 38c (1.0 mg, 4 μmol) was dissolved in deuterated PBS buffer (pH 7.4, 800 μL), placed in an NMR tube, and incubated at 37 °C. ¹H NMR spectra were taken at 30 min, 1 h, 4 h, 24 h, and 48 h to determine its stability in PBS buffer. The decomposition was less than 5% after 48 h on the basis of the integration ratio of impurities and 38c.

Evaluation of stability of all other promoieties tested in PBS buffer (pH 7.4)

A stock solution of the test compound (10 mM in acetonitrile, 50 μL) was added to PBS buffer (pH 7.4, 450 μL), and the mixture was incubated at 37 °C. The final incubation volume was 0.5 mL, Aliquots (10 μL) of the samples were taken from the incubation solution at various times and were immediately injected into the HPLC. These tests were conducted twice for each compound. The t_1/2 (the time for disappearance of half of the starting material) values were calculated using Prism Version 5 a one-phase decay model with the equation t_1/2= ln(2)/b. Here b is the slope in the linear fit of the natural logarithm (ln) of the remaining fraction of the parent molecule in HPLC verses incubation time.

Evaluation of stability in human plasma

The stability of compounds in human plasma was performed after slight modification of a referenced protocol²⁴; human blood plasma (Aldrich) was diluted with distilled water until the indicated volume, and then the solution was pre-incubated for 5 min at 37 °C. The stock solution of the test compound (100 mM in acetonitrile, 20 μL) was added to the human plasma (480 μL), and the mixture was incubated at 37 °C. The incubations were terminated at 1, (5), (10), 15, 30, and 60 min, by removing aliquots (80 μL) of the plasma samples and mixing them with an equal volume of acetonitrile. For stable compounds, 48 h of incubation and analysis was also performed. The mixture was stirred vigorously and centrifuged (5500 rpm, 5 min). The supernatant was filtered, and the filtrate was analyzed by HPLC. These tests were conducted two times for each compound. HPLC analysis was performed on a Phenomenex^® Luna C18 column (250 × 4.6 mm) eluting with a gradient of acetonitrile and H₂O (90% to 10% water over 30 min) Detection was by UV absorbance at 254 nm or 220 nm. The flow rate was 1.0 mL/min. Data analysis was similar to that of PBS buffer stability, performed using Prism Version 5, a non-linear fit one-phase decay model.

Minimal inhibitory concentration (MIC) assay

The MICs of the prodrugs required to inhibit growth of Streptococcus pneumoniae strain TIGR4 was determined using a previously described method with minor modifications.²⁷ Overnight S. pneumoniae culture was diluted 1:10 into fresh THB and grown to logarithmic phase (OD600 of 0.4). The bacterial culture was washed and diluted in PBS. Bacteria (5 μL) was added to individual wells of a 96-well plate containing 195 μL of THB and the prodrugs (12.5 to 1600 μM) were serially diluted. The final concentration of bacteria was 1 × 105 CFU/well. The plate was incubated for 24 h at 37 °C, and the absorbance of the samples at 600 nm was read with a spectrophotometric plate reader. The MIC was defined as the lowest concentration of the drugs that inhibited bacterial growth.

Highlights.

• Prodrug analogues of mevalonate, 6-fluoromevalonate, and 6,6,6-trifluoromevalonate synthesized and characterized

• Human blood plasma stabilities of ester, amide, carbonate, acetal, and ketal promoieties of these analogues determined

• A wide range of half-lives were obtained

• MIC values determined to evaluate antibacterial activity of select prodrugs

• Two of the prodrug analogues exhibited weak antibacterial activity

ABBREVIATIONS

DMP

diphosphomevalonate

MK

mevalonate kinase

PMK

phosphomevalonate kinase

DPM

6-fluoro- and 6,6,6-trifluoro-5-diphosphomevalonate

DPM-DC

diphosphomevalonate decarboxylase

IPP

Isopentenyl diphosphate

EDCI

N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride

HBTU

N,N,N,N-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate

MIC

Minimal inhibitory concentration (MIC)

THB

tissue homogenization buffer