Synthesis of mevalonate- and fluorinated mevalonate prodrugs and their in vitro human plasma stability

Article abstract of DOI:10.1016/j.ejmech.2014.11.040

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 analogs are not bioavailable. To increase cellular permeability of mevalonate analogs, 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 analogs 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 analogs 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.

Full text of DOI:10.1016/j.ejmech.2014.11.040

Accepted Manuscript
Synthesis of Mevalonate- and Fluorinated Mevalonate Prodrugs and Their in vitro
Human Plasma Stability
Soosung Kang , Mizuki Watanabe , J.C. Jacobs , Masaya Yamaguchi , Samira
Dahesh , Victor Nizet , Thomas S. Leyh , Richard B. Silverman
PII:
S0223-5234(14)01072-1
10.1016/j.ejmech.2014.11.040
EJMECH 7533
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 July 2014
Revised Date: 19 November 2014
Accepted Date: 21 November 2014
Please cite this article as: S. Kang, M. Watanabe, J. Jacobs, M. Yamaguchi, S. Dahesh, V. Nizet,
T.S. Leyh, R.B. Silverman, Synthesis of Mevalonate- and Fluorinated Mevalonate Prodrugs and
Their in vitro Human Plasma Stability, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.11.040.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Synthesis of Mevalonate- and Fluorinated Mevalonate Prodrugs
and Their in vitro Human Plasma Stability
1
1
1
2
2
Soosung Kang, Mizuki Watanabe, JC Jacobs, Masaya Yamaguchi, Samira Dahesh, Victor
2
3
1,
Nizet, Thomas S. Leyh, Richard B. Silverman *
1
Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes
Institute, and Center for Molecular Innovation and Drug Discovery, Northwestern University,
Evanston, Illinois, United States
2
Department of Pediatrics and Skaggs School of Pharmacy and Pharmaceutical Sciences,
University of California, San Diego, La Jolla, California
3
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva
University, Bronx, New York, United States
KEYWORDS: mevalonate, 6-fluoromevalonate, 6,6,6-trifluoromevalonate, prodrugs, plasma
stability
*
Correspondence Author: Richard B. Silverman, Department of Chemistry, Northwestern
University, Phone: 847-491-5653; Email: Agman@chem.northwestern.edu
1

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Graphical Abstract
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
2

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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.
3

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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
1
molecules, including cholesterol, steroid hormones, and vitamin A. Therefore, it is not
surprising that enzymes in the mevalonate pathway are targets for a variety of drug discovery
2
programs. The statin cholesterol-lowering drugs target 3-hydroxy-3-methylglutaryl CoA
3
reductase, the enzyme that produces mevalonate; the osteoporosis drug alendronate inhibits the
4
synthesis of farnesyl diphosphate from IPP; enzymes in the mevalonate pathway are also
5
6
targeted for cancer and parasites. Leyh and co-workers discovered that the mevalonate pathway
7
in S. pneumoniae is regulated by 5-diphosphomevalonate (DPM). They showed that DPM is a
8
feedback inhibitor of mevalonate kinase (MK), and binds tightly to an allosteric site of the
pneumococcal MK. However, human MK is not inhibited by DPM at concentrations that
9
effectively inhibit the S. pneumoniae system. Therefore, DPM can be a lead compound for the
development of new anti-pneumococcal antibiotics that do not perturb human metabolism.
4

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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.
1
0
In early studies of DPM analogues, it was found that 6,6,6-trifluoromevalonate was
converted into the corresponding diphosphate enzymatically, which inhibited DPM
1
1
decarboxylase (DPM-DC) and led to the accumulation of DPM in rat liver homogenates.
Moreover, 6-fluoromevalonate causes the accumulation of phosphorylated mevalonates and
1
2
completely blocks the related bioactivity of mevalonate at 200 µM concentration. 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
1
3
drugs, such as bisphosphonates, which block farnesyltransferase. 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
1
4
charged bacterial cell membrane. 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
1
5
moiety that are transformed into the active form in vivo, 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
5

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absorption and bacterial cell permeation, the two hydroxyl groups of the mevalonate were
1
6
17
18
converted to carbonate, acetal, and ketal prodrugs. These esters, lactones, amides,
1
9
20,21
22
carbonates, acetals,
and ketals, 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
2
3
fluorinated mevalonates can be enzymatically converted to the phosphorylated forms. 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
6

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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 ºC,
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.
a
Scheme 1. Synthesis of compound 4
a
Reagents 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
7

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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.
a
Scheme 2. Synthesis of 9a-b and 10a-c
a
Reagents and Conditions: (i) Pd/C, H , quantitative; (ii) EDCI, DMAP, CH Cl , ROH R = Ph
2
2
2
(
9a, 54%), 4-F-Ph (9b, 65%); (iii) R-NH , HBTU, DIEA, DMF, R = Ph (10a, 55%), Bn (10b,
2
6
1%), 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 ºC 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
H O gave dicarboxylic acid 13. The benzylation of crude product 13 was conducted to give
2
2
diester 14 in more than 50% yield for four steps. Partial reduction of 14 with DIBAL-H (3-4
equiv) at 0 ºC 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
8

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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.
a
Scheme 3 Synthesis of Compounds 18a-b
a
Reagents and Conditions: (i) allylmagnesium bromide (1.95 equiv), Et O, 0 °C, < 30 min. (ii)
2
(
1) ozone, CH Cl , -78 °C (2) aq. H O , AcOH, H SO , reflux, (iii) BnBr, K CO , DMF, 54% for
2 2 2 2 2 4 2 3
4
steps (69% for 2 steps), (iv) DIBAL-H, THF, 0 °C, (v) (Cl CO) CO, pyridine, CH Cl , 0 °C
3 2 2 2
(
37% for 2 steps), (vi) H , Pd/C, AcOEt (99%), (vii) NaHCO , DMF, R-X; R= 4-F-BnBr (18a,
2
3
7
7%), 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
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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.
a
Scheme 4. Synthesis of 25a-b
a
Reagents and Conditions: (i) benzyl acetate, LDA, THF, -78 °C (88%), (ii) aq. HCl/acetone,
0
°C (80%), (iii) NaBH(OAc) , benzene, (iv) triphosgene, pyridine, CH Cl , 0 °C (68% for 2
3
2
2
steps), (v) H , Pd/C, AcOEt (98%), (vi) NaHCO , DMF, R-X = 4-F-BnBr (25a, 88%), 2,4-diF-
2
3
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 ºC,
the degradation of the compounds over time was monitored. The half-life (t ) of 4, which is a
1
/2
mevalonate prodrug, was more than 48 h (Table 1). The t1/2 of the 4-monofluoromethyl
analogues (16, 18a-b) were around 25 h, and the t1/2 of the 4-trifluoromethyl analogues (23, 25a-
10

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b) were around 5 h. As the 4-substituent became increasingly more electron deficient, the t1/2
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 R - substituent was constant (CH , CH F, or CF ).
2
3
2
3
Table 1. Half-lives of the diverse carbonates in PBS buffer at 37 ºC.
#
R1
R2
t1/2 (h)
>48
4
Bn
CH3
1
6
Bn
4-F-Bn
CH2F
CH2F
CH2F
CF3
25
26
22
5
1
8a
1
8b
2,4-di-F-Bn
Bn
2
3
2
5a
5b
4-F-Bn
CF3
6
2
2,4-di-F-Bn
CF3
5
1
HPLC and H 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,
1
which results from decomposition of the carbonate moiety, was not observed by HPLC.
H
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
1
prospective intermediates were carried out. By monitoring the H NMR spectrum of synthesized
2
4 in PBS buffer it was found that the t1/2 of 24 was about 3 h, shorter than the t1/2 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 t1/2 for 22
11

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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 (t1/2 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
,
24
were evaluated 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
2
5
haloperidol. The mixture of test compounds and human plasma was incubated at 37 ºC, and the
degradation of the compounds was monitored by HPLC. Slight differences in the t1/2 were
observed for the various ester groups, but the t1/2 for 4 was 4 min and the t1/2 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 t1/2 of these benzyl and
phenyl ester analogues was very short. The t1/2 for N-phenyl amide 10a was also less than 3 min.
12

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Interestingly, the t1/2 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 ºC.
#
R1
BnO
R2
t1/2 (min)
4
CH3
CH3
CH3
CH3
4
9
a
PhO
< 3
< 3
< 3
9
b
4-F-PhO
PhNH
1
0a
0b
a
1
BnNH
4-F-BnNH
BnO
CH3
CH3
CH2F
CH2F
CH2F
CF3
8
a
1
0c
8
1
6
2
2
1
8a
8b
4-F-BnO
2,4-di-F-BnO
BnO
1
1
2
3
< 1
< 1
<1
2
5a
5b
4-F-BnO
2,4-di-F-BnO
CF3
2
CF3
a
Hydrolysis 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
2
6
stereochemistry on the hydrolysis rate. At t and t , a portion of the incubation mixtures of 4
1
/4
1/2
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
13

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4
1/59 at 4 min. In the case of compound 10b, the enantiomeric ratio was 48/52 at 4 min and
4
6/54 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 BF •Et O, gave methylene acetals 30a-b. Reaction
3
2
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
CH Cl gave diastereomeric mixtures 32a-b. Diol 29b was converted to the p-
2
2
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 Boc O in anhydrous acetonitrile.
2
a
Scheme 5. Synthesis of Compounds 30-33
14

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a
Reactions and Conditions: (i) BnNH , DMF, 80 °C (80-88%), (ii) (a) MOMCl, CH Cl , 0 °C to
2
2
2
room temperature, (b) BF OEt , CH Cl , 0 °C to room temperature, 77% yield for 30a, 69%
3
2
2
2
yield for 30b, (iii) 2,2-dimethoxypropane, CSA, CH Cl , 27% yield, (iv) benzaldehyde
2
2
dimethylacetal, CSA, CH Cl , 34% yield for 32a, 42% yield for 32b, (v) anisaldehyde dimethyl
2
2
acetal, CSA, CH Cl , reflux, 60% yield for 32c; 2,4-dimethoxybenzaldehyde, CSA, MS 4Å,
2
2
benzene, reflux, 6% yield for 32d, (vi) Boc O, DMAP, acetonitrile, reflux, 88% yield for 33a, 68%
2
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 t1/2 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 ºC
15

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#
R1
CH3
CH3
CH3
CF3
CF3
CF3
CF3
CH3
CF3
R2
R3
H
t
1/2 (h)
3
0a
H
> 48
> 48
> 48
> 48
> 48
> 48
> 48
> 48
> 48
3
1
CH3
CH3
H
3
2a
Ph
3
3
0b
2b
H
Ph
H
H
3
2c
4-MeO-Ph
H
32d
33a
33b
2,4-(MeO) -Ph
H
2
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,
3
6). 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 LiBH , and
4
addition of one equivalent of acetic anhydride in pyridine yielded mono-acetylated intermediate
3
7. 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).
a
Scheme 6
16

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a
Reagents and Conditions: (i) 2,3,4,5,6-pentafluorobenzylamine, DMF, 80 °C, 53% yield, (ii)
Ac O, pyridine, 41% yield for 35, 20% yield for 36
2
a
Scheme 7
a
Reagents and Conditions: (i) LiBH (3 eq.), THF, 0 °C (70%) (ii) Ac O, DMAP, pyridine (45%),
4
2
(
iii) PDC, DMF, 24 h, room temperature (iv) RNH ,EDCI, K CO , or R-X, K CO , DMF, two
2 2 3 2 3
steps 11~25%.
Stability of the ester promoiety in PBS buffer. The stability of ethyl ester analogue 38c in
1
deuterated PBS buffer (pH 7.4) at 37 °C was monitored by H NMR spectroscopy as a control to
determine the decomposition of the ester prodrug by the medium. The half-life (t ) of 38c in
1
/2
PBS was > 48 h.
17

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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 ºC
t1/2
#
R1
R2
R3
R4
(
min)
3
4
5
6
C F CH NH
CH3
CH3
H
H
H
> 120
6
5
2
a
3
3
C F CH NH
Ac
Ac
Ac
Ac
Ac
20
6
5
2
C F CH NH
CH3
Ac
H
12
6
5
2
a
3
8a
8b
BnNH
MeO
EtO
CH2F
CH2F
CH2F
30
3
H
10
11
3
8c
H
a
Hydrolysis 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
2
7
against S. pneumoniae (Table 5) in THB medium using a well-established technique, 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
18

ACCEPTED MANUSCRIPT
inactive (16, 18a). Moderately stable (10-20 min) ester prodrugs (38a-c) and a quite stable acetal
2
8
prodrug (32b) were found to lack activity. Lactones (26 and 28 ), 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 =
2
00 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
a
MIC
name
(mM)
19

ACCEPTED MANUSCRIPT
1
6
>1.6
>1.6
1.6
18a
5a
2
26
0.8
28
0.2
3
3
3
2b
8a
8b
>1.6
>1.6
>1.6
>1.6
3
8c
Vancomycin
a
Minimum inhibitory concentration (MIC): lowest drug concentration that reduced growth by
0% or more.
8
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 of 10-20 min in human plasma. Although the cyclic carbonate
1
/2
decomposes relatively rapidly (4-8 min), and cyclic ketal, acetal, and amide moieties decompose
relatively slowly in human plasma, this study shows that the t1/2 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.
20

ACCEPTED MANUSCRIPT
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
1
13
positive pressure of nitrogen. H NMR and C 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
1
3
uncorrected. C 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 CDCl or CD OD,
3
3
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 ꢀm 200
Å, 4.6 × 250 mm.
Synthesis and characterization of compounds
21

ACCEPTED MANUSCRIPT
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 H O (4 mL). The solution was stirred at
2
4
0 ºC 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 ºC for 4 h. After the mixture was diluted with AcOEt
and brine, the organic layer was partitioned, dried (Na SO ), and evaporated. The residue was
2
4
purified by silica gel column chromatography (50% AcOEt in hexane) to give 3 (329 mg, 69%)
1
as a colorless oil. H NMR (500 MHz, CDCl ) δ 7.33-7.38 (m, 5H, aromatic), 5.17 (s, 2H,
3
benzyl-CH ), 4.04 (br s, 1H, OH), 3.88 (m, 1H, CH CH O), 3.81 (m, 1H, CH CH O), 2.90 (br s,
2
2
2
2
2
1
1
1
H, OH), 2.69 (d, 1H, OC(O)CH , J = 15.5 Hz), 2.52 (d, 1H, OC(O)CH , J = 15.5 Hz), 1.80 (m,
2 2
1
3
H, CH CH O), 1.74 (m, 1H, CH CH O), 1.32 (s, 3H, CH ); C NMR (125 MHz, CDCl ) δ
2
2
2
2
3
3
72.7, 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] C H NaO : 261.1103, found: 261.1105.
1
3
18
4
Benzyl 2-(4-methyl-2-oxo-1,3-dioxan-4-yl)acetate (4) To a solution of 3 (436 mg, 1.83 mmol)
in CH Cl (15 mL) was added pyridine (224 µL, 2.75 mmol), and the mixture was stirred at 0 ºC
2
2
for 15 min. A solution of triphosgene (98%, 665 mg, 2.20 mmol) in CH Cl (5 mL) was added to
2
2
the mixture, and the resulting mixture was stirred at 0 ºC for 30 min. The reaction was quenched
with saturated aqueous NH Cl and extracted with AcOEt. The organic layer was washed with
4
brine, dried (Na SO ), and evaporated. The residue was purified by silica gel column
2
4
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
22

ACCEPTED MANUSCRIPT
solution in THF, 2.20 mL, 2.2 mmol) at 0 ºC, and the mixture was stirred at 0 ºC for 24 h. The
mixture was diluted with AcOEt and washed with aqueous NaHCO . The organic layer was
3
washed with brine, dried with Na SO , and evaporated. The residue was purified quickly by
2
4
silica gel column chromatography (50% to 80% AcOEt in hexane) to give compound 6. After the
residue was dissolved in CH Cl (8 mL), pyridine (163 µL, 2.0 mmol) was added to the solution,
2
2
and the mixture was cooled at 0 ºC. To the mixture was added a solution of triphosgene (98%,
06 mg, 1.0 mmol) in CH Cl (1 mL), and the resulting mixture was stirred at 0 ºC for 30 min.
3
2
2
The reaction was quenched with addition of saturated aqueous NH Cl and extracted with AcOEt.
4
The organic layer was washed with brine, dried with Na SO , and evaporated. The residue was
2
4
purified by silica gel column chromatography (33% to 50% AcOEt in hexane) to give 4 (155 mg,
1
6
4% for 2 steps) as a light yellow oil, and 1 was recovered (39 mg, 12%). H NMR (500 MHz,
CDCl ) δ 7.34-7.40 (m, 5H, aromatic), 5.14 (s, 2H, benzyl-CH ), 4.42 (m, 2H, CH CH O), 2.83
3
2
2
2
13
(
s, 2H, OC(O)CH ), 2.36 (m, 1H, CH CH O), 2.08 (m, 1H, CH CH O), 1.57 (s, 3H, CH ); C
2 2 2 2 2 3
NMR (125 MHz, CDCl ) δ 168.7, 148.5, 135.1, 128.6, 128.5, 128.4, 81.0, 66.9, 64.5, 44.8, 30.4,
3
+
2
5.8; HRMS (pos. ion ESI) m/z calcd for [M+Na] C H NaO : 287.0890, found: 287.0899.
14
16
5
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 ºC, and the mixture was stirred at room
temperature for 12 h. After the addition of MeOH, the mixture was diluted with Et O and washed
2
with H O (x 3). The organic layer was washed with brine, dried with Na SO , and evaporated.
2
2
4
The residue was purified by silica gel column chromatography (5% AcOEt in hexane) to give 6
1
(
548 mg, 90%) as a colorless liquid. H NMR (500 MHz, CDCl ) δ 3.88 (t, 2H, CH CH O, J =
3
2
2
6
.3 Hz), 2.62 (t, 2H, CH CH O, J = 6.3 Hz), 2.19 (s, 3H, CH ), 0.88 (s, 9H, C(CH ) ), 0.05 (s,
2 2 3 3 3
23

ACCEPTED MANUSCRIPT
13
6
H, Si(CH ) ); C NMR (125 MHz, CDCl ) δ 208.2, 58.8, 46.5, 30.9, 25.8, 18.2, –5.5; HRMS
3 2 3
+
(
ESI) m/z calcd for [M+Na] C H NaO Si: 225.1287, found: 225.1280.
10 22 2
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 ºC, and the mixture
was stirred at –78 ºC 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 ºC, and the resulting mixture was stirred at –78 ºC for 1 h. After
addition of sat. aq. NH Cl, the mixture was warmed to room temperature and evaporated. The
4
residue was partitioned between AcOEt and sat. aq. NH Cl. The organic layer was washed with
4
brine, dried (Na SO ), and evaporated. The residue was purified by silica gel column
2
4
1
chromatography (8% AcOEt in hexane) to give 7 (3.92 mg, 93%) as a colorless oil. H NMR
(
500 MHz, CDCl ) δ 7.31-7.35 (m, 5H, aromatic), 5.17 (d, 1H, benzyl-CH , J = 7.5 Hz), 5.12 (d,
3
2
1
H, benzyl-CH , J = 7.5 Hz), 4.20 (s, 1H, OH), 3.86 (t, 2H, CH CH O, J = 3.6 Hz), 2.66 (d, 1H,
2 2 2
OC(O)CH , J = 15.6 Hz), 2.60 (d, 1H, OC(O)CH , J = 15.6 Hz), 1.82 (m, 2H, CH CH O), 1.30
2
2
2
2
+
(
s, 3H, CH ), 0.89 (s, 9H, C(CH ) ), 0.07 (s, 6H, Si(CH ) ); HRMS (ESI) m/z calcd for [M+Na]
3 3 3 3 2
C H NaO Si: 375.1962, found: 375.1965.
19
32
4
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 H gas at room
2
temperature for 30 min. The resulting mixture was filtered through Celite with acetone, and the
1
filtrate was evaporated to give 8 (224 mg, quant.) as a colorless solid; mp. 83-85 ºC; H NMR
(
500 MHz, CD OD) δ 4.48 (m, 2H, CH CH O), 2.82 (d, 1H, OC(O)CH , J = 15.5 Hz), 2.78 (d,
3 2 2 2
1
H, OC(O)CH , J = 15.5 Hz), 2.48 (m, 1H, CH CH O), 2.12 (m, 1H, CH CH O), 1.57 (s, 3H,
2 2 2 2 2
24

ACCEPTED MANUSCRIPT
1
3
CH ); C NMR (125 MHz, CD OD) δ 172.5, 151.9, 83.3, 66.2, 45.2, 31.3, 26.1; HRMS (ESI)
3
3
+
m/z calcd for [M+Na] C H NaO : 197.0420, found: 197.0426.
7
10
5
Phenyl 2-(4-methyl-2-oxo-1,3-dioxan-4-yl)acetate (9a). To a solution of 8 (35 mg, 0.20
mmol) in CH CN/CH Cl (1/1, 1.5 mL) was added phenol (85 mg, 0.90 mmol), N-(3-
3
2
2
dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (38 mg, 0.20 mmol), and DMAP (20
mg, 0.16 mmol) at 0 ºC, 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. NaHCO , brine, dried with Na SO , and evaporated. The residue was
3
2
4
purified by silica gel column chromatography (33% to 50% AcOEt in hexane) to give 9a (27 mg,
1
5
7
4%) as a white solid; mp. 61-63 ºC; H NMR (500 MHz, CDCl ) δ 7.40 (t, 2H, aromatic, J =
3
.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,
CH CH O, J = 5.1 Hz), 3.05 (s, 2H, OC(O)CH ), 2.47 (m, 1H, CH CH O), 2.19 (m, 1H,
2
2
2
2
2
1
3
CH CH O), 1.68 (s, 3H, CH ); C NMR (125 MHz, CDCl ) δ 167.6, 150.0, 148.4, 129.6, 126.3,
2
2
3
3
+
1
21.3, 81.0, 64.5, 44.9, 30.6, 25.9; HRMS (ESI) m/z calcd for [M+Na] C H NaO : 273.0733,
13 14 5
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-
1
fluorophenol instead of phenol; mp. 68-69 ºC; H NMR (500 MHz, CDCl ) δ 7.06-7.08 (m, 4H,
3
aromatic), 4.48 (m, 2H, CH CH O), 3.03 (s, 2H, OC(O)CH ), 2.48 (m, 1H, CH CH O), 2.17 (m,
2
2
2
2
2
1
3
1
1
H, CH CH O), 1.67 (s, 3H, CH ); C NMR (125 MHz, CDCl ) δ 167.6, 160.4(d), 148.4, 145.8,
2 2 3 3
+
22.8(d), 116.3(d), 80.9, 64.5, 44.9, 30.6, 25.9; HRMS (ESI) m/z calcd for [M+Na]
C H FNaO : 291.0639, found: 291.0648.
13
13
5
25

ACCEPTED MANUSCRIPT
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 ºC, 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. NaHCO , brine, dried with Na SO , and
3
2
4
evaporated. The residue was purified by silica gel column chromatography (50% AcOEt in
1
hexane) to give 10a (23 mg, 55%) as a colorless oil; H NMR (500 MHz, CDCl ) δ 8.03 (br s,
3
1
H, 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, CH CH O), 2.85 (s, 2H, OC(O)CH ), 2.52 (m, 1H,
2
2
2
13
CH CH O), 2.20 (m, 1H, CH CH O), 1.64 (s, 3H, CH ); C NMR (125 MHz, CDCl ) δ 166.1,
2
2
2
2
3
3
1
48.9, 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] C H NNaO : 272.0893, found: 272.0894.
1
3
15
4
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
1
benzylamine instead of aniline. H NMR (500 MHz, CDCl ) δ 7.26-7.35 (m, 5H, aromatic), 6.47
3
(
br s, 1H, NH), 4.38-4.46 (m, 4H, CH CH O and benzyl-CH ), 2.68 (d, 1H, OC(O)CH , J = 14.5
2 2 2 2
Hz), 2.63 (d, 1H, OC(O)CH , J = 14.5 Hz), 2.46 (m, 1H, CH CH O), 2.09 (m, 1H, CH CH O),
2
2
2
2
2
1
3
1
.55 (s, 3H, CH ); C NMR (126 MHz, CDCl ) δ 167.7, 148.7, 137.8, 128.8, 127.8, 127.7, 82.0,
3 3
+
6
4.9, 47.5, 43.8, 30.7, 25.7; HRMS (ESI) m/z calcd for [M+Na] C H NNaO : 286.1055,
14 17 4
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
26

ACCEPTED MANUSCRIPT
1
using 4-fluorobenzylamine instead of aniline. H NMR (500 MHz, CDCl ) δ 7.24-7.27 (m, 2H,
3
aromatic), 6.99-7.02 (m, 2H, aromatic), 6.70 (br s, 1H, NH), 4.37-4.44 (m, 4H, CH CH O and
2
2
benzyl-CH ), 2.66 (s, 2H, OC(O)CH ), 2.45 (m, 1H, CH CH O), 2.10 (m, 1H, CH CH O), 1.54
2
2
2
2
2
2
1
3
(
s, 3H, CH ); C NMR (126 MHz, CDCl ) δ 170.8, 162.2(d), 147.8, 134.0(d), 127.46 , 115.6(d),
3
3
+
8
3
1.8, 64.2, 46.5, 43.1, 29.2, 25.0; HRMS (ESI) m/z calcd for [M+Na] C H FNNaO :
14 16 4
04.0956, found: 304.0960.
Dibenzyl 3-(fluoromethyl)-3-hydroxypentanedioate (14). To a solution of ethyl
fluoroacetate (1.94 mL, 20.0 mmol) in Et O (120 mL) was added allylmagnesium bromide (1.0
2
M solution in Et O, 39.0 mL, 39.0 mmol) at 0 ºC, and the mixture was stirred at 0 ºC for 20 min.
2
After addition of saturated aqueous NH Cl, the organic layer was separated, washed with brine,
4
dried with Na SO , and evaporated. The residue (12) was used in the next reaction without
2
4
further purification. The crude product 12 (2.60 g) was dissolved in CH Cl (40 mL) and cooled
2
2
to –78 ºC. Ozone was bubbled into the solution at –78 ºC 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), H O (15 mL), conc .H SO (0.40 mL),
2
2
4
and aq. H O (30%, 9.0 mL), and the mixture was stirred under reflux for 4 h. After cooling to
2
2
room temperature, the mixture was neutralized with BaCO (1.5 g) and filtered through Celite
3
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 H O . The mixture was filtered through Celite with acetone
2
2
to remove the Pd/C, and the filtrate was evaporated. The residue was co-evaporated with H O (x
2
2
) and toluene (x 3) to give crude dicarboxylic acid 13 as a brown oil. To a solution of crude
27

ACCEPTED MANUSCRIPT
product 13 in DMF (80 mL) was added benzyl bromide (98%, 4.85 mL, 40 mmol) and K CO
2
3
(5.53 g, 40 mmol), and the mixture was stirred at room temperature for 8 h. After filtration
through Celite to remove K CO , the solvent was evaporated. The residue was partitioned
2
3
between AcOEt and aq. HCl (0.5 M). The organic layer was washed with sat. aq. NaHCO and
3
brine, dried (Na SO ), and evaporated. The residue was purified by silica gel column
2
4
chromatography (10% to 20% AcOEt in hexane) to give diester 14 (3.87 g, 54% for 4 steps) as a
1
light yellow oil. H NMR (500 MHz, CDCl ) δ 7.32-7.38 (m, 10H, aromatic), 5.14 (s, 4H,
3
benzyl-CH x2), 4.42 (d, 2H, CH F, J = 47 Hz), 4.25 (s, 1H, OH), 2.78 (m, 4H, C(O)CH x2);
2
2
2
1
3
C NMR (125 MHz, CDCl ) δ 171.0, 135.3, 128.6, 128.5, 128.3, 86.4(d), 71.1, 66.8, 39.9;
3
+
HRMS (ESI) m/z calcd for [M+Na] C H FNaO : 383.1265, found: 383.1276.
2
0
21
5
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 ºC, and the mixture was stirred at 0 ºC for 10 min. DIBAL-H (1.0 M solution in THF,
.86 mL, 1.86 mmol) was added to the mixture at 0 ºC, and the resulting mixture was stirred at 0
1
ºC 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. NaHCO , and brine, and then dried (Na SO ) and
3
2
4
evaporated. After the residue (15) was dissolved in CH Cl (60 mL), pyridine (285 µL, 3.50
2
2
mmol) was added to the solution and the mixture was cooled at 0 ºC. To the mixture was added a
solution of triphosgene (706 mg, 2.33 mmol) in CH Cl (4 mL), and the resulting mixture was
2
2
stirred at 0 ºC for 30 min. The reaction was quenched with addition of sat. aq. NH Cl and
4
extracted with AcOEt. The organic layer was washed with brine, dried with Na SO , and
2
4
evaporated. The residue was purified by silica gel column chromatography (33% to 50% AcOEt
28