Scholarly article on ertapenem bis(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl ester 1446142-21-7 from Journal of Medicinal Chemistry p. 8421

Article

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Design, Synthesis, Structure−Function Relationship, Bioconversion,

and Pharmacokinetic Evaluation of Ertapenem Prodrugs

Sheo B. Singh,*^,†,#Diane Rindgen,^†Prudence Bradley,^†Takao Suzuki,^‡Nengxue Wang,^‡Hao Wu,^‡

Basheng Zhang,^‡Li Wang,^‡Chongmin Ji,^‡Hongshi Yu,^‡Richard M. Soll,^‡David B. Olsen,^§

Peter T. Meinke,^†and Deborah A. Nicoll-Griﬃth^†

^†Merck Research Laboratories, Rahway, New Jersey 07065, United States

^‡WuXi AppTec, 288 Fute Zhong Road, Shanghai 200131, People’s Republic of China

^§Merck Research Laboratories, West Point, Pennsylvania 19486, United States

S

* Supporting Information

ABSTRACT: Described here are synthesis and biological evaluations of diversiﬁed groups of over 57 ertapenem prodrugs which

include alkyl, methylenedioxy, carbonate, cyclic carbonate, carbamate esters, and esters containing active transport groups (e.g.,

carboxyl, amino acid, fatty acids, cholesterol) and macrocyclic lactones linking the two carboxyl groups. Many of the prodrugs

were rapidly hydrolyzed in rat plasma but not in human plasma and were stable in simulated gastrointestinal ﬂuid. The diethyl

ester prodrug showed the best total absorption (>30%) by intredeudenal dosing in dogs, which could potentially be improved by

formulation development. However, its slow rate of the hydrolysis to ertapenem also led to the presence of large amounts of

circulating monoester metabolites, which pose signiﬁcant development challenges. This study also suggests that the size of

susbtituents at C-2 of carbapenem (e.g., benzoic acid of ertapenem) has signiﬁcant impact on the absorption and the hydrolysis

of the prodrugs.

antibiotic for treatment of serious bacterial infections in

arbapenems are a class of β-lactam antibiotics useful for

C_{treatment of serious and life-threatening Gram-positive}hospital setting. In vivo, imipenem is hydrolyzed by renal

and Gram-negative infections.¹Thienamycin (1) was the ﬁrst

dehydropeptidase (DHP). Cilastatin is an inhibitor of DHP

that stabilizes imipenem in vivo by preventing its hydrolysis.

Subsequently, it was discovered that introduction of a β-methyl

group at C-1 of carbapenem renders them resistant to DHP

while retaining most of the antibiotic activity and spectrum.

Because of these useful properties, all subsequent carbapenem

antibiotics, excluding panipenem (3)/betamipron, that were

developed contain a β-methyl group at C-1. There have been

six carbapenem antibiotics approved after imipenem (Chart 1).

Panipenem/betamipron was approved in Japan in 1993, and

that was followed by the U.S. FDA approval of ertapenem (4,

2001) and by Japanese approval of biapenem (5, 2001).

Meropenem (6) and doripenem (7) were approved by the U.S.

FDA in 2005 and 2007, respectively. Orapenem (8), a prodrug

member of the carbapenem class of antibiotics discovered from

Streptomyces cattleya in 1976. At the time, thienamycin

discovery and development presented unique challenges and

opportunities. It was unstable, particularly at higher solution

concentrations in the fermentation broth, making scale-up by

fermentation inherently challenging. Perhaps the most critical

factor impacting thienamycin’s stability was the intramolecular

ring opening of the β-lactam by its pendent primary amine. As

direct sequelae to these stability deﬁcits, total synthesis was the

only viable option to enable discovery and development of a

stable derivative with commercial viability. After tremendous

eﬀorts, Merck scientists synthesized imipenem by total

synthesis during which they masked the primary amine as an

amidine; this compound retained useful antibacterial activity

and spectrum while conferring adequate stability. In 1985, an

imipenem (2) and cilastatin combination was approved as an

Received: June 10, 2014

Published: September 29, 2014

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Chart 1. Structures of Carbapenem Antibiotics

of tebipenem (9), was the last carbapenem antibiotic that was

approved by Japanese regulators in 2009.

2 with either a net positive charge or net zwitterionic, with the

exception of ertapenem, which consists of a prolyl-3-amino-

benzoic acid. Clearly, the net acidic charge of ertapenem

contributes to higher protein binding (92−94%), longer plasma

half-life, and longer duration of action. Therefore, ertapenem

was selected for prodrug studies with the expectation that once-

daily dosing of the prodrug will be maintained. It is envisaged

that the prodrug will rapidly hydrolyze in plasma to produce

ertapenem with minimal or no circulating metabolites, leading

to clear and abbreviated path for development and registration.

In order to accomplish this goal, a careful research operating

plan was designed and executed in which prodrugs were

evaluated for plasma hydrolysis, stability in simulated gastric

and intestinal ﬂuids, and ﬁt for purpose intraduodenal (ID)

pharmacokinetic rat and dog models. In addition, carbapenem

chemistry is synthetically demanding not only because of their

poor stability at both ends of the pH ranges leading to

puriﬁcation challenges but also because of high polarity and

high degree of chemical reactivity. Therefore, special care needs

to be applied for synthesis of carbapenem analogs. Details of

the design, synthesis, structure−function relationship, bio-

conversion, and pharmacokinetic evaluation of a series of

prodrugs of ertapenem are described herein.

As noted, carbapenems are highly eﬀective antibiotics,

typically administered by intravenous infusion in a hospital

setting. Once the bacterial burden of a patient is reduced and

their symptoms are improved, the patient is sent home with a

non-carbapenem oral antibiotic. While this is not an issue for

most patients, it does present a challenge in some cases wherein

patients return to hospital because of lack of eﬀectiveness or for

tolerance issues. Carbapenem antibiotics are not orally

absorbed because of their high polarity, large polar suface

area, and negative log D. We envisaged that a prodrug approach

could improve lipophilicity (increasing clogD) and absorption.

All carbapenems, at a minimum, contain at least one carboxyl

moiety and a secondary hydroxyl group. Ester derivatives

derived from these carbapenem substituents potentially could

confer a viable prodrug strategy for oral formulation that would

allow for step down therapy. Step down therapy represents an

attractive opportunity to eﬀectively treat opportunistic

infections outside of a clinical setting. When we initiated this

program, there was a paucity of information in the literature

related to carbapenem prodrugs. Orapenem (tebipenem pivoxil,

8) was an exception. It is a pivaloxymethyl ester prodrug of

tebipenem (9).²⁻⁶It has been approved for clinical use only in

Japan. In addition, during the course of our studies, syntheses

of several meropenem (6) prodrugs were reported.⁷

RESULTS AND DISCUSSIONS

■

Design. Ertapenem possesses four polar groups amenable to

derivatization by prodrug moieties. Speciﬁcally, 4 is substituted

with a C-8 hydroxyl, a secondary pyrrolidine amine, and two

carboxyl groups. We focused our major attention on the

synthesis of ester prodrugs of both carboxyl groups to increase

log D substantially. Examples of prodrugs available clinically

provided the initial basis for the selection of prodrug moieties.

Since ertapenem is a potent antibiotic with potential to inhibit

Ertapenem (4) is a prominent member of the carbapenem

class of antibiotics and is used broadly. Importantly, ertapenem

is the only carbapenem antibiotic administered once daily.^1,8,9

The core structure of ertapenem and other β-methyl antibiotics,

including tebipenem, is common. They are diﬀerentiated only

by the structure of their side chain at C-2. Carbapenem

antibiotics, including tebipenem, possess basic side chains at C-

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Chart 2. General Structures of Five Classes of Prodrugs

growth of commensurate bacteria in the gut, our design strategy

required that the prodrugs must have reduced or no

antibacterial activity. As a free carboxyl group at C-3 is essential

for potent antibacterial activity¹⁰by all carbapenems, we

posited that a prodrug with an esteriﬁed C-3 carboxyl group

would be devoid of antibacterial activity. Thus, our main focus

was to synthesize bis-esters and lactone prodrugs. Lesser

emphasis was directed toward the synthesis of acyl derivatives

of the C-8 secondary hydroxy group or carbamate derivatives of

the pyrrolidine amino group of diesters to evaluate their

potential role in absorption. Prodrug ester moieties that are part

of successful clinical agents include pivaloxymethyl (e.g.,

adefovir dipivoxil, cefetamet pivoxil),^11,12carbonate esters

(e.g., tenoforvir disoproxil fumarate),¹¹(5-methyl-2-oxo-1,3-

dioxol-4-yl)methyl (e.g., olmesartan medoxomil),^11,13ethyl

(e.g., xemiloﬁban),¹⁴and lactones (e.g., lovastatin and

simvastatin).¹⁵While preparing ertapenem prodrugs, we have

incorporated the concepts of all of the promoieties and

synthesized ﬁve (10−14) groups of prodrugs (Chart 2). The

prodrugs are bis-acyloxylmethyl esters (10), bis-

(alkoxycarbonyl)oxy methyl esters (11), bis(alkoxy-2-oxo-1,3-

dioxol-4-yl) methyl esters (12), bis-alkyl esters (13), and

macrocyclic-lactones (14). We recently reported a preliminary

account of the synthesis and evaluation of a member (10a, 12a,

13a, and 14a) of each group except for the carbonate series

11.¹⁶Prodrugs with varying lypophilicity (clogD), calculated

polar surface area (cPSA), and calculated apparent permeability

coeﬃcient (cPapp) were synthesized to study structure−

function relationships. Despite low and unfavorable clogD or

calculated permeability numbers, in each ester series, we also

generated C-3 monoesters and in some cases both C-3 and

benzoate monoesters to test the potential role of intestinal

organic acid transporters (OATP) for the absorption of

monoacids. These esters also served as bioanalytical reference

standards for metabolites originating from bis-esters.

and smallest group (methyl ester 10k), intermediate isopropyl

(10f), cyclohexyl (10i), and POM methyl analog of 10a (10m).

Both monoesters of tert-butyl (10b and 10c) and isopropyl

(10g and 10h) and a C-3 cyclohexyl monoester (10j) were also

prepared. Similarly, bis- and monoesters in the carbonate series

(11a−j) were also synthesized (Table 2). As expected, the

carbonate esters showed increased polar surface area compared

to corresponding acyloxylmethyl esters. A limited number of

analogs (12a−d) were also prepared in the bis(alkoxy-2-oxo-

1,3-dioxol-4-yl) methyl ester series as represented by isopropyl

and tert-butyl groups (Table 3). An extensive study was

undertaken to study SFR of the alkyl ester series, since the

diethyl ester 13a had shown the best absorption.¹⁶Twenty

compounds (13a−t) representing this series are presented in

Table 4. We prepared a range of esters with varied properties,

including those with electron withdrawing groups such as

triﬂuoroethyl esters (13l−n), small chain alkyl n-propyl (13e),

n-butyl (13i), medium chain fatty acid (e.g., octanoate, 13j),

larger chain fatty acid (e.g., palmitate, 13k), and cholesteryl

ester (13o). To test the role of the remaining polar groups of

13a for absorption, we generated the C-8 acetate (13p),

propionate (13q), and valinyl (13r) analogs. In addition, we

also studied the alkyl (13s) and carbamate (13t) analogs of

pyrrolidine with (alkoxy-2-oxo-1,3-dioxol-4-yl)methyl group.

The latter group was selected for carbamate derivatization

because of its rapid hydrolysis (e.g., 12a) in the human

plasma.¹⁶Addition of two prodrug moieties in the bis-ester

series unfortunately increased the molecular size of the prodrug

substantially. This MW increase is expected to present

challenges for absorption, especially for ertapenem prodrugs,

as ertapenem is already endowed with relatively high molecular

weight. We envisaged circumventing this challenge by macro-

cyclization of the two carboxyl groups with appropriate smaller

linkers. Therefore, we prepared n-propyl linked macrocycle

(14a), extended the macrocycle with one carbon and made the

n-butyl linked analog (14b), and rigidiﬁed the linker by making

Z-butene analog (14c). In addition, we synthesized the cyclic

carbonate macrocyclic analog 14d to test whether the cyclic

carbonate displays a faster rate of hydrolysis such as that

In order to elucidate the structure−function relationship

(SFR) of acyloxylmethyl esters, the experience of tert-butyl

group of (POM) acyloxylmethyl esters (10a) was extended

(Table 1) by synthesis to include analogs bearing the simplest

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Table 1. Results of Plasma Hydrolysis and Calculated Properties of Acyloxylmethyl Esters

a

b

c

R = remaining, C = conversion to ertapenem from respective prodrugs in column 1. ACD = clogP at pH7.4. Disodium salt.

observed for 12a. We also extended the N-carbamate analogs of

the oleﬁnic macrocyles 14e−g. Since we had detected a poor or

incomplete plasma hydrolysis of 14a, we envisaged and

designed macrocyclic analog 14h in which the hydrolysis of

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Table 2. Results of Plasma Hydrolysis and Calculated Properties of (Alkoxycarbonyl)oxy Methyl Esters

a

b

R = remaining, C = conversion to ertapenem from respective prodrugs in column 1. ACD = clogP at pH 7.4.

either ester group is expected to collapse the molecule to

ertapenem. To reduce the polarity further, we also acetylated

C-8 hydroxy group of 14h to produce C-8 acetate analog 14i,

which exhibited one of the best permeability cPapp values

(calculated) of 11. Synthesis and systematic structure−function

relationship exploration of each of the ﬁve prodrug classes are

described herein.

Chemistry. Synthesis of Bis-ester Prodrugs of Ertapenem

(10−13). All bis-esters were synthesized starting from

ertapenem sodium by ﬁrst protecting the pyrrolidine secondary

amine by reaction with allyl chloroformate in phosphate buﬀer

at room temperature to yield an N-allylcarbamate (15) in over

95% yield (Scheme 1). The intermediate 15 was lyophilized at

pH 7 and used directly without further puriﬁcation to avoid

decomposition. This material was reacted with 2−5 equiv of

appropriate halides (RCO₂CH₂X for 10, ROCO₂CH₂X for 11,

(alkoxy-2-oxo-1,3-dioxol-4-yl)CH₂X for 12, and alkyl iodide for

13) using N-ethyldiisopropylamine and phase transfer catalyst

benzyltriethylammonium chloride to aﬀord respective N-

protected esters (16) which was deprotected with phenylsilane

and puriﬁed by reversed phase HPLC to furnish ﬁnal

deprotected esters 10−13 (Tables 1−4). Similar reaction of

15 with 1-pivoloxylethyl iodide ((CH₃)₃CCO₂CH(CH₃)I)

followed by deprotection with phenylsilane aﬀorded the

pivoloxyl-1-ethyl ester of ertapenem 10m in 19% overall two-

step yield (Scheme 1, Table 1).

Synthesis of Macrocyclic Lactone Prodrugs of Ertapenem

(14). Macrocyclic lactones were synthesized following a similar

reaction sequence as described for the synthesis of bis-esters

(Scheme 2). The allylcarbamate protected ertapenem sodium

salt was reacted with appropriate dihalide to yield protected

macrocyclic lactones (17), which upon deprotection by

phenylsilane and tetrakis(triphenylphosphine)palladium or by

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Table 3. Results of Plasma Hydrolysis and Calculated Properties of (Alkoxy-2-oxo-1,3-dioxol-4-yl) Methyl Esters

a

b

R = remaining, C = conversion to ertapenem from respective prodrugs in column 1. ACD = clogP at pH 7.4.

hydrogenolysis followed by reversed phase HPLC puriﬁcation

aﬀorded 14 (Table 5).

chloride to yield acetylated product 22a, which was N-

deprotected by phenylsilane to produce 13p (Scheme 5).

Propionyl ertapenem diethyl ester was prepared by EDCI

coupling of propionic acid and the N-protected diethyl ester

(16a₁₃) to give 22b, which was deprotected with phenylsilane

to aﬀord 13q. Coupling of allylcarbamate-(S)-valine with 16a₁₃

produced 22c which was treated with phenylsilane to remove

both allyl carbanates, aﬀording 13r.

Synthesis of N-Derivatized Diesters. The bis-ethyl ester

(13a) was alkylated with a reaction of (methyl-2-oxo-1,3-

dioxol-4-yl)methyl chloride and DIEA in DMF to give N-

alkylated derivative 13s. A similar reaction of diethyl ester

(13a) with (methyl-2-oxo-1,3-dioxol-4-yl)methyl (4-nitrophen-

yl) carbonate produced carbamate 13t (Scheme 6, Table 4).

Synthesis of 8-Acylated Ertapenem Macrocyclic Lactone.

The macrocyclic lactone (17h₁₄) was acetylated by coupling of

acetic acid using EDCI to give acetylated derivative 23 which

was deprotected by reaction with phenylsilane catalyzed by

tetrakis(triphenylphosphine)palladium to aﬀord acetylated

macrocyclic compound 14i (Scheme 7).

Synthesis of N-Carbamate Substituted Macrocylic Com-

pounds (14e−g). N-Carbamate substituted macrocyles were

synthesized by reacting ertapenem with the 5-methyl-2-oxo-1,3-

dioxol-4-yl)methyl (4-nitrophenyl) carbonate to give a

common intermediate 24 which was cyclized with requisite

dihalides to give carbamate substituted macrocycles (14e−g)

(Scheme 8).

Overview of Research Operating Plan. The program

goal was to ﬁnd a compound that is stable in the

gastrointestinal (GI) tract, absorbed through the intestinal

walls, and rapidly hydrolyzed to parent ertapenem in blood. A

thorough and careful evaluation strategy was developed for

prodrugs as a part of the research-operating plan. Carbapenems

are generally inherently unstable. Therefore, all prodrugs were

tested for their stability in phosphate buﬀer (pH 7), simulated

Synthesis of Monoesters. The C-3 pivaloxylmethyl ester

(10b) was prepared by reacting pivaloxylmethyl iodide with

ertapenem sodium in DMF at 0 °C for 30 min followed by

puriﬁcation by reverse phase HPLC. The free acid tended to

decompose and was stabilized by storing it as a sodium salt.

The benzoate ester 10c was prepared by selective alkylation of

C-3 p-nitrobenzyl ester (18) intermediate obtained from

ertapenem synthesis.¹⁷The pyrrolidine NH was protected as

allylcarbamate to give 19, which was then reacted with

pivaloxylmethyl iodide to give 20c₁₀. Phenylsilane deprotection

gave 21c₁₀, which was hydrogenolyzed and lyophilized to aﬀord

sodium salt 10c (Scheme 3). The other benzoate monoesters

were prepared similarly using Scheme 3. In most instances,

hydrogenolysis of intermediate 20 directly gave the ﬁnal

product.

The monoalkyl esters were prepared by following Scheme 1.

The reactions were closely monitored and stopped in about 4 h

when both monoesters (16, R, R₁= H, ester) were

predominantly present. Intermediate esters 16 were puriﬁed

directly on reversed phase HPLC and deprotected by

phenylsilane and puriﬁed by HPLC to aﬀord monoesters

(10−13, Tables 1−4).

Synthesis of 3-Triﬂuoroethyl-cholesteryl-acetyl-ertape-

nem-benzoate. Ertapenem N-allylcarbamate (15) was reacted

with triﬂuoroethyl triﬂate under phase transfer conditions and

DIEA producing monoester 16m₁₃. This monoester was

alkylated with cholesteryl iodoacetate (prepared from choles-

teryl chloroacetate and NaI) in similar alkylation conditions

except for using Na₂CO₃as a base producing the protected

diester, which was immediately deprotected by phenylsilane to

give highly lipophilic cholesteryl diester 13o (Scheme 4).

Synthesis of 8-Acylated Ertapenem Diesters. N-protected

diethyl ester of ertapenem (16a₁₃) was acetylated with acetyl

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Table 4. Results of Plasma Hydrolysis and Calculated Properties of Alkyl Esters

a

b

R = remaining, C = conversion to ertapenem from respective prodrugs in column 1. ND = not determined. ACD = clogP at pH 7.4.

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a

Scheme 1. Synthesis of Bis-esters

a

Reagents: (i) allyl chloroformate, water, acetone, phosphate buﬀer (pH 7), room temperature, 30 min; (ii) appropriate halide,

benzyltriethylammonium chloride, DIEA, DMF, 40−45 °C, 4−18 h; (iii) Pd(PPh₃)₄, phenylsilane, DMF, room temperature, 10−180 min, or

H₂, Pd/C, THF, room temperature.

a

Scheme 2. Synthesis of Macrocylic Ester Prodrugs of Ertapenem

a

Reagents: (i) alkyl dihalide, benzyltriethylammonium chloride, Na₂CO₃, DMF, 50 °C, 4−18 h; (ii) Pd(PPh₃)₄, phenylsilane, DMF, room

temperature, 10−180 min, or H₂, Pd/C, THF, water, room temperature, 1 h.

gastrointestinal ﬂuid (FaSSIF, pH 6.5), and simulated gastric

ﬂuid (SGF, pH 2.0). All tested compounds showed instability

in SGF with over 25% loss to β-lactam ring open product in 60

min. Therefore, at the later stage of the program, this test was

abandoned. The stability in the upper intenstine was evaluated

by incubating all compounds in simulated gastrointestinal ﬂuid

(FaSSIF, pH 6.5). Samples from all stability tests were analyzed

by LC−MS−MS. All compounds were soluble and stable

(>95% remaining) in phosphate buﬀer (pH 7) and FaSSIF at

50 μM for greater than 3 h.

After the stability test, all compounds were evaluated for the

hydrolysis of prodrugs to ertapenem by rat and human plasma,

measuring for the disappearance of the prodrug and production

of ertapenem. It is well established that rat plasma has

additional esterases (e.g., carboxylesterases, cholinesterases)

compared to higher species.¹⁸Higher mammalian species,

including humans, express many of these esterases in liver.¹⁹

On the basis of the esterase distribution in diﬀerent tissue types

or body ﬂuid in rat compared to higher species, it is expected

that the in vitro hydrolysis of the prodrugs in rat plasma may be

more pronounced than the hydrolysis in plasmas of the other

mammalian species. Therefore, any prodrug that is stable in the

rat plasma may not be suitable for further evaluation.

clear discernible pattern in these experimental models and were

not followed up further.

A selected group of prodrugs that showed rapid hydrolysis in

the rat plasma with rapid conversion (with a few exceptions) to

ertapenem were selected for rat pharmacokinetic (PK) studies.

As explained above, these classes of compounds are known to

be highly unstable at low pH, which was experimentally

demonstrated by measuring stability in SGF. Therefore, it is

expected that these compounds will not survive stomach acids.

Hence, it became critical to ﬁnd an alternative dosing regimen

for dosing prodrugs that either physically protected drugs from

exposure to stomach acid or bypassed the stomach altogether.

Prevention of exposure of drugs to stomach acid can be

accomplished by appropriate formulation (e.g., enteric coating

of the tablets/capsules). However, it is not practical or cost-

eﬀective to enterically coat each compound in the discovery

phase of a program. This is not only because of high cost/

investment required for formulation eﬀorts but also because of

the large amount of compound required for formulation

development, which is particularly challenging for diﬃcult-to-

synthesize targets. Therefore, it was imperative to identify and

implement an alternative-dosing regimen that bypassed the

stomach for successful execution of this program. Intraduodenal

dosing regimen emerged as a viable option that accomplished

this goal, which was readily adopted for conducting rat and dog

PK experiments for these compounds. A development

compound would be expected to be forumuated by enteric

coating for oral delivery of the prodrug.

Because of expression diﬀerences of esterases in diﬀerent

mammalian species, particularly expression of many estarases

only in the liver in many higher species, selected prodrugs were

evaluated for their hydrolysis in rat, human, dog, and monkey

liver microsomal preparations (with or without NADPH) and

hepatocytes. The results of 13a were described earlier¹⁶and

serve as an illustration. However, these studies did not show a

Evaluation of Prodrugs. As described in the research-

operating plan, all prodrugs that were stable in phosphate buﬀer

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Table 5. Results of Plasma Hydrolysis and Calculated Properties of Macrocyclic Lactones

a

b

R = remaining, C = conversion to ertapenem from respective prodrugs in column 1. ACD = clogP at pH 7.4.

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a

Scheme 3. Synthesis of Pivaloxylmethyl-benzoate Ertapenem (10c)

a

Reagents: (i) allyl chloroformate, water, acetone, phosphate buﬀer (pH 7), room temperature, 3 h; (ii) pivaloxylmethyl iodide, DIEA, DMF, room

temperature, 30 min; (iii) Pd(PPh₃)₄, phenylsilane, DMF, room temperature, 30 min; (iv) H₂, NaHCO₃, Pd/C, THF, water, room temperature, 1.5

h.

a

Scheme 4. Synthesis of Cholesteryl Ester (13o)

a

Reagents: (i) triﬂuoroethyl triﬂate, DIEA, DMF, 45 °C, 3 h; (ii) (a) cholesteryl 2-iodoacetate, Na₂CO₃, benzyltriethylammonium chloride, DMSO,

50 °C, 18 h; (b) Pd(PPh₃)₄, phenylsilane, DMF, room temperature, 10 min.

a

Scheme 5. Synthesis of 8-Acylated Ertapenem Diethyl Esters

a

Reagents: (i) acetyl chloride, DMAP, pyridine; or propionic acid or N-Alloc-(S)-valine, EDCI, DMAP, DCM; (ii) Pd(PPh₃)₄, phenylsilane, DMF,

room temperature, 10−60 min.

and FASSIF were incubated in rat and human plasma followed

by PK in rat and dog by ID route. As a control, ertapenem was

incubated in rat and human plasma and found to be completely

stable in both plasmas for 60 min. Prodrugs were incubated in a

similar fashion. For prodrugs, we monitored the disappearance

of prodrug and concomitant appearance of ertapenem. In

selected cases we also monitored the level of monoesters

formed from partial hydrolysis of the prodrugs.

Plasma Hydrolysis of Prodrugs. Incubation of acyloxyl

methyl esters (10) and linear carbonates (11) series (Tables 1

and 2) in rat plasma showed rapid disappearance of prodrugs in

almost all cases. However, incubation of bis-esters with only

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a

to ertapenem in high yields (Table 3). Essentially equivalent

hydrolysis was also observed in human plasma, but eﬃcient

conversion to ertapenem was only noted for the less hindered

methyl carbonates 12a and 12b. Isopropyl (12c) and tert-butyl

(12d) substituted carbonates showed lower ertapenem

conversions, 32% and 46%, respectively. As we have reported

earlier¹⁶for the bis-ethyl ester 13a, the hydrolysis of bis-alkyl

esters 13b−t (Table 4) with (13b−o, 13s, 13t) or without

(13p−r) free OH group at C-8 by rat plasma was generally

eﬃcient with respect to disappearance of the prodrug but not

for the formation of ertapenem. Exceptions to this observation

included the bis-palmitate (13k) and cholesteryl ester (13o),

which were remarkably stable in rat plasma. Bis-esters with

electron withdrawing groups (e.g., 13l) were rapidly hydrolyzed

in the rat plasma with rapid quantitative conversion to

ertapenem. Similar general hydrolysis patterns and conversions

to ertapenem were also observed for alkyl esters by human

plasma except for octanoyl ester 13j and monoester 13m,

which did not show signiﬁcant hydrolysis in human plasma.

While the bis-triﬂuoroethyl ester (13l) was rapidly hydrolyzed

by human plasma at rates similar to rat plasma, no ertapenem

was produced in contrast to results seen using rat plasma. Most

of the macrocyclic lactones (14a−i, Table 5) rapidly

disappeared when incubated in either rat or human plasma

with the exception of the two cyclic analogs 14h and 14i, which

showed lesser hydrolytic eﬃciency in human plasma.

Conversion eﬃciency to ertapenem from macrolactones was

generally poor in both rat and human plasma hydrolytic

reactions except for 14a, which showed excellent ertapenem

conversion in rat plasma. The cyclic carbonate (14d, 14g)

showed moderate ertapenem conversion. Surprisingly, the

cyclic compound 14h showed better conversion to ertapenem

in human plasma than in rat plasma. The C-8 acetate analog of

the cyclic compound 14i was rapidly hydrolyzed in rat plasma

without conversion to any ertapenem in 60 min similar to the

parent 14h. The rate of hydrolysis of 14i in human plasma was

somewhat better than the parent 14h, but conversion to

ertapenem was not observed.

Scheme 6. Synthesis of N-Derivatized Diethyl Esters

a

Reagents: (i) (methyl-2-oxo-1,3-dioxol-4-yl)methyl chloride or

(methyl-2-oxo-1,3-dioxol-4-yl)methyl (4-nitrophenyl) carbonate,

DIEA, DMF, 50 °C, 2 h.

smaller groups (e.g., methyl (10k, 11i), ethyl (10l, 11g))

produced >90% ertapenem in 60 min. Relatively lower

percentages of conversion of ertapenem were observed from

the prodrugs containing hindered prodrug moieties (10a, 10d,

10f, 10i, 10m, 11a) in both the 10 and 11 series, but the eﬀect

was much more pronounced for acyloxyl methyl ester (10)

series as evidenced by the results of 11c and 10f. The isopropyl

carbonate ester 11c showed rapid disappearance of the prodrug

moieties with rapid and proportional conversion to ertapenem,

whereas incubation of 10f showed rapid disappearance of the

prodrug but only 57% conversion to ertapenem. The hydrolysis

pattern of the monoesters was less clear-cut. We could not

acertain which of the two ester groups (C-3 or benzoate)

hydrolyzed faster. The hydrolysis of the 10 and 11 series

prodrugs by human plasma was generally less eﬃcient other

than for really small esters such as the bis-methyl (10k) and bis-

ethyl (10l) esters of acyloxyl methyl esters. These two

compounds showed rapid disappearance of the prodrug, but

only 34 and 60% ertapenem was produced in 60 min from 10k

and 10l, respectively (Table 1). While hydrolysis and

corresponding conversion to ertapenem of linear carbonates

were highly eﬃcient in rat plasma, it was generally less eﬃcient

in the human plasma (Table 2). The cyclic carbonates (12a−d)

were eﬃciently hydrolyzed by rat plasma with rapid conversion

Pharmacokinetics. As per selection criteria layed out in the

research-operating plan, a selected group of the prodrugs were

evaluated for their PK characteristics using intraduodenal

dosing in rat. It has been shown earlier¹⁶from the studies of

compounds 10a, 12a, and 13a that the Sprague−Dawley (SD)

rat ID PK model, which was ﬁrst selected for these studies

because of obvious reasons of lower cost and lower compound

requirement, was not ideal for evaluation because of unveiling

poor absorption (Table 6). However, before abandoning the rat

PK meaurment for prioritization of compounds, we elected to

a

Scheme 7. Synthesis of 8-Acetyl Macrocyclic Lactone (14i)

a

Reagents: (i) acetic acid, EDCI, DMAP, DCM, −5 °C, 1 h; (ii) Pd(PPh₃)₄, phenylsilane, DMF, room temperature, 10 min.

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a

Scheme 8. Synthesis of N-Substituted Macrocycles (14e−g)

a

Reagents: (i) (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl (4-nitrophenyl) carbonate, NaHCO₃, acetone, water, room temperature, 1 h, 50 °C, 2 h; (ii)

dihalide, Na₂CO₃, DMF, 50−55 °C, 4−18 h.

evaluate several new analogs in this model to determine

whether the earlier observation was a compound speciﬁc

phenomenon. Therefore, two additional compounds with

diﬀering characteristics (hydrolysis and/or physical properties)

in each of the three series were evaluated for rat ID PK. The

two isopropyl esters 10f and 10g were selected from the

acyloxylmethyl series. The former compound showed rat

plasma hydrolysis and cPapp, clogD, and cPSA properties

similar to 10a but exhibited slightly improved human plasma

hydrolysis (Table 1). The latter compound exhibited overall

better plasma hydrolysis properties than other compounds from

this series while evincing less favorable calculated properties

(cPSA, clogD, and cPapp), indicative of potentially poor

absorption.^20,21However, 10g contained a free benzoic acid

that allowed for testing the hypothesis as to whether the

benzoid acid can act as substrate for intestinal organic acid

anion uptake transporters (OATPs). Both of these compounds

were dosed at 10 mg/kg by ID route, and the presence of the

parent prodrug, monoester metabolites, and ertapenem was

measured in the rat plasma at various time intervals for 8 h.

These compounds showed only marginal, if any, improvement

from 10a in total absorption (Table 6), suggesting that smaller

ester groups or potential anion transporters do not aﬀect

overall absorption in rats, at least for these compounds. The

compounds 12c and 12d of the cyclic carbonate series were

tested for rat PK to supplement information gleaned from 12a.

Bulkier isopropyl and tert-butyl groups in 12c and 12d

substituted the methyl group of 12a, leading to larger molecular

weights but improved clogD and similar cPapp values (Table

3). Unfortunately, total production of ertapenem from 12c and

12d was marginally decreased compared to 12a (Table 6). As

reported earlier,¹⁶the most promising prodrug series continued

to be bis-alkyl series of 13a type. Two additional bis-esters, the

bis-isopropyl ester 13f and bis-triﬂuoroethyl ester 13l, with

improved cPapp and similar clogD values (Table 4) were

evaluated in rat PK by ID routes of administration. The total

absorption of ertapenem was below the level of quantiﬁcation

for the hindered ester 13f and was about 4-fold lower for the

ester-containing electron withdrawing group 13l (Table 6).

Ertapenem showed 156-fold lower absorption by ID dosing in

rat compare to iv dosing (Table 6). These studies with diverse

group of nine compounds conﬁrm that poor rat absoption of

the original three compounds 10a, 12a, and 13a was not

speciﬁc to just those compounds but to the entire ertapenem

prodrug series. Since the absorption of prodrugs in rat by ID

dosing was very poor, no in vitro/in vivo correlation could be

established in rats. Therefore, rat PK model was abandoned in

favor of beagle dog for further evaluations.

We have reported earlier that the absorption of many of

these compounds was species dependent.¹⁶For example, the

total absorption of bis-acyloxylmethyl ester 10a and bis-ethyl

ester 13a was improved by approximately 8× and 5×,

respectively, in beagle dogs when dosed by ID administration

(Table 7). We extended the dog PK studies with additional

compounds from each of the prodrug series to more fully

elucidate absorption structure function relationship. ID dosing

of the intermediate sized bis-isoproyl acyloxylmethyl ester 10f

resulted in 3.6% total absorption all in the form of ertapenem

with 2× improvement from rat (F_T= 1.8%), which happens to

be the same level improvement observed for 10a (Table 7).

The total absoption of the smaller bis-ethyl acyloxylmethyl

ester 10l was 2.9% similar to the large (bulky) bis-ester 10a,

suggesting that the bis-isopropyl ester may be of optimal size

for this series. The cyclic carbonate tert-butyl ester 12d, with

the best clogD and cPapp values of the series, showed very poor

total absoption (F_T= 0.4%) in the dog PK study (Table 7).

None of the other carbonates were tested for dog PK.

The simple alkyl esters appeared to be most eﬀective

prodrugs of ertapenem, as exempliﬁed by bis-ethyl ester 13a.

Therefore, series 13 compounds that showed rapid hydrolysis

in rat plasma with or without rapid conversion to ertapenem

was systematically studied for beagle dog ID PK starting with

the smaller bis-methyl ester 13d, bis-n-propyl ester 13e,

moderately hindered bis-isopropyl ester 13f, bis-n-butyl ester

13i, a mid-chain fatty acid ester bis-octanoate ester 13j, and bis-

triﬂuoroethyl ester 13l. Of this group, the smallest methyl ester

(13d) showed the best total absorption of 24.2%, bis-n-propyl

ester (13e) 11.0%, bis-n-butyl ester (13i) 16.9%, bis-isopropyl

ester (13f) 5.1%, bis-octanoyl ester (13j) 1.1%, and bis-

triﬂuoroethy ester (13l) 12.0% (Table 7). The bis-ethyl ester

(13a) remained the best compound exhibiting 31.3% total

absorption. However, the bis-ester prodrugs formed long

lasting circulating monoesters in vivo (Table 7). Since the

monoesters formed after dosing of the bis-ester prodrugs were

not measured for all compounds, precise structure−function

relationship cannot be ascertained but a general trend can be

established. C₁−C₄linear esters are reasonably absorbed with

ethyl being the best. Isopropyl ester is less favored, and the

medium chain (C₈) fatty esters are very poorly absorbed. The

long chain (C₁₆) fatty ester (13k) was insoluble in the vehicles

and was not dosed. While bis-triﬂuoroethyl ester (13l) showed

quantitative conversion to ertapenem in rat plasma but not

human plasma (Table 4), it failed to improve overall absorption

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in dog compared to the bis-ethyl ester 13a (Table 7). Addition

of acetate (13p) or propionate (13q) at C-8 of bis-ethyl ester

(13a) did not improve total absorption and exhibited total

absorption of only 23.3% and 20.2%, respectively. The

cholesterol ester (13o) showed very poor total absorption of

only <0.1% and all intact prodrug. No ertapenem or monoester

13m was detected. Clearly, cholesterol transporters played no

role in the absorption of this prodrug. Likewise, the amino acid

transporters played no role in the absorption of the C-8 valinyl

ester (13r). It showed about half (14.9%) of the total

absorption compared with its parent 13a. Substitution of

proton of pyrrolidine NH by a cyclic carbonate carbamate

(13t) of 13a with cPapp of 13 led to about 2-fold reduction in

the total absorption (14.4% vs 31.3%).

The absorption of the macrocyclic esters containing a propyl

group (14a) or cyclic carbonate linked macrocycle (14d) was

generally less than 1%. However, the cyclic acetal acetate 14i,

with an intermediate cPapp value of 11, showed signiﬁcantly

better total absortion of 16%. These studies did not provide

good correlation between rat and human plasma hydrolyses as

well as correlation in vivo. ID dog PK performed well for the

measurement of absorption of these prodrugs and could be

used for absorption measurement of compounds that show

instability in gastric ﬂuid.

Careful analysis of the physical properties and absorption

(Figures 1 and 2) allowed for formulation of some general

conclusions. The molecular size of the prodrugs has a more

substantial impact on absorption than other parameters such as

cPapp values as evident from XY plots shown in Figures 1 and

2. The data suggest that molecular weights of the bis-ester

prodrugs lower than 600 show better absorption. The esters

(13e and 13f) with odd number of carbons in alkyl chain

appear to be exceptions. For example, the ester with the even

alkyl chain (C-4, 13i) showed better absorption than esters

with odd alkyl chain even if they were of a smaller size (C-3,

13e and 13f) (Figure 1). A careful correlation of the total

absorption and cPapp values indicated that cPapp values of 7 or

higher showed absorption of over 10% except when either

steric factors were involved (e.g., isopropyl ester 13f) or the

compound was a carbamate derivative (13s). As expected,

prodrugs with lower cPapp values show lower absorptions than

those with higher cPapp values for this series of compounds

(Figure 2). Unfortunately ertapenem, the parent, is endowed

with high molecular weight (475.5 Da), larger polar surface area

(156), and very low clogD (−5.9) and low cPapp (1.8), so it

fares rather poorly when compared to tebipenem (MW = 383.5

Da, cPSA = 93, clogD = 2.4, and cPapp = 5.2). Diﬀerences in

these properties are likely attributed to favorable absorption of

tebipenem pivoxil. Meropenem POM ester also showed >25%

absorption in rat even with MW greater than 643.⁷This would

suggest benzoic acid structural moiety present as part of

substitution at C-2 in ertapenem negatively impacted the

absorption regardless of the class of prodrugs.

Additionally, tebipenem pivoxil is transported by intestinal

uptake transporters OATB1A2 and OATBB1, which positively

impacts absoption.²²To build transporter aﬃnity in a molecule

is challenging; however, we did attempt to design molecules

that can exploit amino acid transporters (e.g., valinyl ester, 13r),

fatty acid esters (13j and 13k), cholestryl ester (13o), and free

carboxyl monoester (13f). These compounds failed to show

improved absorption. The bis-ethyl ester (13a) remained the

best compound of ertapenem prodrugs showing 31.3% total

absorption. The current study established that only simple alkyl

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prodrugs in this study showed no correlation between rat and

human plasma hydrolysis as well as in vivo in rat or dog. This

study did demonstrate that the benzoic acid substitution at C-2

of ertapenem played a detrimental role in prrodrug absorption

and hydrolysis. In addition, the study suggested the smaller C-2

carbapenem substitution is better for prodrug absorption and

hydrolysis (e.g., tebipenem and meropenem).

EXPERIMENTAL SECTION

■

Solvents, reagents, and intermediates that are commercially available

were used as received. Reagents and intermediates that were not

commercially available were prepared in the manner as described

1

below. H NMR spectra were measured on either a Varian VNMR

Figure 1. Percentage of total absorption versus molecular weights of

prodrugs in beagle dogs by intradeuodenal dosing.

system 400 or Bruker Avance 400 spectrometer at 400 MHz and are

reported as ppm downﬁeld from Me₄Si with number of protons,

multiplicities, and coupling constants in hertz indicated parenthetically.

Where LC/MS data are presented, analyses were performed using an

Agilent 6110A MSD or an Applied Shimadzu 2020MSD. The parent

ion is given. Puriﬁcation was conducted with reversed phase HPLC

(Phenomenex Gemini C₁₈(250 mm × 21.2 mm, 5 μm) or

Phenomenex Synergi C₁₈(250 mm × 50 mm, 10 μm) column,

elution of acetonitrile/water from 100% acetonitrile to 0% acetonitrile)

or silica gel column chromatography (SAN PONT, ZXC II). Animal

hepatocytes were prepared at WuXi AppTec Co., Ltd. All other

reagents were from commercial sources. All compounds (with the

exception of a few compounds) tested in the bioassays were of ≥95%

1

purity as measured by HPLC (A = 220 nm) and H NMR. Speciﬁc

HPLC % purity of all compounds is listed with the compound

synthesis.

Synthesis of N-Allyloxycarbonyl-ertapenem (15). To a

suspension of ertapenem 4 (20 g, 40 mmol) in a mixture of H₂O

(20 mL), acetone (250 mL), and NaH₂PO₄−Na₂HPO₄buﬀer (pH 7,

7.20 mL) was slowly added allyl chloroformate (5.0 mL, 48 mmol).

The mixture was stirred at room temperature for 30 min and diluted

with water. The reaction mixture was adjusted to pH 7 by addition of

aqueous solution of 1 N NaHCO₃. The solution was lyophilized to

give 15 as a yellow solid which was directly used in the next reaction

Figure 2. Percentage of total absorption versus cPapp of prodrugs in

beagle dogs by intradeuodenal dosing.

esters with small and unhindered groups with relatively smaller

molecular weight are favored for absorption. Going forward, a

streamlined research-operating plan could be envisaged for

evaluation of the compounds of this class involving measure-

ment of disappearance of prodrug and appearance of

carbapenem in rat plasma followed by ID dog PK with a

special emphasis given to smaller molecular size, preferably less

than 600 Da, and cPapp > 10.

1

without further puriﬁcation (23 g, 94.8% yield). H NMR (400 MHz,

CD₃OD) δ 7.92−8.02 (m, 1H), 7.75−7.80 (m, 1H), 7.68 (d, J = 7.8

Hz, 1H), 7.30 (dd, J₁= J₂= 7.6 Hz, 1H), 5.75−6.05 (m, 1H), 5.05−

5.40 (m, 2H), 3.35−4.60 (m, 9H), 3.20−3.25 (m, 1H), 2.70−2.85 (m,

1H), 1.95−2.05 (m, 1H), 1.15−1.30 (m, 6H). ESI MS m/z 560.5 [M

+ H]⁺. HPLC purity: 93.6%.

Synthesis of Ertapenem Bis(pivaloyloxy)methyl Ester (10a).

Iodomethyl pivalate (5.1 g, 20.4 mmol) was slowly added to a solution

of compound 15 (3.0 g, 5.1 mmol), N-ethyldiisopropylamine (1.38 g,

10.2 mmol), and benzyltriethylammonium chloride (392 mg, 1.72

mmol) in DMF (10 mL), and the mixture was heated at 45 °C for 18

h. After addition of EtOAc (300 mL), the combined organic layers

were washed with H₂O (100 mL × 3) and brine (100 mL), dried

(Na₂SO₄), and ﬁltered. The ﬁltrate was concentrated under reduced

pressure and the residue was puriﬁed by silica gel column

chromatography, eluting with DCM/MeOH (40:1). Fractions

containing the product were concentrated under vacuo to give the

desired bis(pivaloyloxy)methyl ester 16a₁₀(R = R₁= CH₂OCO-t-Bu)

(2.0 g, 49.3%) as a pale yellow oil. ESI MS m/z 788.5 [M + H]⁺.

To a solution of 16a₁₀(2.0 g, 2.54 mmol) in DMF (8.0 mL) were

added phenylsilane (213 mg, 5.08 mmol) and tetrakis(triphenyl-

phosphine)palladium (80 mg, 0.069 mmol), and the mixture was

stirred at room temperature for 10 min. EtOAc (100 mL) was added,

and the organic solution was washed with H₂O (40 mL × 3) and brine

(100 mL), dried (Na₂SO₄), and ﬁltered. The ﬁltrate was concentrated

under reduced pressure. The residue was puriﬁed by reversed phase

HPLC on a GILSON 281 instrument ﬁtted with a Phenomenex

Gemini C₁₈(250 mm × 21.2 mm, 5 μm) column eluted with a water

and acetonitrile gradient (mobile phase A, water; mobile phase B,

acetonitrile; gradient 52−72% B, 0−8 min; 100% B, 8.5−10.5 min; 5%

B, 11−12 min) followed by lyophilization to aﬀord the desired

compound 10a (600 mg, 33.7%) as a colorless powder. ¹H NMR (400

In summary, we have described a detailed study of the

synthesis and evaluation of ertapenem prodrugs to address the

lack of oral absorption of ertapenem. Five classes of prodrugs

were studied using intraduodenal dosing to bypass and avoid

the eﬀect of stomach acid and approximately mimic oral

absorption. The simplest bis-ethyl and bis-methyl ester

prodrugs were shown to be the most promising, providing

31.3% and 24.2% total absorption in dog, respectively, versus

the 1.6% absorption observed when dosing ertapenem.

Unfortunately, the best compound 13a showed slow hydrolysis

and conversion to parent ertapenem in vivo in dog and rats and

showed the presence of signiﬁcant amounts of long-lived

circulating monoester metabolites. These properties of the

prodrugs were undesirable for a development compound and

did not meet the developmental objective, leading to the

termination of 13a and the program from further development.

Therefore, no eﬀorts were expended to develop expensive

enteric-coated formulation and testing of 13a by oral dosing.

The macrocyclic lactone acetate acetal prodrug 14i showed

some promise with 16.0% absorption of ertapenem in dogs and

warrants additional investigation. The study showed that

intradeudenal dosing is a very eﬀective method for PK

measurement for compounds unstable in gastric ﬂuid. The

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MHz, CD₃OD) δ 8.30 (s, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.76 (d, J =

7.6 Hz, 1H), 7.45 (dd, J₁= J₂= 7.6 Hz, 1H), 6.00 (s, 2H), 5.75−5.85

(m, 2H), 3.75−4.25 (m, 4H), 3.40−3.58 (m, 2H), 3.25−3.30 (m, 1H),

2.65−2.95 (m, 2H), 1.82−1.90 (m, 1H), 1.25 (t, J = 6.4 Hz, 6H), 1.15

(s, 18H). ESI MS m/z 704.3 [M + H]⁺. HPLC purity: 95.1%.

Synthesis of Ertapenem C-3-Methyl Pivalate (10b). Iodo-

methyl pivalate (2.42 g, 10 mmol) was added dropwise to a solution of

ertapenem 4 (5.0 g, 10 mmol) in DMF (30 mL) at 0 °C for 30 min.

The mixture was directly puriﬁed by reversed phase HPLC on a

GILSON 281 instrument ﬁtted with a Phenomenex Synergi C₁₈(250

mm × 50 mm, 10 μm) using water (0.1% formic acid) and acetonitrile

as eluents (mobile phase A, water (0.1% formic acid); Mmobile phase

B, acetonitrile; gradient, B from 25% to 50% in 25 min; ﬂow rate, 80

mL/min; detective wavelength, 220 nm) followed by freeze-drying to

aﬀord free acid of C-3 monoester 10b (250 mg, 4.2%) as a white solid.

Structure was determined by HMBC. ¹H NMR (400 MHz, CD₃OD) δ

8.18 (s, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.40

(dd, J₁= J₂= 7.6 Hz, 1H), 5.83 (d, J = 5.6 Hz, 1H), 5.77 (d, J = 5.6 Hz,

1H), 4.19−4.22 (m, 1H), 3.99−4.12 (m, 2H), 3.79−3.85 (m, 1H),

3.48−3.53 (m, 2H), 3.22−3.30 (m, 1H), 2.95−2.99 (m, 1H), 2.73−

2.81 (m, 1H), 1.84−1.91 (m, 1H), 1.24−1.27 (m, 6H), 1.15 (s, 9H).

ESI MS m/z 590.2 [M + H]⁺. HPLC purity: 96.5%.

Free acid form of 10b (250 mg, 0.42 mmol) was dissolved in MeCN

(20 mL)−H₂O (20 mL), and 0.01 N NaHCO₃aqueous solution (42

mL, 0.42 mmol) was added dropwise. The mixture was concentrated

by freeze-drying. The residue was puriﬁed by reversed phase HPLC

followed by freeze-drying to give sodium salt 10b (206 mg, 80.0%) as

a white solid. ¹H NMR (400 MHz, CD₃OD) δ 8.18 (s, 1H), 7.85 (d, J

= 7.6 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.40 (dd, J₁= J₂= 7.6 Hz,

1H), 5.83 (d, J = 5.6 Hz, 1H), 5.77 (d, J = 5.6 Hz, 1H), 4.19−4.22 (m,

1H), 3.99−4.12 (m, 2H), 3.79−3.85 (m, 1H), 3.48−3.53 (m, 2H),

3.22−3.30 (m, 1H), 2.95−2.99 (m, 1H), 2.73−2.81 (m, 1H), 1.84−

1.91 (m, 1H), 1.24−1.27 (m, 6H), 1.15 (s, 9H). ESI MS m/z 590.2

[M + H]⁺, 612.2 [M + Na]⁺. HPLC purity: 95.0%.

Synthesis of Ertapenem C-3-Sodium Carboxyl Methyl

Pivalate (10c). To a solution of 18 (8.54g, 14.0 mmol) in H₂O

(12 mL), acetone (110 mL), and NaH₂PO₄−Na₂HPO₄buﬀer (pH 12

mL) was added allyl chloroformate (2.4 g, 0.02 mol) dropwise, and

then the mixture was stirred at room temperature for 3 h. After

concentration by vacuo followed by dilution with H₂O, the resulting

solid was ﬁltered and dried to give 19 (9.5 g, 97.7%) as a pale white

solid which was directly used in the next reaction without further

puriﬁcation. ESI MS m/z 695 [M + H]⁺.

To a solution of compound 19 (4.5 g, 6.5 mmol) and iodomethyl

pivalate (3.1 g, 12.8 mmol) was added N-ethyldiisopropylamine (0.84

g, 6.5 mmol), and the mixture was stirred at room temperature for 30

min. The reaction was quenched with H₂O (500 mL), extracted with

EtOAc (80 mL × 3), and the organic layer was concentrated and

puriﬁed by silica gel columm chromatography, eluting with DCM/

MeOH (40:1). It was concentrated by vacuo to give 20c₁₀(R₁=

CH₂OCO-t-Bu) (1.8 g, 34.4%) as a pale yellow oil. ESI MS m/z 809

[M + H]⁺.

1H), 1.50−1.57 (m, 1H), 1.21 (s, 9H), 1.07−1.12 (m, 3H), 1.02 (d, J

= 6.8 Hz, 3H). ESI MS m/z 590.2 [M + H]⁺, 612.2 [M + Na]⁺. HPLC

purity: 95.7%.

Synthesis of Ertapenem Bis((2-ethylbutanoyl)oxy)methyl

Ester (10d). Reaction of 2-ethylbutanoyl oxymethylchloride with 15

in a similar manner gave 16d₁₀(R = R₁= CH₂OCOCH(Et)₂) as

described above, which was followed by deprotection and puriﬁcation

by reversed phase HPLC to yield the desired diester 10d in 32.2%

1

yield as a colorless powder. H NMR (400 MHz, CDCl₃) δ 9.69 (s,

1H), 8.09 (d, J = 7.6 Hz, 1H), 8.05 (s, 1H), 7.85 (d, J = 7.6 Hz, 1H),

7.43 (dd, J₁= J₂= 7.6 Hz, 1H), 6.01 (s, 2H), 5.89 (s, 2H), 4.20−4.25

(m, 2H), 3.95−4.05 (m, 1H), 3.60−3.68 (m, 1H), 3.45−3.50 (m, 1H),

3.30−3.38 (m, 1H), 3.23−3.25 (m, 1H), 2.88−2.91 (m, 1H), 2.75−

2.83 (m, 1H), 2.22−2.30 (m, 3H), 1.96−2.02 (m, 1H), 1.49−1.70 (m,

8H), 1.34 (d, J = 6.4 Hz, 3H), 1.26 (d, J = 7.6 Hz, 3H), 0.84−0.89 (m,

12H). ESI MS m/z 732.3 [M + H]⁺. HPLC purity: 99.9%.

Synthesis of Ertapenem C-3-Methyl 2-Ethylbutanoate

Sodium Salt (10e). Reaction of iodomethyl 2-ethylbutanoate with

ertapenem 4 followed by formation of sodium salt and puriﬁcation by

reversed phase HPLC gave 5.2% yield of desired C-3 monoester

sodium salt 10e (R = CH₂OCOCH(Et)₂, R₁= Na) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 10.06 (s, 1H), 8.24 (s, 1H), 7.77

(d, J = 7.6 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.35 (dd, J₁= J₂= 7.6 Hz,

1H), 5.80 (d, J = 6.0 Hz, 1H), 5.75 (d, J = 6.0 Hz, 1H), 5.10 (brs, 1H),

4.19−4.22 (m, 1H), 3.94−3.97 (m, 1H), 3.83−3.86 (m, 1H), 3.40−

3.60 (m, 3H), 3.23−3.25 (m, 2H), 2.59−2.67 (m, 2H), 2.18−2.24 (m,

1H),1.65−1.68 (m,1H), 1.39−1.52 (m, 4H), 1.14−1.17 (m, 6H),

0.77−0.80 (m, 6H). ESI MS m/z 604.3 [M + H]⁺, 626.3 [M + Na]⁺.

HPLC purity: 99.4%.

Synthesis of Ertapenem Bis(isobutanoyloxy)methyl Ester

(10f). Chloromethyl isobutyrate was reacted with N-allyloxy

carbamate 15 to give 16f₁₀(R = R₁= CH₂OCO-i-Pr) followed by

deprotection with phenylsilane and puriﬁcation by reversed phase

HPLC to provide 2.9% yield of the diester 10f as a colorless powder.

¹H NMR (400 MHz, CDCl₃) δ 9.66 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H),

7.99 (s, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.37 (dd, J₁= J₂= 8.0 Hz, 1H),

5.93 (s, 2H), 5.85 (d, J = 5.6 Hz, 1H), 5.77 (d, J = 5.6 Hz, 1H), 4.15−

4.19 (m, 2H), 3.93−3.95 (m, 1H), 3.57−3.61 (m, 1H), 3.40−3.44 (m,

1H), 3.26−3.30 (m, 1H), 3.16−3.19 (m, 1H), 2.83−2.87 (m, 1H),

2.70−2.76 (m, 1H), 2.48−2.59 (m, 2H), 1.92−1.98 (m, 1H), 1.28 (d, J

= 6.4 Hz, 3H), 1.20 (d, J = 7.6 Hz, 3H), 1.09−1.13 (m, 12H). ESI MS

m/z 676.3 [M + H]⁺. HPLC purity: 99.3%.

Synthesis of Ertapenem C-3-Methyl Isobutyratesodium Salt

(10g). Reaction of iodomethyl isobutyrate with ertapenem 4 followed

by formation of sodium salt and puriﬁcation by reversed phase HPLC

gave desired C-3 monoester sodium salt 10g (R = CH₂OCO-i-Pr, R₁=

Na) in 2.2% yield as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ

9.99 (s, 1H), 8.12 (s, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 7.6

Hz, 1H), 7.31 (dd, J₁= 8.0 Hz, J₂= 7.6 Hz, 1H), 5.81 (d, J = 5.6 Hz,

1H), 5.71 (d, J = 5.6 Hz, 1H), 5.11 (brs, 1H), 4.20−4.23 (m, 1H),

3.94−3.98 (m, 1H), 3.81−3.85 (m, 1H), 3.24−3.66 (m, 5H), 2.67−

2.70 (m, 2H), 1.63−1.70 (m, 1H), 1.04−1.18 (m, 12H). ESI MS m/z

576.2 [M + H]⁺, 598.2 [M + Na]⁺. HPLC purity: 95.8%.

A solution of N-allyoxycarbonyl 20c₁₀(1.8 g, 2.2 mmol),

phenylsilane (475 mg, 4.4 mmol), and Pd(PPh₃)₄(100 mg) in

DMF (20 mL) was stirred at room temperature for 30 min. After

ﬁltration, the ﬁltrate was concentrated and puriﬁed by silica gel column

chromatography, eluting with DCM/MeOH (40:1) and concentrated

by vacuo to give des-allyloxy compound 21c₁₀(R₁= CH₂OCO-t-Bu)

(900 mg, 55.8%) as a pale yellow solid. ESI MS m/z 725 [M + H]⁺.

10 % Pd−C (110 mg) was added to a solution of 21c₁₀(1.1 g, 1.5

mmol) and NaHCO₃(63 mg, 0.75 mmol) in THF (50 mL)−H₂O (50

mL), and the mixture was stirred at room temperature under H₂for

1.5 h. After ﬁltration, the ﬁltrate was concentrated by freeze-drying.

The residue was puriﬁed by reversed phase-HPLC followed by freeze-

drying to give 10c (R = Na, R₁= CH₂OCO-t-Bu) (268 mg, 30%) as a

white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.24 (s, 1H), 8.37 (s,

1H), 7.92 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.44 (dd, J₁=

J₂= 8.0 Hz, 1H), 5.92 (s, 2H), 4.95 (brs, 1H), 3.93−3.95 (m, 1H),

3.85−3.88 (m, 1H), 3.76−3.80 (m, 1H), 3.38−3.44 (m, 1H), 3.19−

3.25 (m, 1H), 3.07−3.11 (m, 1H), 2.98−3.01 (m, 1H), 2.60−2.64 (m,

Synthesis of Ertapenem C-3-Sodium Carboxyl (Isobutyloxy)-

methylbenzoate (10h). Reaction of 1.2 equiv of chloromethyl

isobutyrate (705 mg, 5.19 mmol) with compound 19 (3.0 g, 4.32

mmol) followed by puriﬁcation by silica gel columm chromatography

aﬀorded desired benzoate 20h₁₀(R₁= CH₂OCOi-Pr) (1.3 g, 37.9%)

as a red oil. To a solution of 20h₁₀(124 mg, 0.17 mmol) and NaHCO₃

(12 mg, 0.14 mmol) in THF (6.0 mL)−H₂O (6.0 mL) was added 10%

Pd−C (110 mg), and the mixture was stirred at room temperature

under H₂for 1 h. After ﬁltration, the ﬁltrate was concentrated by

freeze-drying. The residue was puriﬁed by reversed phase HPLC on a

GILSON 281 instrument ﬁtted with a Phenomenex Gemini C₁₈(250

mm × 21.2 mm, 5 μm) using water (0.2% HCOONH₄) and

acetonitrile as eluents (mobile phase A, water (0.2% HCOONH₄);

mobile phase B, acetonitrile; gradient, 52−72% B, 0−8 min; 100% B,

8.5−10.5 min; 5% B, 11−12 min) followed by freeze-drying to give

1

10h (6.8 mg, 7.2%) as a white powder. H NMR (400 MHz, DMSO-

d₆) δ 10.22 (s, 1H), 8.37 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.67 (d, J =

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8.0 Hz, 1H), 7.49 (dd, J₁= J₂= 8.0, 1H), 5.95 (s, 2H), 5.06 (brs, 1H),

4.14−4.17 (m, 1H), 3.94−3.96 (m, 1H), 3.85−3.89 (m, 1H), 3.58−

3.62 (m, 2H), 3.19−3.21 (m, 2H), 2.58−2.73 (m, 3H), 1.64−1.74 (m,

1H), 1.14−1.16 (m, 12H). ESI MS m/z 576.2 [M + H]⁺, 598.2 [M +

Na]⁺. HPLC purity: 95.3%.

1H), 3.46−3.51 (m, 1H), 3.23−3.46 (m, 2H), 2.75−2.86 (m, 2H),

1.93−2.05 (m, 1H), 1.61 (d, J = 5.2 Hz, 3H), 1.52−1.53 (m, 3H),

1.33−1.36 (m, 3H), 1.16−1.27 (m, 21H). ESI MS m/z 732.3 [M +

H]⁺. HPLC purity: 98.3%.

Synthesis of Ertapenem Bis((tert-butoxycarbonyl)oxy)-

methyl Ester (11a). tert-Butyl (chloromethyl)carbonate was reacted

with N-allyloxy carbamate 15, which provided N-protected dicar-

bonate 16a₁₁(R = R₁= -CH₂OCOO-t-Bu), which was likewise

deprotected with phenylsilane followed by reversed phase HPLC

puriﬁcation and lyophilization to aﬀord the desired dicarbonate 11a in

Synthesis of Ertapenem Bis((cyclohexanecarbonyl)oxy)-

methyl Ester (10i). Reaction of chloromethyl cyclohexanecarboxylate

with N-protected 15 produced 16i₁₀(R = R₁= CH₂OCOC₆H₁₁)

followed by deprotection of allylcarbamate and puriﬁcation by

reversed phase HPLC aﬀorded diester 10i in 13.8% yield as a

colorless powder. ¹H NMR (400 MHz, CDCl₃) δ 9.69 (s, 1H), 8.06−

8.09 (m, 2H), 7.81 (d, J = 8.0 Hz, 1H), 7.43 (dd, J₁= J₂= 8.0 Hz, 1H),

5.98 (s, 2H), 5.89 (d, J = 5.6 Hz, 1H), 5.82 (d, J = 5.6 Hz, 1H), 4.22−

4.25 (m, 2H), 4.02−4.05 (m, 1H), 3.63−3.68 (m, 1H), 3.48−3.51 (m,

1H), 3.33−3.37 (m, 1H), 3.23−3.25 (m, 1H), 2.88−2.92 (m, 1H),

2.77−2.82 (m, 1H), 2.28−2.39 (m, 2H), 1.98−2.03 (m, 1H), 1.55−

1.96 (m, 11H), 1.12−1.46 (m, 17H). ESI MS m/z 756.3 [M + H]⁺.

HPLC purity: 99.4%.

1

14.0% yield as a colorless powder. H NMR (400 MHz, DMSO-d₆) δ

10.69 (s, 1H), 8.30 (s, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 7.6

Hz, 1H), 7.54 (dd, J₁= 8.0 Hz, J₂= 7.6 Hz, 1H), 5.89 (s, 2H), 5.76 (d,

J = 6.0 Hz, 1H), 5.67 (d, J = 6.0 Hz, 1H), 5.13 (brs, 1H), 4.23−4.30

(m, 2H), 3.90−3.97 (m, 2H), 3.70−3.75 (m, 1H), 3.26−3.53 (m, 2H),

3.04−3.09 (m, 1H), 2.86−2.89 (m, 1H), 1.87−1.94 (m, 1H), 1.42 (s,

9H), 1.40 (s, 9H), 1.12−1.18 (m, 6H). ESI MS m/z 736.3 [M + H]⁺.

HPLC purity: 97.7%.

Synthesis of Ertapenem C-3-((Cyclohexanecarbonyl)oxy)-

methyl Ester Sodium Salt (10j). Reaction of 1.0 equiv of

chloromethyl cyclohexanecarboxylate (1.76 g, 9.96 mmol) with

compound 15 (5.81 g, 10.0 mmol) followed by puriﬁcation by

reversed phase HPLC aﬀorded desired C-3-((cyclohexanecarbonyl)-

oxy)methyl ester 16j₁₀(R = CH₂OCOc-Hex, R₁= H) (450 mg, 6.5%)

as white solid. 16j₁₀was deprotected by using phenylsilane and

puriﬁed by reversed phase HPLC to aﬀord 10j (240 mg, 60.9%) as

white powder, which was converted to sodium salt using NaHCO₃and

puriﬁed by neutral HPLC as described earlier to give 35.1% yield of

Synthesis of Ertapenem C-3-((tert-Butylcarbonyl)oxy)methyl

Ester Sodium Salt (11b). Reaction of 1.0 equiv of tert-butyl

(chloromethyl)carbonate (2.90 g, 17.5 mmol) with compound 15

(10.0 g, 17.2 mmol) followed by puriﬁcation by reversed phase HPLC

aﬀorded desired C-3-((tert-butylcarbonyl)oxy)methyl ester 16b₁₁(R =

-CH₂OCOO-t-Bu, R₁= H) (820 mg, yield 6.5%) as a white solid.

16b₁₁was deprotected by using phenylsilane and puriﬁed by reversed

phase HPLC to aﬀord 11b (700 mg, 51.8%) as white powder, which

was converted to sodium salt using NaHCO₃to give 55.7% yield of the

1

desired sodium salt 11b (113 mg) as a white solid. H NMR (400

1

the desired sodium salt 10j (40 mg) as a white solid. H NMR (400

MHz, DMSO-d₆) δ 10.09 (s, 1H), 8.30 (s, 1H), 7.85 (d, J = 7.6 Hz,

1H), 7.63 (d, J = 7.6 Hz, 1H), 7.42 (dd, J₁= J₂= 7.6 Hz, 1H), 5.76 (d,

J = 6.0 Hz, 1H), 5.65 (d, J = 6.0 Hz, 1H), 5.09 (brs, 1H), 4.21−4.23

(m, 1H), 3.92−3.99 (m, 1H), 3.83−3.87 (m, 1H), 3.63−3.67 (m, 1H),

3.50−3.55 (m, 1H), 3.24−3.41 (m, 2H), 2.55−2.71 (m, 2H), 1.62−

1.72 (m, 1H), 1.41 (s, 9H), 1.12−1.20 (m, 6H). ESI MS m/z 606.2

[M + H]⁺, 628.2 [M + Na]⁺. HPLC purity: 95.2%.

Synthesis of Ertapenem Bis((isopropoxycarbonyl)oxy)-

methyl Ester (11c). N-Ethyldiisopropylamine (690 mg, 5.34

mmol) and benzyltriethylammonium chloride (1.2 g, 5.26 mmol)

were added to a solution of compound 15 (3.0 g, 5.16 mmol) and

chloromethyl isopropyl carbonate (1.6 g, 10.48 mmol) in DMF (10

mL), and the mixture was stirred at 45 °C for 4 h. After cooling to

room temperature and addition with EtOAc, the combined organic

layers were washed with H₂O and brine, dried (Na₂SO₄), and ﬁltered.

The ﬁltrate was concentrated under reduced pressure and the residue

was puriﬁed by reversed phase HPLC to give the desired N-protected

dicarbonate ester 16a₁₁(R = R₁= -CH₂OCOO-i-Pr) as a white solid

(1.4 g, 34.3%). ESI MS m/z 792 [M + H]⁺.

MHz, DMSO-d₆) δ 9.99 (s, 1H), 8.10 (s, 1H), 7.76 (d, J = 7.6 Hz,

1H), 7.58 (d, J = 7.6 Hz, 1H), 7.30 (dd, J₁= J₂= 7.6 Hz, 1H), 5.81 (d,

J = 6.0 Hz, 1H), 5.71 (d, J = 6.0 Hz, 1H), 5.12 (brs, 1H), 4.20−4.23

(m, 1H), 3.95−3.98 (m, 1H), 3.81−3.85 (m, 1H), 3.24−3.65 (m, 5H),

2.58−2.70 (m, 2H), 1.76−1.79 (m, 2H), 1.60−1.69 (m, 3H), 1.48−

1.51 (m, 1H), 1.10−1.36 (m, 11H). ESI MS m/z 616.2 [M + H]⁺,

638.3 [M + Na]⁺. HPLC purity: 96.8%.

Synthesis of Ertapenem Bis-acetoxymethyl Ester (10k).

Reaction of acetoxymethyl chloride with 15 aﬀorded 16k₁₀(R = R₁

= CH₂OCOMe) followed by upon deprotection and puriﬁcation by

reversed phase HPLC yielded the diacetoxy diester 10k in 2.3% yield

1

as a colorless powder. H NMR (400 MHz, CDCl₃) δ 9.64 (s, 1H),

8.09 (d, J = 8.4 Hz, 1H), 8.04 (s, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.43

(dd, J₁= 8.4 Hz, J₂= 7.6 Hz, 1H), 5.97 (s, 2H), 5.86 (d, J = 5.6 Hz,

1H), 5.81 (d, J = 5.6 Hz, 1H), 4.20−4.23 (m, 2H), 3.97−4.01 (m,

1H), 3.60−3.67 (m, 1H), 3.45−3.49 (m, 1H), 3.30−3.37 (m, 1H),

3.22−3.24 (m, 1H), 2.88−2.93 (m, 1H), 2.73−2.81 (m, 1H), 2.38

(brs, 1H), 2.12 (s, 3H), 2.08 (s, 3H), 1.98−2.08 (m, 1H), 1.79 (brs,

1H), 1.34 (d, J = 6.4 Hz, 3H), 1.25 (d, J = 7.2 Hz, 3H). ESI MS m/z

620.2 [M + H]⁺. HPLC purity: 95.9%.

To a solution of compound 16a₁₁(600 mg, 0.75 mmol) in DMF

(2.0 mL) were added phenylsilane (162 mg, 1.50 mmol) and

tetrakis(triphenylphosphine)palladium (60 mg, 0.052 mmol), and the

mixture was stirred at room temperature for 10 min. EtOAc was

added, and the organic solution was washed with H₂O and brine, dried

(Na₂SO₄), and ﬁltered. The ﬁltrate was concentrated under reduced

pressure. The residue was puriﬁed by reverse phase HPLC on a

GILSON 281 instrument ﬁtted with a Phenomenex Gemini C₁₈(250

mm × 21.2 mm, 5 μm) column, eluting with a water and acetonitrile

gradient (mobile phase A, water; mobile phase B, acetonitrile;

gradient, 52−72% B, 0−8 min; 100% B, 8.5−10.5 min; 5% B, 11−

12 min) followed by lyophilization to give the desired compound 11c

Synthesis of Ertapenem Bis(propionyloxy)methyl Ester

(10l). Propionyloxyl methylchloride was reacted with N-allyloxy

carbamate 15, which furnished 16l₁₀(R = R₁= CH₂OCOEt),

followed by deprotection with phenylsilane and puriﬁcation by

reversed phase HPLC to provide the desired diester 10l in 4.7%

1

yield as a colorless powder. H NMR (400 MHz, CDCl₃) δ 9.66 (s,

1H), 8.03−8.09 (m, 2H), 7.80 (d, J = 7.6 Hz, 1H), 7.42 (dd, J₁= J₂

=7.6 Hz, 1H), 5.98 (s, 2H), 5.88 (d, J = 5.6 Hz, 1H), 5.82 (d, J = 5.6

Hz, 1H), 4.18−4.24 (m, 2H), 3.99−4.03 (m, 1H), 3.60−3.68 (m, 1H),

3.45−3.50 (m, 1H), 3.32−3.36 (m, 1H), 3.22−3.24 (m, 1H), 2.89−

2.93 (m, 1H), 2.73−2.81 (m, 1H), 2.33−2.42 (m, 4H), 1.98−2.05 (m,

1H), 1.33 (d, J = 6.0 Hz, 3H), 1.25 (d, J = 7.6 Hz, 3H), 1.09−1.16 (m,

6H). ESI MS m/z 648.2 [M + H]⁺. HPLC purity: 95.5%.

Synthesis of Ertapenem Bis-1-(pivaloyloxy)ethyl Ester

(10m). Reaction of 1-chloroethyl pivalate with 15 provided 16m₁₀

(R = R₁= -CH(Me)OCO-t-Bu), which was followed by phenylsilane

deprotection and reversed phase HPLC puriﬁcation to furnish the

diester 10m in 4.4% yield as a colorless powder. ¹H NMR (400 MHz,

CDCl₃) δ 9.68 (d, J = 5.6 Hz, 1H), 8.02−8.13 (m, 2H), 7.78 (d, J = 7.6

Hz, 1H), 7.42 (dd, J₁= J₂= 7.6 Hz, 1H), 7.06−7.09 (m, 1H), 6.88−

6.96 (m, 1H), 4.20−4.25 (m, 2H), 4.01−4.04 (m, 1H), 3.58−3.68 (m,

1

(210 mg, 39.6%) as a white powder. H NMR (400 MHz, CDCl₃) δ

9.65 (s, 1H), 8.08−8.10 (m, 2H), 7.82 (d, J = 7.6 Hz, 1H), 7.44 (dd, J₁

= J₂= 7.6 Hz, 1H), 5.99 (s, 2H), 5.89 (d, J = 5.6 Hz, 1H), 5.83 (d, J =

5.6 Hz, 1H), 4.87−4.95 (m, 2H), 4.21−4.26 (m, 2H), 3.99−4.03 (m,

1H), 3.63−3.67 (m, 1H), 3.46−3.50 (m, 1H), 3.33−3.37 (m, 1H),

3.23−3.25 (m, 1H), 2.89−2.94 (m, 1H), 2.75−2.80 (m, 1H), 1.99−

2.06 (m, 1H), 1.25−1.35 (m, 18H). ESI MS m/z 708.2 [M + H]⁺.

HPLC purity: 95.1%.

Synthesis of Ertapenem C-3-((Isopropoxycarbonyl)oxy)-

methyl Estersodium Salt (11d). To a solution of compound 15

(5.81 g, 10.0 mmol) in DMF (60 mL) were added DIPEA (1.26 g,

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9.76 mmol), NaI (1.49 g, 9.94 mmol), and chloromethyl isopropyl

carbonate (1.52 g, 9.96 mmol), and then the mixture was stirred at 45

°C for 5 h. The mixture was directly puriﬁed by reversed phase HPLC

on a GILSON 281 instrument ﬁtted with a Phenomenex Synergi C₁₈

(250 mm × 50 mm, 10 μm) using water (0.2% formic acid) and

acetonitrile as eluents (mobile phase A, water (0.2% formic acid);

mobile phase B, acetonitrile; gradient, B from 25% to 50% in 25 min;

ﬂow rate, 80 mL/min; detective wavelength, 220 nm.) followed by

freeze-drying to give desired product 16d₁₁(R = CH₂OCOO-i-Pr, R₁

= H) (380 mg, 5.6%) as a white solid.

1H), 8.08−8.09 (m, 2H), 7.82 (d, J = 7.6 Hz, 1H), 7.44 (dd, J₁= 8.8

Hz, J₂= 7.6 Hz, 1H), 6.00 (s, 2H), 5.89 (d, J = 5.6 Hz, 1H), 5.84 (d, J

= 5.6 Hz, 1H), 4.21−4.29 (m, 6H), 3.98−4.02 (m, 1H), 3.63−3.66 (m,

1H), 3.45−3.49 (m, 1H), 3.30−3.37 (m, 1H), 3.23−3.25 (m, 1H),

2.89−2.91 (m, 1H), 2.74−2.80 (m, 1H), 1.98−2.05 (m, 1H), 1.25−

1.34 (m, 12H). ESI MS m/z 680.2 [M + H]⁺. HPLC purity: 99.3%.

Synthesis of Ertapenem C-3-Methyl Propionate Carbonate

Sodium Salt (11h). Reaction of iodomethyl propionate with

ertapenem 4 followed by formation of sodium salt and puriﬁcation

by reversed phase HPLC gave 0.43% yield of desired C-3 monoester

1

sodium salt 11h (R = CH₂OCOOEt, R₁= Na) as a white solid. H

The mixture of compound 16d₁₁(380 mg, 0.56 mmol),

phenylsilane (120 mg, 1.12 mmol), and Pd(PPh₃)₄(45 mg) in

DMF (4 mL) was stirred at room temperature for 10 min and then

puriﬁed by reversed phase HPLC followed by freeze-drying to aﬀord

11d (200 mg, 60.1%, R₁= H) as a white powder. To a solution of the

free acid of 11d (200 mg, 0.34 mmol) in CH₃CN (20 mL) was slowly

added 100 mL of H₂O containing 28 mg of NaHCO₃(0.33 mmol) at

0−5 °C. Freeze-drying followed by puriﬁcation by reversed phase

HPLC on a GILSON 281 instrument ﬁtted with a Phenomenex

Gemini C₁₈(250 mm × 21.2 mm, 5 μm) using water and acetonitrile

as eluents (mobile phase A, water; mobile phase B, acetonitrile;

gradient, 52−72% B, 0−8 min; 100% B, 8.5−10.5 min; 5% B, 11−12

min) followed by freeze-drying aﬀorded the desired sodium

carboxylate 11d (88 mg, 42.5%, R₁= Na) as a white solid. ¹H

NMR (400 MHz, DMSO-d₆) δ 10.18 (s, 1H), 8.37 (s, 1H), 7.91 (d, J

= 7.6 Hz, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.50 (dd, J₁= J₂= 7.6 Hz,

1H), 5.86 (d, J = 6.0 Hz, 1H), 5.76 (d, J = 6.0 Hz, 1H), 5.16 (d, J = 5.2

Hz, 1H), 4.84−4.89 (m, 1H), 4.27−4.30 (m, 1H), 4.02−4.06 (m, 1H),

3.90−3.94 (m, 1H), 3.71−3.74 (m, 1H), 3.56−3.62 (m, 1H), 3.27−

3.50 (m, 2H), 3.05−3.13 (m, 1H), 2.60−2.80 (m, 1H), 1.72−1.79 (m,

1H), 1.27−1.32 (m, 12H). ESI MS m/z 592.2 [M + H]⁺, 614.2 [M +

Na]⁺. HPLC purity: 98.4%.

NMR (400 MHz, DMSO-d₆) δ 9.84 (s, 1 H), 7.89 (s, 1H), 7.64 (d, J =

7.6 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.17 (dd, J₁= 8.0 Hz, J₂= 7.6

Hz, 1H), 5.77 (d, J = 5.6 Hz, 1H), 5.69 (d, J = 5.6 Hz, 1H), 5.11 (brs,

1 H), 4.17−4.20 (m, 1H), 3.82−3.93 (m, 1H), 3.76−3.79 (m, 1H),

3.62−3.64 (m, 1H), 3.46−3.52 (m, 1H), 3.20−3.22 (m, 1H), 2.67−

2.70 (m, 2H), 2.28−2.38 (m, 3H), 1.53−1.60 (m, 1H), 1.15−1.18 (m,

6H), 0.87−0.92 (m, 3H). ESI MS m/z 562.2 [M + H]⁺, 584.2 [M +

Na]⁺. HPLC purity: 97.0%.

Synthesis of Ertapenem Bis((methoxycarbonyl)oxy)methyl

Ester (11i). Chloromethyl methyl carbonate was reacted with N-

allyloxy carbamate 15, which aﬀorded 16i₁₁(R = R₁

=

-CH₂OCOOMe), followed by deprotection with phenylsilane and

puriﬁcation by reversed phase HPLC to provide the desired dimethoxy

1

carbonate 11i in 11.8% yield as a colorless powder. H NMR (400

MHz, CDCl₃) δ 9.66 (s, 1H), 8.07−8.09 (m, 2H), 7.82 (d, J = 7.6 Hz,

1H), 7.44 (dd, J₁= J₂= 7.6 Hz, 1H), 6.00 (s, 2H), 5.89 (d, J = 5.6 Hz,

1H), 5.84 (d, J = 5.6 Hz, 1H), 4.22−4.26 (m, 2H), 3.98−4.02 (m,

1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.63−3.66 (m, 1H), 3.45−3.49 (m,

1H), 3.33−3.37 (m, 1H), 3.23−3.25 (m, 1H), 2.82−2.94 (m, 1H),

2.74−2.79 (m, 1H), 1.99−2.06 (m, 1H), 1.32 (d, J = 6.4 Hz, 3H), 1.24

(d, J = 7.2 Hz, 3H). ESI MS m/z 652.2 [M + H]⁺. HPLC purity:

98.5%.

Synthesis of Ertapenem C-3-Methyl Acetate Carbonate

Sodium Salt (11j). Reaction of iodomethyl acetate with ertapenem

4 followed by formation of sodium salt and puriﬁcation by reversed

phase HPLC gave desired C-3 monoester sodium salt 11j (R =

CH₂OCOMe, R₁= Na) in 0.44% yield as a white solid. ¹H NMR (400

MHz, DMSO-d₆) δ 10.08 (s, 1H), 8.27 (s, 1H), 7.81 (d, J = 7.6 Hz,

1H), 7.60 (d, J = 7.6 Hz, 1H), 7.40 (dd, J₁= J₂= 7.6 Hz, 1H), 5.72 (d,

J = 6.0 Hz, 1H), 5.68 (d, J = 6.0 Hz, 1H), 5.07 (d, J = 4.4 Hz, 1H),

4.17−4.20 (m, 1H), 3.91−3.94 (m, 1H), 3.79−3.83 (m, 1H), 3.60−

3.64 (m, 1H), 3.47−3.51 (m, 1H), 3.21−3.23 (m, 2H), 2.55−2.70 (m,

2H), 2.07 (s, 3H),1.63−1.70 (m, 1H), 1.04−1.18 (m, 6H). ESI MS m/

z 548.1 [M + H]⁺, 570.1 [M + Na]⁺. HPLC purity: 97.6%.

Synthesis of Ertapenem Bis((cyclohexylcarbonyl)oxy)methyl

Ester (11e). Reaction of chloromethyl cyclohexyl carbonate with 15

by using NaI and DIEA in DMF at 40 °C for 4 h gave 16e₁₁(R = R₁=

CH₂OCOOC₆H₁₃), which was followed by deprotection and

puriﬁcation by reversed phase HPLC to yield the desired dicarbonate

1

11e in 1.4% yield as a white powder. H NMR (400 MHz, CDCl₃) δ

9.80 (s, 1H), 8.10 (s, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.81 (d, J = 7.6

Hz, 1H), 7.43 (dd, J₁= J₂= 7.6 Hz, 1H), 5.99 (s, 2H), 5.89 (d, J = 5.6

Hz, 1H), 5.83 (d, J = 5.6 Hz, 1H), 4.63−4.71 (m, 2H), 4.24−4.26 (m,

2H), 3.54−3.72 (m, 2H), 3.31−3.39 (m, 1H), 3.24−3.25 (m, 1H),

2.98−3.08 (brs, 1H), 2.78−2.88 (m, 1H), 1.98−2.08 (m, 1H), 1.68−

1.96 (m, 9H), 1.13−1.58 (m, 20H). ESI MS m/z 788.3 [M + H]⁺,

810.2 [M + Na]⁺. HPLC purity: 99.7%.

Synthesis of Ertapenem Bis(5-methyl-2-oxo-1,3-dioxol-4-

yl)methyl Ester (12a). After addition of 4-(chloromethyl)-5-

methyl-1,3-dioxol-2-one (2.29 g, 15.5 mmol) to a solution of

compound 15 (3.0 g, 5.1 mmol) in DMF (15 mL) at room

temperature, N-ethyldiisopropylamine (2.0 g, 15.5 mol) was added to

the mixture. The mixture was stirred at 40 °C for 4 h. After cooling to

room temperature and addition with EtOAc, the combined organic

layers were washed with H₂O and brine, dried (Na₂SO₄), and ﬁltered.

The ﬁltrate was concentrated in vacuo and the residue was puriﬁed by

reversed phase HPLC to give the desired N-protected dicyclic

carbonate ester 16a₁₂(R = R₁= -CH₂-c-(C(OCOO)C-Me) as a

white solid (600 mg, 14.8%). ESI MS m/z 784 [M + H]⁺.

To a solution of 16a₁₂(600 mg, 0.77 mmol) in DMF (3.0 mL) was

added phenylsilane (166 mg, 1.54 mmol) and tetrakis(triphenyl-

phosphine)palladium (112 mg, 0.097 mmol), and the mixture was

stirred at room temperature for 10 min. EtOAc was added, and the

organic solution was washed with H₂O and brine, dried (Na₂SO₄), and

ﬁltered. The ﬁltrate was concentrated under reduced pressure. The

residue was puriﬁed by reversed phase HPLC on a GILSON 281

instrument ﬁtted with a Phenomenex Gemini C₁₈(250 mm × 21.2

mm, 5 μm) column, eluting with a water and acetonitrile gradient

(mobile phase A, water; mobile phase B, acetonitrile; gradient, 52−

72% B, 0−8 min; 100% B, 8.5−10.5 min; 5% B, 11−12 min) followed

by lyophilization to aﬀord the desired compound 12a (91 mg, 17%) as

a colorless powder. ¹H NMR (400 MHz, CDCl₃) δ 9.60 (s, 1H), 8.08

Synthesis of Ertapenem C-3-Methyl Cycloxexyl Carbonate

Sodium Salt (11f). To a solution of chloromethyl cyclohexyl

carbonate (1.92 g, 10 mmol) in acetone (60 mL) was added NaI

(4.47 g, 30 mmol), and the mixture was stirred at reﬂux for 18 h. The

mixture was ﬁltered, concentrated, and then diluted with DCM and

ﬁltered again. The ﬁltrate was concentrated to give the desired product

cyclohexyl (iodomethyl) carbonate (4.0 g, 91.5% yield) as a brown oil

which was used directly in the next step without further puriﬁcation.

Reaction of cyclohexyl (iodomethyl) carbonate with ertapenem 4

followed by formation of sodium salt and puriﬁcation by reversed

phase HPLC gave 4.1% yield of desired C-3 monocarbonate sodium

salt 11f (R = -CH₂OCOOc-C₆H₁₃, R₁= Na) as a white solid. ¹H NMR

(400 MHz, DMSO-d₆) δ 10.26 (s, 1H), 8.28 (s, 1H), 7.85 (d, J = 8.0

Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.45 (dd, J₁= J₂= 8.0 Hz, 1H), 5.81

(d, J = 6.0 Hz, 1H), 5.71 (d, J = 6.0 Hz, 1H), 5.10 (d, J = 5.2 Hz, 1H),

4.52−4.60 (m, 1H), 4.22−4.25 (m, 1H), 3.97−4.00 (m, 2H), 3.70−

3.80 (m, 1H), 3.48−3.52 (m, 2H), 2.58−2.85 (m, 2H), 1.61−1.83 (m,

6H), 1.22−1.46 (m, 7H), 1.13−1.18 (m, 6H). ESI MS m/z 632.2 [M

+ H]⁺, 654.2 [M + Na]⁺. HPLC purity: 95.0%.

Synthesis of Ertapenem Bis((ethoxycarbonyl)oxy)methyl

Ester (11g). Chloromethyl ethyl carbonate was reacted with

compound 15, which furnished 16g₁₁(R = R₁= CH₂OCOOEt),

which was followed by phenylsilane deprotection and reversed phase

HPLC puriﬁcation to furnish 11.3% yield of the diethoxy carbonate

1

11g as a colorless powder. H NMR (400 MHz, CDCl₃) δ 9.66 (s,

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(s, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.74 (d, J = 7.6 Hz, 1H), 7.40 (dd, J₁

= J₂= 7.6 Hz, 1H), 5.05 (s, 2H), 4.82−4.96 (m, 2H), 4.15−4.22 (m,

2H), 3.90−4.00 (m, 1H), 3.15−3.60 (m, 4H), 2.70−2.92 (m, 2H),

2.18 (s, 3H), 2.12 (s, 3H), 1.95−2.04 (m, 1H), 1.28 (d, J = 6.4 Hz,

3H), 1.18 (d, J = 7.2 Hz, 3H). ESI MS m/z 700.2 [M + H]⁺. HPLC

purity: 95.1%.

Synthesis of Ertapenem C-3-Sodium Carboxyl(4-methyl-5-

methyl-1,3-dioxol-2-one)benzoate (12b). Reaction of 1.0 equiv of

4-(chloromethyl)-5-methyl-1,3-dioxol-2-one (300 mg, 2.0 mmol) with

compound 19 (1.46 g, 2.0 mmol) followed by puriﬁcation by silica gel

columm chromatography aﬀorded desired benzoate 20b₁₂(R = CH₂-c-

(C(OCOO)C-Me) (400 mg, 23.7%) as a white solid. ESI MS m/z

807.2 [M + H]⁺. 20b₁₂was deprotected by hydrogenolysis and

puriﬁed by reversed HPLC to aﬀord 12b (11 mg, 1.3%) as a white

solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.23 (s, 1H), 7.77 (d, J = 7.6

Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.42 (dd, J₁= J₂= 7.6 Hz, 1H), 4.96

(s, 2H), 4.13−4.16 (m, 1H), 3.88−3.94 (m, 2H), 3.37−3.48 (m, 2H),

3.20−3.22 (m, 1H), 2.73−2.78 (m, 1H), 2.60−2.67 (m, 2H), 2.07 (s,

3H), 1.69−1.74 (m, 1H), 1.09−1.18 (m, 6H). ESI MS m/z 588.1 [M

+ H]⁺, 610.2 [M + Na]⁺. HPLC purity: 95.2%.

acetonitrile; gradient, 52−72% B, 0−8 min; 100% B, 8.5−10.5 min; 5%

B, 11−12 min) followed by lyophilization to aﬀord the desired diethyl

1

ester 13a (80 mg, 11.3%) as a white powder. H NMR (400 MHz,

CDCl₃) δ 9.65 (s, 1H), 8.07 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.79 (d,

J = 7.6 Hz, 1H), 7.41 (dd, J₁= 8.0 Hz, J₂= 7.6 Hz, 1H), 4.20−4.40 (m,

6H), 3.95−4.06 (m, 1H), 2.74−3.64 (m, 6H), 1.98−2.07 (m, 1H),

1.23−1.42 (m, 12H). ESI MS m/z 532.2 [M + H]⁺. HPLC purity:

96.8%.

Synthesis of Ertapenem C-3-Ethyl Ester (13b) and Ethyl

Benzoate (13c). N-Ethyldiisopropylamine (433 mg, 3.44 mol) was

slowly added to a solution of compound 15 (5.8 g, 10.0 mmol) and

iodoethane (1.4 g, 10.0 mmol) in DMF (30 mL), and the mixture was

stirred at 45 °C for 4 h. The mixture was directly puriﬁed by reversed

phase HPLC on a GILSON 281 instrument ﬁtted with a Phenomenex

Synergi C₁₈(250 mm × 50 mm, 10 μm) using water (0.1% formic

acid) and acetonitrile as eluents (mobile phase A, water (0.1% formic

acid); mobile phase B, acetonitrile; gradient, B from 25% to 50% in 25

min; ﬂow rate, 80 mL/min; detective wavelength, 220 nm) followed

by freeze-drying to give 16b₁₃(R = Et, R₁= H) (460 mg, 7.8%) and

1

16c₁₃(R = H, R₁= Et) (420 mg, 6.9%) as a white solid. 16b₁₃: H

NMR (400 MHz, DMSO-d₆) δ 10.26 (s, 1H), 8.22−8.25 (m, 1H),

7.84−7.86 (m, 1H), 7.64 (d, J = 7.6 Hz, 1H), 7.43 (dd, J₁= J₂= 7.6

Hz, 1H), 5.72−5.92 (m, 1H), 4.94−5.36 (m, 3H), 3.71−4.61 (m,

11H), 3.23−3.33 (m, 2H), 2.71−2.87 (m, 1H), 1.71−1.88 (m, 1H),

1.14−1.33 (m, 9H). ESI MS m/z 588 [M + H]⁺. 16c₁₃: ¹H NMR (400

MHz, DMSO-d₆) δ 10.32 (s, 1H), 8.23−8.27 (m, 1H), 7.87 (d, J = 7.6

Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.46 (dd, J₁= J₂= 7.6 Hz, 1H),

5.72−5.94 (m, 1H), 5.00−5.36 (m, 3H), 4.28−4.52 (m, 5H), 3.77−

4.20 (m, 4H), 3.45−3.52 (m, 2H), 3.23−3.33 (m, 2H), 2.67−2.70 (m,

1H), 1.76−1.88 (m, 1H), 1.32 (t, J = 7.2 Hz, 3H), 1.05−1.22 (m, 6H).

ESI MS m/z 588 [M + H]⁺.

Synthesis of Ertapenem Bis(5-isopropyl-2-oxo-1,3-dioxol-4-

yl)methyl Ester (12c). Reaction of 4-(bromomethyl)-5-isopropyl-1,3-

dioxol-2-one²³with 15 provided 16c₁₂(R = R₁= -CH₂-c-

(C(OCOO)C-i-Pr), which was followed by deprotection and

puriﬁcation by reversed phase HPLC to yield the desired dicyclic

1

carbonate ester 12c in 9.4% yield as a white powder. H NMR (400

MHz, DMSO-d₆) δ 8.28 (s, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.68 (d, J =

7.6 Hz, 1H), 7.48 (dd, J₁= J₂= 7.6 Hz, 1H), 5.20 (s, 2H), 5.00−5.09

(m, 3H), 4.19−4.21 (m, 1H), 3.92−4.04 (m, 2H), 3.71−3.77 (m, 1H),

3.46−3.59 (m, 2H), 3.23−3.25 (m, 1H), 3.08−3.15 (m, 1H), 2.98−

3.06 (m, 1H), 2.79−2.85 (m, 1H), 1.76 (brs, 1H), 1.06−1.20 (m,

18H). ESI MS m/z 756.2 [M + H]⁺, 778.2 [M + Na]⁺. HPLC purity:

95.4%.

N-Allyloxycarbonyl 16b₁₃(200 mg, 0.34 mmol) was hydro-

genolyzed as described for 10c, aﬀording 13b (10 mg, 5.9%) as a

1

Synthesis of Ertapenem Bis(5-tert-butyl-2-oxo-1,3-dioxol-4-

yl)methyl Ester (12d). Reaction of 4-(bromomethyl)-5-(tert-butyl)-

1,3-dioxol-2-one²³with 15 gave 16d₁₂(R = R₁= -CH₂-c-

(C(OCOO)C-t-Bu), which was followed by deprotection and

puriﬁcation by reversed phase HPLC to yield the desired dicyclic

white powder. H NMR (400 MHz, DMSO-d₆) δ 10.15 (s, 1H), 8.30

(s, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.64 (d, J = 7.6 Hz, 1H), 7.43 (dd, J₁

= J₂= 7.6 Hz, 1H), 5.09 (d, J = 3.6 Hz, 1H), 4.08−4.20 (m, 3H),

3.90−3.97 (m, 1H), 3.82−3.88 (m, 1H), 3.61−3.66 (m, 1H), 3.22−

3.24 (m, 2H), 2.60−2.75 (m, 3H), 1.69−1.81 (m, 1H), 1.12−1.39 (m,

9H). ESI MS m/z 504.1 [M + H]⁺. HPLC purity: 91.3%.

1

carbonate ester 12d in 14.3% yield as a white powder. H NMR (400

MHz, CDCl₃) δ 9.65 (s, 1H), 8.06 (s, 1H), 8.01 (d, J = 7.6 Hz, 1H),

7.77 (d, J = 7.6 Hz, 1H), 7.42 (dd, J₁= J₂= 7.6 Hz, 1H), 5.18 (s, 2H),

5.07 (d, J = 14.0 Hz, 1H), 5.02 (d, J = 14.0 Hz, 1H), 4.20−4.22 (m,

2H), 3.98−4.08 (m, 1H), 3.58−3.68 (m, 1H), 3.45−3.52 (m, 1H),

3.28−3.38 (m, 1H), 3.20−3.23 (m, 1H), 2.82−2.94 (m, 1H), 2.75−

2.80 (m, 1H), 1.97−2.00 (m, 1H), 1.33 (s, 9H), 1.28 (s, 9H), 1.23−

1.36 (m, 6H). ESI MS m/z 784.2 [M + H]⁺, 806.3 [M + Na]⁺. HPLC

purity: 96.8%.

Synthesis of Ertapenem Diethyl Ester (13a). Iodoethane (805

mg, 5.16 mmol) was added to a solution of compound 15 (1.0 g, 1.66

mmol), N-ethyldiisopropylamine (430 mg, 3.32 mmol), and

benzyltriethylammonium chloride (378 mg, 1.66 mmol) in DMF

(10 mL), and the mixture was stirred at 45 °C for 18 h. After addition

of EtOAc (300 mL), the combined organic layer was washed with

H₂O (100 mL × 3) and brine (100 mL), dried (Na₂SO₄), and ﬁltered.

The ﬁltrate was concentrated under reduced pressure, and the residue

was puriﬁed by silica gel column chromatography, eluting with DCM/

MeOH (40:1). Fractions containing the product were combined and

concentrated under vacuo to give the desired diethyl ester 16a₁₃(R =

R₁= Et) as a pale yellow solid (820 mg, 80.4%). ESI MS m/z 616 [M

+ H]⁺.

To a solution of 16a₁₃(820 mg, 1.33 mmol) in DMF (4.0 mL) were

added phenylsilane (288 mg, 2.66 mmol) and tetrakis(triphenyl-

phosphine)palladium (80 mg, 0.069 mmol), and the mixture was

stirred at room temperature for 10 min. EtOAc (100 mL) was added,

and the organic solution was washed with H₂O (40 mL × 3) and brine

(100 mL), dried (Na₂SO₄), and ﬁltered. The ﬁltrate was concentrated

under reduced pressure. The residue was puriﬁed by reversed phase

HPLC on a GILSON 281 instrument ﬁtted with a Phenomenex

Gemini C₁₈(250 mm × 21.2 mm, 5 μm) column, eluting with a water

and acetonitrile gradient (mobile phase A, water; mobile phase B,

Similar deprotection of N-allyloxycarbonyl 16_c13(200 mg, 0.34

mmol) by hydrogenolysis aﬀorded 13c (25 mg, 14.7%) as a white

1

powder. H NMR (400 MHz, DMSO-d₆) δ 10.51 (s, 1 H), 8.32 (s, 1

H), 7.87 (d, J = 8.0 Hz, 1 H), 7.61−7.68 (d, J = 7.6 Hz, 1H), 7.48 (dd,

J₁= 8.0 Hz, J₂= 7.6 Hz, 1 H), 5.12 (brs, 1H), 4.31 (q, J = 7.2 Hz, 2H),

4.02−4.18 (m, 2H), 3.88−3.97 (m, 2H), 3.74−3.76 (m, 1H), 3.21−

3.23 (m, 2H), 2.87−2.91 (m, 1H), 2.70−2.76 (m, 1H), 1.74−1.81 (m,

1H), 1.31 (t, J = 7.2 Hz, 3H), 1.14−1.18 (m, 6H). ESI MS m/z 504.2

[M + H]⁺. HPLC purity: 90.1%.

Synthesis of Ertapenem Dimethyl Ester (13d). Reaction of the

carboxylic acid 15 (1.9 g, 18 mmol) and iodomethane (5.1 g, 36

mmol) gave dimethyl ester 16d₁₃(R = R₁= Me) (1.4 g, 27.7%) as a

colorless oil. ESI MS m/z 588 [M + H]⁺. The N-allyoxycarbonyl

carbamate 16d₁₃was deprotected by phenylsilane followed by reversed

phase HPLC puriﬁcation to aﬀord the desired dimethyl ester 13d

(400.0 mg, 33.3%) as a colorless powder. ¹H NMR (400 MHz,

CDCl₃) δ 9.64 (s, 1H), 8.09 (s, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.77 (d,

J = 7.6 Hz, 1H), 7.41 (dd, J₁= J₂= 7.6 Hz, 1H), 4.20−4.27 (m, 2H),

3.99−4.03 (m, 1H), 3.90 (s, 3H), 3.77 (s, 3H), 3.60−3.65 (m, 1H),

3.45−3.50 (m, 1H), 3.23−3.36 (m, 2H), 2.90−2.95 (m, 1H), 2.73−

2.82 (m, 1H), 2.00−2.07 (m, 1H), 1.34 (d, J = 6.4 Hz, 3H), 1.24 (d, J

= 7.6 Hz, 3H). ESI MS m/z 504.2 [M + H]⁺. HPLC purity: 99.3%.

Synthesis of Ertapenem Di-n-propyl Ester (13e). Reaction of

1-iodopropanewith the N-allyloxy carbonate 15 produced the di-n-

propyl ester 16e₁₃(R = R₁= n-C₃H₇), which was deprotected

accordingly by phenylsilane and puriﬁed by reversed phase HPLC to

1

give 4.5% yield of di-n-propyl ester 13e as a white powder. H NMR

(400 MHz, CDCl₃) δ 9.65 (s, 1H), 8.07 (s, 1H), 8.01 (d, J = 7.6 Hz,

1H), 7.79 (d, J = 7.6 Hz, 1H), 7.42 (dd, J₁= J₂= 7.6 Hz, 1H), 4.10−

4.30 (m, 6H), 3.99−4.03 (m, 1H), 3.60−3.63 (m, 1H), 3.46−3.50 (m,

1H), 3.23−3.33 (m, 2H), 2.90−2.95 (m, 1H), 2.75−2.80 (m, 1H),

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1.99−2.05 (m, 1H), 1.50−1.90 (m, 4H), 1.36 (d, J = 6.0 Hz, 3H), 1.26

(d, J = 6.8 Hz, 3H), 1.03 (t, J = 7.6 Hz, 3H), 0.94 (t, J = 7.6 Hz, 3H).

ESI MS m/z 560.2 [M + H]⁺. HPLC purity: 99.0%.

Synthesis of Ertapenem Ditriﬂuoroethyl Ester (13l). Reaction

of 15 with 1,1,1-triﬂuoro-2-iodoethane gave 16l₁₃(R = R₁= CH₂CF₃),

which was treated with phenylsilane and puriﬁed by reversed phase

HPLC to aﬀord 13l in 8.5% yield as a colorless powder. ¹H NMR (400

MHz, DMSO-d₆) δ 10.22 (s, 1H), 8.40 (s, 1H), 7.95 (d, J = 8.0 Hz,

1H), 7.69 (d, J = 8.0 Hz, 1H), 7.51 (dd, J₁= J₂= 8.0 Hz, 1H),), 5.11

(d, J = 5.2 Hz, 1H), 4.97−5.04 (m, 2H), 4.77−4.83 (m, 2H), 4.24 (d, J

= 9.6 Hz, 1H), 3.82−4.02 (m, 2H), 3.87 (brs, 1H), 3.26−3.72 (m,

3H), 2.55−2.72 (m, 2H), 1.70−1.74 (m, 1H), 1.14−1.19 (m, 6H). ESI

MS m/z 640.1 [M + H]⁺. HPLC purity: 99.1%.

Synthesis of Ertapenem C-3-Triﬂuoroethyl Ester (13m) and

Triﬂuoroethyl Benzoate Ester (13n). Reaction of 2,2,2-triﬂuor-

oethyl triﬂuoromethanesulfonate with compound 15 gave 16m₁₃(R =

CH₂CF₃, R₁= H) and 16n₁₃(R = H, R₁= CH₂CF₃), which were

separated as described for the synthesis of 13b and 13c. Deprotection

of 16m₁₃by hydrogenolysis followed by puriﬁcation by reversed

HPLC provided 2.8% yield of 13m as a white powder. ¹H NMR (400

MHz, DMSO-d₆) δ 10.97 (s, 1H), 8.40 (s, 1H), 7.94 (d, J = 7.6 Hz,

1H), 7.74 (d, J = 7.6 Hz, 1H), 7.56 (dd, J₁= J₂= 7.6 Hz, 1H), 5.10

(brs, 1H), 4.98−5.05 (m, 2H), 4.32−4.44 (m, 1H), 4.19−4.21 (m,

1H), 3.65−3.96 (m, 4H), 3.22−3.24 (m, 1H), 3.04−3.08 (m, 1H),

2.82−2.92 (m, 1H), 1.85−1.90 (m, 1H), 1.10−1.20 (m, 6H). ESI MS

m/z 558.1 [M + H]⁺. HPLC purity: 76.5%.

Deprotection of 16n₁₃by hydrogenolysis followed by puriﬁcation

by reversed HPLC provided 0.41% yield of 13n as a white powder. ¹H

NMR (400 MHz, DMSO-d₆) δ 10.20 (s, 1H), 8.29 (s, 1H), 7.84 (d, J

= 7.6 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.44 (dd, J₁= 8.0 Hz, J₂= 7.6

Hz, 1H), 5.11 (d, J = 4.8 Hz, 1H), 4.77−4.85 (m, 2H), 4.24−4.27 (m,

1H), 3.88−3.99 (m, 2H), 3.68−3.74 (m, 2H), 3.50−3.56 (m, 2H),

2.75−2.80 (m, 1H), 2.62−2.70 (m, 1H), 1.20−1.28 (m, 1H), 1.16−

1.22 (m, 6H). ESI MS m/z 558.2 [M + H]⁺. HPLC purity: 82.7%.

Synthesis of C-3-Triﬂuoroethyl-cholesteryl-acetyl-ertape-

nem-benzoate (13o). To a solution of compound 15 (11.6 g, 20.0

mmol) and 2,2,2-triﬂuoroethyl triﬂuoromethanesulfonate (4.6 g, 20.0

mmol) in DMF (30 mL) was slowly added N-ethyldiisopropylamine

(5.0 g, 30.0 mmol), and the mixture was heated at 45 °C for 3 h. The

mixture was puriﬁed by reversed phase HPLC on a GILSON 281

instrument ﬁtted with a Phenomenex Synergi C₁₈(250 mm × 50 mm,

10 μm) using water (0.1% formic acid) and acetonitrile as eluents

(mobile phase A, water (0.1% formic acid); mobile phase B,

acetonitrile; gradient, B from 25% to 50% in 25 min; ﬂow rate, 80

mL/min; detective wavelength, 220 nm) followed by lyophilization to

aﬀord the desired C-3-triﬂuoroethyl 16m₁₃(2.0 g, 15.6%) as a yellow

solid.

Synthesis of Ertapenem Diisopropyl Ester (13f). Isopropyl

iodide was reacted with N-allyloxycarbonyl ertapenem 15 to give

diisoproyl ester 16f₁₃(R = R₁= i-CH(CH₃)₂) which was deprotected

with phenylsilane and puriﬁed by reversed phase HPLC to furnish

16.1% overall yield of 13f as a colorless powder. ¹H NMR (400 MHz,

CDCl₃) δ 9.64 (s, 1H), 8.02 (s, 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.76 (d,

J = 7.6 Hz, 1H), 7.38 (dd, J₁= J₂= 7.6 Hz, 1H), 5.20−5.26 (m, 1H),

5.06−5.12 (m, 1H), 4.18−4.25 (m, 2H), 3.97−4.01 (m, 1H), 3.54−

3.62 (m, 1H), 3.45−3.49 (m, 1H), 3.20−3.32 (m, 2H), 2.87−2.92 (m,

1H), 2.73−2.80 (m, 1H), 1.97−2.04 (m, 1H), 1.23−1.36 (m, 18H).

ESI MS m/z 560.2 [M + H]⁺. HPLC purity: 97.6%.

Synthesis of Ertapenem C-3-Isopropyl Ester (13g) and

Isopropyl Benzoate (13h). Reaction of 2-iodopropane with

compound 15 followed by separation aﬀorded 16g₁₃(R = i-Pr, R₁=

H) and 16h₁₃(R = H, R₁= i-Pr) as described for the synthesis of 13b

and 13c. Hydrogenolysis of 16g₁₃followed by reversed HPLC

1

provided 0.23% yield of 13g as a white powder. H NMR (400 MHz,

DMSO-d₆) δ 10.34 (s, 1 H), 8.28 (s, 1H), 7.88 (d, J = 8.0 Hz, 1H),

7.66 (d, J = 7.6 Hz, 1H), 7.47 (dd, J₁= 8.0 Hz, J₂= 7.6 Hz, 1H), 5.09−

5.17 (m, 2H), 4.15−4.18 (m, 1H), 3.80−4.03 (m, 2H), 3.66−3.77 (m,

2H), 3.21−3.22 (m, 2H), 2.81−2.89 (m, 1H), 2.67−2.72 (m, 1H),

1.71−1.78 (m, 1H), 1.16−1.36 (m, 12H). 3.20−4.18 (m, 8H), 2.67−

2.89 (m, 2H), 1.71−1.78 (m, 1H), 1.28−1.36 (m, 6H), 1.16−1.18 (m,

6H). ESI MS m/z 518.2 [M + H]⁺. HPLC purity: 84.5%.

Hydrogenolysis of 16h₁₃followed by reversed HPLC furnished 13h

1

(0.54%) as a white powder. H NMR (400 MHz, DMSO-d₆) δ 10.19

(s, 1H), 8.30 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 7.6 Hz,

1H), 7.43 (dd, J₁= 8.0 Hz, J₂= 7.6 Hz, 1H), 5.09 (brs, 1H), 4.88−4.97

(m, 1H), 4.18−4.20 (m, 1H), 3.89−3.98 (m, 2H), 3.63−3.68 (m, 2H),

3.22−3.24 (m, 2H), 2.74−2.79 (m, 1H), 2.59−2.67 (m, 1H), 1.72−

1.78 (m, 1H), 1.10−1.28 (m, 12H). ESI MS m/z 518.2 [M + H]⁺.

HPLC purity: 81.6%.

Synthesis of Ertapenem Di-n-butyl Ester (13i). Reaction of 1-

iodobutane with 15 furnished 16i₁₃(R = R₁= n-Bu), which was

followed by deprotection and puriﬁcation by reversed phase HPLC to

yield the desired di-n-butyl ester 13i in 1.7% yield as a colorless

1

powder. H NMR (400 MHz, CDCl₃) δ 9.58 (s, 1H), 8.00 (s, 1H),

7.95 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.35 (dd, J₁= J₂=

7.6 Hz, 1H), 4.08−4.27 (m, 6H), 3.92−4.00 (m, 1H), 3.52−3.58(m,

1H), 3.38−3.45 (m, 1H), 3.17−3.26 (m, 2H), 2.84−2.88 (m, 1H),

2.68−2.75 (m, 1H), 1.92−1.98 (m, 1H), 1.25−1.78 (m, 9H), 1.29 (d, J

= 6.0 Hz, 3H), 1.19 (d, J = 7.2 Hz, 3H), 0.91 (t, J = 7.6 Hz, 3H), 0.82

(t, J = 7.6 Hz, 3H). ESI MS m/z 588.3 [M + H]⁺. HPLC purity:

96.6%.

To a solution of cholesteryl 2-chloroacetate (10.5 g, 22.68 mmol) in

acetone (300 mL) was added NaI (6.758 g, 45.36 mmol), and the

mixture was reﬂuxed for 18 h. After ﬁltration, the ﬁltrate was

concentrated under reduced pressure to aﬀored the desired 2-

iodoacetate which was used directly in the next step without further

puriﬁcation (10 g, 80% yield).

Synthesis of Ertapenem Dioctyl Ester (13j). Reaction of 1-

iodooctane with 15 gave 16j₁₃(R = R₁= n-C₈H₁₇), which was

followed by deprotection and puriﬁcation by reversed phase HPLC to

The mixture of 16m₁₃(2.0 g, 3.12 mmol), cholesteryl 2-iodoacetate

(1.72 g, 3.12 mmol), Na₂CO₃(0.66 g, 6.24 mmol), and

benzyltriethylammonium chloride (0.70 g, 3.12 mmol) in DMSO

(40 mL) was stirred at 50 °C for 18 h. The reaction was quenched

with water (150 mL), extracted with DCM (100 mL × 3), dried

(Na₂SO₄), and ﬁltered. The ﬁltrate was concentrated under reduced

pressure. The residue was puriﬁed by silica gel column chromatog-

raphy, eluting with DCM/MeOH (40:1) and lyophilized to aﬀord 1.0

g of cholesteryl-acetyl-ertapenem-benzoate 16o₁₃(R = CH₂CF₃, R₁=

CH₂CO-cholesteryl) as a yellow solid. N-Allyoxycarbonyl cholesteryl-

acetyl-ertapenem-benzoate 16o₁₃(1.0 g, 0.94 mmol) was deprotected

with phenylsilane and puriﬁed by reversed phase HPLC to provide

1

give 17.1% yield of diester 13j as a colorless powder. H NMR (400

MHz, DMSO-d₆) δ 10.11 (s, 1H), 8.30 (s, 1H), 7.86 (d, J = 7.6 Hz,

1H), 7.60 (d, J = 7.6 Hz, 1H), 7.41 (dd, J₁= J₂= 7.6 Hz, 1H), 5.05 (d,

J = 5.2 Hz, 1H), 4.10−4.26 (m, 3H), 3.90−4.08 (m, 3H), 3.71−3.82

(m, 1H), 3.51−3.58 (m, 1H), 3.35−3.48 (m, 1H), 3.12−3.19 (m, 1H),

2.45−2.70 (m, 2H), 1.62−1.67 (m, 3H), 1.10−1.52 (m, methylene),

0.65−0.75 (m, 6H). ESI MS m/z 700.4 [M + H]⁺. HPLC purity:

99.7%.

Synthesis of Ertapenem Dihexadecyl Ester (13k). Reaction of

15 with hexadecyl iodide gave 16k₁₃(R = R₁= n-C₁₆H₃₃), which was

followed by deprotection with phenylsilane and reversed phase HPLC

puriﬁcation to produce 1.7% yield of diester 13k as a colorless powder.

¹H NMR (400 MHz, DMSO-d₆) δ 10.12 (s, 1H), 8.34 (s, 1H), 7.88

(d, J = 7.6 Hz, 1H), 7.63 (d, J = 7.2 Hz, 1H), 7.43 (dd, J₁= J₂= 7.6 Hz,

1H), 5.07 (d, J = 4.8 Hz, 1H), 4.16−4.36 (m, 3H), 3.95−4.07 (m,

3H), 3.82−3.86 (m, 1H), 3.58−3.68 (m, 2H), 3.11−3.15 (m, 1H),

2.67−2.72 (m, 2H), 1.67−1.74 (m, 3H), 1.14−1.52 (m, methylenes),

0.80−0.90 (m, 6H). ESI MS m/z 925.5 [M + H]⁺. HPLC purity:

98.7%.

1

13o (300 mg, 8.9% in 2 steps) as a pale brown solid. H NMR (400

MHz, CDCl₃) δ 9.69 (s, 1H), 8.14 (s, 1H), 8.02 (d, J = 7.6 Hz, 1H),

7.81 (d, J = 7.6 Hz, 1H), 7.45 (dd, J₁= J₂= 7.6 Hz, 1H), 5.30−5.35

(m, 1H), 4.60−4.80 (m, 4H), 4.23−4.26 (m, 2H), 3.98−4.04 (m, 1H),

3.63−3.70 (m, 1H), 3.45−3.52 (m, 1H), 3.32−3.40 (m, 1H), 3.24−

3.28 (m, 1H), 2.92−2.95 (m, 1H), 2.74−2.82 (m, 1H), 2.28−2.32 (m,

2H), 1.76−2.06 (m, 8H), 0.84−1.70 (m, 36H), 0.98 (s, 3H), 0.65 (s,

3H). ESI MS m/z 985.3 [M + H]⁺. HPLC purity: 92.7%.

8440

dx.doi.org/10.1021/jm500879a | J. Med. Chem. 2014, 57, 8421−8444

Journal of Medicinal Chemistry

Article

Synthesis of 8-Acetyl-ertapenem Diethyl Ester (13p). Acetyl

chloride (0.32 g, 4.8 mmol) was added to a solution of compound

16a₁₃(1.0 g, 1.6 mmol) and DMAP (20 mg) in pyridine (25 mL), and

the mixture was stirred at room temperature for 4 h. After addition of

water, the mixture was extracted with EtOAc, washed with H₂O, dried

(Na₂SO₄), and ﬁltered. The ﬁltrate was concentrated under reduced

pressure. The residue was puriﬁed by silica gel column chromatog-

raphy, eluting with DCM/MeOH (40:1), and then it was concentrated

under vacuo to give the desired product 8-acylated compound 22a

(460 mg, 43.8%) as a pale yellow solid. ESI MS m/z 658 [M + H]⁺.

The N-allyloxycarbonyl group of compound 22a was removed by

upon deprotection with phenylsilane followed by reversed phase

HPLC puriﬁcation and lyophilization to aﬀord the 8-acetyl diethyl

(645 mg, 5.0 mmol) and 4-(chloromethyl)-5-methyl-1,3-dioxol-2-one

(555 mg, 3.75 mmol), and the mixture was stirred at room

temperature for 2 h. The mixture was puriﬁed by reversed phase

HPLC on a GILSON 281 instrument ﬁtted with a Phenomenex

Gemini C₁₈(250 mm × 21.2 mm, 5 μm) using water (0.2%

HCOONH₄) and acetonitrile as eluents (mobile phase A, water;

mobile phase B, acetonitrile; gradient, 52−72% B, 0−8 min; 100% B,

8.5−10.5 min; 5% B, 11−12 min) followed by lyophilization to aﬀord

the desired compound 13s (336 mg, 20.8%) as a white solid. ¹H NMR

(400 MHz, CDCl₃) δ 9.20 (s, 1H), 8.24 (s, 1H), 7.91 (d, J = 8.0 Hz,

1H), 7.81 (d, J = 8.0 Hz, 1H), 7.43 (dd, J₁= J₂= 8.0 Hz,1H), 4.38 (q, J

= 7.2 Hz, 2H), 4.24−4.32 (m, 4H), 3.84 (brs, 1H), 3.68 (d, J = 15.2

Hz, 1H), 3.57 (d, J = 15.2 Hz, 1H), 3.36−3.40 (m, 1H), 3.20−3.28

(m, 3H), 3.05−3.09 (m, 1H), 2.82−2.90 (m, 1H), 2.10 (s, 3H), 2.01−

2.07 (m, 1H), 1.51−1.53 (m, 1H), 1.35−1.44 (m, 6H), 1.23−1.30 (m,

1

ester 13p (199 mg, 37.8%) as a colorless amorphous. H NMR (400

MHz, DMSO-d₆) δ 10.12 (s, 1 H), 8.31 (s, 1H), 7.87 (d, J = 8.0 Hz,

1H), 7.63 (d, J = 8.0 Hz, 1H), 7.43 (dd, J₁= J₂= 8.0 Hz, 1H), 5.08−

5.15 (m, 1H), 4.29 (q, J = 7.2 Hz, 2H), 4.18−4.21 (m, 1H), 4.04−4.11

(m, 2H), 3.80−3.84 (m, 1H), 3.55−3.62 (m, 2H), 3.46−3.51 (m, 1H),

3.28−3.38 (m, 1H), 2.50−2.69 (m, 2H), 2.00 (s, 3H), 1.61−1.71 (m,

1H), 1.30 (t, J = 7.2 Hz, 3H), 1.24 (d, J = 6.4 Hz, 3H), 1.11−1.18 (m,

6H). ESI MS m/z 574.2 [M + H]⁺. HPLC purity: 98.3%.

Synthesis of 8-Propyonyl-ertapenem Diethyl Ester (13q). To

a solution of 16a₁₃(1.5 g, 2.5 mmol) in DCM (25 mL) were added 1-

ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI,

3.8 g, 19.8 mmol), DMAP (300 mg, 2.46 mmol), and then propionic

acid (740 mg, 10 mmol). The mixture was stirred at −20 °C for 1 h.

The reaction was quenched with water (80 mL), and it was extracted

with DCM (30 mL × 3), washed with brine, dried over Na₂SO₄, and

ﬁltered. After concentration of ﬁltrate by vacuo, it was puriﬁed by

reversed phase HPLC on a GILSON 281 instrument ﬁtted with a

Phenomenex Gemini C₁₈(250 mm × 21.2 mm, 5 μm) using water

(0.2% HCOONH₄) and acetonitrile as eluents (mobile phase A, water

(0.2% HCOONH₄); mobile phase B, acetonitrile; gradient, 52−72%

B, 0−8 min; 100% B, 8.5−10.5 min; 5% B, 11−12 min) followed by

freeze-drying to give the desired propionate 22b (1.5 g. 94%) as a

yellow solid. ESI MS m/z 672 [M + H]⁺.

+

6H). ESI MS m/z 644.3 [M + H] , 666.2 [M + Na]⁺. HPLC purity:

97. 1%.

Synthesis of Ertapenem (5-Methyl-2-oxo-1,3-dioxol-4-yl)-

methyl N-carbamate Diethyl Ester (13t). Reaction of 1.0 equiv (5-

methyl-2-oxo-1,3-dioxol-4-yl)methyl (4-nitrophenyl) carbonate (pre-

pared as described by Alexander et al.²⁴) (885 mg, 3.0 mmol) with

ertapenem diethyl ester 13a (1.6 g, 3.0 mmol) followed by reversed

phase HPLC puriﬁcation and lyophilization aﬀorded the desired

compound 13t (605 mg, 29.2%) as a white solid. ¹H NMR (400 MHz,

CDCl₃) δ 8.82 (brs, 1H), 8.01 (brs, 1H), 7.93 (d, J = 7.6 Hz, 1H), 7.80

(d, J = 7.6 Hz, 1H), 7.41 (dd, J₁= J₂= 7.6 Hz,1H), 4.80−5.04 (m,

2H), 4.45−4.58 (m, 1H), 4.37 (q, J = 7.2 Hz, 2H), 4.20−4.30 (m,

4H), 3.92−4.02 (m, 1H), 3.75−3.85 (m, 1H), 3.38−3.64 (m, 1H),

3.25−3.31 (m, 2H), 2.57−2.78 (m, 2H), 2.02−2.22 (m, 4H), 1.30−

1.41 (m, 6H), 1.23−1.29 (m, 6H). ESI MS m/z 688.2 [M + H]⁺, 710.2

[M + Na]⁺. HPLC purity: 96.9%.

Synthesis of Ertapenem Macrocyclic Propyl-bis-lactone

(14a). Reaction of 1,3-dibromopropane with N-allylcarbamate 15

gave protected cyclic product 17a (R = CH₂CH₂CH₂), which was

followed by deprotection with phenylsilane followed by reversed phase

HPLC puriﬁcation to aﬀord the macrocyclic bis-lactone 14a in 3.7%

1

yield as a colorless powder. H NMR (400 MHz, DMSO-d₆) δ 10.07

N-Allyloxycarbonyl group of compound 22b was removed by upon

deprotection with phenylsilane, followed by reversed phase HPLC

puriﬁcation and lyophilization to aﬀord the 8-propyonyl ertapenem

diethyl ester 13q (1.0 g, 77%) as a colorless amorphous compound. ¹H

NMR (400 MHz, CDCl₃) δ 9.65 (s, 1H), 8.06 (s, 1H), 8.02 (d, J = 8.0

Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.41 (dd, J₁= J₂= 8.0 Hz,1H),

5.25−5.29 (m, 1H), 4.38 (q, J = 7.2 Hz, 2H), 4.26 (q, J = 7.2 Hz, 2H),

4.16−4.19 (m, 1H), 3.98−4.02 (m, 1H), 3.58−3.64 (m, 1H), 3.45−

3.50 (m, 1H), 3.36−3.38 (m, 1H), 3.24−3.30 (m, 1H), 2.89−2.94 (m,

1H), 2.72−2.82 (m, 1H), 2.34 (q, J = 7.6 Hz, 2H), 2.01−2.05 (m,

1H), 1.38−1.42 (m, 6H), 1.24−1.30 (m, 6H), 1.15 (t, J = 7.6 Hz, 3H).

ESI MS m/z 588.2 [M + H]⁺, 610.2 [M + Na]⁺. HPLC purity: 98.3%.

Synthesis of 8-(S)-Valinyl-ertapenem Diethyl Ester (13r).

Condensation of (S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanoic

acid (1.4 g, 6.4 mmol) with compound 16a₁₃(1.0 g, 1.6 mmol) by

using the above procedure followed by silica gel column puriﬁcation,

eluting with DCM/MeOH (40:1), and concentration by vacuo

aﬀorded the desired 8-(S)-N-allyloxycarbonyl-valinyl-ertapenem 22c

(1.0 g, 72%) as a yellow solid. ESI MS m/z799 [M + H]⁺. The two

allyloxycarbonyl groups in compound 22c (1.0 g, 1.2 mmol) were

removed upon deprotection with phenylsilane followed by reversed

phase HPLC puriﬁcation and lyophilization to aﬀord the 8-(S)-valinyl-

(s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.61−7.67 (m, 2H), 7.46 (dd, J₁= J₂

= 7.6 Hz, 1H), 5.06 (d, J = 4.7 Hz, 1H), 4.56−4.65 (m, 1H), 4.38−

4.41 (m, 1H), 4.23−4.26 (m, 1H), 3.80−4.03 (m, 5H), 3.38−3.45 (m,

2H), 3.18−3.24 (m, 2H), 2.54−2.65 (m, 2H), 2.24−2.30 (m, 1H),

1.82−1.91 (m, 2H), 1.11−1.14 (m, 6H). ESI MS m/z 516.2 [M + H]⁺.

HPLC purity: 97.0%.

Synthesis of Ertapenem Macrocylic butyl-bis-lactone (14b).

1,4-Dibromobutane was reacted with intermediate 15 and aﬀorded

17b (R = CH₂CH₂CH₂CH₂), which was followed by deprotection

with phenylsilane and puriﬁcation by reversed phase HPLC

puriﬁcation to furnish the desired macrocyclic bis-lactone 14b in

1

0.92% yield as an amorphous powder. H NMR (400 MHz, DMSO-

d₆) δ 10.02 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.77 (s, 1H), 7.62 (d, J =

7.2 Hz, 1H), 7.46 (dd, J₁= 8.0 Hz, J₂= 7.2 Hz, 1H), 5.05 (d, J = 4.0

Hz, 1H), 4.34−4.43 (m, 2H), 4.17−4.22 (m, 2H), 3.88−3.96 (m, 2H),

3.78−3.80 (m, 2H), 3.38−3.45 (m, 2H), 3.20−3.21 (m, 1H), 3.05−

3.08 (m, 1H), 2.50−2.66 (m, 2H), 1.63−1.90 (m, 5H), 1.08−1.15(m,

6H). ESI MS m/z 530.2 [M + H]⁺. HPLC purity: 98.1%.

Synthesis of Ertapenem Macrocylic (Z)-But-2-ene-bis-lac-

tone (14c). Reaction of (Z)-1,4-dichlorobut-2-ene with 15 furnished

17f (R = CH₂CHCH-CH₂), which was followed by deprotection

with phenylsilane and puriﬁcation by reversed phase HPLC to give

0.96% yield of desired bis-lactone 14c as white powder. ¹H NMR (400

MHz, DMSO-d₆) δ 10.05 (s, 1H), 8.17 (brs, 1H), 8.08 (brs, 1H), 7.68

(d, J = 7.6 Hz, 1H), 7.51 (dd, J₁= J₂= 7.6 Hz, 1H), 5.78−5.93 (m,

2H), 5.33−5.38 (m, 1H), 5.09−5.10 (m, 1H), 4.88−4.94 (m, 1H),

4.74−4.78 (m, 1H), 4.45−4.49 (m, 1H), 4.21−4.24 (m,1H), 3.89−

3.97 (m, 3H), 3.46−3.49 (m, 2H), 3.14−3.24 (m, 3H), 2.67−2.74 (m,

1 H), 1.86−1.92 (m, 1H), 1.10−1.20 (m, 6H). ESI MS m/z 528.2 [M

+ H]⁺. HPLC purity: 88.8%.

1

ertapenem diethyl ester 13r (100 mg, 13%) as a white powder. H

NMR (400 MHz, CDCl₃) δ 9.65 (s, 1H), 8.05 (s, 1H), 7.99 (d, J = 7.6

Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.39 (dd, J₁= J₂= 7.6 Hz, 1H),

5.28−5.35 (m, 1H), 4.36 (q, J = 7.2 Hz, 2H), 4.24 (q, J = 7.2 Hz, 2H),

4.13−4.16 (m, 1H), 3.96−4.04 (m, 1H), 3.52−3.64 (m, 1H), 3.46−

3.50 (m, 1H), 3.26−3.36 (m, 3H), 2.89−2.93 (m, 1H), 2.72−2.82 (m,

1H), 1.98−2.05 (m, 2H), 1.36−1.41 (m, 6H), 1.22−1.28 (m, 6H),

0.98 (d, J = 6.8 Hz, 3H), 0.90 (d, J = 6.8 Hz, 3H). ESI MS m/z 631.3

[M + H]⁺, 653.3 [M + Na]⁺. HPLC purity: 92.7%.

Synthesis of Ertapenem N-(5-Methyl-2-oxo-1,3-dioxol-4-

yl)methyl Diethyl Ester (13s). To a solution of ertapenem diethyl

ester 13a (1.33 g, 2.5 mmol) in DMF (10 mL) were added DIPEA

Synthesis of Ertapenem Macrocyclic (5-Methyl-2-oxo-1,3-

dioxol-4-yl)methyl-bis-lactone (14d). To a solution of 4-

(chloromethyl)-5-methyl-1,3-dioxol-2-one (1.48 g, 10 mmol) in

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CCl₄(60 mL) were added AIBN (164 mg, 1.0 mmol) and NBS (1.78

g, 10 mmol). Then the mixture was stirred at reﬂux for 12 h. After

ﬁltration, the ﬁltrate was concentrated under reduced pressure and the

residue was puriﬁed by silica gel column, eluting with PE/EtOAc

(50:1) to give the desired product 4-(bromomethyl)-5-(chlorometh-

yl)-1,3-dioxol-2-one (1.13 g, 50.2% yield) as yellow oil. ¹H NMR (400

MHz, CDCl₃) δ 4.38 (s, 2H), 4.21 (s, 2H).

8.04−8.07 (m, 1H), 7.68−7.72 (m, 2H), 7.47−7.53 (m, 1H), 5.56−

5.61 (m, 1H), 5.29−5.42 (m, 2H), 4.81−5.11 (m, 4H), 4.45−4.48 (m,

1H), 4.24−4.26 (m, 1H), 3.97−4.09 (m, 3H), 3.64−3.68 (m, 1H),

3.38−3.47 (m, 1H), 3.22−3.24 (m, 1H), 2.84−2.98 (m, 1H), 2.04−

2.15 (m, 4H), 1.02−1.18 (m, 6H). ESI MS m/z 742.2 [M + H]⁺.

HPLC purity: 96.0%.

Synthesis of Ertapenem Macrocyclic Methyleneoxymethy-

lene-bis-lactone (14h). To a solution of N-allyloxy carbamate 15

(8.0 g, 15 mmol) in DMF (100 mL) were added bromo-

(bromomethoxy)methane²⁵(3.0 g, 15 mmol) and DIEA (4.7 g, 38

mmol) at room temperature, and it was stirred at 50 °C for 4 h. After

the solution was cooled to 0 °C, the reaction was quenched by

addition of saturated aqueous NaCl (1500 mL), extracted with EtOAc

(300 mL × 5), dried (Na₂SO₄), and concentrated in vacuo. The

residue was puriﬁed by reversed phase HPLC on a GILSON 281

instrument ﬁtted with a Phenomenex Gemini C₁₈(250 mm × 21.2

mm, 5 μm) using water (0.2% HCOONH₄) and acetonitrile as eluents

(mobile phase A, water (0.2% HCOONH₄); mobile phase B,

acetonitrile; gradient, 52−72% B, 0−8 min; 100% B, 8.5−10.5 min;

5% B, 11−12 min) followed by freeze-drying to give N-protected

macrocyclic bis-lactone 17h₁₄(R = -CH₂OCH₂-) (1.0 g, 11%) as a

white solid. ESI MS m/z 602 [M + H]⁺.

Reaction of 4-(bromomethyl)-5-(chloromethyl)-1,3-dioxol-2-one

with the N-protected intermediate 15 gave the desired bis-lactone

17c (R = CH₂-c-(C(OCOO)C-)CH₂) which was hydrogenolyzed

over 5% Pd/C with 1 atm of hydrogen in THF−water at room

temperature for 1 h. The mixture was ﬁltered and puriﬁed by reversed

1

phase HPLC to aﬀord 14d in 2.7% yield as a colorless powder. H

NMR (400 MHz, DMSO-d₆) δ 9.95 (s, 1H), 8.10 (d, J = 8.4 Hz, 1H),

7.75 (s, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.53 (dd, J₁= J₂= 8.0 Hz, 1H),

5.61 (d, J = 14.0 Hz, 1H), 5.30−5.38 (m, 2H), 5.10 (d, J = 5.2 Hz,

1H), 4.85 (d, J = 14.0 Hz, 1H), 4.21 (d, J = 5.2 Hz, 1H), 4.05 (s, 1H),

3.88−4.01 (m, 2H), 3.76−3.78 (m, 1H), 3.49−3.53 (m, 1H), 3.21−

3.23 (m, 1H), 3.10−3.13 (m, 1H), 2.67−2.75 (m, 2H), 1.77−1.81 (m,

1H), 1.14−1.18 (m, 6H). ESI MS m/z 586.1 [M + H]⁺. HPLC purity:

98.2%.

Synthesis of Ertapenem Macrocyclic (Z)-1,4-But-2-ene (5-

methyl-2-oxo-1,3-dioxol-4-yl)methyl N-Carbamate (14e). A

mixture of ertapenem sodium carboxylate 4 (4.75g, 10 mmol), (5-

methyl-2-oxo-1,3-dioxol-4-yl)methyl (4-nitrophenyl) carbonate²⁴

(2.95g, 10 mmol), and NaHCO₃(840 mg, 10 mmol) in acetone

(25 mL)−H₂O (25 mL) was stirred at room temperature for 1 h, and

it was heated at 50 °C for 2 h. The reaction solvent was evaporated

and the mixture was directly puriﬁed by reversed phase HPLC to give

ertapenem (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl N-carbamate 24

(500 mg, 7.4%) as a white solid. To a solution of compound 24

(200 mg, 0.32 mmol) in DMF (10 mL) was slowly added Na₂CO₃(74

mg, 0.70 mmol), and it was stirred at room temperature for 20 min.

After addition of (Z)-1,4-dichlorobut-2-ene (37 mg, 0.30 mmol), it

was stirred at 50−55 °C for 18 h, and then it was ﬁltered. The ﬁltrate

was puriﬁed by reversed phase HPLC on a GILSON 281 instrument

ﬁtted with a Phenomenex Gemini C₁₈(250 mm × 21.2 mm, 5 μm)

using water (0.2% HCOONH₄) and acetonitrile as eluents (mobile

phase A, water; mobile phase B, acetonitrile; gradient, 52−72% B, 0−8

min; 100% B, 8.5−10.5 min; 5% B, 11−12 min) followed by

lyophilization to aﬀord the desired macrocycle 14e (20 mg, 10%) as a

To a solution of N-allyloxycarbonyl bis-lactone 17h₁₄(200 mg, 0.33

mmol) in DMF (2.0 mL) were added phenylsilane (72 mg, 0.66

mmol) and tetrakis(triphenylphosphine)palladium (140 mg, 0.12

mmol), and the mixture was stirred at room temperature for 3 h. The

reaction was quenched with saturated brine, extracted with EtOAc, and

the combined organic extracts were dried (Na₂SO₄) and ﬁltered. The

ﬁltrate was concentrated under reduced pressure. The residue was

puriﬁed by reversed phase HPLC on a GILSON 281 instrument ﬁtted

with a Phenomenex Gemini C₁₈(250 mm × 21.2 mm, 5 μm) column,

eluting with a water and acetonitrile gradient (mobile phase A, water;

mobile phase B, acetonitrile; gradient, 52−72% B, 0−8 min; 100% B,

8.5−10.5 min; 5% B, 11−12 min) followed by lyophilization to give

the desired compound 14h (38 mg, 24.5%) as a white solid. ¹H NMR

(400 MHz, CDCl₃) δ 9.93 (brs, 1H), 7.96−8.00 (m, 1H), 7.81−7.84

(m, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.36−7.41 (m, 1H), 5.73 (d, J = 6.8

Hz, 1H), 5.68 (d, J = 4.8 Hz, 1H), 5.35 (d, J = 6.8 Hz, 1H), 5.24 (d, J

= 4.8 Hz, 1H), 4.19−4.22 (m, 1H), 4.01−4.11 (m, 2H), 3.79−3.81 (m,

1H), 3.54−3.56 (m, 1H), 3.29−3.34 (m, 2H), 3.13−3.15 (m, 1H),

2.50−2.75 (m, 1H), 2.13−2.17 (m, 1H), 1.26 (d, J = 6.0 Hz, 3H), 1.19

(d, J = 7.2 Hz, 3H). ESI MS m/z 518.1 [M + H]⁺. HPLC purity:

96.1%.

Synthesis of 8-Acetyl Macrocyclic Methyleneoxymethylene

Bis-lactone (14i). Acetic acid (0.40 g, 6.66 mmol) was condensated

with N-allyoxycarbonyl macrocyclic methyleneoxymethylene bis-

lactone 17h₁₄(1.0 g, 1.67 mmol) by EDCI and DMAP method

followed by silica gel column puriﬁcation, eluting with DCM/MeOH

(40:1), and concentration in vacuo to give the desired 8-acetyl

ertapenem 23 (0.83 g, 78.1%) as a pale yellow solid.

N-Allyloxycarbonyl group of compound 23 was removed by

phenylsilane followed by reversed phase HPLC puriﬁcation and

lyophilization to aﬀord the 8-acetyl macrocyclic methyleneoxy-

methylene bis-lactone 14i (172 mg, 23.8%) as a white powder.¹H

NMR (400 MHz, CDCl₃) δ 9.80 (s, 1H), 8.22 (d, J = 8.0 Hz, 1H),

7.77 (d, J = 8.0 Hz, 1H), 7.76 (s, 1H), 7.42 (dd, J₁= J₂= 8.0 Hz, 1H),

5.78 (d, J = 6.4 Hz, 1H), 5.73 (d, J = 5.2 Hz, 1H), 5.40 (d, J = 6.4 Hz,

1H), 5.32 (d, J = 5.2 Hz, 1H), 5.20−5.28 (m, 1H), 4.16−4.19 (m,

1H), 3.92−3.97 (m, 1H), 3.75−3.78 (m, 1H), 3.48−3.51 (m, 1H),

3.24−3.35 (m, 3H), 2.66−2.74 (m, 1H), 2.08−2.14 (m, 1H), 2.05 (s,

3H), 1.38 (d, J = 6.3 Hz, 3H), 1.21 (d, J = 7.0 Hz, 3H). ESI MS m/z

560.2 [M + H]⁺. HPLC purity: 100.0%.

1

white solid. H NMR (400 MHz, DMSO-d₆) δ 9.98−10.02 (m, 1H),

8.00−8.04 (m, 1H), 7.78−7.82 (m, 1H), 7.64−7.68 (m, 1H), 7.40−

7.49 (m, 1H), 5.75−5.90 (m, 2H), 5.32−5.42 (m, 1H), 5.06−5.07 (m,

1H), 4.72−5.01 (m, 4H), 4.33−4.44 (m, 2H), 4.21−4.23 (m, 1H),

3.91−3.98 (m, 3H), 3.56−3.59 (m, 1H), 3.36−3.42 (m, 1H), 3.21−

3.23 (m, 1H), 2.78−2.88 (m, 1H), 2.00−2.12 (m, 4H), 1.10−1.14 (m,

6H). ESI MS m/z 684.2 [M + H]⁺, 706.2 [M + Na]⁺. HPLC purity:

92.2%. Tautomer is observed.

Synthesis of Ertapenem Macrocyclic (E)-1,4-But-2-ene-bis-

lactone (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl N-Carbamate

(14f). Reaction of 1.0 equiv (E)-1,4-dichlorobut-2-ene (37 mg, 0.30

mmol) with ertapenem (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl N-

carbamate 24 followed by reversed phase HPLC puriﬁcation and

lyophilization aﬀorded the desired compound 14f (20 mg, 10%) as a

white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.19 (s, 1H), 8.47 (s,

1H), 7.61−7.70 (m, 2H), 7.45−7.49 (m, 1H), 6.00−6.02 (m, 2H),

5.06−5.15 (m, 2H), 4.68−5.02 (m, 3H), 4.53−4.60 (m, 2H), 4.45−

4.48 (m, 1H), 4.18−4.21 (m, 1H), 3.92−4.02 (m, 3H), 3.60−3.66 (m,

1H), 3.37−3.44 (m, 1H), 3.19−3.21 (m, 1H), 2.82−2.95 (m, 1H),

1.98−2.12 (m, 4H), 1.06−1.13 (m, 6H). ESI MS m/z 684.1 [M + H]⁺,

706.1 [M + Na]⁺. HPLC purity: 93.2%.

Plasma Stability Assay. Plasma was prewarmed at 37 °C in water

bath for 5 min. Prodrugs were incubated in plasma at 50 μM with an

incubation volume of 100 μL for each time point sample. The reaction

solutions were kept at 37 °C. Time samples (0, 5, 15, 30, and 60 min)

were removed from the water bath and immediately mixed with 500

μL of acetonitrile containing internal standard. Some prodrugs were

quenched by ethanol containing internal standard. After centrifugation

the supernatant was collected and diluted with mobile phase or

Synthesis Ertapenem Macrocyclic (5-Methyl-2-oxo-1,3-diox-

ol-4-yl)methyl-bis-lactone (5-Methyl-2-oxo-1,3-dioxol-4-yl)-

methyl N-Carbamate (14g). Reaction of 4-(bromomethyl)-5-

(chloromethyl)-1,3-dioxol-2-one (110 mg, 0.48 mmol) with ertape-

nem (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl N-carbamate 24 (300

mg, 0.47 mmol) followed by reversed phase HPLC puriﬁcation and

lyophilization aﬀorded the desired compound 14g (13 mg, 3.6%) as a

white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.06−10.10 (m, 1H),

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ultrapure water for LC−MS/MS analysis. Tebipenem pivoxil was used

as positive control. Standard curves were used to calculate the

concentrations of the prodrugs, monoesters, and parent drugs. One of

the following LC−MS/MS conditions was used for quantitative

analysis. (i) The LC−MS/MS system consisted of HPLC (Shimadzu

LC 20AD) and API 4000 (Applied Biosystems) instruments with

autosampler of CTC PAL. The HPLC mobile phases consisted of 2

mM ammonium acetate in water and acetonitrile. Chromatographic

separation was achieved on a pHlex ODS column (5 μm, 2.1 mm × 50

mm, Boston Separations). (ii) The LC−MS/MS system consisted of

UPLC (Waters Acquity) and API 4000 (Applied Biosystems)

instruments. The UPLC mobile phases consisted of 2 mM ammonium

acetate in water and acetonitrile. Chromatographic separation was

achieved on a pHlex ODS column (5 μm, 2.1 mm × 50 mm, Boston

Separations). (iii) The LC−MS/MS system consisted of HPLC

(Shimadzu LC 20AD) and API 4000 (Applied Biosystems) instru-

ments with autosampler of CTC PAL. The HPLC mobile phases

consisted of 2 mM ammonium acetate in water and acetonitrile.

Chromatographic separation was achieved on Synergi, Hydro-RP, C₁₈

column (4 μm, 2.10 mm × 30 mm, Phenomenex). (iv) The LC−MS/

MS system consisted of HPLC (Shimadzu LC 20AD) and API 4000

(Applied Biosystems) instruments with autosampler of CTC PAL.

The HPLC mobile phases consisted of 0.1% formic acid in water and

0.1% formic acid in acetonitrile. Chromatographic separation was

achieved on Synergi, Hydro-RP, C₁₈column (4 μm, 2.10 mm × 30

mm, Phenomenex). All analytes were detected using either positive or

negative ESI (electrospray ionization) with both Q1 and Q3 operated

under unit resolution. The ion source conditions were optimized as

the following: 600 °C source temperature; 5500 V for positive

ionspray voltage and −4200 V for negative ionspray voltage; 3 L/min

nebulizer gas; 15 L/min drying gas.

Stability Measurement of Prodrugs in Simulated Gastric

Fluid (SGF), Simulated Gastrointestinal Fluid (FaSSIF), and

Phosphate Buﬀer. Prodrugs and parent were dissolved in DMSO,

and a 10 mM stock solution was prepared and stored at −20 °C. A 25

μL aliquot was added to 4975 μL of simulated gastric ﬂuid (pH 2.0),

simulated intestinal ﬂuid (pH 6.5), phosphate buﬀer (pH 7.0), and

acetonitrile−water (1:1) to make 50 μM concentration samples. A 50

μL aliquot of each solution was further diluted 50-fold by addition of

2450 μL of the corresponding media to provide 1 μM concentration

samples. DMSO control was prepared similarly from 25 μL of DMSO

without prodrug. Acetonitrile−water samples served as a stability

control for the prodrug and parent, and DMSO control served as blank

control. Each sample was incubated at 37 °C for 3−4 h, and 5 μL

aliquot was removed (SGF, 10, 20, 60, 90, 120 min; FaSSIF and

phosphate buﬀer, 15, 30, 60, 120, 180 min) from each incubated

sample and diluted with 5 μL of acetonitrile and 990 μL of 1:1

acetonitrile−water containing 10 ng/mL of tolbutamide and 1 ng/mL

of buspirone (internal standards). Samples were analyzed by LC−MS/

MS using the experimental condition (iv) described for plasma

hydrolysis. Prodrugs and parent were quantiﬁed using calibration

curves based on peak area ration of analyte and two internal standards.

Dosing Solution Preparation for Rat and Dog Studies.

Dosing solutions were prepared and administered within an hour of

formulation. The iv formulations were ﬁltered with 0.22 μM ﬁlter

before dosing. The vehicles were 30% captisol for iv and 0.5%

methylcellulose for ID for rat (Table 6). For dog studies, evaluations

of solubility and redispersibility in a variety of potential vehicles

including PEG 400, 40% PEG400/10% Tween 80/50% water, 30%

Captisol, 10% Tween 80, 50% Imwitor/50% Tween were conducted

to select the optimal vehicle for the prodrugs (Table 7).

720192, 0.4 mm internal diameter; jugular vein, Harvard-598322, 0.28

mm internal diameter) according to generally accepted procedures.

Rats for intraduodenal (ID) dosing were implanted with a catheter

(Access Technologies-BC-3P French size 3, gauge 20) which was

inserted into the duodenum according to generally accepted

procedures. The animals were also cannulated at the carotid artery

for blood sampling as described above. The rats were allowed to

recover at least 3 days after surgery. Male beagle dogs (6−10 kg) were

obtained from Marshall Bioresources (Beijing, China) and were fed

certiﬁed dog diet (Beijing Vital Keao Feed Co., Ltd. Beijing, P. R.

China). For the dogs dosed via ID administration, a cannula made of

silicone tubing (0.025 in. i.d. × 0.047 in. o.d., catalog no. SIL-3.5-25,

Instech Solomon) was placed within the ﬁrst 1.5−2 cm of duodenum

after the pylorus, as close as possible to pylorus, according to generally

accepted procedures. The cannula was enveloped and placed outside

with 1−2 cm exterior to the skin. The dogs were allowed to recover 7

days following surgery.

Pharmacokinetic Evaluation in Rat (Dosing and Sample

Processing). Rats were fasted overnight prior to drug administration

and had access to water ad libitum and access to chow 4 h postdose.

For iv studies, the jugular vein cannula was used for administration at 1

mpk in a dose formulation of 0.5 mg/mL and the carotid artery

catheter was used for blood sampling. For ID studies, the duodenal

catheter was used for dosing at 10 mpk in a dose formulation of 2 mg/

mL at a rate of 5 mL/h using an infusion pump (Harvard Apparatus

PHD 2000) and the carotid artery cannula used for blood sampling.

Approximately 0.20 mL of blood was collected at 0 (predose to serve

as a blank), 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h postdose from

carotid artery via a catheter after iv and ID infusion administration of

each compound. All blood samples were transferred into plastic

microcentrifuge tubes containing 4 μL of 0.5 M K₂-EDTA

anticoagulant, placed on wet ice, and then immediately centrifuged

to get plasma. To plasma, an equal volume of 1 M MOPS buﬀer, pH

7.0, was added under ice-cold conditions to stabilize the carbapemens

and then mixed. After that, the mixed samples were immediately

quenched by addition of a 4-fold volume of acetonitrile containing

internal standard. The processed plasma samples were stored in

polypropylene tubes, quick-frozen over dry ice, and kept at −70 10

°C until LC−MS/MS analysis.

Pharmacokinetic Evaluation in Dogs. Dogs were fasted

overnight prior to drug administration and had access to water ad

libitum and access to chow 4 h postdose. For iv studies, the cephalic

vein was used for administration at 1 mpk in a dose formulation of 1

mg/mL. For ID studies, the duodenal catheter was used for dosing at

10 mpk in a dose formulation of 2 or 5 mg/mL for at least 15 min

using an infusion pump (Harvard Apparatus PHD 2000). Approx-

imately 0.20 mL of blood was collected at 0 (predose to serve as a

blank), 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h postdose from the

cephalic vein or saphenous vein after iv and ID infusion administration

of each compound. All blood samples were transferred into plastic

microcentrifuge tubes containing 4 μL of 0.5 M of K₂-EDTA

anticoagulant, placed on wet ice, and then immediately centrifuged

to get plasma. To plasma, an equal volume of 1 M MOPS buﬀer, pH

7.0, was added under ice-cold conditions to stabilize the carbapemens

and then mixed. After that, the mixed samples were immediately

quenched by addition of a 4-fold volume of precipitant (80% ACN/

EtOH) containing internal standard. The processed samples were

stored in polypropylene tubes, quick-frozen over dry ice, and kept at

−70 10 °C until LC−MS/MS analysis.

Quantitative Analysis. Dose formulation retains were assayed in

duplicate for each dose using LC/UV or LC−MS/MS methodologies

with a calibration curve at least six points. Each processed plasma

sample was assayed using LC−MS/MS with an internal standard using

a minimum of eight-point standard curves. No formal method

validation was conducted, but a minimum of six QC samples were

included in each analytical run and needed to be within 25% of their

nominal values when back-calculated from the standard curve linear

regression analysis to ensure assay performance. The BA methods for

PK samples’ analysis were optimized individually for diﬀerent

compounds.

Husbandry and Surgical Preparation of Rats and Dogs. All

animal studies were approved by the Animal Care Committees of

Merck and WuXi. Male Sprague−Dawley Rats (200−300 g) were

obtained from SLAC Laboratory Animal Co. Ltd. (Shanghai, China).

They were housed in stainless steel cages and had free access to water

and certiﬁed rodent diet (catalog no. M-01F, SLAC Laboratory Animal

Co. Ltd., Shanghai, China). Rats for intraveneous (iv) bolus dosing

were surgically implanted with catheters in both the jugular vein and

carotid artery using polyethylene tubing (carotid artery, Harvard-

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Data Analysis and Calculations. Plasma concentrations versus

time data were analyzed by noncompartmental approaches using the

WinNonlin software program (version 5.2, Pharsight Corporation,

Mountain View, CA).

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ASSOCIATED CONTENT

■

S

* Supporting Information

¹H NMR spectra of all ﬁnal prodrugs. This material is available

free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*E-mail: sheo.singh.215@gmail.com. Phone: (908) 930-5757.

■

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Present Address

^#S.B.S.: SBS Pharma Consulting, Edison, NJ 08820.

Author Contributions

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

Notes

The authors declare no competing ﬁnancial interest.

ABBREVIATIONS USED

■

ID, intradeuodenal; OATP, organic acid transporter;

OATB1A2, organic acid transporter B1A2; OATBB1, organic

acid transporter BB1

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dx.doi.org/10.1021/jm500879a | J. Med. Chem. 2014, 57, 8421−8444

Article Doi

Design, synthesis, structure-function relationship, bioconversion, and pharmacokinetic evaluation of ertapenem prodrugs

DOI: 10.1021/jm500879a

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Article abstract of DOI:10.1021/jm500879a

Full text of DOI:10.1021/jm500879a

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