Improved total synthesis and biological evaluation of potent apratoxin S4 based anticancer agents with differential stability and further enhanced activity

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Open Access on 03/24/2015

Improved Total Synthesis and Biological Evaluation of Potent

Apratoxin S4 Based Anticancer Agents with Diﬀerential Stability and

Further Enhanced Activity

Qi-Yin Chen,^†,‡Yanxia Liu,^†,‡Weijing Cai,^†and Hendrik Luesch*^,†,‡

^†Department of Medicinal Chemistry and ^‡Center for Natural Products, Drug Discovery and Development (CNPD3), University of

Florida, Gainesville, Florida, United States

S

* Supporting Information

ABSTRACT: Apratoxins are cytotoxic natural products

originally isolated from marine cyanobacteria that act by

preventing cotranslational translocation early in the secretory

pathway to downregulate receptor levels and inhibit growth

factor secretion, leading to potent antiproliferative activity.

Through rational design and total synthesis of an apratoxin A/

E hybrid, apratoxin S4 (1a), we have previously improved the

antitumor activity and tolerability in vivo. Compound 1a and

newly designed analogues apratoxins S7−S9 (1b−d), with various degrees of methylation at C34 (1b,c) or epimeric

conﬁguration at C30 (1d), were eﬃciently synthesized utilizing improved procedures. Optimizations have been applied to the

synthesis of key intermediate aldehyde 7 and further include the application of Leighton’s silanes and modiﬁcations of Kelly’s

methods to induce thiazoline ring formation in other crucial steps of the apratoxin synthesis. Apratoxin S9 (1d) exhibited

increased activity with subnanomolar potency. Apratoxin S8 (1c) lacks the propensity to be deactivated by dehydration and

showed eﬃcacy in a human HCT116 xenograft mouse model.

formation, Scheme S1).⁷Unfortunately, the only natural

apratoxin without that α,β-unsaturated carbonyl system has

INTRODUCTION

■

Apratoxins are potent cytotoxins derived from marine

cyanobacteria.¹Because of their biological activity and

been apratoxin E, which, however, exerts much reduced activity,

presumably due to the dehydration of the tertiary alcohol at

intriguing structures, they have been subject to several total

C35.^1cTherefore, we designed and synthesized apratoxin S4

syntheses and SAR studies.²We recently established that

apratoxins prevent cotranslational translocation and thereby

downregulate various receptors, including receptor tyrosine

(1a), which represents a hybrid of the natural products

apratoxins A and E, containing the hydroxy group as in

apratoxin A that is important for potent activity and lacking the

kinases (RTKs), and inhibit traﬃcking of other secretory

Michael acceptor as in apratoxin E (Figure 1).^2aCompound 1a

molecules, including growth factors that act on RTKs.^2a,3RTKs

showed greater potency and eﬃcacy in a colorectal tumor

xenograft model than the parent compound (apratoxin A) and

such as epidermal growth factor receptors and corresponding

ligands such as vascular endothelial growth factor A (VEGF-A)

was much better tolerated in vivo, providing evidence that the

individually are validated drug targets, which resulted in the

apratoxin scaﬀold can serve as a new modality to treat cancer

approval of small molecules and antibodies against these

proteins for colorectal cancer and other cancers.⁴The

but also validating the mechanism of action as a new

therapeutic strategy.^2a

combined indirect inhibition of both classes of molecules by

apratoxins has proven very powerful and an alternative to the

speciﬁc targeting of selected secretory proteins in cancers that

In our previous work,^2asome fragments of 1a were prepared

with modiﬁed methods that paralleled those by other

groups.^2b−dTo enable the large-scale synthesis of 1a for

rigorous preclinical assessment, several steps needed to be

improved or replaced due to their low eﬃciency or harsh

operation. An improved synthesis would also provide easier

access to additional apratoxin analogues, which we designed

based on earlier observations with the goal of improving

metabolic stability and reducing complexity of the molecule. In

apratoxin A, the chiral center at C34 bears the risk of

epimerization; however, the C34 epimer has the same potency

rely on autocrine loops^2asuch as colorectal cancer.⁵Apratoxin

A was rigorously proﬁled and shown to possess broad-spectrum

yet diﬀerential in vitro activity;⁶however, it also showed

irreversible toxicity in vivo and was not well tolerated.^2aOur

initial investigation of the structure−activity relationships

indicated that the irreversible toxicity may be an oﬀ-target

eﬀect rather than mechanism-based and may be related to the

presence of the Michael acceptor in the molecule that might be

prone to nonspeciﬁc addition of cellular nucleophiles.^2aIndeed,

in vitro experiments showed that Michael addition at C29 of

apratoxin A can occur with thiol-containing compounds such as

glutathione, cysteine, and N-acetylcysteine (Supporting In-

Received: December 30, 2013

Published: March 24, 2014

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Figure 1. Natural apratoxins A and E and synthetic apratoxin S4.

Scheme 1. Retrosynthetic Analysis of 1a and Analogues 1b−d

as apratoxin A^2c,eand the same holds true for 1a and 34-epi-1a

(apratoxin S6).^2aIt is also known that the hydroxy group at

C35 in apratoxin A is sensitive toward acid-induced

dehydration, leading to the double bond between C34 and

C35,^1b,2cand the product has greatly decreased activity,^1b,cthus

presenting a potential major deactivation pathway for 1a as

well. On the basis of the above, it was necessary to explore and

investigate analogues at C34: (1) nonmethyl-C34, to remove

the chiral center, thereby simplifying the molecule (apratoxin

S7, 1b), and (2) gem-dimethyl-C34, to remove the chiral center

to simplify the molecule and reduce the possibility to dehydrate

to form a conjugated system with the thiazoline (apratoxin S8,

1c). Both analogues were expected to retain potent activity

based on our previous results with 34-epi-1a.^2aTo continue our

systematic SAR investigation in the nonpeptide portions, we

also aimed to explore the synthesis and activity of 30-epi-1a

(apratoxin S9, 1d).

of this improved synthesis, several new reactions were applied,

key reactions were modiﬁed to meet diﬀerent substrate

requirements, and many steps were optimized, resulting in an

eﬃcient synthesis that enables the generation of large-scale

quantities of apratoxins for preclinical evaluation. We studied

the SAR by subjecting all new synthetic apratoxins to a

multidimensional assay platform, measuring cell growth,

receptor tyrosine kinase receptor (RTK) levels, and growth

factor secretion. We also investigated the in vitro stability of

these compounds and tested the compound with the least

propensity to be deactivated via dehydration (1c) for in vivo

antitumor eﬃcacy.

RESULTS AND DISCUSSION

■

Synthesis. The main strategies and procedures in the

synthesis of all analogues we designed paralleled those of

reported procedures but with notable modiﬁcations.^2a−dOn the

basis of the retrosynthetic analysis depicted in Scheme 1, 1a

and its analogues 1b−d can be synthesized from two parts:

tripeptide 6 and thiazole-containing long chain aliphatic acid 2.

Here we show the details of an improved synthesis of 1a and

the synthesis of analogues with diﬀerent C34 methylation status

(1b,c) and conﬁguration of C30 (1d). During the development

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Scheme 2. Synthesis of Aldehyde 7

Scheme 3. Synthesis of 4a−c

The key step for the synthesis of each analogue is the formation

of the thiazoline ring in 2 from the open-chain precursor 3.

Experiments below showed that diﬀerent degrees of methyl-

ation at C34 or diﬀerent conﬁguration of C30 aﬀected the ring

formation to diﬀerent extents. Compound 3 can be

disconnected into modiﬁed cysteine 5 and proline ester based

carboxylic acid 4, which in turn can be synthesized from

aldehyde 7 through a series of known reactions.

Initially, we prepared aldehyde 7,^2a−da public and key

intermediate. Scheme 2 details the steps which were improved

from published procedures^2b−hto meet practical large-scale

production. β-Hydroxy ketone 8 was prepared via ^D-proline

catalyzed aldol reaction of pivalaldehyde with acetone. After

protection with TBS to 9, reduction with NaBH₄to 10, and

elimination through mesylate, 8 was transformed into allyl TBS

ether 11. The cleavage of the TBS from 11 aﬀorded allyl

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Scheme 4. Synthesis of 5a and 5b

alcohol 12, which is the most critical step in the preparation of

12 due to its volatile property.⁸The solvent (Et₂O and THF)

in 12-containing fractions can be removed by distillation using

a Vigreux fractionation column for small-scale reactions under

normal pressure; however, for large-scale reactions, this was not

feasible and we used the combination of cooling-concentration

method under ambient reduced pressure (see Supporting

Information, Figure S1) and Vigreux fraction concentration.

Even though our reaction sequence consisted of more steps to

prepare 12 than Brown’s method using IPC₂BOMe,^2dthe latter

caused puriﬁcation problems which we did not encounter

utilizing our optimized method.^2g,hWe found that this is indeed

a practical route to the large-scale production of 12, which we

could easily achieve on 10 g scale.

synthesized utilizing a similar strategy (Scheme 3a). In our

previous work, we constructed the C34(Me)−C35(OH) chiral

unit of 19a through Roush’s crotylation with Roush’s (E)-

crotylborate at −78 °C in toluene^2afollowing published

procedures.^2c,10However, Roush’s (E)-crotylborate is not

commercially available, it is laborious to purify crude Roush’s

(E)-crotylborate prepared in the lab by distillation and, if crude

product was used without puriﬁcation, the impurity reduced the

yield to as low as 55%. Here we now applied Leighton’s silanes

18a,b instead of Roush’s crotylborate to construct the C34−

C35 units in 19a,b. Leighton’s silanes 18a and 18b are

commercially available and inexpensive; they are solid and easy

to handle.¹¹Compound 19a and 19b were obtained smoothly

in high yields when aldehyde 7 was treated with 18a and 18b

along with Sc(OTf)₃in CH₂Cl₂at 0 °C, respectively.

Compound 19a was converted into Troc ester 20a with

2,2,2-trichloroethoxycarbonyl chloride (TrocCl) in the pres-

ence of pyridine and DMAP. The Troc ester of 19b was not

stable and partly dehydrated to form a conjugate double bond,

which led to low yield and caused problems with puriﬁcation.

Therefore, we protected hydroxy group 19b with TBS and

smoothly obtained the stable TBS ether 20b.

The removal of the PMB group and subsequent esteriﬁcation

with Fmoc-Pro-OH by Yamaguchi’s method^2b,c,12provided

prolyl ester 21a and 21b. The combination of oxidants, OsO₄/

oxone and NaIO₄, was eﬀective to oxidize 21a to carboxylic

acid 4a; however, when 21b was exposed to the same

conditions, the TBS group was cleaved simultaneously and

gave carboxylic acid 4b with a free hydroxy group. The cleavage

of the TBS group probably resulted from the acidic property of

oxone which is a triple salt 2KHSO₅·KHSO₄·K₂SO₄. Both 4a

and 4b were obtained in good yields (83% and 80%,

respectively).

According to published procedures^2dwith minor modiﬁca-

tions, aldehyde 7 was prepared starting from allyl alcohol 12

through formation of acryloyl ester 13, Grubbs’ catalyst-eﬀected

RCM reaction (14), methylation with Me₂(CuCN)Li₂(15),

Weinreb amide formation (16), protection of hydroxy group

with PMB (17), and reduction with DIBAL-H (7). The yield

was low (10−25%) when we used p-methoxybenzyl bromide

(PMBBr) to protect the hydroxy group of 16 in the presence of

NaH/tetra-n-butyl-ammonium iodide (TBAI) in THF or NaH

in DMF. Fortunately, we found that the PMBOC(NH)CCl₃/

TfOH method^2bwas eﬀective in smoothly converting 16 into

PMB ether in moderate yield (56%). Furthermore, unreacted

starting material 16 was recovered quantitatively and could be

used in the next cycle.

It is noteworthy that, to shorten the synthetic route, we also

tried to apply a Grignard reaction to the syn-cyclic sulfate

derivative of 8 to aﬀord aldehyde 7 (Supporting Information,

Scheme S2).^9aUsing this strategy, 8 should be stereoselectively

reduced to the syn-diol. We tried both Et₃BOMe/NaBH₄^9aand

DIBAL-H^9bas reducing systems. When we performed small-

scale reactions (<2 g), we obtained adequate results; however,

for large-scale reactions (>4 g), both methods provided

unsatisfactory results: low yields, low diastereoselectivity, and

most of the product was obtained as an inseparable mixture.

We speculate that an extremely slow rate of addition of

reducing reagents is crucial for the success of this reaction.^9b

With aldehyde 7 in hand, we prepared proline ester-based

aliphatic acid 4a−c using modiﬁed published procedures^2a−c

(Scheme 3). C34-Methyl 4a and C34-nonmethyl 4b were

The enantioselective chiral borane-mediated aldol reaction

developed by Kiyooka¹³was used to construct the β-hydroxy-

α,α-dimethyl acid part of C34-gem-dimethyl acid 4c (Scheme

3b). Treatment of aldehyde 7 with methyl trimethylsilyl

dimethylketene acetal at −78 °C in the presence of chiral

oxazaborolidinone 22 (derived from ^D-Val) provided (S)-β-

hydroxy ester 23. With 23 in hand, we ﬁrst tried to protect the

hydroxy with the Troc group and then hydrolyze the methyl

ester with LiOH. However, we found that TrocO-23 was not

compatible to the LiOH aq solution during hydrolysis in which

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Scheme 5. Synthesis of 3a−d and Further 2a−d

a complex mixture including Troc-cleaved acid was generated.

We therefore hydrolyzed 23 with LiOH in the MeOH−H₂O

solution to give β-hydroxy acid 24 smoothly. We planned to

couple 24 with modiﬁed cysteine (moCys) unit 5a ﬁrst and

then protect the hydroxy group with Troc and ﬁnally remove

PMB to form proline ester. However, we found the

esteriﬁcation from this reaction sequence provided a very low

yield and caused diﬃculty in the puriﬁcation. Therefore, we

adjusted the reaction sequence to esterify with Fmoc-Pro-OH

ﬁrst and then install the moCys unit. Hydroxy acid 24 was

converted into allyl ester 25 with allylBr in the presence of

K₂CO₃in 93% yield. Similarly to the preparation of 21a,b,

hydroxy ester 25 was protected with Troc and the PMB group

was removed followed by esteriﬁcation with Fmoc-Pro-OH to

provide prolyl ester 27, which was transformed into the

carboxylic acid 4c by treatment with Pd⁰catalyst and N-

methylaniline. The yield for each step was greater than 90%.

The synthesis of modiﬁed cysteine unit 5a and its

enantiomer 5b was achieved by following published procedur-

es^2awith minor modiﬁcations as depicted in Scheme 4. The

Weinreb amide 28 from S- (or R-) N-Boc-Cys(S-Trt)-OH was

reduced to amino aldehyde 29, which was selectively converted

into chain-extended modiﬁed cysteine (E)-30 by Wittig

oleﬁnation. During this conversion, the choice of reductant

was of importance because α-amino aldehydes are easily

racemized.¹⁴The method using lithium aluminum hydride

(LiAlH₄) in THF at 0 °C decreased the racemization rate

compared with that using DIBAL-H in toluene at −78 °C in

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this preparation.^2g,14−16The reduction with LiAlH₄probably

proceeds through a stable lithium-chelated intermediate.^14a,b

Subsequent reduction of the conjugated double bond in 30a

and 30b using NaBH₄in 95% ethanol aﬀorded saturated

compounds 31a and 31b, respectively, but only in 35% yield for

both of them.^2aTo increase this yield, we also attempted

hydrogenation with RhCl(PPh₃)₃/H₂in toluene at 50 °C.¹⁷

This reaction, however, gave a similar yield but also posed

problems during the chromatographic puriﬁcation of the

product, which was considered a greater problem. Fortunately,

our apratoxin synthesis is convergent and this low yield is still

not detrimental because it is not part of the longest linear

sequence. Finally, ethyl esters 31 were transformed into allyl

ester 5 through hydrolysis and subsequent alkylation.

Table 2. Exploring the Conditions of Thiazoline Formation

of 33c from 3c

yield

d

a

c

entry

reagents

solvent

temp (°C)

time (h)

(%)

1

2

3

4

5

6

7

8

Ph₃PO/Tf₂O

TiCl₄

CH₂Cl₂

0

0.5

24

6

trace

29

CH₂Cl₂

0

CH₂Cl₂

25

60

25

(ClCH₂)₂

CH₂Cl₂

15

5

34

b

TiCl₄

CH₂Cl₂

25−40

60

24−40

2.5

30

TiCl₄

(ClCH₂)₂

72

a

Reactions were carried out using Ph₃PO (8 equiv) and Tf₂O (4

equiv) for entries 1−5. TiCl₄(5 equiv) for entries 6−8 (10 mg scale

reactions) or 2.5−3.0 equiv TiCl₄used for >30 mg scale reactions).

Using modiﬁed reported procedures,^2a−c3a−d and 2a−d

were synthesized as depicted in Scheme 5. The N-Boc groups

in 4a−c were selectively removed with TMSOTf in the

presence of 2,6-lutidine and subsequent coupling of depro-

tected 4a−c with 5a or 5b gave 3a−d. First, we screened

several coupling reagents for the preparation of 3a (Table 1,

b

This reaction ﬁrst was carried out at 25 °C for 24 h, when large

amounts of starting material were still found by TLC and MS, and

then this reaction was heated under reﬂux for another 16 h. Reactions

c

were monitored by MS, the bands of starting material and product on

d

TLC were very close. Products were isolated using preparative TLC

plates.

Table 1. Screening of Coupling Reagents for Preparation of

3a, 3c, and 3d (% Yield)

(Table 2, entry 8). Thiazoline-containing intermediates 33a−d

were immediately treated with Zn-NH₄OAc to remove the

Troc group to avoid elimination of the O-Troc group,

providing 34a−d in 55−90% yield. In the preparation of

34a−b, the percentage of their dehydration products was:

dehyd-34b = 15.5% ≫ dehyd-34d trace > dehyd-34a trace ≫

dehyd-34c = 0. Dehyd-34b was isolated, and dehyd-34a and

dehyd-34d were detected by MS. The removal of allyl esters of

34a−d using Pd(PPh₃)₄/N-methylaniline provided acids 2a−d.

With 2a−d in hand, we synthesized the ﬁnal targets 1a−d

(Scheme 6). Fmoc-protected tripeptide 6^2a−cwas treated with

Et₂NH in MeCN to liberate the corresponding amine and then

coupled with acids 2a−d to provide 36a−d in yields of 71−

95%. PyAOP was chosen as the coupling reagent in the

preparation of 36a,c,d with acceptable results.^2e,fHowever, for

C34-nonmethyl-acid 2b (Table 3), when PyAOP or HATU

were used as coupling reagents, product 36b was obtained in

only low yield (10−35%) along with dedydration product

(dehyd-36b) (5−20% isolated yield). One of the reasons for

the low yield of 36b was that starting material 2b was

consumed by intramolecular dehydration (via elimination to

form a double bond at C34−C35 or intramolecular cyclization

to form lactone between COOH and C35-OH of 2b). When

we used DEPBT, a speciﬁc reagent for amine coupling,¹⁹the

desired product 36b was obtained in 72% yield and 13% of

dehyd-36b was isolated, while only trace amount of intra-

molecular dehydration compound of 2b was detected.

The cleavage of O-allyl ester by Pd(PPh₃)₄/N-methylaniline

and the removal of Fmoc by Et₂NH/MeCN from linear

precursors 36a−d gave unmasked reactive precursors, which

were then macrocyclized with PyAOP or DEPBT in diluted

solution. Subsequent puriﬁcation by semipreparative HPLC

aﬀorded ﬁnal targets 1a−d in yields of 60%, 25%, 70%, and

45%, respectively. Along with 1b and 1d, there were 10% and

5% dehydrated cyclized compounds isolated, respectively. The

yields of cyclization and ﬁnal total yields for the longest linear

sequence from pivalaldehyde are summarized in Table 4. The

most optimized yield (5.0%) was obtained for 1a. The lower

yields of 1b and 1d resulted mainly from their dehydration

propensity at C35 during the formation of the thiazoline ring

and macrocyclization steps. However, the lower total yield of 1c

EDCI

(%)

HATU

(%)

BOP

(%)

BEP

(%)

PyAOP

(%)

entry

1 (4a→3a)

2 (4c→3c)

3 (4d→3d)

63

a

50

20

80

a

87

a

b

47

89

91

a

62

a

b

Coupling reagent was not tried. ¹H NMR is complex.

entry 1). The coupling reaction with BEP gave the highest yield

of 87%. Even though HATU did not provide a good yield in the

preparation of 3a, the yield was as high as 82% when it was

used in the preparation of 3b.

Acknowledging that it may be diﬃcult to prepare 3c due to

the steric hindrance of α-gem-dimethyl in acid 4c, we also

screened coupling reagents for its preparation (Table 1, entry

2): PyAOP gave the highest yield of 89% and surprisingly the

BEP coupling product gave a complex proton NMR as well as

low yield while HATU gave an even lower yield of 20%.

Considering the similarity of 3d to 3a and the problem with

BEP to obtain 3c, we used BOP as coupling reagent but only

obtained product in moderate yield (62%); however, PyAOP

provided a high yield of 91% (Table 1, entry 3).

With 3a−d in hand, we synthesized thiazoline-based acids

2a−d (Scheme 5). The formation of thiazoline ring is the key

step in the preparation 2a−d. Replicating published procedur-

es^2a−cusing Kelly’s method,^18aPh₃PO/Tf₂O induced

thiazoline ring formation smoothly for 3a,b to give 33a,b in

CH₂Cl₂at 0 °C within 30 min. However, for 3d, it took 3 h to

consume all starting material and to ﬁnally form cyclized

compound 33d. For gem-dimethyl 3c, it was surprising to us

that Ph₃PO/Tf₂O was unable to induce thiazoline formation

eﬀectively (Table 2), despite extended reaction time or

increased reaction temperature. Solely the reaction at 60 °C

for 24 h gave 29% of 33c (Table 2, entry 5). Under other

conditions, we only detected trace amounts of product (Table

2, entries 1−4). Then we turned to Kelly’s TiCl₄method^18b

using conditions as shown in Table 2. For TiCl₄mediated

thiazoline formation, at 25−40 °C in 5−40 h, only 30−34%

yield was obtained (entries 6−7); however, the yield improved

to 72% when the reaction temperature was increased to 60 °C

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Scheme 6. Synthesis of Targets 1a−d

Table 3. Screening of Coupling Reagents for Reaction of 2b

and 6 to Form 36b

Table 5. Activities of Synthetic Apratoxins on HCT116 Cell

Viability and VEGF-A Secretion

a

b

coupling

reagent

yield of 36b yield of dehyd-36b

ratio 2b to

dehyd-2b

IC₅₀(nM) cell

IC₅₀(nM) VEGF-A

entry

(%)

apratoxin

S4 (1a)

viability

secretion

1

2

3

HATU

PyAOP

DEPBT

10

35

72

5

20

13

1:1

3:1

1.43

1.25

1.99

0.32

0.30

0.47

0.12

S7 (1b)

20:1

S8 (1c)

S9 (1d = C30-epi-1a)

0.69

a

b

Table 4. Summary of Yields of Final Products 1a−d

Determined after 48 h (n = 4). Determined after 12 h (n = 3).

apratoxin

S4 (1a)

apratoxin

S7 (1b)

apratoxin

S8 (1c)

apratoxin

S9 (1d)

measured representative key members of these protein classes.

The low-nanomolar antiproliferative activity is paralleled by a

similar potency in reducing levels of met proto-oncogene

receptor (MET) (Figure 2), representative for receptor tyrosine

kinases (RTKs) overexpressed in cancers. We also measured

secretion of the angiogenic VEGF-A, which is potently

inhibited even at subnanomolar concentrations by 1b and 1c

a

c

yield of

60.0

25.0

70.0

45.0

cyclization (%)

b

d

total yield (%)

5.0

1.2

3.0

2.4

a

b

Yield over 3 steps. Based on the reaction sequence: pivalaldehyde →

7 → 4 → 3 → 2 → 1. Previous yield^2awas 52%. Previous yield^2awas

c

d

0.8%.

is due to the lower eﬃciency of the thiazoline ring formation,

presumably as a result of steric hindrance of gem-dimethyl at

C34. Nevertheless, the prevention of dehydration of gem-

dimethyl in the formation of the thiazoline ring (34c) and the

best yield (70%) during macrocyclization (1c) supported the

utility and synthetic accessibility of the gem-dimethyl analogue

at C34.

Structure−Activity Relationships. Compounds 1b−d

retained potent activity compared with 1a in all biological

assays. As hypothesized at the onset of this study and consistent

with our previous data that the cytotoxic activity is independent

of the conﬁguration of methyl-C34, we found that the

nonmethylated compound (1b) as well as the gem-dimethyl

analogue (1c) have similar antiproliferative activity between 1

and 2 nM (Table 5). This indicates that the complexity of

apratoxins by eliminating one chiral center (C34) can be

somewhat reduced. We have previously shown that apratoxins

inhibit cotranslational translocation of secretory molecules,

including receptors and growth factors,^2a,3and therefore we

Figure 2. SAR for synthetic apratoxins by immunoblot analysis for

RTK (MET) levels.

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Figure 3. In vitro stability of apratoxins under various conditions. Apratoxins were incubated as indicated and extracted with ethyl acetate, subjected

to LC-MS and monitored by using compound-speciﬁc MRM mode with harmine as internal standard. (A) Stability in aqueous solution, pH 7.40.

(B) Stability in aqueous solution, pH 4.88. (C) Stability in mouse serum. (D) Cellular stability upon exposure to HCT116 protein lysate (0.7 mg/

mL).

a

Table 6. Microsomal Stability Studies

apratoxin S4 (1a)

apratoxin S7 (1b)

apratoxin S8 (1c)

apratoxin S9 (1d)

time

(min)

microsomes

microsomes +

NADPH

microsomes

microsomes +

NADPH

microsomes

microsomes +

NADPH

microsomes

microsomes +

NADPH

only

0

100

3

87.05 1.63

70.80 23.48

87.20 2.83

73.85 15.49

87.10 1.41

55.25 12.09

39.55 1.06

32.20 16.12

10.07 1.46

9.58 1.03

10.00 0.71

6.02 2.11

66.60 12.30

60.50 2.26

76.55 9.55

54.80 1.56

54.50 0.00

53.85 9.40

54.95 4.74

18.70 1.41

52.40 2.40

6.10 1.96

5.41 1.09

4.30 0.82

74.10 9.62

77.45 3.18

63.45 3.61

74.75 8.70

81.25 1.91

70.05 0.78

37.20 1.27

17.30 2.55

8.28 2.86

4.83 1.05

3.55 0.91

5.01 0.23

83.10 0.42

71.40 1.27

45.25 5.30

56.05 1.06

48.45 0.92

56.25 0.21

51.40 8.06

17.80 0.28

3.55 0.25

3.73 0.25

6.06 2.40

5.63 0.18

5

15

30

60

120

a

Assays were done in triplicate. Values are expressed as % remaining. Mean values are shown SD.

with IC₅₀values of 300 and 470 pM, respectively. These eﬀects

are comparable to the activity of 1a (Table 5). We had been

unable to predict the biological consequences of inverting the

C30 conﬁguration of the thiazoline ring. Interestingly, the 30-

epi-1a, compound 1d, showed superior, picomolar (subnano-

molar) potency in all three assays, outperforming the other

apratoxins by 2−3-fold (Table 5, Figure 2).

In Vitro Stability. Compounds 1a−d were remarkably

stable (t_1/2> 24 h) under aqueous conditions at physiological

(pH 7.4) and lysosomal pH (4.88) (Figures 3A,B) and

possessed excellent plasma and cellular stability (Figures

3C,D). Under these conditions, the C34−C35 dehydrated

and hydrated compounds were probably also not interconvert-

ing because we also did not observe any hydration at C28−C29

of the conjugated system in apratoxin A. We had been

concerned about the tendency of apratoxins A, 1a, 1b, and 1d

to dehydrate during synthesis as well as upon prolonged

exposure to acidic organic solvents (e.g., chloroform),^1b,cwhich

could represent a major deactivation pathway for the apratoxins

because the dehydrated compounds possess much reduced

activity. While an issue during synthesis (see above) and in

organic solvents, however, this appeared to not be a major

concern in our biological in vitro systems. Nevertheless, 1a and

1b were generally somewhat less stable than 1c, as expected

(Figure 3), especially under acidic conditions (Figure 3B). The

stability of 1d was also slightly enhanced compared with that of

1a, suggesting that the conﬁguration at C30 may aﬀect the

tendency to dehydrate (possibly by changing the macrocycle

conformation), assuming that dehydration is indeed the mode

of primary apratoxin modiﬁcation. In the synthesis, however,

the dehydration propensity at C35 during the formation of the

thiazoline ring and macrocyclization steps was slightly greater

for 1d compared with 1a. Microsomal metabolism of all

apratoxins was strongly accelerated by NADPH and stability

was found to be low (t_1/2< 5 min, Table 6), which may suggest

that certain apratoxin biotransformation products could also

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retain activity, considering that 1a was extremely potent and

active in vivo as well.^2a

ments, and (5) the optimized steps are eﬃcient for the

synthesis of 1a and viable to generate 1c and 1d. As

hypothesized, SAR studies demonstrated that 1b and 1c retain

activity compared with 1a. Unexpectedly, we identiﬁed 1d as

the most potent apratoxin to date, exhibiting 2−3-fold

improved in vitro activity, even though 1d still has the

potential to be deactivated by dehydration. However,

compound 1d was found to be stable during repuriﬁcations

and stability tests. The propensity of apratoxins to dehydrate to

form a C34−C35 double bond is decreased with each

methylation at C34; this is possibly a reason why the

methylene-C34 has not been isolated from cyanobacteria but

only the dehydrated form, apratoxin E (Figure 1),^1cwhich

could have been formed enzymatically or nonenzymatically.

While dehydration appears to not be a problem during in vitro

incubations and stability studies, it may lead to apratoxin

deactivation in vivo so that the corresponding gem-dimethyl

compound 1c was further characterized and shown to possess

in vivo antitumor eﬃcacy. The low microsomal stability

combined with the potent in vivo activity may suggest that

apratoxin biotransformation products could play a role in the

overall activity, and future experiments are aimed at character-

izing the in vivo biotransformation products. Overall, our study

further corroborates that the development of this class of

anticancer agents is warranted.

In Vivo Activity of 1c. Even though 1c had slightly lower

activity than our other synthetic apratoxins, it was still very

potent in vitro and had the potential advantage that it cannot

dehydrate to form a conjugated system. Therefore, we tested 1c

for eﬃcacy in the same HCT116 xenograft mouse model as

previously performed for 1a^2aand administered 1c at the

previous eﬃcacious dose for 1a (0.25 mg/kg, ∼5 μg/mouse)

and a 2.5-fold lower dose (0.1 mg/kg, ∼2 μg/mouse) daily via

intraperitoneal (ip) injection for 16 days. There was a clear

dose-dependent tumor growth inhibition (Figure 4A). The

EXPERIMENTAL SECTION

■

Synthesis. General Procedures. All commercial reagents were

used without further puriﬁcation unless otherwise noted. Solvents

were puriﬁed according to the guidelines in ref 21. Tetrahydrofuran

(THF) and diethyl ether (Et₂O) were distilled from sodium chips in

the presence of a small amount of benzophenone; CH₂Cl₂and toluene

were distilled from CaH₂; MeCN, N,N-dimethylformamide (DMF)

were dried with 4 Å molecular sieves (MS) and MeOH dried with 3 Å

MS; 4 M hydrochloric acid (HCl) solution in ethyl acetate was

prepared by dissolving HCl gas (yielding by dropping aqueous

hydrochloric acid (34%) to concentrated sulfuric acid (98%)) to ethyl

acetate. All reactions were performed in heat-gun dried ﬂasks (400 °C

under reduced pressure) under an inert atmosphere of anhydrous Ar

unless otherwise noted. Thin layer chromatography was performed on

EMD silica gel 60 Å F₂₅₄glass plates and preparative thin layer

chromatography was performed on Whatman silica gel 60 Å F₂₅₄glass

plates (layer thick 1000 μm). Flash column chromatography was

performed with Fisher 170−400 mesh silica gel. Nuclear magnetic

resonance (NMR) spectra were recorded on a Varian Mercury 400

MHz, Bruker Avance II 600, Bruker Avance III 600 MHz, or Agilent

VNMR 600 MHz spectrometer as indicated in the data list. Chemical

shifts for proton nuclear magnetic resonance (¹H NMR) spectra are

reported in parts per million relative to the signal residual CDCl₃at

7.26 ppm. Chemicals shifts for carbon nuclear magnetic resonance

(¹³C NMR) spectra are reported in parts per million relative to the

center line of the CDCl₃triplet at 77.16 ppm. The abbreviations s, d,

dd, ddd, dddd, t, q, br, and m stand for the resonance multiplicity

singlet, doublet, doublet of doublets, doublet of doublet of doublets,

doublet of doublet of doublet of doublets, triplet, quartet, broad and

multiplet, respectively. Optical rotation was measured on a Perkin-

Elmer 341 polarimeter (Na D line) using a microcell of 1 dm path

length. High resolution mass spectra (HRMS) data were obtained

using an Agilent-LC-TOF mass spectrometer with an APCI/ESI

multimode ion source detector. LR-MS data was obtained using a

3200 QTrap triple quadrupole mass spectrometer and detection by

electrospray ionization-MS in the positive ion mode. The purity

(>95%) of all tested compounds was determined by HPLC analysis

(Shimadzu) equipped with a Phenomenex Ultracarb ODS column

(250 mm × 10 mm, 5 μm) and a Shimadzu SPD-M20A detector at

200/220 nm wavelength. The mobile phase was a MeCN/H₂O

mixture. All tested compounds were at least 95% pure.

Figure 4. Dose-dependent in vivo activity of 1c using a HCT116

xenograft mouse model. (A) Subcutaneous tumor-bearing mice were

injected daily ip with 1c (n = 7) or vehicle (n = 6), and tumor volumes

were monitored over time to assess eﬃcacy. Error bars indicate SEM

(B) At the end of the eﬃcacy study, tumors were analyzed by

immunoblot analysis for levels of an RTK.

higher dose strongly retarded the tumor growth, paralleling the

eﬀects of 1a.^2aThe lower dose showed a weaker yet

pronounced eﬀect (Figure 4A); however, the tumor growth

could only be inhibited by about 50% using 0.1 mg/kg. It is

noteworthy that we did not observe signs of toxicity based on

lack of both weight loss and abnormal behavior. Although

VEGFR2 expression in HCT116 xenografts was found to be

low (consistent with literature data²⁰), at the higher dose of 1c,

there was a detectable reduction in VEGFR2 expression levels,

indicating target engagement in vivo (Figure 4B).

CONCLUSION

■

Apratoxin S4 (1a) along with its hitherto unknown C30 epimer

(1d) and two analogues with achiral C34 (1b,c) were

synthesized using improved procedures. Most steps have

been further optimized compared to our previous apratoxin

S4 synthesis. The innovative points in the synthesis are: (1)

preparation of homoallylic alcohol 12 from its TBS ether

directly working up by cooling-concentration, which is critical

to the scale up of aldehyde 7, (2) Leighton’s silanes were

introduced to eﬃciently prepare β-hydroxy acid 4, one key

segment of composing apratoxins, (3) LiAlH₄was used instead

of DIBAL-H to prepare amino aldehyde in the preparation of

moCys part to greatly decrease the racemization, (4) Kelly’s

thiazoline formation methods (both Ph₃PO/Tf₂O and

TiCl₄) were developed to meet diﬀerent substrate require-

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(S)-2,2-Dimethyl-5-hexen-3-ol (12).^2d−h,8The solution of tetra-n-

butylammonium ﬂoride trihydrate (TBAF) (82.07 g, 260.116 mmol)

in THF (220 mL) was added to the mixture of compound 11 (21.0 g,

86.705 mmol) and 4 Å molecular sieves (22 g, predried at 450 °C

under reduced pressure 1 h) in anhydrous THF (300 mL) at 0 °C.

Then the reaction mixture was stirred at room temperature overnight

and ﬁltered through a small pad of Celite (washed with diethyl ether).

The ﬁltrate was quenched with 200 mL of water, extracted with diethyl

ether (300 mL × 3), washed with brine (200 mL × 2), dried over

anhydrous MgSO₄, concentrated with cooling/condensing fraction

distillation system under moderate vacuum, and further concentrated

by Vigreux fraction distillation column. The concentrated mixture was

puriﬁed by column chromatography eluted by 3−5% diethyl ether in

pentane. The eluted product fractions were also concentrated by

cooling/condensing fraction distillation system under moderate

vacuum to give product 12 and further distilled by Vigreux fraction

21.1 ppm. HRMS (ESI) m/z calcd for C₂₁H₃₄O₃Na (M + Na)⁺

357.2400, found 357.2409.

tert-Butyl [(4S,6S,8S)-8-(4-Methoxybenzyloxy)-6,9,9-tetramethyl-

dec-1-en-4-yloxy]dimethyl-silane (20b). To the solution of 19b

(178.6 mg, 0.534 mmol) in CH₂Cl₂(8 mL) were added 2,6-lutidine

(310 μL, 2.672 mmol) and tert-butyldimethylsilyl triﬂuoromethanesul-

fonate (TBSOTf) (368.2 μL, 1.603 mmol) at 0 °C under Ar. After

being stirred at the same temperature for 1.5 h, the reaction was

quenched with MeOH (5 mL) and saturated aq NH₄Cl (7 mL),

extracted with ethyl acetate (10 mL × 4), washed with brine (10 mL ×

2), dried with anhydrous MgSO₄, evaporated in vacuo, and puriﬁed by

column chromatography (eluted by 3.6% ethyl acetate in hexane) to

give product 20b (224.4 mg, 94%) as a colorless oil; [α]²⁰−49.0 (c

D

1

0.31, CH₂Cl₂). H NMR (400 MHz, CDCl₃): δ 7.28 (d, J = 8.4 Hz,

2H), 6.86 (d, J = 8.8 Hz, 2H), 5.86−5.76 (m, 1H), 5.07−5.03 (m,

2H), 4.65 (d, J = 10.8 Hz, 1H), 4.45 (d, J = 10.8 Hz, 1H), 3.86−3.80

(m, 1H), 3.80 (s, 3H), 3.08 (dd, J = 7.2, 3.6 Hz, 1H), 2.31−2.20 (m,

2H), 1.87 (br m, 1H), 1.66 (ddd, J = 13.2, 9.2, 2.8 Hz, 1H),1.46−1.34

(m, 2H), 1.06 (d, J = 6.8 Hz, 3H), 0.91 (s, 9H), 0.86 (s, 9H) ppm. ¹³C

NMR (100 MHz, CDCl₃): δ 159.0, 135.2, 131.9, 128.9, 117.0, 113.7,

85.6, 73.9, 69.8, 55.4, 43.9, 43.4, 40.5, 36.2, 26.8, 26.6, 26.1, 21.0, 18.2,

−4.1, −4.3 ppm. HRMS (ESI) m/z calcd for C₂₇H₄₈O₃SiNa (M +

Na)⁺471.3265, found 471.3264.

Compound 21a,b. To a solution of 20 (0.806 mmol) in the

mixture of CH₂Cl₂(5.0 mL) and H₂O (0.5 mL) was added 2,3-

dichloro-5,6-dibenzoquinone (DDQ) (220 mg, 0.968 mmol) at 0 °C.

The reaction mixture was stirred at the same temperature for 1 h,

quenched with saturated aqueous NaHCO₃(10 mL), and ﬁltered in

vacuo. The organic layer was separated and the water layer was

extracted with CH₂Cl₂(30 mL × 3). The organic phase was combined

and washed with brine (20 mL × 2), dried with anhydrous MgSO₄,

ﬁltered, and concentrated in vacuo. This residue was used for the next

reaction without further puriﬁcation.

To the suspended solution of Fmoc-Pro-OH (549.5 mg, 1.629

mmol) in toluene (5.0 mL) were added N,N-diisopropylethylamine

(DIEA) (435 μL, 2.5 mmol) and 2,4,6-trichlorobenzoyl chloride (390

μL, 2.5 mmol) at room temperature under argon, and the mixture was

stirred at the same temperature for 30 min. Then the crude alcohol in

toluene (3.0 mL) and DMAP (350 mg, 2.862 mmol) were added to

the above mixture at 10 °C. After being stirred at room temperature

for 3 h, the reaction mixture was quenched and extracted with diethyl

ether (10 mL × 3). The combined organic layer was washed with

saturated NH₄Cl (15 mL × 2), saturated NaHCO₃(15 mL × 2), and

brine (15 mL), dried over MgSO₄, and concentrated in vacuo. The

residue was puriﬁed by silica gel column chromatography (eluted by

10% ethyl acetate in hexane) to give ester 21.

1

distillation column to provide product 12 (10.5 g, 95%). H NMR

(400 MHz, CDCl₃): δ 5.86 (dddd, J = 14.4, 10.4, 8.8, 6.0 Hz, 1H),

5.14 (m, 2H), 3.25 (dd, J = 10.4, 2.0 Hz, 1H), 2.39−2.33 (m, 1H),

1.98 (ddd, J = 13.6, 9.6, 9.6 Hz, 1H), 0.91 (s, 9H) ppm. ¹³C NMR

(100 MHz, CDCl₃): δ 136.7, 117.9, 78.2, 36.7, 34.7, 25.9 ppm.

(3R,5S)-5-(4-Methoxy-benzyloxy)-3,6,6-trimethylheptanoic Acid

Methoxy-methyl-amide (17).^2d4-Methoxybenzyl-2,2,2-trichloroaceti-

midate (3.7 mL, 17.8 mmol) and triﬂuoromethane sulfonic acid

(TfOH) (7.9 μL, 0.089 mmol) was added sequentially to the solution

of 16 (2.05 g, 8.9 mmol) in THF (20 mL) at 0 °C. The resulting

mixture was stirred at room temperature overnight and was then

diluted with ethyl acetate (20 mL), quenched with saturated NaHCO₃

(20 mL), extracted with ethyl acetate (20 mL × 3), dried with

anhydrous MgSO₄, and evaporated in vacuo. Hexane (100 mL) was

added to the residue, which resulted in the precipitation of a white

solid (2,2,2-trichloroacetimidate). The solid was ﬁltered oﬀ, and the

ﬁltrate was concentrated and puriﬁed by column chromatography

(eluted by 20−50% ethyl acetate in hexane) to give product 17 (1.74

g, 56%, 100% BRSM) (recovered starting material 1.08 g and used in

next cycle).

(3R,4S,6S,8S)-8-(4-Methoxybenzyloxy)-3,6,9,9-tetramethyldec-1-

en-4-ol (19a).^2a,cTo the solution of 7 (234.1 mg, 0.801 mmol) in

CH₂Cl₂(8 mL) was added (S,S)-trans EZ-CrotylMix (mixture of 18a

and Sc(OTf)₃) (943 mg, 1.602 mmol) at room temperature. This

mixture was stirred vigorously at the same temperature for 2.5 h, and

then it was treated with 12 mL of Et₂O and 12 mL of 1N aq HCl. After

this quenched mixture was stirred at room temperature for 30 min, it

was ﬁltered oﬀ to remove precipitated solid, extracted with Et₂O (15

mL × 3), washed with saturated NaHCO₃(20 mL × 2) and brine (20

mL), dried with anhydrous MgSO₄, evaporated in vacuo, and puriﬁed

by chromatography column (eluted by 3.5−4.5% ethyl acetate in

hexane) to give product 19a (245.7 mg, 88%) as a colorless oil.

(4S,6S,8S)-8-(4-Methoxybenzyloxy)-6,9,9-tetramethyldec-1-en-4-

ol (19b). To the solution of 7 (203.3 mg, 0.696 mmol) in CH₂Cl₂(7

mL) was added (S,S)-18b (772.1 mg, 1.392 mmol) and Sc(OTf)₃

(28.5 mg, 0.058 mmol) at 0 °C. This mixture was stirred vigorously at

the same temperature for 2.0 h, and then it was treated with 12 mL of

Et₂O and 12 mL of 1N aq HCl. Afterwards, the quenched mixture was

stirred at room temperature for 30 min, ﬁltered to remove the

precipitate, extracted with Et₂O (15 mL × 3), washed with saturated

NaHCO₃(20 mL × 2) and brine (20 mL), dried with anhydrous

MgSO₄, evaporated in vacuo, and puriﬁed by column chromatography

Pyrrolidine-1,2-dicarboxylic Acid-(2S)-2-[(4S,6S,8S)-8-tert-Butyl-4-

(tert-butyldimethylsilyl-oxy)-6-dimethyloct-1-en-8-yl]ester 1-(9H-

Fluoren-9-ylmethyl)ester (21b) (from 20b). Yield 100%; [α]²⁰

D

−66.4 (c 0.11, CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃, mixture of

rotamers): δ 7.78−7.75 (m,2 H), 7.67−7.55 (m, 2H), 7.42−7.38 (m,

2H), 7.33−7.30 (m, 2H), 5.85−5.71 (m, 1H), 5.05−4.99 (m, 2H),

4.80 (dd, J = 8.4, 2.0 Hz, 1H), 4.53−4.40 (m, 2H), 4.35−4.17 (m,

2H), 3.81−3.71 (m, 1H), 3.70−3.62 (m, 1H), 3.60−3.50 (m, 1H),

2.32−2.15 (m, 3H), 2.14−1.93 (m, 3H), 1.62−1.49 (m, 3H), 1.39−

1.30 (m, 1H), 1.20−1.03 (m, 1H), 0.95 (d, J = 6.4 Hz, 2H), 0.87 (s, 9

H), 0.87 (s, 9 H), 0.82 (d, J = 6.4 Hz, 1H), 0.05−0.02 (m, 6H) ppm.

¹³C NMR (100 MHz, CDCl₃, mixture of rotamers): δ 172.2, 172.1,

154.7, 154.5, 144.3, 144.2, 144.1, 143.8, 141.4, 141.4, 141.3, 135.6,

135.3, 127.8, 127.8, 127.2, 127.1, 127.1, 125.5, 125.3, 125.2, 120.0,

116.9, 116.8, 80.6, 80.4, 77.5, 77.2, 76.8, 70.2, 70.0, 67.8, 67.5, 59.8,

59.6, 47.4, 47.0, 46.4, 44.2, 44.0, 42.6, 42.5, 39.3, 39.1, 35.2, 35.1, 31.3,

30.1, 27.1, 27.0, 26.1, 26.0, 26.0, 24.4, 23.4, 20.9, 20.6, 18.2 ppm.

HRMS (ESI) m/z calcd for C₃₉H₅₇NO₅SiNa (M + Na)⁺670.3898,

found 670.3920.

(eluted by 4.2% ethyl acetate in hexane) to give product 19b (197.6

1

mg, 85%) as a colorless oil; [α]²⁰−57.8 (c 0.32, CH₂Cl₂). H NMR

D

(400 MHz, CDCl₃): δ 7.30 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz,

2H), 5.89−5.79 (m, 1H), 5.17−5.13 (m, 2H),4.63 (d, J = 10.4 Hz,

1H), 4.52 (d, J = 10.4, 1H), 3.79 (s, 3H), 3.79−3.74 (m, 1H), 3.11

(dd, J = 9.2, 2.4 Hz, 1H), 2.30−2.24 (m, 1H), 2.21−2.14 (m, 1H),

2.00−1.90 (br m, 1H), 1.65 (ddd, J = 13.6, 10.6, 2.8 Hz, 1H), 1.46

(ddd, J = 14.0, 8.8, 4.0 Hz, 1H), 1.35 (ddd, J = 14.0, 9.2, 2.4 Hz, 1H),

1.09 (ddd, J = 13.6, 10.4, 2.0 Hz, 1H), 0.97 (d, J = 6.4 Hz, 3H), 0.94

(s, 9H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 159.0, 135.0, 131.7,

129.2, 118.1, 113.7, 85.3, 74.3, 68.4, 55.3, 43.5, 43.3, 39.8, 36.2, 26.6,

Compound 4a,b. To the solution of 21 (0.646 mmol) in DMF (6.0

mL) were added Oxone (1.588 g, 2.583 mmol), NaHCO₃(217.0 mg,

2.583 mmol), and OsO₄(2.5% solution in tert-BuOH) (81 μL, 6.5

μmol) at room temperature. After being stirred at the same

temperature for 15 h, the reaction mixture was diluted with water (4

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mL) and tert-BuOH (7.5 mL), and then NaIO₄(276.3 mg, 1.292

mmol) was added. The reaction mixture was stirred at room

temperature for an additional 5 h and poured into aqueous HCl (1

M for 4a, 0.5 M for 4b to pH 1; 25 mL) and CH₂Cl₂(25 mL). The

water layer was extracted with CH₂Cl₂(50 mL × 3). The combined

CH₂Cl₂layer was washed with 10 wt % Na₂S₂O₃(50 mL × 3) and

brine (50 mL × 1), dried over MgSO₄, and concentrated in vacuo. The

residue was puriﬁed by silica gel column chromatography to give

product 4 as a white solid (4a eluted by 17−25% ethyl acetate in

hexane; 4b eluted by 2.5−10% MeOH in CH₂Cl₂).

Allyl (3S,5S,7S)-3-Hydroxy-7-(4-methoxybenzyloxy)-2,2,5,8,8-

pentamethylnonanoate (25). To the solution of compound 24

(222.4 mg, 0.585 mmol) in dimethyl sulfoxide (DMSO) (5.0 mL)

were added K₂CO₃(242.5 mg, 1.754 mmol) and allyl bromide

(AllylBr) (99 μL, 1.170 mmol) at room temperature. After being

stirred at the same temperature overnight, the reaction was quenched

with water (10 mL) and extracted with ethyl acetate (20 mL × 4). The

combined organic layer was washed with brine (10 mL × 5), ﬁltered,

concentrated in vacuo, and puriﬁed by silica gel column chromatog-

raphy (eluted by 6.5% ethyl acetate in hexane) to give product 25

(228.6 mg, 93%) as a colorless oil; [α]²⁰_D−56.0 (c 0.175, CH₂Cl₂). ¹H

NMR (400 MHz, CDCl₃): δ 7.30 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.4

Hz, 2H), 5.96−5.86 (m, 1H), 5.35−5.22 (m, 2H), 4.64 (d, J = 10.4

Hz, 1H), 4.60 (d, J = 5.6 Hz, 2H), 4.50 (d, J = 10.4 Hz, 1H), 3.78 (br

m, 1H), 3.77 (s, 3H), 3.10 (dd, J = 8.0, 2.0 Hz, 1H), 2.54 (br, 1H), 2.0

(br m, 1H), 1.61−1.55 (m, H), 1.49 (ddd, J = 17.6, 9.6, 4.0 Hz, 1H),

1.37−1.31 (m, 1H), 1.21 (s, 6H), 1.05−0.98 (m, 1H), 0.97 (d, J = 6.8

Hz, 3H), 0.94 (s, 9H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 177.3,

158.9, 132.0, 131.5, 129.1, 118.2, 113.6, 85.0, 74.3, 74.2, 65.2, 55.2,

47.3, 39.7, 38.1, 36.1, 26.6, 26.5, 21.9, 20.9, 20.7 ppm. HRMS (ESI)

m/z calcd for C₂₅H₄₀O₅Na (M + Na)⁺443.2768, found 443.2769.

Allyl (3S,5S,7S)-3-(2,2,2-Trichloroethoxycarbonyloxy)-7-(4-me-

thoxybenzyloxy)-2,2,5,8,8-pentamethylnonanoate (26). To the

solution of 25 (220.0 mg, 0.523 mmol) in CH₂Cl₂(5.0 mL) at 0

°C under Ar were added 4-dimethylaminopyridine (DMAP) (1.3 mg,

10.5 μmol), pyridine (423 μL, 5.234 mmol), and 2,2,2-trichloroethox-

ylcarbonyl chloride (Troc-Cl) (554.5 mg/360.0 μL, 2.617 mmol).

After being stirred at 0 °C for 10 min and room temperature for 1 h,

the reaction was quenched with water (10 mL) and extracted with

ethyl acetate (15 mL × 4). The combined organic layer was washed

with saturated NaHCO₃and brine, dried with MgSO₄, concentrated in

vacuo, and puriﬁed by silica gel column chromatography (2.5−10−

Pyrrolidine-1,2-dicarboxylic Acid (2S)-2-[(1S,3S,5S)-1-tert-Butyl-6-

carboxy-5-hydroxy-3-methylhept-1-yl]ester 1-(9H-ﬂuoren-9-

ylmethyl)ester (4b) (from 21b). Yield 80%; [α]²⁰−46.2 (c 0.119,

D

CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃, mixture of rotamers, major): δ

7.75 (d, J = 7.6 Hz, 2H), 7.62−7.55 (m, 2H), 7.39 (dd, J = 7.6, 7.4 Hz,

2H), 7.33−7.29 (m, 2H), 5.40 (br, 1H), 4.91 (d, J = 10.8 Hz, 1H),

4.42−4.38 (m, 1H), 4.34−4.29 (m, 1H), 4.12−4.06 (m, 1H), 3.68−

3.62 (m, 1H), 3.52−3.47 (m, 1H), 2.44−2.43 (m, 2H), 2.33−2.23 (m,

1H), 2.08−1.91 (m, 3H), 1.86−1.80 (m, 1H), 1.75 (dd, J = 13.6, 2.4

Hz, 1H), 1.66 (dd, J = 14.4, 2.0 Hz, 1H), 1.33−1.26 (m, 1H), 1.04−

0.98 (m, 1H), 0.98 (d, J = 6.4 Hz, 3H), 0.89 (s, 9H) ppm. ¹³C NMR

(100 MHz, CDCl_3,mixture of rotamers, major): δ 174.6, 172.4,155.4,

144.0, 143.7, 127.9, 127.8, 127.3, 127.2, 125.3, 125.2, 120.1, 120.1,

78.5, 68.0, 66.0, 59.6, 47.2, 46.7, 42.7, 41.7, 37.2, 34.8, 30.0, 26.1, 25.0,

24.6, 20.6 ppm. HRMS (ESI) m/z calcd for C₃₂H₄₁NO₇Na (M + Na)⁺

574.2775, found 574.2970.

Methyl (3S,5S,7S)-3-Hydroxy-7-(4-methoxybenzyloxy)-2,2,5,8,8-

pentamethylnonanoate (23). To the solution of N-tosyl-^D-Val-OH

(145.6 mg, 0.537 mmol) in CH₂Cl₂(7 mL) at 0 °C under argon was

added the solution of BH₃in THF (1M, 537 μL, 0.537 mmol)

dropwise. The resulting mixture was stirred at room temperature for

20 min and then cooled to −78 °C. Aldehyde 7 (157.0 mg, 0.537

mmol) and methyl trimethylsilyl dimethylketene acetal (120.0 μL,

0.591 mmol) were added successively to the above mixture at −78 °C.

After being stirred at −78 °C for 3 h, the reaction mixture was

quenched with buﬀer (pH 7; 8 mL) and then it was warmed to room

temperature and another 5 mL of buﬀer (pH 7) was added. The

quenched reaction mixture was extracted with Et₂O (25 mL × 3),

washed with saturated NaHCO₃(15 mL × 2) and brine (15 mL × 2),

dried with anhydrous MgSO₄, and puriﬁed by column chromatography

15% ethyl acetate in hexane) to give 26 (295.1 mg, 95.0%); [α]²⁰

D

1

−7.6 (c 0.37, CH₂Cl₂). H NMR (400 MHz, CDCl₃): δ 7.27 (d, J =

8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 5.96−5.86 (m, 1H), 5.37−5.23

(m, 3H), 4.78 (d, J = 12.0 Hz, 1H), 4.59 (d, J = 5.6 Hz, 2H), 4.55 (d, J

= 10.8 Hz, 1H), 4.49 (d, J = 10.8 Hz, 1H), 4.30 (d, J = 12.0 Hz, 1H),

3.78 (s, 3H), 3.05 (dd, J = 9.6, 2.0 Hz, 1H),1.99−1.93 (m, 1H), 1.75

(br m, 1H), 1.50 (ddd, J = 17.2, 9.2, 3.0 Hz, 1H), 1.35−1.29 (m, 1H),

1.27 (s, 3H), 1.25 (s, 3H), 1.08−1.01 (m, 4H), 0.92 (s, 9H) ppm. ¹³C

NMR (100 MHz, CDCl₃): δ 174.8, 159.0, 154.4, 131.9, 131.5, 128.7,

118.6, 113.7, 94.6, 85.0, 80.9,76.6, 74.4, 65.6, 55.3, 47.0, 39.8, 36.8,

36.1, 26.5, 26.4, 22.4, 20.8, 19.7 ppm. HRMS (ESI) m/z calcd for

C₂₈H₄₁Cl₃O₇Na (M + Na)⁺617.1810, found 617.1832.

(eluted by 8.5% ethyl acetate in hexane) to give product 23 as a

1

colorless oil (171.5 mg, 81%); [α]²⁰−55.8 (c 0.12, CH₂Cl₂). H

D

NMR (400 MHz, CDCl₃): δ 7.29 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.4

Hz, 2H), 4.63 (d, J = 10.4 Hz, 1H), 4.50 (d, J = 10.4 Hz, 1H), 3.77 (s,

3H), 3.74 (br m, 1H), 3.69 (s, 3H), 3.09 (dd, J = 9.2, 2.4 Hz, 1H), 2.47

(br, 1H), 2.03−1.95 (br m, 1H), 1.58−1.45 (m, 2H), 1.37−1.30 (m,

1H), 1.18 (s, 6H), 1.03−1.00 (m, 1H), 0.96 (d, J = 6.4 Hz, 3H), 0.93

(s, 9H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 178.3, 159.0, 131.6,

129.2, 113.7, 85.2, 74.4, 74.2, 55.3, 51.9, 47.3, 39.7, 38.2, 36.2, 26.8,

26.6, 22.1, 20.9, 20.6 ppm. HRMS (ESI) m/z calcd for C₂₃H₃₈O₅Na

(M + Na)⁺417.2611, found 417.2628.

Pyrrolidine-1,2-dicarboxylic Acid (2S)-2-[(1S,3S,5S)-1-tert-Butyl-6-

allyloxycarbonyl-5-(2,2,2-trichloroethoxycarbonyloxy)-3,6-dime-

thylhept-1-yl]ester 1-(9H-Fluoren-9-ylmethyl)ester (27). To a sol-

ution of 26 (291.5 mg, 0.491 mmol) in a mixture of CH₂Cl₂(5.0 mL)

and H₂O (0.5 mL) was added 2,3-dichloro-5,6-dibenzoquinone

(DDQ) (133.6 mg, 0.589 mmol) at 0 °C. The reaction mixture was

stirred at the same temperature for 1 h, quenched with saturated

aqueous NaHCO₃(6 mL), and ﬁltered in vacuo. The organic layer was

separated, and the water layer was extracted with CH₂Cl₂(15 mL ×

4). The organic phase was combined and washed with brine (3 × 15

mL), dried with anhydrous MgSO₄, ﬁltered, and concentrated in

vacuo. This residue was used for the next reaction without further

puriﬁcation.

To the suspended solution of Fmoc-Pro-OH (334.3 mg, 0.991

mmol) in toluene (3.0 mL) were added N,N-diisopropylethylamine

(DIEA) (0.26 mL, 1.49 mmol) and 2,4,6-trichlorobenzoyl chloride

(233 μL, 1.491 mmol) at room temperature under argon, which was

stirred at the same temperature for 10 min. Then the crude alcohol in

toluene (1.5 mL) and DMAP (212.8 mg, 1.742 mmol) were added to

the above mixture. After being stirred at room temperature overnight,

the reaction mixture was quenched with water and extracted with

diethyl ether (20 mL × 4). The combined organic layer was washed

with saturated NH₄Cl (20 mL × 2), saturated NaHCO₃(20 mL × 2),

and brine (20 mL), dried over MgSO₄, and concentrated in vacuo. The

residue was puriﬁed by silica gel column chromatography (eluted by

10% ethyl acetate in hexane) to give ester 27 (360.8 mg, 90.4%) as a

(3S,5S,7S)-3-Hydroxy-7-(4-methoxybenzyloxy)-2,2,5,8,8-pentam-

ethylnonanoic Acid (24). To the solution of 23 (223.8 mg, 0.568

mmol) in the mixture of THF−MeOH−H₂O (7 mL−3.5 mL−0.7

mL) was added aq LiOH (119.1 mg in 2.8 mL H₂O, 2.838 mmol).

After being stirred at room temperature for 5 h, the reaction mixture

was diluted with 10 mL of water, acidiﬁed by addition of aq HCl (2M)

to pH 2, extracted with ethyl acetate (10 mL × 4), washed with brine

(10 mL × 2), dried with anhydrous MgSO₄, and puriﬁed by column

chromatography (eluted by 8.5−17% acetone in hexane) to give 24

1

(225 mg, 100%) as a white solid; [α]²⁰−52.3 (c 0.26, CH₂Cl₂). H

D

NMR (400 MHz, CDCl₃): δ 7.30 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4

Hz, 2H), 4.63 (d, J = 10.4 Hz, 1H), 4.53 (d, J = 10.4 Hz, 1H), 3.80 (d,

J = 13.0, 1H), 3.78 (s, 3H),3.13 (dd, J = 8.0, 2.0 Hz, 1H), 1.80 (br m,

1H), 1.62−1.49 (m, 2H), 1.40−1.34 (m, 1H), 1.21 (s, 3H), 1.19 (s,

3H), 1.09 (dd, J = 12.4, 12.4), 0.98 (d, J = 6.4 Hz, 3H), 0.95 (s, 9H)

ppm. ¹³C NMR (100 MHz, CDCl₃): δ 182.9, 159.0, 131.4, 129.3,

113.7, 85.2, 74.3, 74.3, 55.3, 47.1, 39.4, 37.9, 36.2, 26.8, 26.6, 22.3,

20.8, 20.2 ppm. HRMS (ESI) m/z calcd for C₂₂H₃₆O₅Na (M + Na)⁺

403.2455, found 403.2472.

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1

colorless oil; [α]²⁰−58.9 (c 0.124, CH₂Cl₂). H NMR (400 MHz,

28.5, 14.2 ppm. HRMS (ESI) m/z calcd for C₃₁H₃₅NO₄SNa ((M +

Na)⁺540.2210, found 540.2223.

D

CDCl₃, mixture of rotamers): δ 7.77−7.75 (m, 2H), 7.67−7.62 (m,

2H), 7.40 (dd, J = 7.4, 7.2 Hz, 2H), 7.34−7.29 (m, 2H), 6.00−5.79

(m, 1H), 5.34−5.12 (m, 3H), 4.97 (d, J = 12.0 Hz, 0.6H), 4.87 (d, J =

12.0 Hz, 0.4H), 4.78 (d, J = 10.0 Hz, 1H), 4.69 (d, J = 12.0 Hz, 0.4H),

4.64 (d, J = 12.0 Hz, 0.6H), 4.59−4.16 (m, 6H), 3.67−3.61 (m, 1H),

3.59−3.48 (m, 1H), 2.38−2.28 (m, 0.6H), 2.24−2.16 (m, 0.4H),

2.15−2.06 (m, 1H), 2.04−1.93 (m, 2H), 1.92−1.80 (m, 1H), 1.55−

1.48 (m, 1H), 1.46−1.42 (m, 1H), 1.38−1.29 (m, 1H), 1.25−1.19 (m,

6H), 1.15−1.07 (m, 0.6H), 1.00−0.96 (m, 1.6H), 0.89 (s, 5.4H), 0.86

(s, 3.6H), 0.67(d, J = 6.4 Hz, 1.8H) ppm. ¹³C NMR (100 MHz,

CDCl₃, mixture of rotamers): δ 175.0,175.0, 172.9, 172.4, 154.7, 154.5,

154.3, 154.2, 144.4, 144.3, 144.0, 143.9, 141.4, 141.4, 141.3, 141.3,

132.0, 131.9, 127.8, 127.2, 127.1, 125.5, 125.4, 125.3, 125.2, 120.0,

120.0, 118.6, 94.9, 94.8, 80.8, 80.6, 79.8, 79.5, 77.1, 76.9, 68.0, 67.5,

65.7, 65.6, 59.9, 59.4, 47.3, 47.3, 47.2, 47.1, 46.5, 38.2, 38.0, 37.3, 36.8,

34.9, 34.8, 34.7, 31.7, 31.4, 30.1, 26.9, 26.7, 25.9, 25.9, 25.4, 23.5, 22.8,

22.0, 22.0, 20.5, 20.4, 19.8, 19.7, 14.2 ppm. HRMS (ESI) m/z calcd for

C₄₀H₅₀Cl₃NO₉Na (M + Na)⁺816.2443, found 816.2420.

Pyrrolidine-1,2-dicarboxylic Acid (2S)-2-[(1S,3S,5S)-1-tert-Butyl-6-

carboxy-5-(2,2,2-trichloroethoxycarbonyloxy)-3,6-dimethylhept-1-

yl]ester 1-(9H-Fluoren-9-ylmethyl)ester (4c). To a solution of 27

(349.3 mg, 0.440 mmol) in THF (8 mL) were added Pd(PPh₃)₄(76.3

mg, 0.066 mmol) and N-methyl aniline (144.3 μL, 1.321 mmol) at

room temperature under argon. This reaction was protected with

aluminum foil. After being stirred at room temperature for 1 h, the

reaction mixture was concentrated in vacuo and puriﬁed by column

Compound 31a^2aand 31b. To the solution of compound 30

(3.379 g, 6.532 mmol) in 95% EtOH (35 mL) was added NaBH₄

(247.1 mg, 6.532 mmol) at room temperature. The resulting mixture

was stirred at room temperature for 30 h and then quenched with

water (50 mL) and extracted with Et₂O (50 mL × 4). The combined

organic layer was dried with anhydrous MgSO₄, evaporated in vacuo,

and puriﬁed by column chromatography (eluted with the mixture of

CH₂Cl₂/hexane/acetone from 50:148:2 → 50:98:2 → 50:48:2) to give

product 31 (1.187 g, 35%) as a thick colorless oil.

Ethyl (R)-4-tert-Butoxycarbonylamino-5-(triphenylmethylthio)-

1

pentanonate (31b). [α]²⁰+4.5 (c 0.20, CH₂Cl₂). H NMR (400

D

MHz, CDCl₃₎: δ 7.42−7.40 (m, 6H), 7.30−7.27 (m, 6H), 7.23−7.19

(m, 3H), 4.49 (d, J = 9.2 Hz, 1H), 4.10 (q, J = 7.2 Hz, 2H), 3.64 (br,

1H), 2.34 (br m, 2H), 2.21 (t, J = 7.6 Hz, 2H), 1.78−1.59 (m, 2H),

1.43 (s, 9H), 1.23 (t, J = 7.2 Hz, 3H) ppm. ¹³C NMR (100 MHz,

CDCl₃): δ 173.3, 155.3, 144.7, 129.7, 128.0, 126.8, 79.4, 66.7, 60.5,

49.6, 37.2, 31.1, 29.7, 28.5, 14.3 ppm. HRMS (ESI) m/z calcd for

C₃₁H₃₇NO₄SNa (M + Na)⁺542.2336, found 542.2331.

Compound 5a^2aand 5b. To the solution of 31 (1.045g, 2.013

mmol) in 95% ethanol (7.5 mL) was added aq NaOH (1M, 4.0 mL) at

room temperature. After being stirred at the same temperature for 2 h,

the reaction mixture was diluted with water (10 mL), acidiﬁed with 1−

0.5 M aq HCl to pH 4−5, and extracted with diethyl ether (20 mL ×

3). The combined organic layer was dried over MgSO₄, ﬁltered, and

concentrated in vacuo to give crude acid 32, which was used in the

next step without further puriﬁcation. To the above crude acid 32

solution in DMSO (15 mL) was added K₂CO₃(556.5 mg, 4.027

mmol) and allyl bromide (255.5 μL, 3.020 mmol) at room

temperature. After being stirred at room temperature 5 h, the reaction

was quenched with water (30 mL) and extracted with ethyl acetate (50

mL × 3). The combined organic layer was washed with brine, ﬁltered,

and concentrated in vacuo, and the residue was puriﬁed by column

chromatography on silica gel (eluted by 10% ethyl acetate in hexane)

to give product 5 as a colorless oil.

chromatography (eluted by acetone/hexane 1:3) to give acid 4c (354.1

mg, 95%) as a pale-yellow solid; [α]²⁰−51.1 (c 0.131, CH₂Cl₂). H

1

D

NMR (400 MHz, CDCl₃, mixture of rotamers): δ 9.90 (br, 1H), 7.77−

7.74 (m, 2H), 7.68−7.63 (m, 2H), 7.42−7.38 (m, 2H), 7.34−7.29 (m,

2H), 5.23 (d, J = 10.4 Hz, 0.4H), 5.13 (d, J = 10.4 Hz, 0.6H), 5.01 (d, J

= 12.0 Hz, 0.6H), 4.90 (d, J = 12.0 Hz, 0.4H), 4.85−4.79 (m, 1H),

4.70 (d, J = 12.0 Hz, 0.4H), 4.63 (d, J = 12.0 Hz, 0.6H), 4.52−4.17 (m,

4H), 3.67−3.48 (m, 1H), 2.37−2.27 (m, 0.6H), 2.24−2.19 (m, 0.4H),

2.16−2.10 (m, 1H), 2.01−1.83 (m, 3H), 1.78−1.69 (m, 0.4H), 1.62−

1.49 (M, 1.6H), 1.43−1.35 (m, 1H), 1.31−1.20 (m, 6H), 1.09−1.00

(m, 1H), 1.01 (d, J = 6.4 Hz, 1.2 H), 0.94 (s, 5.4H), 0.87 (s, 3.6H),

0.69 (d, J = 6.4 Hz, 1.8H) ppm. ¹³C NMR (100 MHz, CDCl₃, mixture

of rotamers): δ 181.1, 180.7, 172.8, 172.3, 154.8, 154.4, 154.2, 144.3,

144.1, 143.9, 143.7, 141.3, 141.3, 141.2, 141.2, 135.1, 135.0, 135.0,

130.6, 128.1, 128.1, 128.0, 127.7, 127.1, 127.1, 127.0, 125.5, 125.3,

125.2, 125.1, 120.0, 119.9, 94.9, 94.8, 80.5, 80.3, 79.8, 79.4, 77.0, 76.8,

68.0, 67.6, 59.8, 59.3, 47.2, 47.1, 46.9, 46.4, 38.1, 37.5, 37.1, 36.7, 34.9,

34.7, 34.6, 34.5, 31.6, 31.3, 30.0, 29.1, 29.1, 26.7, 26.5, 25.9, 25.3, 24.3,

23.5, 22.7, 22.2, 21.9, 20.8, 20.4, 19.9, 19.6, 19.4, 18.8, 14.2, 11.5 ppm.

HRMS (ESI) m/z calcd for C₃₇H₄₆Cl₃NO₉Na (M + Na)⁺776.2130,

found 776.2149.

Allyl (R)-4-tert-Butoxycarbonylamino-5-(triphenylmethylthio)-

pentanoate (5b). Yield 0.75 g, 70%: [α]²⁰+10.0 (c 0.16, CH₂Cl₂).

D

¹H NMR (400 MHz, CDCl₃): δ 7.42−7.41 (m, 6H), 7.31−7.27 (m,

6H), 7.23−7.20 (m, 3H), 5.95−5.85 (m, 1H), 5.32−5.21(m, 2H), 4.55

(d, J = 5.6 Hz, 2H), 4.49 (d, J = 8.8 Hz, 1H), 3.65 (br m, 1H), 2.34 (br

m, 2H), 2.26 (t, J = 7.6 Hz, 2H), 1.80−1.62 (m, 2H), 1.44 (s, 9H)

ppm. ¹³C NMR (100 MHz, CDCl₃): δ 172.9, 155.3, 144.7, 132.2,

129.6, 128.1, 126.8, 118.3, 79.4, 66.7,65.3, 49.5, 37.2, 31.0, 29.7, 28.5

ppm. HRMS (ESI) m/z calcd for C₃₂H₃₇NO₄SNa ((M + Na)⁺

554.2336, found 554.2341.

General Procedure A: Synthesis of 3a,c,d and 34. To the solution

of 5 (169.6 mg, 0.319 mmol) in CH₂Cl₂(4 mL) were added 2,6-

lutidine (556 μL, 4.790 mmol) and trimethylsilyl triﬂuoro-

methanesulfonate (TMSOTf) (694 μL, 3.832 mmol) dropwise at

room temperature under argon. After being stirred at the same

temperature for 1.5 h, the reaction mixture was quenched with MeOH

(6 mL) and water (10 mL) at 0 °C and extracted with CH₂Cl₂(15 mL

× 4). The combined organic layer was washed with brine, dried over

anhydrous MgSO₄, and evaporated in vacuo to give the crude free

amine of 5, which was used in next step without further puriﬁcation.

To the crude free amine of 5 (1.3 equiv) in CH₂Cl₂(4 mL) were

added corresponding acid 4 (based in Scheme 5) (0.245 mmol, 1

equiv), coupling reagent (CR, 0.368 mmol, 1.5 equiv), and DIEA (140

μL, 0.735 mmol, 3.0 equiv) at room temperature or 0 °C. After being

stirred at room temperature for 2.5−24 h, the resulting reaction

mixture was evaporated in vacuo and puriﬁed by column

chromatography, eluting with ethyl acetate in hexane to give product

3a,c,d or 34 as a colorless oil.

Compound 30a^2a−cand 30b. To the solution of Weinreb amide

28 (2.505 g, 4.945 mmol) in THF (50 mL) was added LiAlH₄(234.6

mg, 6.182 mmol) in one portion at 0 °C. After being stirred at 0 °C for

30 min, the reaction mixture was quenched with 0.2N aq KHSO₄(30

mL) and extracted with Et₂O (50 mL × 3). The combined organic

layer was washed with 1N aq HCl (30 mL × 3) and brine (30 mL ×

3), dried with anhydrous MgSO₄, and evaporated in vacuo to give the

crude aldehyde 29, which was used in the next step without further

puriﬁcation. To the crude aldehyde 28 in toluene (30 mL) was added

Ph₃P=CHCO₂Et (3.101 g, 8.901 mmol) at 0 °C under Ar. After being

stirred at room temperature for 3 h, the reaction mixture was

concentrated in vacuo and puriﬁed by column chromatography (eluted

by 12−17% ethyl acetate in hexane) to give product(s) 30 as a

colorless oil.

Ethyl (R,E-)-4-tert-Butoxycarbonylamino-5-(triphenylmethyl-

thio)-penta-2-enoate (30b). Yield 88%, 2 steps; [α]²⁰+6.0 (c

D

1

0.20, CH₂Cl₂). H NMR (400 MHz, CDCl₃₎: δ 7.44−7.42 (m, 6H),

7.31−7.27 (m, 6H), 7.24−7.20 (m, 3H), 6.72 (dd, J = 15.6, 4.4 Hz,

1H), 5.84 (d, J = 15.6 Hz, 1H), 4.80 (d, J = 8.4 Hz, 1H), 4.29 (br, 1H),

4.17 (q, J = 7.2 Hz, 2H), 2.53−2.41 (m, 2H), 1.45 (s, 9H), 1.27 (t, J =

7.2 Hz, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 169.9, 154.7,

146.6, 144.3, 129.5, 128.0, 126.8, 121.5, 79.7, 67.0, 60.4, 50.5, 36.2,

Pyrrolidine-1,2-dicarboxylic Acid (2S)-2-{(1S,3S,5S,6S)-6-[(1S)-3-

Allyloxycarbonyl-1-(triphenylmethylthio)methyl-propylcarbamoyl]-

1-tert-butyl-5-(2,2,2-trichloroethoxy-carbonyloxy)-3-methylhept-1-

yl}ester 1-(9H-Fluoren-9-ylmethyl)ester (3a).^2aFrom 4a, 5a; CR BEP

added at 0 °C; reaction time 2.5 h; yield 245.6 mg, 87%).

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Article

Pyrrolidine-1,2-dicarboxylic Acid (2S)-2-{(1S,3S,5S)-6-[(1S)-3-Allyl-

oxycarbonyl-1-(triphenylmethylthio)methyl-propylcarbamoyl]-1-

tert-butyl-5-hydroxy-3-methylhex-1-yl}ester 1-(9H-Fluoren-9-

30.1, 29.8, 29.2, 29.2, 26.2, 26.1, 26.0, 25.9, 25.4, 24.4, 23.5, 22.8, 20.6,

20.2, 14.3, 14.0, 13.8 ppm. HRMS (ESI) m/z calcd for

C₆₃H₇₁Cl₃N₂O₁₀SNa (M + Na)⁺1175.3787, found 1175.3805.

ylmethyl)ester (35). From 4b, 5a; CR HATU added at rt, reaction

Pyrrolidine-1,2-dicarboxylic Acid (2S)-2-{(1S,3S,5S)-6-[(1S)-3-Ally-

loxycarbonyl-1-(triphenylmethylthio)methyl-propylcarbamoyl]-1-

tert-butyl-3-methyl-5-(2,2,2-trichloroethoxycarbonyloxy)hex-1-yl}-

ester 1-(9H-Fluoren-9-ylmethyl)ester (3b). To the solution of

compound 35 (166.3 mg, 0.172 mmol) and pyridine (140 μL, 1.742

mmol) in CH₂Cl₂(5.0 mL) were added 2,2,2-trichloroethoxylcarbonyl

chloride (Troc-Cl) (182.6 mg/118.7 μL, 0.862 mmol) and 4-

dimethylaminopyridine (DMAP)(0.36 mg, 2.93 μmol) at 0 °C. After

being stirred at the same temperature for 1 h, the reaction was

quenched with water (15 mL). The water layer was extracted with

ethyl acetate (20 mL × 4). The combined organic layer was washed

with 5% NaHCO₃(20 mL × 2) and brine (20 mL), dried with

MgSO₄, and concentrated in vacuo. The residue was puriﬁed by

chromatography column on silica gel (20−30% ethyl acetate in

hexane) to give 3b (156.5 mg, 80%); [α]²⁰_D−45.0 (c 0.111, CH₂Cl₂).

¹H NMR (400 MHz, CDCl₃, mixture of rotamers): δ 7.77−7.74 (m,

2H), 7.67 (d, J = 7.6 Hz, 0.4H),7.60 (d, J = 7.6 Hz, 1.6H), 7.41−7.37

(m, 8H), 7.33−7.17 (m, 11H), 6.13 (d, J = 8.4 Hz, 0.6H), 5.92−5.80

(m, 1H), 5.62 (d, J = 8.4 Hz, 0.4H), 5.30−5.18 (m, 2.4H), 4.85−4.80

(m, 1.2H), 4.77 (d, J = 12.0 Hz, 0.4H), 4.61 (d, J = 12.0 Hz, 0.4H),

4.54 (d, J = 12.0 Hz, 0.6H), 4.53−4.45 (m, 3H), 4.40−4.30 (m, 1.4H),

4.26−4.18 (m, 1.6H), 3.93−3.82 (m, 1H), 3.66−3.60 (m, 1H), 3.58−

3.45 (m, 1H), 2.54−2.56 (m, 4H), 2.22−2.10 (m, 3H), 2.00−1.85 (m,

3H), 1.75−1.60 (m, 2H), 1.55−1.24 (m, 3H), 0.99 (d, J = 6.4 Hz,

1.8H), 0.88 (s, 3.6H), 0.87 (s, 5.4H), 0.80 (d, J = 6.4 Hz, 1.2 H) ppm.

¹³C NMR (100 MHz, CDCl₃, mixture of rotamers): δ 172.8, 172.7,

172.7, 172.3, 168.7, 168.3, 154.9, 154.4, 153.6, 153.5, 144.6, 144.6,

144.2, 144.1, 143.9, 143.8, 141.3, 141.3, 132.2, 132.1, 129.6, 128.0,

128.0, 127.8, 127.8, 127.1, 126.9, 126.8, 125.5, 125.4, 125.2, 120.0,

120.0, 118.4, 118.3, 94.8, 94.8, 79.5, 79.1, 76.7, 76.6, 75.3, 74.8, 67.8,

67.5, 65.3, 65.2, 59.8, 59.5, 48.4, 47.3, 47.0, 46.5, 41.6, 41.0, 39.8, 39.4,

38.1, 37.7, 36.4, 36.3, 35.0, 34.7, 31.7, 31.3, 29.8, 29.1, 25.9, 25.9, 25.4,

24.4, 23.4, 22.7, 20.8, 20.7, 14.2 ppm. HRMS (ESI) m/z calcd for

C₆₂H₆₉Cl₃N₂O₁₀SNa (M + Na)⁺1161.3631, found 1161.3634.

General Procedure B: Synthesis of 34a,b,d. To the solution of

triphenylphosphine oxide (223.3 mg, 0.802 mmol) in CH₂Cl₂(1 mL)

was added dropwise triﬂuoromethanesulfonic anhydride (Tf₂O) (68

μL, 0.401 mmol) at 0 °C under argon. After being stirred at the same

temperature for 10 min, compound 3 (0.100 mmol) in CH₂Cl₂(0.5

mL) was added at 0 °C. The reaction mixture was stirred at the same

temperature for 30 min for 3a/3b and 3 h for 3d. This reaction was

monitored by mass spectrometry and was quenched with saturated

NaHCO₃(6 mL) at 0 °C when starting material disappeared. The

aqueous layer was extracted with ethyl acetate (10 mL × 4), washed

with brine (10 mL), dried with MgSO₄, ﬁltered, and concentrated in

vacuo to give Troc protected thiazoline intermediate 33a, b, or d. The

residue was used in the next step immediately without further

puriﬁcation.

The above residue 33 was dissolved in THF (4 mL), and then

aqueous NH₄OAc (1 M, 1.0 mL) and zinc powder (freshly activated

with 1 M aqueous HCl) (100 mg) were added at room temperature.

After being stirred at the same temperature for 30 min, ethyl acetate (5

mL) and brine (5 mL) and were added to the reaction mixture. The

aqueous layer was extracted with ethyl acetate (5 mL × 4). The

combined organic layer was dried with MgSO₄, ﬁltered, concentrated

in vacuo, and puriﬁed by column chromatography on silica gel (eluted

by ethyl acetate/hexane 1:3, v/v) to give thiazoline ring product 34a,b,

or d as a colorless oil.

1

time 3.0 h; yield 193.8 mg, 82%; [α]²⁰−31.2 (c 0.24, CH₂Cl₂). H

D

NMR (400 MHz, CDCl₃, mixture of rotamers): δ 7.74 (d, J = 7.6 Hz,

2H), 7.63−7.54 (m, 2H), 7.41−7.36 (m, 8H), 7.30−7.15 (m, 11H),

6.69 (d, J = 8.8 Hz, 0.84H), 6.06 (d, J = 8.4 Hz, 0.16H), 5.92−5.77 (m,

1H), 5.30−5.15 (m, 2H), 4.89 (d, J = 11.2 Hz, 0.84H), 4.79 (d, J =

10.4 Hz, 0.16H), 4.53−4.44 (m, 2H), 4.39−4.29 (m, 2H), 4.23−4.16

(m, 1H), 4.01(br m, 1H), 3.95−3.85 (m, 1H), 3.79 (br, 1H), 3.66−

3.61 (m, 1H), 3.58−3.44 (m, 1H), 2.40−2.15 (m, 6H), 2.11−2.01 (m,

3H), 1.98−1.88 (m, 2H), 1.85−1.75 (m, 2H), 1.71−1.60 (m, 3H),

1.55−1.47 (m, 1H), 1.40−1.30 (m, 1H), 1.04−0.97 (m, 1H), 0.96 (d, J

= 6.8 Hz, 2.52H), 0.88 (s, 9H), 0.77 (d, J = 6.8 Hz, 0.48H) ppm. ¹³C

NMR (100 MHz, CDCl₃, mixture of rotamers): δ 172.7, 172.3, 172.0,

155.2, 144.8, 144.7, 144.0,143.9, 141.4, 132.3, 129.7, 128.1, 128.0,

127.8, 127.3, 127.2, 126.9, 126.8, 125.2, 125.2, 120.1, 120.1, 118.3,

78.7, 67.7, 66.6, 66.3, 65.2, 59.6, 47.9, 47.3, 46.6, 44.3, 42.9, 42.2, 41.4,

38.0, 37.1, 36.8, 34.9, 34.6, 31.7, 31.3, 31.0, 30.0, 29.8, 29.3, 26.1, 25.2,

24.5, 23.5, 22.8, 20.7, 14.3 ppm. HRMS (ESI) m/z calcd for

C₅₉H₆₈N₂O₈SNa (M + Na)⁺987.4589, found 987.4628.

Pyrrolidine-1,2-dicarboxylic Acid (2S)-2-{(1S,3S,5S)-6-[(1S)-3-Allyl-

oxycarbonyl-1-(triphenylmethylthio)methyl-propylcarbamoyl]-1-

tert-butyl-3,6-dimethyl-5-(2,2,2-trichloroethoxycarbonyloxy)hept-1-

yl}ester 1-(9H-Fluoren-9-ylmethyl)ester (3c). From 4c, 5a; CR

PyAOP added at rt, reaction time 24 h; yield 254.3 mg, 89%; [α]²⁰

D

−48.5 (c 0.20, CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃, mixture of

rotamers): δ 7.78−7.75 (m, 2H), 7.67 (d, J = 7.6 Hz, 0.6H), 7.62 (d, J

= 7.2 Hz, 1.4H), 7.41−7.38 (m, 8H), 7.34−7.27 (m, 8H), 7.22−7.20

(m, 3H), 6.02 (d, J = 8.0 Hz, 0.4H), 5.96 (d, J = 8.4 Hz, 0.6 H), 5.92−

5.80 (m, 1H), 5.30−5.18 (m, 2H), 5.13 (d, J = 10.0 Hz, 0.4H), 5.04 (d,

J = 10.0 Hz, 0.6H), 4.95 (d, J = 12.0 Hz, 0.6H), 4.83 (d, J = 12.0 Hz,

0.4H), 4.79−4.75 (m, 1H), 4.70 (d, J = 12.0 Hz, 0.4H), 4.66 (d, J =

12.0 Hz, 0.6H), 4.52−4.34 (m, 4.4H), 4.28−4.23 (m, 1H), 4.20−4.16

(m, 0.6H), 3.99−3.87 (m, 1H), 3.67−3.45 (m, 2H), 2.36−2.27 (m,

2.6H), 2.24−2.16 (m, 2.4H), 2.12 (br m, 1H), 2.01−1.90 (m, 2H),

1.85−1.74 (m, 3H), 1.53−1.44 (m, 2H), 1.37−1.27 (m, 1H), 1.19−

1.16 (m, 6H), 1.06−1.00 (m, 1H), 0.97 (d, J = 6.4 Hz, 1.8H), 0.88 (s,

5.4H), 0.85 (s, 3.6H), 0.69 (d, J = 6.4 Hz, 1.2H) ppm. ¹³C NMR (100

MHz, CDCl₃, mixture of rotamers): δ 174.2, 172.7, 172.6, 172.2,

154.6, 154.3, 154.2, 154.1, 144.5, 144.5, 144.3, 144.1, 143.9, 143.7,

141.3, 141.2, 132.1, 132.0, 129.5, 128.0, 127.7, 127.1, 127.0, 126.8,

125.4, 125.3, 125.1, 125.1, 120.0, 119.9, 118.3, 94.8, 94.8, 81.6, 81.4,

79.6, 79.4, 79.3, 76.7, 67.8, 67.4, 66.5, 65.2, 59.8, 59.3, 48.3, 47.2, 47.0,

46.8, 46.3, 38.1, 37.8, 36.8, 36.3, 36.3, 34.8, 34.7, 34.5, 31.6, 31.3, 30.8,

30.0, 26.8, 26.5, 25.8, 25.8, 25.3, 24.4, 23.4, 23.1, 22.7, 20.4, 20.3, 20.1,

14.2 ppm. HRMS (ESI) m/z calcd for C₆₄H₇₃Cl₃N₂O₁₀SNa (M +

Na)⁺1189.3944, found 1189.3964.

Pyrrolidine-1,2-dicarboxylic Acid (2S)-2-{(1S,3S,5S,6S)-6-[(1R)-3-

Allyloxycarbonyl-1-(triphenylmethylthio)methyl-propylcarbamoyl]-

1-tert-butyl-3-methyl-5-(2,2,2-trichloro-ethoxycarbonyloxy)hept-1-

yl}ester 1-(9H-Fluoren-9-ylmethyl)ester (3d). From 4a, 5b; CR

PyAOP added at rt, reaction time 24 h; yield 257.0 mg, 91%; [α]²⁰

D

−40.0 (c 0.05, CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃, mixture of

rotamers): δ 7.87−7.78 (m, 2H), 7.71−7.67 (dd, J = 8.4, 8.0 Hz,

2H),7.51−7.46 (m, 8H), 7.40−7.28 (m, 11H), 6.17 (d, J = 8.4 Hz,

0.6H), 6.02−5.92 (m, 1H), 5.51 (d, J = 8.4 Hz, 0.4H), 5.40−5.30 (m,

2H), 5.10 (ddd, J = 7.6, 7.4, 7.2 Hz, 1H), 4.97−4.80 (m, 2.4H), 4.75

(d, J = 12.0 Hz, 0.6H), 4.62−4.30 (m, 6H), 4.00 (br m, 1H), 3.74−

3.55 (m, 2H), 2.60−2.57 (m, 0.4H), 2.42−2.23 (m, 6.6 H), 2.06 (br

m, 2H), 1.98−1.86 (m, 1H), 1.82−1.70 (m, 3H), 1.66−1.58 (m, 2H),

1.55−1.30 (m, 1H), 1.24 (d, J = 6.8 Hz, 1.8H), 1.20 (d, J = 7.2 Hz, 1.2

H), 1.07 (d, J = 6.0 Hz, 1.8H), 0.98 (s, 3.6H), 0.96 (s, 5.4H), 0.88 (d, J

= 6.4 Hz, 1.2H) ppm. ¹³C NMR (100 MHz, CDCl₃, mixture of

rotamers): δ 172.8, 172.8, 172.6, 172.5, 172.4, 172.3, 154.9, 154.5,

153.7, 153.7, 144.6, 144.5, 144.2, 144.0, 143.8, 141.4, 141.2, 132.2,

129.8, 129.6, 128.1, 128.0, 127.8, 127.3, 127.2, 127.1, 126.9, 126.9,

125.7, 125.5, 125.2, 120.0, 118.6, 118.5, 79.5, 79.2, 79.0, 78.7, 76.7,

68.0, 67.5, 66.8, 66.7, 65.4, 65.4, 59.8, 59.5, 48.1, 47.3, 47.3, 47.1, 46.4,

45.7, 45.4, 38.3, 37.6, 37.1, 36.6, 36.5, 35.1, 34.8, 34.8, 31.7, 31.4, 29.9,

Pyrrolidine-1,2-dicarboxylic Acid (2S)-2-{(1S,3S,5S)-6-[(5S)-5-(2-

Allyloxycarbonylethyl)-4,5-dihydro-thiazol-2-yl]-1-tert-butyl-5-hy-

droxy-3-methylhex-1-yl}ester 1-(9H-Fluoren-9-ylmethyl)ester (34b).

1

Yield 53.2 mg, 76%; [α]²⁰−78.0 (c 0.10, CH₂Cl₂). H NMR (400

D

MHz, CDCl₃, mixture of rotamers): δ 7.74 (d, J = 7.6 Hz, 2H), 7.63

(dd, J = 5.6, 6.0 Hz, 1.6 H), 7.56 (d, J = 7.6 Hz, 0.4H), 7.37 (dd, J =

7.2, 7.2 Hz, 2H), 7.32−7.27 (m, 2H), 5.93−5.83 (m, 1H), 5.30−5.19

(m, 2H), 4.90 (d, J = 10.8 Hz, 0.8H), 4.83 (d, J = 9.6 Hz, 0.2H), 4.58−

4.51 (m, 2H), 4.46−4.34 (m, 3.8H), 4.28−4.18 (m, 1.2H), 3.97 (br m,

3023

dx.doi.org/10.1021/jm4019965 | J. Med. Chem. 2014, 57, 3011−3029

Journal of Medicinal Chemistry

Article

1H), 3.68−3.61 (m, 2H), 3.57−3.47 (m, 1H), 3.33 (dd, J = 10.4, 8.8

Hz, 0.2H), 3.24 (dd, J = 10.4, 8.8 Hz, 0.8H), 2.88 (dd, J = 10.0, 8.4 Hz,

0.2H), 2.81 (dd, J = 10.4, 8.4 Hz, 0.8H), 2.55−2.48 (m, 4H), 2.29−

2.19 (m, 1H), 2.08−1.80 (m, 5H), 1.75−1.58 (m, 2H), 1.48−1.25 (m,

2H), 1.04−0.97 (m, 1H), 0.96 (d, J = 6.4 Hz, 2.4H), 0.87 (s, 9H), 0.79

(d, J = 6.4 Hz, 0.6H) ppm. ¹³C NMR (100 MHz, CDCl₃, mixture of

rotamers): δ 172.9, 172.9, 172.8, 172.3, 169.2, 169.1, 154.9, 154.3,

144.3, 144.1, 144.0, 143.8, 141.3, 141.3, 141.2, 132.2, 132.2, 127.6,

127.6, 127.1, 127.0, 127.0, 127.0, 125.4, 125.3, 125.3, 125.2, 79.5, 78.5,

76.0, 75.8, 67.8, 67.7, 67.2, 66.7, 65.2, 65.1, 59.5, 47.2, 47.1, 47.0, 46.5,

42.9, 42.2, 42.0, 41.9, 38.0, 37.9, 37.4, 34.8, 34.7, 34.6, 31.6, 31.5, 31.4,

31.2, 30.4, 29.9, 26.0, 25.9, 25.7, 25.3, 25.1, 24.5, 23.3, 22.7, 20.6, 20.5,

14.2 ppm. HRMS (ESI) m/z calcd for C₄₀H₅₂N₂O₇SNa (M + Na)⁺

727.3387, found 727.3399.

0.9H), 1.19−1.18 (m, 5.1H), 0.96 (d, J = 6.4 Hz, 2.1H), 0.87 (s, 9H),

0.79 (d, J = 6.8 Hz, 0.9H) ppm. ¹³C NMR (100 MHz, CDCl₃, mixture

of rotamers): δ 178.8, 178.3, 173.1, 173.0, 172.9, 172.5, 154.9, 154.4,

144.4, 144.3, 144.0, 143.9, 141.4, 141.3, 141.2, 132.3, 127.7, 127.7,

127.1, 127.1, 125.5, 125.4, 125.3, 125.2, 120.0, 118.3, 118.3, 79.4, 78.6,

76.0, 75.8, 75.0, 74.9, 67.9, 67.7, 65.2, 65.2, 59.7, 59.7, 47.3, 47.2, 47.0,

46.5, 45.9, 45.6, 38.0, 37.8, 37.0, 37.0, 36.9, 36.6, 34.8, 34.6, 31.6, 31.4,

31.3, 30.3, 30.2, 30.0, 29.8, 26.1, 25.8, 25.3, 24.5, 24.3, 23.7, 23.4, 23.1,

20.6, 20.5 ppm. HRMS (ESI) m/z calcd for C₄₂H₅₆N₂O₇SNa (M +

Na)⁺755.3700, found 755.3718.

General Procedure C: Synthesis of Compounds 2a−d. To a

solution of 34 (0.054 mmol) in THF (1.5 mL) were added Pd(PPh₃)₄

(7.1 mg, 0.005 mmol) and N-methylaniline (0.015 mL, 0.136 mmol)

at room temperature under argon. This reaction was protected with

aluminum foil. After being stirred at the same temperature for 1 h, the

reaction mixture was concentrated in vacuo and puriﬁed by preparative

TLC (20 cm × 20 cm plate) to give acid 2.

Pyrrolidine-1,2-dicarboxylic Acid (2S)-2-{(1S,3S,5S,6S)-6-[(5R)-5-

(2-Allyloxycarbonylethyl)-4,5-dihydro-thiazol-2-yl]-1-tert-butyl-5-

hydroxy-3-methylhept-1-yl}ester 1-(9H-Fluoren-9-ylmethyl)ester

(34d). Yield 57.0 mg, 79%; [α]²⁰−22.5 (c 0.12, CH₂Cl₂). ¹H

D

Pyrrolidine-1,2-dicarboxylic Acid (2S)-2-{(1S,3S,5S)-6-[(5S)-5-(2-

Carboxylethyl)-4,5-dihydro-thiazol-2-yl]-1-tert-butyl-5-hydroxy-3-

methylhex-1-yl}ester 1-(9H-Fluoren-9-ylmethyl)ester (2b). Yield 31.8

mg, 89%; [α]²⁰_D−90.0 (c 0.04, CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃,

mixture of rotamers): δ 7.74 (d, J = 7.6 Hz, 2H), 7.63(d, J = 7.2 Hz,

1.8H), 7.57 (d, J = 7.6 Hz, 0.2H), 7.38 (dd, J = 7.6, 7.6 Hz, 2H), 7.32−

7.28 (m, 2H), 6.22 (br, 1H), 4.90 (d, J = 10.0 Hz, 0.8H), 4.84 (d, J =

9.2 Hz, 0.2H), 4.53−4.34 (m, 3.8H), 4.29−4.17 (m, 1.2H), 4.00−3.92

(m, 1H), 3.68−3.61 (m, 1H), 3.57−3.45 (m, 1H), 3.35 (dd, J = 10.8,

8.8 Hz, 0.2H), 3.23 (dd, J = 10.8, 8.4 Hz, 0.8H), 2.91 (dd, J = 10.8, 8.4

Hz, 0.2H), 2.80 (dd, J = 10.8, 8.4 Hz, 0.8H), 2.60−2.43 (m, 4H),

2.35−2.17 (m, 1H), 2.08−1.82 (m, 5H), 1.74−1.59 (m, 2H), 1.48−

1.25 (m, 2H), 1.03−0.96 (m, 1H), 0.96 (d, J = 6.4 Hz, 2.4H), 0.87 (s,

9H), 0.77 (d, J = 6.4 Hz, 0.6H) ppm. ¹³C NMR (100 MHz, CDCl₃,

mixture of rotamers): δ 176.7, 176.5, 173.1, 172.5, 171.8, 170.5, 155.1,

154.5, 144.3, 144.1, 144.0, 143.8, 141.4, 141.4, 141.3, 141.3, 127.7,

127.7, 127.2, 127.1, 127.1, 125.5, 125.4, 125.3, 120.0, 79.8, 75.7, 75.3,

67.9, 67.8, 67.4, 67.0, 59.6, 47.2, 47.2, 47.0, 46.6, 42.7, 42.3, 42.1, 41.9,

38.2, 38.1, 38.0, 37.4, 36.8, 34.8, 34.7, 32.0, 31.7, 31.3, 30.1, 30.0, 30.0,

26.1, 26.0, 25.7, 25.1, 24.6, 23.4, 20.7, 20.5 ppm. HRMS (ESI) m/z

calcd for C₃₇H₄₈N₂O₇SNa (M + Na)⁺687.3074, found 687.3070.

Pyrrolidine-1,2-dicarboxylic Acid (2S)-2-{(1S,3S,5S)-6-[(5S)-5-(2-

Carboxylethyl)-4,5-dihydro-thiazol-2-yl]-1-tert-butyl-3,6-dimethyl-

NMR (400 MHz, CDCl₃, mixture of rotamers): δ 7.76 (d, J = 7.2 Hz,

2H), 7.63 (dd, J = 6.6, 6.4 Hz, 1.7 H), 7.57 (d, J = 7.2 Hz, 0.3H), 7.39

(dd, J = 7.2, 7.2 Hz, 2H), 7.30 (dd, J = 7.4, 7.2 Hz, 2H), 5.94−5.85 (m,

1H), 5.31−5.20 (m, 2H), 4.88 (d, J = 10.0 Hz, 0.7H), 4.81 (d, J = 8.8

Hz, 0.3H), 4.59−4.51 (m, 2.3H), 4.45−4.33 (m, 3H), 4.31−4.18 (m,

1.7H), 3.82−3.72 (m, 0.7H), 3.68−3.62 (m, 1.3H), 3.58−3.49 (m,

1.3H), 3.35−3.24 (m, 1H), 2.90−2.85 (m, 0.3H), 2.81 (dd, J = 10.8,

7.6 Hz, 0.7H), 2.72−2.65 (m, 0.7H), 2.61−2.44 (m, 2.3H), 2.34−2.18

(m, 1.7H), 2.10−1.89 (m, 4.4H), 1.82 (m, 0.7H), 1.70−1.57 (m, 2H),

1.37−1.29 (m, 1H), 1.19 (d, J = 7.0 Hz, 0.9H), 1.17 (d, J = 7.0 Hz,

2.1H), 1.06−1.00 (m, 1H), 0.96 (d, J = 6.4 Hz, 2.1H), 0.87 (s, 9H),

0.79 (d, J = 6.4 Hz, 0.9H) ppm. ¹³C NMR (100 MHz, CDCl₃, mixture

of rotamers): δ 174.2, 173.1, 173.0, 172.8, 172.5, 155.0, 154.5, 144.4,

144.3, 144.1, 143.9, 141.4, 141.4, 141.3, 132.3, 132.3, 128.0, 127.7,

127.4, 127.2, 127.1, 125.6, 125.4, 125.3, 125.3, 121.0, 120.0, 118.4,

118.3, 79.6, 78.6, 77.4, 76.2, 75.7, 71.5, 71.5, 70.7, 67.9, 67.8, 65.3,

65.3, 59.7, 59.7, 47.3, 47.2, 47.1, 46.6, 46.0, 45.3, 40.4, 39.3, 38.2, 37.8,

37.4, 37.3, 34.8, 34.7, 31.7, 31.5, 31.3, 30.3, 30.2, 30.1, 29.8, 26.1, 26.1,

25.9, 25.5, 25.3, 24.6, 23.4, 20.7, 20.6, 16.3, 15.8 ppm. HRMS (ESI)

m/z calcd for C₄₁H₅₄N₂O₇SNa (M + Na)⁺741.3544, found 741.3567.

Pyrrolidine-1,2-dicarboxylic Acid (2S)-2-{(1S,3S,5S)-6-[(5S)-5-(2-

Allyloxycarbonylethyl)-4,5-dihydro-thiazol-2-yl]-1-tert-butyl-3,6-di-

methyl-5-hydroxyhept-1-yl}ester 1-(9H-Fluoren-9-ylmethyl)ester

(34c). To the solution of 3c (97.8 mg, 0.084 mmol) in 1,2-

dichloroethane (5 mL) was added TiCl₄(1 M in CH₂Cl₂, 0.294 mL,

0.293 mmol, 3.5 equiv) at room temperature. The resulting solution

was heated to 60 °C and stirred at this temperature for 2.5 h. This

reaction was monitored by mass spectrometry. When the starting

material was consumed completely, the reaction was cooled to 0 °C

and quenched with saturated aq NaHCO₃(7 mL), stirred at room

temperature for another 10 min, extracted with ethyl acetate (10 mL ×

4), dried over anhydrous MgSO₄, ﬁltered, and evaporated in vacuo to

give crude intermediate 33c, which was used in the next step without

further puriﬁcation.

5-hydroxyhept-1-yl}ester 1-(9H-Fluoren-9-ylmethyl)ester (2c). Yield

1

31.2 mg, 84%; [α]²⁰−57.7 (c 0.052, CH₂Cl₂). H NMR (400 MHz,

D

CDCl₃, mixture of rotamers): δ 7.76 (d, J = 7.6 Hz, 2H), 7.65−7.58

(m, 2H), 7.39 (dd, J = 7.4, 7.2 Hz, 2H), 7.32−7.28 (m, 2H), 4.88 (d, J

= 12.0, 0.7H), 4.80 (d, J = 12.0 Hz, 0.3H), 4.51 (dd, J = 8.8, 2.8 Hz,

0.3H), 4.46 (d, J = 7.2 Hz, 0.3H), 4.43 (d, J = 7.2 Hz, 0.7H), 4.41−

4.32 (m, 2H), 4.30−4.25 (m, 1.7H), 3.65−3.56 (m, 2H), 3.53−3.45

(m, 1H), 3.30 (dd, J = 11.0, 8.4 Hz, 0.3H), 3.19 (dd, J = 11.0, 8.4 Hz,

0.7H), 2.88 (dd, J = 10.8, 10.8 Hz, 1H), 2.70−2.53 (m, 2H), 2.37−

2.14 (m, 1H), 2.08−1.90 (m, 5H), 1.79 (br m, 1H), 1.68−1.60 (m,

2H), 1.35−1.26 (m, 2H), 1.26 (s, 0.9H), 1.19 (s, 2.1H), 1.18 (s, 3H),

0.95 (d, J = 6.6 Hz, 2.1H), 0.87 (s, 9H), 0.74 (d, J = 6.6 Hz, 0.9H)

ppm. ¹³C NMR (100 MHz, CDCl₃, mixture of rotamers): δ 182.4,

180.5, 175.7, 175.5, 173.2, 172.7, 155.1, 154.7, 144.5, 144.2, 144.1,

143.8, 141.5, 141.4, 141.3, 127.8, 127.8, 127.2, 127.1, 127.1, 125.5,

125.4, 125.4, 125.3, 120.0, 79.7, 78.6, 76.0, 75.8, 75.4, 75.1, 68.1, 67.9,

59.7, 59.6, 47.3, 47.2, 47.1, 46.6, 46.5, 45.8, 38.0, 37.8, 37.5, 37.4, 37.1,

36.3, 34.8, 34.6, 33.4, 32.7, 32.7, 31.7, 31.3, 30.2, 30.0, 29.8, 29.7 26.1,

25.6, 25.1, 24.9, 24.7, 23.8, 23.7, 23.5, 22.8, 20.7, 20.4, 14.3 ppm.

HRMS (ESI) m/z calcd for C₃₉H₅₂N₂O₇SNa (M + Na)⁺715.3387,

found 715.3397.

The above crude 33c was dissolved in THF (4 mL), and then

aqueous NH₄OAc (1 M, 1.0 mL) and zinc powder (freshly activated

with 1 M aqueous HCl) (80 mg) were added at room temperature.

After being stirred at the same temperature for 30 min, to the reaction

were added ethyl acetate (5 mL) and brine (5 mL). The aqueous layer

was extracted with ethyl acetate (7 mL × 4). The combined organic

layer was dried with MgSO₄, ﬁltered, concentrated in vacuo, and

puriﬁed by column chromatography on silica gel (eluted by ethyl

acetate/hexane 1:3, v/v) to give thiazoline ring product 34c (101.3

1

mg, 55%) as a colorless oil; [α]²⁰−80.8 (c 0.12, CH₂Cl₂). H NMR

D

Pyrrolidine-1,2-dicarboxylic Acid (2S)-2-{(1S,3S,5S,6S)-6-[(5R)-5-

(400 MHz, CDCl₃, mixture of rotamers): δ 7.76−7.74 (m, 2H), 7.66−

7.61 (m, 1.7H), 7.57 (d, J = 7.2 Hz, 0.3H), 7.39 (dd, J = 7.4, 7.2 Hz,

2H), 7.31−7.28 (m, 2H), 5.94−5.84 (m, 1H), 5.31−5.19 (m, 2H),

4.88 (dd, J = 11.6, 1.6 Hz, 0.7H), 4.81 (dd, J = 11.6, 1.6 Hz, 0.3H),

4.58−4.54 (m, 2H), 4.52−4.38 (m, 3H), 4.35−4.19 (m, 2H), 3.66−

3.59 (m, 2H), 3.58−3.48 (m, 2H), 3.30−3.23 (m, 1H), 2.86−2.81 (m,

1H), 2.59−2.46 (m, 2H), 2.36−2.19 (m, 1H), 2.12−1.91 (m, 5H),

1.78 (br m, 1H), 1.67−1.56 (m, 2H), 1.37−1.27 (m, 2H), 1.22 (s,

(2-Carboxylethyl)-4,5-dihydro-thiazol-2-yl]-1-tert-butyl-5-hydroxy-

3-methylhept-1-yl}ester 1-(9H-Fluoren-9-ylmethyl)ester (2d). Yield

1

33.0 mg, 90%; [α]²⁰−60.0 (c 0.04, CH₂Cl₂). H NMR (400 MHz,

D

CDCl₃, mixture of rotamers): δ 7.75 (d, J = 7.6 Hz, 2H), 7.67−7.57

(m, 2H), 7.39 (dd, J = 7.4, 7.2 Hz, 2H), 7.30 (dd, J = 7.6, 7.4 Hz, 2H),

5.90 (br, 1H), 4.91(dd, J = 12.8, 12.4 Hz, 0.7H), 4.86−4.82 (m, 0.3H),

4.54−4.50 (m, 0.3H), 4.45−4.40 (m, 1H), 4.38−4.33 (m, 1.7H),

4.30−4.27 (m, 1.3H), 4.25−4.16 (m, 0.7H), 3.82−3.80 (m, 0.7H),

3024

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Journal of Medicinal Chemistry

Article

3.75−3.70 (m, 0.3H), 3.64 (br m, 1H), 3.51−3.49 (m, 1H), 3.36−3.30

(m, 0.3H), 3.26−3.15 (m, 0.7H), 2.91−2.85 (m, 0.3H), 2.84−2.72 (m,

0.7H), 2.68−2.50 (m, 3H), 2.35−2.21 (m, 1H), 2.21−2.15 (m, 0.3H),

2.05−1.89 (m, 5H), 1.84−1.79 (m, 0.7H), 1.75−1.70 (m, 1H), 1.66−

1.60 (m, 1H), 1.54−1.45 (m, 0.7H), 1.45−1.40 (m, 0.3H), 1.19−1.16

(m, 3H), 1.03−1.00 (m, 1H), 0.96 (d, J = 6.0 Hz, 2.1H), 0.88 (s, 9H),

0.74 (d, J = 6.0 Hz, 0.9H) ppm. ¹³C NMR (100 MHz, CDCl₃, mixture

of rotamers): δ 177.8, 177.6, 175.9, 173.4, 173.4, 173.3, 172.7, 155.1,

155.1, 154.6, 144.4, 144.4, 144.2, 144.2, 144.2, 144.0, 143.9, 141.4,

141.4, 141.4, 141.3, 141.3, 128.0, 128.0, 127.8, 127.8, 127.5, 127.2,

127.1, 125.5, 125.4, 125.4, 125.3, 121.0, 121.0, 120.0, 120.0, 80.0, 80.0,

79.2, 78.7, 77.4, 76.2, 76.0, 75.6, 75.5, 71.8, 70.8, 70.4, 68.0, 67.9, 67.9,

67.7, 67.5, 59.7, 59.6, 47.3, 47.3, 47.1, 46.6, 46.0, 45.9, 45.4, 40.7, 39.7,

39.4, 39.2, 38.1, 38.0, 37.8, 37.6, 37.0, 34.8, 34.7, 32.9, 32.9, 32.3, 31.7,

31.3, 31.1, 30.2, 30.0, 30.0, 29.8, 29.4, 29.2, 28.7, 26.1, 26.0, 25.8, 25.4,

25.2, 25.1, 24.6, 23.4, 22.8, 20.8, 20.8, 20.6, 20.5, 16.6, 16.0, 14.3, 14.2

ppm. HRMS (ESI) m/z calcd for C₃₈H₅₀N₂O₇SNa (M + Na)⁺

701.3231, found 701.3253.

General Procedure D: Synthesis of Cyclic Precursors 36a−d. To a

solution of Fmoc protected tripeptide 6^2a−c(28.1 mg, 0.042 mmol) in

MeCN (1.2 mL) was added diethylamine (0.6 mL) at room

temperature. After being stirred at the same temperature for 30 min,

the reaction mixture was evaporated in vacuo, then azeotroped with

toluene and CH₂Cl₂two times, respectively, and dried under reduced

pressure for 1 h to give the free amine tripeptide, which was used in

the next coupling reaction without further puriﬁcation.

1.55 (m, 2.3H), 1.50−1.45 (m, 0.7H), 1.35−1.17 (m, 13H), 0.97−0.91

(m, 6H), 0.91−0.82 (m, 14.1H), 0.76 (d, J = 6.4 Hz, 0.9H) ppm. ¹³C

NMR (100 MHz, CDCl₃, mixture of rotamers): δ 178.8, 178.3, 173.0,

172.7, 172.3, 172.2, 171.9, 171.6, 171.6, 171.4, 170.8, 169.7, 158.6,

154.9, 154.6, 144.6, 144.3, 144.0, 143.9, 141.4, 141.4, 141.3, 131.8,

130.5, 130.5, 130.4, 128.4, 128.3, 127.8, 127.7, 127.1, 125.5, 125.4,

125.4, 125.3, 120.0, 120.0, 118.8, 113.9, 79.4, 78.7, 77.4, 76.1, 75.7,

75.2, 75.0, 68.0, 67.7, 66.1, 65.5, 64.6, 60.5, 59.8, 59.6, 55.3, 53.6, 50.4,

49.8, 49.3, 47.3, 47.2, 47.1, 46.6, 45.9, 45.4, 38.1, 37.9, 37.0, 36.6, 34.8,

34.7, 34.5, 33.7, 33.5, 33.3, 32.0, 31.7, 31.3, 31.0, 31.0, 30.9, 30.6, 30.1,

29.8, 26.1, 25.6, 25.4, 25.1, 24.6, 24.0, 23.7, 23.5, 22.8, 20.8, 20.7, 20.6,

16.2, 15.9, 15.0, 14.9, 14.4, 14.3, 11.7, 10.7 ppm. HRMS (ESI) m/z

calcd for C₆₃H₈₇N₅O₁₁SNa (M + Na)⁺1144.6015, found 1144.6041.

Cyclic Precursor 36d. Yield 22.0 mg, 71%; [α]²⁰−66.0 (c 0.05,

D

CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃, mixture of rotamers): δ 7.77−

7.74 (m, 2H), 7.65−7.60 (m, 2H), 7.39 (dd, J = 7.6, 7.2 Hz, 2H), 7.30

(dd, J = 7.2, 6.8 Hz, 2H), 7.10−7.07 (m, 2H), 6.78−6.75 (m, 2H),

5.92−5.81 (m, 1H), 5.39−5.35 (m, 1H), 5.31−5.21 (m, 2H), 5.18−

5.11 (m, 1H), 4.93−4.81 (m, 2H), 4.62−4.53 (m, 2H), 4.46−4.21 (m,

5H), 3.81−3.69 (m, 4H), 3.69−3.60 (m, 1H), 3.58−3.49 (m, 1H),

3.22−3.18 (m, 1H), 3.03−2.88 (m, 4H), 2.82−2.76 (m, 3H), 2.71−

2.57 (m, 4H), 2.34−2.21 (m, 4H), 2.11−1.79 (m, 8H), 1.71 (br m,

1H), 1.64−1.58 (m, 1H), 1.34−1.18 (m, 5H), 0.97−0.78 (m, 21H)

ppm. ¹³C NMR (100 MHz, CDCl₃, mixture of rotamers): δ 175.1,

174.6, 173.2, 172.9, 172.7, 172.6, 172.3, 172.2, 172.0, 172.0, 171.9,

171.9, 171.8, 171.6, 171.5, 171.4, 170.8, 169.7, 158.7, 158.6, 155.1,

155.0, 154.5, 144.5, 144.4, 144.3, 144.2, 144.1, 144.0, 143.9, 141.4,

141.4, 141.3, 131.8, 130.5, 128.5, 128.3, 128.0, 127.8, 127.1, 125.5,

125.4, 125.4, 125.3, 120.0, 118.8, 114.0, 113.9, 79.7, 78.8, 77.4, 76.2,

75.9, 75.6, 71.8, 70.9, 70.3, 67.9, 67.8, 67.6, 66.1, 65.5, 60.5, 59.8, 59.7,

59.7, 55.3, 50.4, 49.9, 47.3, 47.3, 47.2, 47.1, 46.6, 45.8, 45.2, 45.0, 40.8,

39.8, 39.6, 38.1, 37.8, 37.7, 37.3, 37.3, 34.8, 34.7, 33.5, 33.4, 32.1, 31.7,

31.4, 31.0, 31.0, 30.7, 30.6, 30.1, 30.1, 29.8, 26.1, 25.7, 25.4, 25.2, 25.1,

24.6, 23.4, 22.8, 20.7, 20.6, 16.8, 16.4, 15.9, 14.4, 14.4, 14.3, 13.9, 11.8,

11.6, 10.7 ppm. HRMS (ESI) m/z calcd for C₆₂H₈₅N₅O₁₁SNa (M +

Na)⁺1130.5859, found 1130.5905.

The above crude free amine tripeptide was dissolved in CH₂Cl₂

(THF for 2b) (2 mL). To this solution were added acid 2 (0.028

mmol), corresponding coupling reagent (0.056 mmol) (PyAOP for

2a,c,d and DEPBT for 2b), and DIEA (0.014 mL, 0.083 mmol) at

room temperature. After being stirred at the same temperature for 15−

24 h, the reaction mixture was concentrated in vacuo and puriﬁed by

preparative TLC plate (developed by acetone/hexane (2:3, v/v)) to

give the precursor 36a−d as a colorless oil.

Cyclic Precursor 36b. Yield 22.0 mg, 72%; [α]²⁰−99.3 (c 0.14,

D

CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃, mixture of rotamers): δ 7.77−

7.74 (m, 2H), 7.64−7.62 (m, 1.7H), 7.56 (d, J = 7.6 Hz, 0.3H), 7.39

(dd, J = 7.6, 7.4 Hz, 2H), 7.32−7.26 (m, 2H), 7.09 (d, J = 8.0 Hz, 2H),

6.76 (d, J = 8.0 Hz, 2H), 6.63 (d, J = 8.0 Hz, 0.3H), 6.49 (br m, 0.3H),

6.34−6.27 (m, 1H), 6.20−6.12 (m, 0.3H), 5.92−5.83 (m, 0.7H),

5.54−5.47 (m, 0.3H), 5.42−5.35 (m, 0.7H), 5.31−5.17 (m, 2.7H),

5.11−5.06 (m, 0.3H), 4.94−4.85 (m, 1.7H), 4.62−4.50 (m, 2H),

4.47−4.35 (m, 2.3H), 4.27 (t, J = 7.2 Hz, 1H), 4.20−4.16 (m, 1.4H),

3.74 (s, 3H), 3.72−3.64 (m, 1.3H), 3.60−3.54 (m, 1H), 3.26−3.07 (m,

1H), 3.02−2.90 (m, 4H), 2.83−2.77 (m, 2H), 2.77−2.60 (m, 4H),

2.50−2.46 (m, 0.7H), 2.39−2.19 (m, 4H), 2.18−1.90 (m, 6.3H),

1.87−1.78 (m, 1H), 1.73 (br m, 1H), 1.61 (br m, 0.7H), 1.55−1.49

(m, 1H), 1.42−1.38 (m, 1H), 1.27−1.21 (m, 3H), 1.00−0.84 (m,

19.1H), 0.68 (d, J = 6.4 Hz, 0.9H) ppm. ¹³C NMR (100 MHz, CDCl₃,

mixture of rotamers): δ 172.7, 172.1, 172.0, 171.9, 171.6, 171.6, 170.8,

158.7, 154.8, 154.4, 144.3, 144.2, 144.1, 143.9, 141.4, 141.3, 131.8,

131.4, 130.5, 130.4, 128.2, 128.2, 127.8, 127.8, 127.2, 127.1, 127.1,

126.4, 125.5, 125.4, 125.3, 120.1, 118.8, 114.0, 79.6, 79.1, 77.4, 75.9,

75.7, 67.9, 67.7, 66.1, 65.5, 60.6, 59.6, 59.4, 55.3, 50.4, 49.8, 49.2, 47.3,

47.1, 46.6, 38.7, 38.2, 37.9, 37.2, 37.1, 36.8, 34.8, 34.5, 33.7, 33.4, 32.1,

31.7, 31.5, 31.1, 31.0, 30.7, 30.2, 29.8, 29.6, 29.5, 28.8, 26.1, 25.1, 24.6,

23.5, 22.8, 21.1, 20.7, 16.2, 15.9, 14.5, 14.4, 14.3, 11.8, 10.7 ppm.

HRMS (ESI) m/z calcd for C₆₁H₈₃N₅O₁₁SNa (M + Na)⁺1116.5702,

found 1116.5742.

General Procedure E: Synthesis of 1a−d. To a solution of cyclic

precursor 36 (11.6 μmol) in THF (1.0 mL) were added Pd(PPh₃)₄

(2.7 mg, 2.32 μmol) and N-methylaniline (6.3 μL, 58.0 μmol) at room

temperature under argon. This reaction was protected with aluminum

foil. After being stirred at the same temperature for 1 h, the reaction

mixture was concentrated in vacuo and puriﬁed by preparative TLC

plate (developed with MeOH/CH₂Cl₂1:9, v/v) to give the free acid

cyclic precursor.

To the solution of free acid cyclic precursor in MeCN (1.5 mL) was

added N,N-diethylamine (0.75 mL). After being stirred at room

temperature for 30 min, the reaction mixture was evaporated in vacuo,

azeotroped with toluene (three times) and CH₂Cl₂(two times), and

then dried under reduced pressure for 1 h to give the unmasked

precursor as a foam solid. Then the unmasked precursor was dissolved

in CH₂Cl₂(DMF for 36b) (20 mL). To this solution were added

DIEA (20.0 μL, 0.116 mmol) and corresponding coupling reagent

(34.8 μmol) at 0 °C (PyAOP for 36a,c,d and DEPBT for 36b). After

being stirred at 0 °C for 30 min, the reaction was allowed to warm up

to room temperature and stirred for additional 15 h. Then the reaction

was concentrated in vacuo and puriﬁed by semipreparative reversed-

phase HPLC (Phenomenex Ultracarb, ODS 250 mm × 10 mm, 5 μm,

3.0 mL/min, UV detection at 200/220 nm) using an isocratic system

of 80% aqueous MeCN for 30 min, 80−100% MeCN for 30−40 min,

and 100% MeCN for 40−60 min to aﬀord 1a−d.

Cyclic Precursor 36c. Yield 28.3 mg, 90%; [α]²⁰−130.0 (c 0.10,

D

CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃, mixture of rotamers): δ 7.76−

7.73 (m, 2H), 7.65−7.59 (m, 2H), 7.40−7.36 (m, 2H), 7.31−7.26 (m,

2H), 7.10−7.08 (m, 2H), 6.85 (d, J = 8.4 Hz, 0.3H), 6.77−6.74 (m,

2H), 6.65 (d, J = 8.0 Hz, 0.7H), 5.92−5.82 (m, 1H), 5.37 (dddd, J =

6.8, 6.8, 6.8, 6.8 Hz, 1H), 5.31−5.20 (m, 2H), 5.20−5.14 (m, 1H),

4.93−4.87 (m, 1.7H), 4.81 (d, J = 10.8 Hz, 0.3H), 4.62−4.51 (m, 3H),

4.46−4.41 (m, 2H), 4.33−4.24 (m, 3H), 3.74 (s, 3H), 3.68−3.62 (m,

1.3H), 3.60−3.48 (m, 1.7H), 3.18 (ddd, J = 18.8, 10.8, 8.8 Hz, 1H),

3.03−2.95 (m, 4H), 2.82−2.73 (m, 2H), 2.68 (s, 2.1H), 2.65 (s, 0.9H),

2.43−2.17 (m, 4H), 2.15−1.86 (m, 5H), 1.85−1.75 (m, 2H), 1.70−

Apratoxin S7 (1b). Yield 2.3 mg, 25% in 3 steps; [α]²⁰−106.2 (c

D

1

0.024, CH₂Cl₂); t_R= 17.5 min. H NMR (600 MHz, CDCl₃, mixture

of rotamers, major and minor (6/4)): δ 7.14 (d, J = 8.4 Hz, 1.2H),

7.10 (d, J = 8.4 Hz, 0.8H), 6.80 (d, J = 8.4 Hz, 0.8H), 6.79 (d, J = 8.4

Hz, 1.2H), 6.17 (d, J = 9.0 Hz, 0.4H), 5.78 (d, J = 10.2 Hz, 0.6H), 5.26

(d, J = 11.4 Hz, 0.6H), 5.20−5.16 (m, 0.4H), 5.15 (ddd, J = 15.0, 10.2,

4.8 Hz, 0.6H), 4.96 (dd, J = 12.6, 2.4 Hz, 0.6H), 4.89 (d, J = 11.4 Hz,

0.4H), 4.87 (dd, J = 12.6, 2.4 Hz, 0.4H), 4.64 (q, J = 6.6 Hz, 0.4H),

4.60 (d, J = 10.8 Hz, 0.6H), 4.38−4.31 (m, 1.2H), 4.24−4.19 (m, 1H),

4.11−4.08 (m, 0.4H), 4.05 (br m, 0.4H), 4.00−3.95 (m, 0.6H), 3.90

3025

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Journal of Medicinal Chemistry

Article

(br m, 0.4H), 3.77 (s, 1.8H), 3.77 (s, 1.2H), 3.70−3.66 (m, 0.6H),

3.65−3.61 (m, 0.4H), 3.39 (dd, J = 10.8, 8.4 Hz, 0.6H), 3.32 (dd, J =

10.8, 8.4 Hz, 0.4H), 3.29 (q, J = 6.6 Hz, 0.6H), 3.09 (dd, J = 12.0, 11.4

Hz, 1H), 3.05−3.01 (m, 1H), 2.94 (dd, J = 12.6, 4.2 Hz, 0.4H), 2.88

(s, 1.2H), 2.80 (s, 1.8H), 2.77 (dd, J = 12.6, 4.8 Hz, 0.6H), 2.74 (s,

1.8H), 2.64−2.58 (m, 1.8H), 2.52 (dd, J = 13.5, 11.4 Hz, 0.6H), 2.45−

2.32 (m, 1.6H), 2.31−2.23 (m, 1.6H), 2.15−1.96 (m, 2.4H), 1.96−

1.83 (m, 3.6H), 1.81−1.69 (m, 4H), 1.62 (ddd, J = 13.8, 9.6, 3.6 Hz,

1H), 1.56−1.51 (m, 1H), 1.31−1.25 (m, 1H), 1.23 (d, J = 7.2 Hz,

1.8H), 1.21−1.17 (m, 0.4H), 1.09 (d, J = 6.6 Hz, 1.2H), 1.03 (t, J = 6.6

Hz, 1.8H), 1.03 (d, J = 6.6 Hz, 1.8H), 1.00 (d, J = 6.6 Hz, 1.2H), 0.98

(d, J = 6.6 Hz, 1.2H), 0.98−0.92 (m, 0.6H), 0.87 (s, 9H), 0.84 (t, J =

7.2 Hz, 1.2H), 0.57 (d, J = 6.6 Hz, 1.2H) ppm. ¹³C NMR (150 MHz,

CDCl₃, mixture of rotamers, major and minor): δ 172.7, 172.1, 171.9,

171.2, 170.7, 170.5, 170.3, 170.2, 170.0, 168.6, 158.8, 158.7, 130.7,

130.6, 128.7, 128.5, 114.2, 114.0, 78.1, 77.6, 76.2, 75.8, 68.6, 67.3, 60.7,

59.9, 59.5, 58.1, 57.9, 57.4, 55.5, 55.4, 54.1, 50.9, 49.8, 47.8, 45.0, 43.9,

42.9, 41.0, 39.8, 38.8, 37.6, 37.5, 37.4, 37.0, 36.9, 34.1, 34.1, 33.2, 33.0,

31.5, 30.6, 30.6, 29.3, 29.3, 29.0, 26.2, 26.2, 25.7, 25.6, 25.3, 25.2, 24.8,

20.9, 19.9, 15.1, 14.3, 14.1, 14.1, 10.0, 10.0 ppm. HRMS (ESI) m/z

calcd for C₄₃H₆₇N₅O₈SNa (M + Na)⁺836.4603, found 836.4607.

(td, J = 11.4, 3.6 Hz, 0.6H), 1.33 (ddd, J = 13.8, 11.1, 3.0 Hz, 0.4H),

1.29−1.26 (m, 1.4H), 1.23 (d, J = 7.2 Hz, 1.8H), 1.12−1.09 (m, 2.2H),

1.07 (d, J = 6.6 H, 1.8H), 1.05 (d, J = 7.2 Hz, 1.8H), 1.01 (t, J = 7.2

Hz, 1.8H), 0.97−0.95 (m, 3.6H), 0.92 (m, 0.4H), 0.87 (s, 5.4H), 0.87

(s, 3.6H), 0.85 (t, J = 7.2 Hz, 1.2H), 0.43 (d, J = 6.6 Hz, 1.2H) ppm.

¹³C NMR (150 MHz, CDCl₃, mixture of rotamers, major and minor):

δ 175.3, 174.9, 172.6, 172.2, 171.9, 171.4, 171.1, 170.7, 170.5, 170.5,

170.3, 170.0, 158.9, 158.7, 130.7, 130.5, 128.7, 128.4, 114.3, 114.0,

78.0, 77.6, 75.5, 74.9, 72.5, 72.1, 60.7, 59.8, 59.3, 58.3, 56.9, 55.5, 55.4,

53.8, 51.6, 49.6, 49.0, 47.8, 47.5, 40.0, 39.4, 38.3, 38.0, 37.8, 37.6, 37.5,

37.1, 36.8, 35.2, 35.2, 34.2, 34.0, 33.2, 32.5, 31.6, 31.1, 30.8, 30.5, 29.3,

29.2, 28.8, 26.2, 25.8, 25.7, 25.3, 25.2, 24.8, 24.7, 20.6, 20.1, 16.5, 16.4,

14.6, 14.3, 14.0, 14.0, 10.1, 9.9 ppm. HRMS (ESI) m/z calcd for

C₄₄H₆₉N₅O₈SNa (M + Na)⁺850.4759, found 850.4773.

Biological Material and Methods. Cell Culture. Human colon

adenocarcinoma HCT116 cells were purchased from ATCC

(Manassas, VA) and cultured in Dulbecco’s Modiﬁed Eagle’s Medium

(Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum

(HyClone, Logan, UT) at 37 °C humidiﬁed air and 5% CO₂.

Cell Viability Assay (MTT). HCT116 cells were seeded at a density

of 1 × 10⁴cells per well in 96-well plates. Then 24 h later, the cells

were treated with various concentrations of apratoxins or solvent

control (1% EtOH). After 48 h of incubation, cell viability was

detected using MTT according to the manufacturer’s instructions

(Promega, Madison, WI).

Apratoxin S8 (1c). Yield 6.8 mg, 70% in 3 steps; [α]²⁰−59.5 (c

D

1

0.037, CH₂Cl₂); t_R= 26.2 min. H NMR (600 MHz, CDCl₃, mixture

of rotamers, major and minor (7/3)): δ 7.14 (d, J = 8.4 Hz, 0.6H),

7.12 (d, J = 8.4 Hz, 1.4H), 6.80 (d, J = 8.4 Hz, 1.4H), 6.78 (d, J = 8.4

Hz, 0.6H), 6.18 (d, J = 9.0 Hz, 0.7H), 5.73 (d, J = 10.2 Hz, 0.3H), 5.27

(d, J = 11.4 Hz, 0.3H), 5.18−5.11 (m, 1H), 4.96 (d, J = 12.0 Hz,

0.3H), 4.89 (d, J = 10.8 Hz, 1H), 4.60 (q, J = 6.0 Hz, 0.7H), 4.45−4.37

(m, 1H), 4.35−4.31 (m, 0.7H), 4.15 (t, J = 8.4 Hz, 0.3H), 4.10−4.06

(m, 0.7H), 3.87 (d, J = 10.8 Hz, 0.7H), 3.76 (s, 3H), 3.70 (t, J = 10.8

Hz, 0.7H), 3.63−3.59 (m, 1H), 3.34 (dd, J = 10.8, 8.4 Hz, 0.3H), 3.31

(q, J = 6.6 Hz, 0.3H), 3.24 (dd, J = 10.8, 8.4 Hz, 0.7H), 3.12 (dd, J =

12.6, 11.4 Hz, 0.3H), 3.09−3.04 (m, 1.4H), 2.95 (dd, J = 12.6, 3.6 Hz,

0.7H), 2.88 (s, 2.1H), 2.87 (s, 0.9H), 2.76 (s, 0.9H), 2.75−2.73 (m,

0.3H), 2.58 (s, 2.1H), 2.41 (ddd, J = 13.2, 13.2, 4.8 Hz, 0.7H), 2.36−

2.26 (m, 2.3H), 2.23−2.18 (m, 1H), 2.14−2.10 (br m, 0.7H), 2.08−

2.02 (m, 1.3H), 1.98−1.91 (m, 3.3H), 1.89−1.82 (m, 2.3H), 1.78−

1.73 (m, 3.3H), 1.60−1.56 (m, 0.7H), 1.51 (m, 1H), 1.29−1.23 (m,

2.8H), 1.17 (s, 0.9H), 1.16 (s, 2.1H), 1.07−1.06 (m, 5.1H), 1.03 (t, J =

7.2 Hz, 0.9H), 0.98 (d, J = 6.6 Hz, 3H),0.87 (s, 9H), 0.83 (t, J = 7.2

Hz, 2.1H), 0.49 (d, J = 6.6 Hz, 2.1H) ppm. ¹³C NMR (150 MHz,

CDCl₃, mixture of rotamers, major and minor): δ 179.6, 178.4, 172.2,

172.0, 172.0, 171.8, 170.9, 170.4, 170.3, 170.2, 170.1, 169.8, 158.9,

158.6, 130.7, 130.6, 128.7, 128.5, 114.2, 113.9, 77.7, 75.8, 75.3, 74.8,

73.9, 60.8, 60.0, 59.3, 57.8, 57.4, 55.5, 55.4, 54.2, 50.9, 49.8, 47.8, 46.7,

46.5, 40.0, 38.2, 38.0, 37.6, 37.3, 37.0, 36.0, 35.2, 35.1, 34.9, 34.6, 34.1,

33.7, 31.7, 31.4, 30.8, 30.8, 30.7, 29.8, 29.5, 29.3, 28.9, 26.3, 26.3, 25.8,

25.6, 25.6, 25.5, 25.3, 25.0, 24.2, 22.8, 20.7, 20.3, 18.7, 18.3, 14.9, 14.6,

14.3, 14.1, 14.1, 10.7, 10.0 ppm. HRMS (ESI) m/z calcd for

C₄₅H₇₁N₅O₈SNa (M + Na)⁺864.4916, found 864.4918.

Measurement of VEGF-A Production. HCT116 cells (1 × 10⁴cells

per well) were seeded in 96-well plates and one day later treated with

various concentrations of apratoxins or solvent control. After 12 h of

incubation, culture supernatants were collected for detection of VEGF-

A by using an alphaLISA kit (PerkinElmer, Waltham, MA) following

the manufacturer’s instructions. Brieﬂy, acceptor bead and anti-VEGF

antibody were incubated ﬁrst with the supernatants for 60 min, donor

beads were added later and incubated for another 30 min, and then

VEGF-A levels were detected using Envision (PerkinElmer).

Immunoblot Analysis. HCT116 cells were seeded in 6-well plates

at a density of 4 × 10⁵cells and the next day treated with various

concentrations of apratoxins or solvent control (1% EtOH). Then 24 h

later, whole cell lysates were collected using PhosphoSafe buﬀer (EMD

Chemicals, Inc., Gibbstown, NJ). The protein concentration was

measured with the BCA Protein Assay kit (Thermo Fisher Scientiﬁc,

Rockford, IL). Lysates containing equal amounts of protein were

separated by SDS polyacrylamide gel electrophoresis (4−12%),

transferred to polyvinylidene diﬂuoride membranes, probed with

primary and secondary antibodies, and detected with the SuperSignal

West Femto Maximum Sensitivity Substrate (Thermo Fisher

Scientiﬁc). Anti-Met, anti-VEGFR2, and secondary antimouse anti-

bodies were from Cell Signaling Technology, Inc. (Danvers, MA).

In Vivo Eﬃcacy Studies. Three−ﬁve week old female nude mice

(nu/nu) were obtained from Charles River Laboratory (Wilmington,

MA). One ×10⁶HCT116 cells in a volume of 100 μL of sterile saline

were injected subcutaneously on the left rear ﬂank of a nude mouse to

establish tumors. Tumor dimensions were measured using calipers

every day, and tumor volumes were calculated using the formula W²×

L × 0.5, where width (W) ≤ length (L). Tumors with a starting

volume bigger than 100 mm³were excluded from the analysis. Mice

were injected intraperitoneally with the doses of 2 μg/mouse (0.1 mg/

kg), 5 μg/mouse (0.25 mg/kg) of 1c or solvent (DMSO) control

every day (25 μL) until the tumor size in one dimension reached 15

mm and tumor tissue was harvested on the following day. Finally, 50

mg of tumor tissue was sonicated in PhosphoSafe lysis buﬀer (EMD

chemicals, Inc.) and used for immunoblot analysis described as the

above. All studies were carried out under the protocol approved by the

Institutional Animal Care and Use Committee at the University of

Florida.

Apratoxin S9 (1d). Yield 4.3 mg, 45% in 3 steps; [α]²⁰−82.6 (c

D

1

0.023, CH₂Cl₂); t_R= 24.8 min. H NMR (600 MHz, CDCl₃, mixture

of rotamers, major and minor (6/4)): δ 7.13 (d, J = 9.0 Hz, 1.2H),

7.13 (d, J = 9.0 Hz, 0.8H), 6.82 (d, J = 9.0 Hz, 0.8H), 6.79 (d, J = 9.0

Hz, 1.2H), 6.23 (d, J = 8.4 Hz, 0.4H), 5.86 (d, J = 9.6 Hz, 0.6H), 5.23

(d, J = 11.4 Hz, 0.6H), 5.19 (ddd, J = 10.5, 10.5, 4.8 Hz, 0.6H), 5.05

(ddd, J = 10.8, 8.4, 4.2 Hz, 0.4H), 4.95 (dd, J = 13.2, 3.0 Hz, 0.6H),

4.88 (dd, J = 12.6, 3.6 Hz, 0.4H), 4.82 (d, J = 11.4 Hz, 0.4H), 4.73 (d, J

= 11.4 Hz, 0.6H), 4.59 (q, J = 6.6 Hz, 0.4H), 4.23 (qd, J = 8.4, 4.8 Hz,

0.4H), 4.36−4.34 (m, 0.4H), 4.31−4.23 (m, 1H), 4.23 (t, J = 7.8 Hz,

0.6H), 4.19 (d, J = 10.2 Hz, 0.4H), 4.13−4.09 (m, 0.4H), 3.77 (s,

1.8H), 3.77 (s, 1.2H), 3.69−3.64 (m, 0.6H), 3.64−3.60 (m, 0.4H),

3.55−3.51 (m, 0.4H), 3.50 (dd, J = 10.8, 7.8 Hz, 0.4H), 3.46 (qd, J =

11.1, 3.6 Hz, 0.6H), 3.38 (dd, J = 10.8, 7.8 Hz, 0.6H), 3.26 (q, J = 6.6

Hz, 0.6H), 3.09−2.98 (m, 1.4H), 2.94 (s, 1.2H), 2.80−2.76 (m, 1.6H),

2.74 (s, 1.8H), 2.74 (s, 1.8H), 2.70 (dd, J = 11.4, 11.4 Hz, 0.6H),

2.63−2.57 (m, 2.2H), 2.44 (ddd, J = 16.8, 5.4, 3.0 Hz, 0.6H), 2.41−

2.34 (m, 1H), 2.30−2.22 (m, 1.2H), 2.20−2.17 (m, 0.4H), 2.16−2.10

(m, 1H), 2.09−2.03 (m, 1.4H), 1.98−1.84 (m, 2.4H), 1.79 (td, J =

13.2, 3.6 Hz, 0.6H), 1.75−1.61 (m, 4.4H), 1.58−1.52 (m, 0.6H), 1.49

Metabolite Analyses. Materials and General Procedures.

HPLC-MS was done on a 3200 QTRAP (Applied Biosystems)

equipped with a Shimadzu (Kyoto, Japan) UFLC System. Mouse

serum and harmine were purchased from Sigma-Aldrich. Pooled CD1

mouse liver (female) microsomes were purchased from XenoTech,

LCC (Lenexa, KS) with protein concentrations of 0.5 mg/mL.

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HCT116 cell lysates were prepared with PhosphoSafe lysis buﬀer

(Novagen, San Diego, CA). Protein concentration was determined by

using the BCA Assay. Analysis was carried out similarly as previously

described.²²

entrance potential (EP) 12, collision energy (CE) 45, collision cell exit

potential (CXP) 6, collision cell entrance potential (CEP) 32; and

harmine, DP 56.0, EP 4.5, CE 44.0, CXP 5, CEP 16.0. Source gas

parameters used were as follows: curtain gas, 15.0; collision gas low,

ionspray voltage 5500; temperature, 600.0; ion source gas 1 50.0; ion

source gas 2 60.0.

Data Analysis. Calibration curves for 1a−d in the presence of

mouse serum, HCT116 cell lysates, and aqueous solutions were

generated by least-squares linear regression analysis of the analyte peak

area and internal standard peak area ratio against the nominal

concentration of the standard solutions. A linear regression analysis

was performed, and the concentration of remaining apratoxins at each

time point was determined through interpolation for plasma, cellular,

and aqueous stability experiments. The amount of remaining

apratoxins with microsome incubation was determined from the

peak area ratio of apratoxins at t_x(3 min to 2 h) and t₀. All calculations

were done by using Analyst 1.4.2 (Applied Biosystems) Quantitate

Mode.

Sample Preparation. Stock solutions of 1a−d were prepared by

dissolving the compounds in ethanol to give a 1 mg/mL solution.

Aliquots of this stock solution were then obtained to aﬀord a 40 μg/

mL solution in acetonitrile. Serial dilution of the 40 μg/mL solution in

acetonitrile gave standard solutions with concentrations of 25, 12.5,

2.5, 1.25, 0.25, 0.125, 0.025, and 0.0125 μg/mL. A 1 mg/mL stock

solution of the internal standard harmine was prepared in ethanol,

which subsequently was used to prepare a 100 μg/mL solution with

ethanol. An aliquot of the 100 μg/mL harmine solution was diluted to

35 ng/mL with ethyl acetate to serve as the working internal standard

solution.

Plasma Stability. In vitro plasma stability of 1a−d was done by

using a modiﬁcation of a published method.²³First, 10 μL of

apratoxins (25 μg/mL) were added to 100 μL of mouse serum, and

the solution was then vortex-mixed for 15 s and incubated for 0.25 min

to 24 h. At the end of each incubation period, 400 μL of ethyl acetate

was added to each tube, followed by 200 μL of harmine to quench the

reaction and extract remaining apratoxin. Samples were further

incubated in a thermomixer at 27 °C (750 rpm, 5 min) and later

centrifuged for 5 min at 1643g. The ethyl acetate layer was collected

and evaporated to dryness under nitrogen. Samples were reconstituted

in 50 μL of acetonitrile. A volume of 10 μL of the reconstituted

solution was injected into the HPLC-MS system.

ASSOCIATED CONTENT

* Supporting Information

■

S

Preparation procedures and characterizations for some known

compounds, cooling concentration equipment for the prepara-

tion of 12, Michael adduct formation of apratoxin A with thiol-

containing compounds, trial route to synthesize 7 from diol

1

derivative of 8, H NMR and ¹³C NMR spectra for new

Microsomal Stability. The stability of 1a−d in the presence of

mouse microsomes was determined by using an adaptation of a

published procedure.²⁴In brief, microsomes were added to prewarmed

phosphate buﬀer (100 mM, pH 7.4) at 37 °C. Apratoxins (3 μL) were

added to the microsomal preparation followed by NADPH cofactor

solution (1.3 mM NADP, 3.3 mM glucose 6-phosphate, 0.4 U/mL

glucose-6-phosphate dehydrogenase, 3.3 mM MgCl₂). The reaction

was allowed to proceed for 3 min to 2 h at 37 °C (Thermomixer, 1050

rpm). The reaction was quenched by addition of ethyl acetate and

subsequently spiked with harmine. The zero time point was deﬁned by

denaturing the microsomes with ethyl acetate before the addition of

apratoxins. Incubation of apratoxins with microsomes alone was also

performed following the same procedure to determine NADPH-

independent metabolism. The ﬁnal concentration of the incubation

mixture contained 0.5 mg/mL protein concentration and 1 μM

apratoxins.

Cellular Stability. Aliquots of HCT116 cell lysates were diluted

with 25 mM Tris-HCl buﬀer, pH 8.0, to give a ﬁnal reaction volume of

100 μL and protein concentration of 0.7 mg/mL. Cell lysate solutions

were incubated with 10 μL of apratoxins (25 μg/mL) for 0.25 min to

24 h. Remaining apratoxins were extracted from the reaction solution

at the end of the incubation periods with ethyl acetate using the same

procedure as described for the plasma stability assay.

Aqueous Stability. The stability of 1a−d in aqueous solution was

determined in 100 mM phosphate buﬀer, pH 4.88, 100 mM phosphate

buﬀer, pH 7.4. Portions of each solution (100 μL) were spiked with 10

μL of apratoxin solution (25 μg/mL) and allowed to incubate for 0.25

min to 24 h. The reaction was quenched at the end of each time point,

and the remaining apratoxins were extracted with ethyl acetate, as in

the plasma stability study.

HPLC-MS Parameters. Analysis of 1a−d was done by using HPLC-

MS [column, Onyx Monolithic C18 (3.0 mm × 100 mm),

Phenomenex (Torrance, CA); solvent, water (solvent A) acetonitrile

(solvent B); ﬂow rate, 0.5 mL/min; detection by electrospray

ionization−MS in positive ion mode (MRM scan)]. A stepwise

gradient elution was used starting at 60% B and 40% A, then increasing

to 80% B at 5 min and maintained at this condition for 5 min.

Parameters were optimized before analysis by using direct syringe

infusion. The retention times (t_R, min; MRM ion pair) of the analytes

and internal standard are as follows: harmine (2.2; 213.1 → 169.9), 1a

(4.1; 828.5 → 432.2), 1b (4.05; 814.5 → 418.2), 1c (5.2; 842.5 →

446.2), 1d (4.9; 828.5 → 432.2). Compound-dependent parameters

used were as follows: apratoxins, declustering potential (DP) 51,

compounds. This material is available free of charge via the

Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Phone: +1-352-273-7738. Fax: +1-352-273-7741. E-Mail:

luesch@cop.u ﬂ .edu.

■

Notes

The authors declare the following competing ﬁnancial

interest(s): Hendrik Luesch is co-founder of Oceanyx

Pharmaceuticals, Inc., which is negotiating licenses for patent

applications related to the subject matter.

ACKNOWLEDGMENTS

■

This work was supported by the Bankhead-Coley Cancer

Research Program, grants 1BG07 and 4BB07, and the National

Institutes of Health, NCI grant R01CA172310.

ABBREVIATIONS USED

■

BCA, bicinchoninic acid; BEP, 2-bromo-1-ethyl-pyridinium

tetraﬂuoroborate; BOP, (benzotriazol-1-yloxy)tris-

(dimethylamino)phosphonium hexaﬂuorophosphate; BRSM,

based on recovered starting material; CEP, collision cell

entrance potential; CXP, collision cell exit potential; CE,

collision energy; DEPBT, 3-(diethoxyphosphoryloxy)-1,2,3-

benzotriazin-4(3H)-one; DIEA, N,N-diisopropylethylamine;

DP, declustering potential; EDCI, N-(3-dimethylaminoprop-

yl)-N′-ethylcarbodiimide hydrochloride; EGFR, epidermal

growth factor receptor; EP, entrance potential; epi, epimer;

G2, generation 2; GSH, glutathione; HATU, 1-[bis-

(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-

pyridinium 3-oxid hexaﬂuorophosphate; HCT116, human

colorectal carcinoma cell line; HOBt, 1-Hydroxybenzotriazole;

ip, intraperitoneal; IPC, diisopinocampheyl; LiAlH₄, lithium

aluminum hydride; moCys, modiﬁed cysteine; MET, met

proto-oncogene (hepatocyte growth factor receptor); MTT, 3-

(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;

NAC, N-acetyl-^L-cysteine; PyAOP, (7-azabenzotriazol-1-yloxy)-

3027

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Journal of Medicinal Chemistry

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tripyrrolidinophosphonium hexaﬂuorophosphate; RTK, recep-

tor tyrosine kinase; SEM, standard error of the mean; TBAI,

tetrabutylammonium iodide; TMSOTf, trimethylsilyl triﬂuoro-

methanesulfonate; TfOH, triﬂuoromethanesulfonic acid; Troc,

2,2,2-trichloroethoxycarbonyl; Trt, trityl/triphenylmethyl; Tris-

HCl, tris(hydroxymethyl)aminomethane hydrochloride; VEGF,

vascular endothelial growth factor; VEGFR, vascular endothelial

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Improved total synthesis and biological evaluation of potent apratoxin S4 based anticancer agents with differential stability and further enhanced activity

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