Synthesis, structurea-activity analysis, and biological evaluation of sanguinamide B analogues

Article

pubs.acs.org/joc

Synthesis, Structure−Activity Analysis, and Biological Evaluation of

Sanguinamide B Analogues

Hendra Wahyudi, Worawan Tantisantisom, Xuechao Liu, Deborah M. Ramsey, Erinprit K. Singh,

and Shelli R. McAlpine*

School of Chemistry, Gate 2 High Street, Dalton 219, University of New South Wales, Sydney, NSW 2052, Australia

S

* Supporting Information

ABSTRACT: We report the ﬁrst synthesis of sanguinamide B

analogues. Substituting N-methylated (N-Me) amino acids,

glycine (Gly), and ^L- or D-phenylalanine (Phe) into the

backbone of sanguinamide B showed that only ^L- and D-Phe

residues controlled the macrocycle conformation. The N-

methylated and glycine analogues all had multiple conforma-

tions, whereas the ^L- and D-Phe derivatives only had a single conformation. Testing of all conformer analogues showed that

inclusion of an ^L- or D-Phe was a superior design element than incorporating the N-Me moiety that is often utilized to control

macrocyclic conformation. Finally, we show that there is an ideal Phe residue (in this case ^L-Phe) for generating compounds that

have the greatest inhibitory eﬀect on bacterial motility. Our data support the hypothesis that the macrocyclic conformation is

dictated by the benzyl moiety requiring a “pseudoequatorial” position, and all other energy considerations are secondary.

The two prolines (Pro) present in 1 also make this natural

product very intriguing. Prolines, such as N-methyl (N-Me)

amino acids, are common structural motifs in bioactive peptides

that dramatically alter the 3D conformation of macrocycles.¹⁶

Speciﬁcally, the rotation about prolyl amide bonds is restricted,

and the proline residue may adopt either a cis or trans

conﬁguration.⁴A cis-amide bond generates a diﬀerent 3D shape

of the macrocycle than a trans-amide bond, where one may

induce a bioactive conformation and the other an inactive

conformation. This phenomena was exhibited with the isolation

and biological evaluation of two conformational isomers, cis,cis-

and trans,trans-ceratospongamide (Figure 2), which were

isolated from a symbiotic sponge (Sigmadocia symbiotica).

Ceratospongamide’s structure consists of a modiﬁed heptapep-

tide with a thiazole, methyloxazoline, and two prolines.¹⁷When

tested for anti-inﬂammatory activity, only the trans,trans

conformer exhibited potent inhibition of sPLA₂expression

(ED₅₀32 nM), and the cis,cis isomer was inactive.¹⁷

INTRODUCTION

■

A proven source of new antibiotics are natural products,¹and

screening compounds isolated from natural sources has

produced eﬀective antimicrobial agents including penicillin,

vancomycin, and erythromycin.²We recently reported the

synthesis of the natural product sanguinamide B (San B)

(Figure 1, 1).³San B (1) was isolated from a nudibranch

collected in the Yasawa Island chain in Figi, Hexabranchus

sanguineus, meaning “six-gilled blood-colored” sea slug with a

spongiverous diet.^4,5H. sanguineus and its egg masses contain

bioactive polyketide macrolides including kabiramides⁶and

ulapualides⁷(Figure 1). In 2009, Molinski and co-workers

determined that the structure of 1 was a modiﬁed macrocyclic

octapeptide containing ﬁve ^L-conﬁguration amino acids, two

thiazoles (Th), and one oxazole (Ox).⁴

STRUCUTURAL FEATURES OF SANGUINAMIDE B

■

Compound 1 falls into a unique class of natural products, as it

bears directly linked azoles: a 4,2-oxazole-thiazole moiety. Over

the last two decades there has been a surge in the discovery of

natural product compounds that contain directly linked azoles,

and many of these compounds are now candidates for drug

development.⁸⁻¹³Mixed 4,2-bisheterocycle tandem pairs,

however, are extremely rare in peptides, and only two natural

products have been reported to date to contain an oxazole-

thiazole subunit:⁸leucamide A¹⁴and microcin B17 (Figure

1).¹⁵Investigation of the two oxazole-thiazole units present in

microcin B17 showed that altering or removing the two

thiazole-oxazole pairs signiﬁcantly reduced antibiotic activity.¹⁵

Given the importance of the mixed tandem bisheterocycle

moiety in multiple natural products, evaluating the structure−

activity relationship of 1 analogues was appealing.

RESULTS AND DISCUSSION

■

Design of Sanguinamide B Analogues. One common

modiﬁcation made when exploring the SAR of cyclic peptides is

the incorporation of an N-methyl moiety within the backbone

of the peptide. N-Methylating peptides control the macrocyclic

conﬁrmation in a manner that is similar to a proline residue.

Nonmethylated peptide bonds sit in a trans orientation about

the amide. Addition of N-Me on the amide nitrogen increases

the propensity for producing the cis-peptide bond about the

amide.^18,19Thus, methylation has a signiﬁcant eﬀect on the

backbone conformation of cyclic peptides, and the presence of

Received: August 20, 2012

Published: October 10, 2012

10596

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

Figure 1. Examples of bioactive polyketide macrolides isolated from H. sanguineus and natural products with tandem bisheterocycles pairs.

We performed an N-Me scan on 1 to evaluate the impact of

this moiety on the biological activity. The N-Me substituent is

applied at two diﬀerent positions within the macrocycle (Figure

3), N-Me Ala (2), and N-Me Leu (3). The third possible

analogue of 1, introduction of the N-methylvaline, was not

synthesized because coupling the N-methylvaline residue to the

proline gave very poor yields.

Contrary to substituting an N-Me into 1, introduction of a

Gly provides ﬂexibility to the macrocycle conformation (Figure

3). A glycine residue scan produced macrocycles whereby

glycine was placed at each of the three amino acids within 1.

Glycine analogues, where Gly replaces Val, Ala, or Leu

Figure 2. Conformational isomers cis,cis- and trans,trans-ceratospon-

gamide.

(compounds 4, 5, and 6, respectively) are shown in Figure 3.

a single N-Me minimizes the number of conformations that a

macrocycle can adopt, thereby enhancing receptor selectivity by

potentially locking the peptide into a single bioactive

conformation.²⁰⁻²⁵Furthermore, incorporating N-Me amino

acids into peptides can result in analogues with improved

biological activity and pharmacological properties for several

reasons, including improvement in the molecule's membrane

permeability and oral availability, a key property for drug

development.²⁶Thus, the N-Me moiety is an important tool in

optimizing potency of cyclic peptides. There are many natural

product cyclic peptides that contain N-Me amino acids with

notable pharmacological proﬁles, IB-01212,²⁷koshikamides,²⁸

and cyclosporine A,²⁹being the most prominent. Cyclosporine

A, a well-known immunosuppressant, is a cyclic undecapeptide

with seven N-methyls,³⁰and its metabolic stability is partly due

to the presence of N-Me groups.^31,32

Another common modiﬁcation made to peptides when

exploring their SAR is exchanging ^L- for D-amino acids. The

conformation of macrocyclic peptides with small ring sizes (e.g.,

four to eight amino acid residues) is dictated by the amino acid

stereochemistry in the peptide sequence.^33,34A single D-amino

acid (^D-AA) substitution within a macrocycle results in a γ-turn

conformation of the ^D-amino acid and the two amino acids on

either side. This γ-turn is in equilibrium with a βII′-turn.³⁵

Thus, inserting a D-amino acid into the macrocyclic backbone

signiﬁcantly impacts the overall 3-D macrocyclic conformation.

Additionally, including ^D-AAs in a peptide sequence stabilizes

the analogue against enzymatic degradation.³³Interestingly,

generating the enantiomer of a peptide sequence has typically

led to compounds losing their biological activity. Thus, a

productive approach to determining the ideal substitution site

10597

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

Figure 3. N-Me series (2,3), glycine series (4−6), aromatic series (7−10) of San B analogues.

Figure 4. Retrosynthetic strategies of San B fragments A and B.

for a D-AA involves a D-scan.³⁶This particular approach was

used to generate the drug cilengitide.³⁷

Utilizing an L-Phe scan and D-Phe scan (Figure 3) allowed us

to evaluate the impact of bulky substituent as well as the eﬀects

of an ^L-AA on the molecule conformation. Substitution of both

Phe stereoisomers provides compounds whereby we can

evaluate the impact of the residues’ stereochemistry. It is

anticipated that one Phe enantiomer will generate a pseudoaxial

moiety, and the other a pseudoequatorial moiety, thus dictating

the overall macrocyclic conformation.

Synthesis of San B Analogues. San B analogues (Figure

4) were synthesized via cyclization of a linear octapeptide

10598

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

precursor, which was generated using a convergent solution

phase approach. The linear octapeptide was obtained by

coupling two fragments: fragment A (a tripeptide containing a

thiazole) and fragment B (a pentapeptide containing the

tandem oxazole-thiazole unit).

Scheme 2. Synthesis of San B Fragment A

Synthesis of fragment A (Figure 4) involved ﬁrst forming the

thiazole moiety using a modiﬁed Hantzsch thiazole synthesis

between a thioamide (where R¹varied depending on the

analogue) and an α-bromo ketone species. The thioamide was

made in three steps via conversion from the acid precursor

(Scheme 1) to the amide and subsequently the thioamide. N-

terminal elongation of the thiazole with another amino acid

carrying an R²substituent generated fragment A.

Scheme 1. Synthesis of Thioamide

Synthesis of fragment B (Figure 4) involved forming the

tandem oxazole-thiazole moiety using the same modiﬁed

Hantzsch thiazole method between a proline thioamide and

an α-bromo ketone oxazole. Subsequent coupling of the core

oxazole-thiazole moiety with another proline and an amino acid

containing an R³substituent generated fragment B. The α-

bromo ketone oxazole was made by performing a cyclization

and dehydration step starting from the serine precursor. The

serine-bromoketal was synthesized by coupling a bromo-ketal

acid and a benzyl-protected serine. Subsequent hydrogenolysis

to remove the benzyl protecting group generated the desired

serine precursor. The proline thioamide was achieved by

converting the acid precursor to the thioamide via the amide

(Scheme 1).

Synthesis of the thioamide started with conversion of the

acid to the methyl ester (11, Scheme 1) using (trimethylsilyl)-

diazomethane (TMSD) in a solvent mixture of benzene and

methanol. The ester 12 was then converted to amide 13 using

ammonium hydroxide in methanol. Utilizing Lawesson’s

reagent on the amide aﬀorded the thioamide 14.

Condensing the thioamide 14 with an ethyl bromopyruvate

in the presence of KHCO₃in DME formed a thiazoline

(Scheme 2). The thiazoline was then subjected to dehydration

conditions using triﬂuoroacetic anhydride (TFAA) and pyridine

in DME to aﬀord the thiazole 15. Amine deprotection of the

thiazole moiety using triﬂuoroacetic acid (TFA) gave a free

amine species, which was then coupled to an amino acid

carrying R²substituents (16) in the presence of O-

(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetraﬂuoro-

borate (TBTU), 2-(7-Aaza-1H-benzotriazol-1-yl)-1,1,3,3-tetra-

methyluronium hexaﬂuorophosphate (HATU), and N,N-

diisopropylethylamine (DIPEA) in CH₂Cl₂to give fragment

A (17).

genolysis of this fragment generated the free serine 21. This

fragment was then subjected to the diethylaminosulfur

triﬂuoride (DAST) ﬂuorinating agent in CH₂Cl₂, followed by

addition of K₂CO₃, which induced cyclization, generating the

oxazoline intermediate. Oxidation of the oxazoline using

BrCCl₃with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in

CH₂Cl₂aﬀorded the oxazole product 22. Deprotection of

ketone using formic acid at reﬂux prepared the α-bromo ketone

oxazole 23 for thiazole formation. The α-bromo ketone oxazole

was then introduced to the same modiﬁed Hantzsch thiazole

synthesis with the premade proline thioamide 24 to form the

tandem oxazole-thiazole moiety 25. Removal of the Boc group

using TFA to this moiety facilitated the coupling of the next

amino acid carrying the R³substituent, 26, in the presence of

TBTU, HATU, and DIPEA in CH₂Cl₂to aﬀord the

pseudotetrapeptide fragment 27. Ester hydrolysis of this

fragment using LiOH in methanol aﬀorded the free acid

species, which was coupled to the second proline residue to

form fragment B 28.

Synthesis of fragment B involved coupling a dimethoxyacetal

bromopyruvic acid (18, Scheme 3) with a benzyl-protected

serine 19 in the presence of TBTU, HATU, and DIPEA in

CH₂Cl₂to form the pseudodipeptide fragment 20. Hydro-

With fragment A (17, Scheme 4) and fragment B, 28, in

hand, coupling of these two fragments in the presence of

TBTU, HATU, and DIPEA in CH₂Cl₂aﬀorded the protected

linear precursor, 29. Subsequent acid deprotection using LiOH

10599

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

Scheme 3. Synthesis of San B Fragment B

a

Scheme 4. Synthesis of Linear Precursor of San B and Cyclization

a

See Supporting Information for speciﬁc coupling agents and conditions.

in ethanol followed by amine deprotection using TFA in

CH₂Cl₂yielded the double deprotected linear precursor. Using

a cocktail of coupling agents and DIPEA in dilute condition in

CH₂Cl₂, cyclization of the double deprotected linear precursor

was taken to form the San B analogues 2−10 (Figures 4−5).³⁸

Structural Analysis of San B Analogues. The cyclic

peptide derivatives were puriﬁed using HPLC and LCMS to

identify unique conformers for each analogue. As previously

noted in the synthesis of 1, we identiﬁed two conformers of 1

upon synthesis.¹⁰The ﬁnal structure and purities of the nine

would be best achieved using a temperature where the structure

was primarily in a single conformation (indicated by sharp

proton NMR peaks). Shown in Figure 5 are the temperature

1

dependence plots of H NMR for biologically active 7 and 8

1

analogues. All temperature-dependence plots of H NMR for

all other derivatives are available in the Supporting Information.

In contrast to 1, where 2-D data was collected at 263 K, 2-D

data was collected at 308 K for 7, while 2-D NMR data for 8

was collected at 318 K.¹⁰

Evaluation of the ideal temperature that generated deﬁned

peaks in each region for each analogue aﬀorded the ideal

temperature to run the HSQC, HMBC, and COSY NMR on

each derivative. Identiﬁcation of the conﬁguration of each San B

isomer about the proline bonds involved examining the

chemical shifts of their respective Pro β and γ carbons. A Pro

amide bond that adopts cis orientation has larger Δβγ than a

Pro with an amide bond in the trans orientation.¹⁸Analysis

using the proline β and γ carbons of each analogue deﬁned its

1

San B analogues and their conformers were established via H

and ¹³C NMR and 2D NMR experiments (¹H−¹H COSY,

1

¹H−¹³C HSQC, H−¹³C HMBC), as well as HPLC, LC/MS,

and HRMS (see Supporting Information). To evaluate the

structure of each analogue, we ran proton NMR temperature

dependence plots to assess which temperature would be ideal

for gathering the 2-D data. Given the potential interconversion

about the prolyl or N-methyl amide bonds, gathering 2-D data

10600

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

1

Figure 5. H NMR temperature dependence plots of 7 and 8 (CDCl₃, 600 MHz).

conformation. A table of the β and γ carbon shifts for each

conformer are shown (Figures 6, 7, and 8).

conformers of 2 and 3. This observation is likely a result of the

N-Me moiety that is placed within the macrocyclic backbone,

whereby the N-Me requires a cis conﬁguration about the prolyl

bond.

In contrast to N-Me moieties, it is anticipated that

incorporating glycine analogues will provide greater ﬂexibility

to the backbone compared to the inclusion of N-Me amino

acids. This is observed by the number of conformers isolated

for each compound with a glycine analogue. We observed three

conformers when the glycine was substituted next to the

proline 1 residue. The prolyl amide conﬁguration of the two

Given that population of a speciﬁc conformation that is

appropriately oriented to bind to a protein or DNA target

dictates the degree of desired biological activity, knowing the

conﬁguration about the prolyl residue is critical. As described

earlier, N-Me moieties rigidify macrocycles, making them less

ﬂexible and allowing fewer conformers than the same

compound without an N-Me. In our San B series, we observed

that only proline 1 was able to interconvert between cis and

trans conﬁguration (Figure 6). Proline 2 remained as cis in both

10601

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

Figure 6. Chemical shift of C_βand C_γprolines of the N-methyl conformers.

Figure 7. Chemical shift of C_βand C_γprolines of the glycine conformers.

conformers were trans at proline 1 and cis at proline 2 (4A and

4C, Figure 7). These conformers have almost identical shifts;

however, they have diﬀerent retention times on the HPLC.

Their identical proline orientation but unique retention times

indicate that they are conformational analogues, each showing a

unique 3-D conformation. The third conformer (4B, Figure 7)

prefers a cis,cis conﬁguration about both proline residues,

suggesting that the glycine residue provides enough ﬂexibility to

allow proline 1 to reorient from trans to cis-.

Analogue 5 also has three conformers, but all three have

distinct conﬁgurations about the prolyl amide bond. The

glycine allows the macrocyclic backbone to be ﬂexible enough

for proline 1 and 2 to ﬂip between cis and trans orientation (5A

and 5B, Figure 7). In contrast to compound 4, this glycine

analogue prefers the trans,trans conﬁguration over the cis,cis

(5C, Figure 7). The third glycine analogue only has two

conformations: trans,cis (6A, Figure 7) and trans,trans (6B,

Figure 7). These data support the hypothesis that modiﬁcation

of a single residue signiﬁcantly alters the 3-D conformation.

Further, the data support the hypothesis that each con-

formation is driven by unpredictable factors: replacing an

amino acid at diﬀerent locations within a macrocycle does not

cause a single uniform eﬀect. Finally, the data strongly reinforce

the concept that the only valid method of determining the eﬀect

of a single modiﬁcation on a macrocycle is to synthesize it and

evaluate its ﬁnal conformation.

Because glycines do not have side chains, they do not have

energy requirements for positioning these side chains. This

10602

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

Figure 8. Chemical shift of C_βand C_γprolines of the L- and D-Phe conformers.

means that glycines allow the macrocycle to respond to other

energy related factors that drive the conformation, which

produced multiple conformations for each glycine analogue. In

contrast, ^L- and D-Phe have large side chains, whereby

positioning of these side chains becomes a factor in

determining the lowest energy conformation for each macro-

cycle. Exchanging the alanine for an ^L-phenylalanine (L-Phe)-

generated compound 7 (Figure 8). Given that the benzyl side

chain was anticipated to play a role in dictating the

conformation, it is not surprising that only a single

conformation was generated for each derivative. The absence

of other conformations in the presence of an ^L-Phe substitution

strongly supports our hypothesis that a single modiﬁcation

alters the 3-D conformation, and that the lowest energy

conformation must be determined by experiment. Further,

these data strongly support the hypothesis that the ^L-Phe

analogue has locked the macrocycle into a single conformation

by requiring it be placed in a “pseudoequatorial” position.

Insertion of a ^D-Phe residue at the same position as the L-Phe

also generated only a single low energy conformation: cis,cis

about the proline orientation (7 and 8, Figure 8). Comparison

of the two Phe series to the corresponding glycine analogues

(Figure 8) conﬁrms the hypothesis that glycine allows the

macrocyclic backbone freedom, while the phenylalanine

analogues do not. Further, it should be noted that the Phe

residues induce a single conformation, whereas the N-Me

moieties induced two conformations. Thus, Phe residues are

potential design elements, providing a superior approach for

locking the molecule into a single conformation than utilizing

the standard N-Me moiety. Assuming that a desirable

conformation (i.e., one that is most eﬀective at binding to

the biological target) is achieved, improvements in the

biological activity will then be observed.

(Figure 8). As observed with compounds 7 and 8, compounds

9 and 10 each had a single conformation. Although the ^L- and

D-Phe in compounds 9 and 10 induce a trans,cis conﬁguration

about the two proline residues, as opposed to cis,cis seen in

compounds 7 and 8, formation of a single conformation

reinforces the concept that Phe is an extremely useful design

element.

In summary, placement of an N-methyl moiety within the

macrocyclic backbone has the reported impact of inducing only

two of the three possible conﬁgurations about the prolyl

residue. In contrast to the N-methyl analogues, glycine-

substituted analogues have three conformations, consistent

with glycine providing ﬂexibility within the macrocyclic

backbone. Finally, we demonstrated that an ^L- or D-Phe locks

the macrocycle into a single conformation, thereby constituting

an ideal design element, superior to N-Me. We hypothesize that

this is due to a “pseudoequatorial” requirement by the benzyl

side chain. Although ^L- and D-Phe’s have been substituted into

other macrocycles, this is the ﬁrst report demonstrating that

they are more eﬀective than N-Me moieties at locking a

macrocycle into a single conformation.

BIOLOGICAL ACTIVITY

■

To measure the antimicrobial eﬃcacy of San B conformers

against Gram-positive and Gram-negative bacteria, we meas-

ured the cell viability of Staphylococcus aureus (S. aureus) and

Escherichia coli (E. coli) following treatment with 50 μM of

compound. The San B conformers did not reduce cell viability

below 50% for either bacterial strain, indicating that the

analogues did not exhibit antimicrobial activity (Supporting

Information Figure S1).

Numerous bacterial pathogens require motility to establish

infections and migrate from the initial site of colonization, and

inhibitors of bacterial motility can dramatically impact other

disease-promoting actions such as bioﬁlm formation.³⁹

Insertion of an ^L- and D-Phe into the backbone between

proline 2 and the thiazole generated compounds 9 and 10

10603

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

Pseudomonas aeruginosa (P. aeruginosa) are rod-shaped, Gram-

negative bacteria with two motility organelles: a single

unsheathed polar ﬂagellum and type IV pili. The polar

ﬂagellum is a long, whip-like structure used in swarming and

swimming motilities. Type IV pili are polar appendages

responsible for adhesion to epithelium, bioﬁlm formation, and

swarming and twitching motilities. We examined the eﬀects of

all San B conformers on twitching motility using a type IV

ﬁmbriae-positive strain of P. aeruginosa (PAO1).

P. aeruginosa PAO1 (expressing type IV pili) or S. aureus

(negative control, nonmodal) were stab-inoculated through a

thin agar plate, containing either no compound (controls) or

with 50 μM of San B analogues. The zone of motility was

measured, and the twitching motility area was expressed as a

percent of the untreated PAO1 control.

inhibits the peptide’s ability to hydrogen bond, whereas Phe

substitution does not, which could explain the observed

reduction in the pilicidal activity compared to Phe-containing

analogues. Not unexpectedly, there is a preferred stereo-

chemistry of the Phe, in this case ^L-Phe, which is likely related

to the “pseudoequatorial” position into which it is placed within

the macrocycle. Finally, our data show that Phe-containing San

B analogues reduce twitching motility by 50%, suggesting these

compounds act as pilicides. Motility inhibitors are an emerging

trend in antimicrobial development because they target novel

systems essential for pathogenesis, and these small molecules

are important candidates for further structure−activity studies.

EXPERIMENTAL SECTION

■

Experimental Procedures: Synthesis. General Remarks. All

reactions were carried out under N₂atmosphere with dry solvent

under anhydrous conditions, unless indicated otherwise. Reagents

were commercially obtained without further puriﬁcation, unless

otherwise stated. Reaction was monitored via thin-layer chromatog-

raphy (TLC) carried out on silica gel plates using UV light at λ = 254

nm for visualization, and potassium permanganate in water with heat

and developing agents. Silica gel was used for ﬂash chromatography.

NMR spectra were obtained at 298 K, while variable temperature

(VT) NMR spectra were taken from 318 K to 238 K. LC/MS was

recorded on an LCMS system connected to a trap running in positive

electrospray ionization (ESI+) mode. The mobile phase was

composed of DDI water with 0.1% (v/v) formic acid (solvent A),

and HPLC grade acetonitrile with 0.1% (v/v) formic acid (solvent B).

The gradient elution was conducted as follows: ﬂow rate 0.5 mL/min;

initial 70% solvent A, 30% solvent B; at 4 min 100% solvent B; at 12

min 70% solvent A, 30% solvent B. Semipreparative reversed-phase

HPLC was carried out on an LCMS system. The mobile phase was

composed of DDI water with 0.1% (v/v) formic acid (solvent A), and

HPLC grade acetonitrile with 0.1% (v/v) formic acid (solvent B). The

gradient elution was conducted as follows: ﬂow rate 2 mL/min; initial

70% solvent A, 30% solvent B hold for 35 min; at 35 min 100% solvent

B hold for 13 min; at 48 min 70% solvent A, 30% solvent B hold for 2

min.

The most eﬀective compound was the phenylalanine

analogue 9 (trans,cis conformer), where the area of twitching

motility was reduced in size by 74% when cells were treated

with 50 μM of compound (Figure 9). Only one analogue

Figure 9. Twitching motility area (expressed as a percent of the

untreated control) for untreated P. aeruginosa (PAO1; positive

control), nonmodal S. aureus (SA; negative control) and P. aeruginosa

PAO1 treated with 50 μM San B analogues (2A-10). The zone of

twitching motility for untreated PAO1 was set at 100%.

General Peptide Synthesis. All peptide coupling reactions were

carried out under N₂atmosphere with dry solvent, using methylene

chloride and/or acetonitrile for peptide couplings. The amine (1.1

−1.5 equiv) and acid (1.0 equiv) were weighed into a dry ﬂask along

with 4−8 equiv of DIPEA and 1.1 equiv of TBTU. (Some coupling

reactions would not go to completion using only TBTU and therefore

0.8−1.0 equiv of HATU, and/or 3-(diethoxyphosphoryloxy)-3H-

benzo[d][1,2,3]triazin-4-one (DEPBT) or 4-(4,6-dimethoxy-1,3,5-

triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) were used.

In a few cases, up to 0.8 equiv of all three coupling reagents were

used.) Anhydrous methylene chloride and/or acetonitrile were added

to generate a 0.1 M solution. The solution was stirred at room

temperature for 1 h and reactions were monitored by TLC upon

completion. The crude reaction was extracted with pH 1 hydrochloric

acid solution, saturated sodium bicarbonate solution, and ﬁnally brine.

The organic layers were combined, dried over sodium sulfate, ﬁltered,

and concentrated in vacuo. The crude product underwent puriﬁcation

via ﬂash column chromatography on silica gel using a gradient of ethyl

acetate−hexane to yield the desired peptide.

General Acid Protection. The acid was purged with N₂and

dissolved in a solvent mixture of benzene/methanol (3:2) to a

concentration of 0.1 M. A solution of (trimethylsilyl)diazomethane

(2.0 M in diethyl ether) was added dropwise to the reaction until the

solution turned lightly yellow, and the mixture was stirred for 30 min.

The reaction was monitored by TLC upon completion, and then the

methyl ester was concentrated in vacuo and taken on to the next

reaction without further puriﬁcation.

compared favorably to the Phe derivatives, and it was

compound 6B (trans,trans). Consistent with our theory, all

analogues containing Phe (7−10, Figure 8) have greater than

50% reduction in twitching motility. These data support the

hypothesis that the Phe residues facilitate biological activity.

Further, in both cases the ^L-Phe analogues are more eﬀective

than the ^D-Phe analogues in reducing twitching motility,

highlighting that the modiﬁcations are not just assisting

bioactivity but are related to an induced conformation from

the Phe residue.

CONCLUSION

■

This is the ﬁrst report describing the synthesis, conformational

analysis, and structure−activity relationship of sanguinamide B

analogues. Furthermore, it is the ﬁrst proof that the exchange of

an amino acid residue for an ^L- or D-Phe is a key design

element. Our data support the fact that not only do both ^L- and

D-Phe lock the macrocycle into a single conformation,

performing better than if one installed the traditional N-Me

moiety, these residues may also enhance the binding activity of

the macrocycle to their molecular target(s). N-Methylation

General Amine Deprotection. Amines (0.1 M) were deprotected

using 20% triﬂuoroacetic acid in methylene chloride with 2.0 equiv of

anisole. Reactions were carried out for 1−2 h and were monitored by

10604

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

TLC upon completion. The amines were concentrated in vacuo and

taken on to the next reaction without further puriﬁcation.

brine (100 mL × 2). The organic layer was dried over sodium sulfate,

ﬁltered, and concentrated in vacuo. The washed reaction underwent a

puriﬁcation with ﬂash column chromatography using a gradient of

ethyl acetate/methylene chloride or ethyl acetate/hexane to give the

desired peptidyl-thiazole. Finally, when necessary, reversed-phase

HPLC was used for additional puriﬁcation using a gradient of

acetonitrile and deionized water with 0.1% formic acid.

General Acid Deprotection. Acids (0.1 M) were deprotected using

2 equiv of lithium hydroxide with 3.4 equiv of hydrogen peroxide in

methanol or ethanol. The peptide was dissolved in methanol or

ethanol and cooled to 0 °C. Hydrogen peroxide was added followed by

lithium hydroxide. The reaction was monitored by TLC and usually

done in 1−2 h. Upon completion, sodium thiosulfate (3.8 equiv) was

added to neutralize the peroxide, and 5% hydrochloric acid was added

till the solution pH was 1. The aqueous solution was extracted ﬁve

times with methylene chloride, and the combined organic layer was

dried, ﬁltered, and concentrated in vacuo and taken on to the reaction

without further puriﬁcation.

Compound 2. MeO-Ala-NMeBoc (12b). MeO-Ala-NMeBoc was

synthesized following the General Acid Protection procedure, utilizing

1.51 g (7.45 mmol, 1.0 equiv) of HO-Ala-NMeBoc (11b) and 5.30 mL

of (trimethylsilyl)diazomethane in 29.8 mL of methanol and 44.7 mL

of benzene. The methyl ester was concentrated in vacuo and taken on

to the next reaction without further puriﬁcation (1.62 g, quantitative

1

Macrocyclization Procedure (with Syringe Pump). Three coupling

agents (DEPBT or DMTMM, HATU, and TBTU) were used at ∼0.5

to 0.8 equiv each. These coupling agents were dissolved in 3/4 of a

calculated volume of dry methylene chloride that would give a 0.001 to

0.0007 M overall concentration when included in the volume used for

the deprotected peptide. The crude, dry, double-deprotected peptide

(free acid and free amine) was dissolved in the other 1/4 solvent

volume of methylene chloride. DIPEA (8 equiv) was then added to the

solution containing coupling reagents dissolved in methylene chloride.

The double-deprotected peptide was then added to the bulk solution

dropwise using a syringe pump at a rate of 30 mL/h. The reaction was

monitored via LCMS and generally complete in 1−2 h. Upon

completion, the reaction was worked up by washing with aqueous

hydrochloric acid (pH 1) and saturated sodium bicarbonate. After

back extraction of aqueous layers with large quantities of methylene

chloride, the organic layers were combined, dried, ﬁltered, and

concentrated. All macrocycles were ﬁrst puriﬁed by ﬂash column

chromatography using an ethyl acetate/hexane gradient on silica gel.

Finally, when necessary, reversed-phase HPLC was used for additional

puriﬁcation using a gradient of acetonitrile and deionized water with

0.1% formic acid.

yield) as clear crystals. R_f: 0.89 (hexanes/ethyl acetate 1:1). H NMR

(300 MHz, CDCl₃): δ 1.45 (t, 3H, CHCH₃); 1.50 (s, 9H, C(CH₃)₃);

2.81−2.92 (m, 3H, NCH₃); 3.75 (s, 3H, OCH₃); 4.37−4.49 (br,

1αH); 4.82−4.93 (br, 1H, NH).

NH₂-Ala-NMeBoc (13b). NH₂-Ala-NMeBoc was synthesized

following the General Amide Formation procedure, utilizing 1.62 g

(7.45 mmol, 1.0 equiv) of MeO-Ala-NMeBoc (12b) dissolved in 37.3

mL of ammonium hydroxide (25% in water) and 37.3 mL of

methanol. The resulting amide was taken on to the next reaction

without further puriﬁcation (1.50 g, quantitative yield) as a white

powder (rotamers 2:1). R_f: 0.15 (hexanes/ethyl acetate 1:1). ¹H NMR

(300 MHz, CDCl₃): δ 1.36 (d, J = 7.04 Hz, 3H, CHCH₃); 1.50 (s, 9H,

C(CH )₃); 2.83 (s, 3H, NCH₃); 4.27−4.95 (br, 2H, αH and N H);

3

5.78−6.34 (br, 2H, NH₂). ¹³C NMR (75 MHz, CDCl₃): δ 13.8, 28.4,

30.9, 80.2, 81.0, 156.4, 174.7.

Thioamide-Ala-NMeBoc (14b). Thioamide-Ala-NMeBoc was syn-

thesized following the General Thioamide Formation procedure,

utilizing 1.50 g (7.45 mmol, 1.0 equiv) of NH₂-Ala-NMeBoc (13b)

and 2.26 g (5.59 mmol, 0.75 equiv) of Lawesson’s Reagent dissolved in

74.5 mL of 1,2-dimethoxyethane. The thioamide was puriﬁed via

column chromatography on silica gel (hexanes/ethyl acetate 1:1 to

3:7) to aﬀord the desired thioamide (797 mg, 49% yield) as white

crystals. R_f: 0.6 (hexanes/ethyl acetate 1:1). ¹H NMR (300 MHz,

CDCl₃): δ 1.43 (s, 9H, C(CH₃)₃), 1.48 (d, J = 7.27 Hz, 3H, CHCH₃),

General Amide Formation. Boc-protected amino ester (1.0 equiv)

was dissolved in 50% ammonium hydroxide and 50% methanol to a

concentration of 0.1 M. The reaction mixture was stirred overnight

and monitored by TLC upon completion. Upon completion, the

solvent was concentrated in vacuo, and the amides were taken on to

the next reaction without further puriﬁcation.

2.80 (s, 3H, NCH ); 4.83−4.95 (m, 1H, αH), 7.86−8.07 (br, 1H,

3

NH_aH_b), 8.07−8.28 (br, 1H, NH_aH_b). ¹³C NMR (75 MHz, CDCl₃): δ

17.4, 28.4, 30.1, 58.5, 81.0, 156.7, 208.8.

General Thioamide Formation. Boc-protected amide (1.0 equiv)

was converted into Boc-protected thioamide using Lawesson’s Reagent

(0.8 equiv) in 0.4 M 1,2-dimethoxyethane at room temperature under

N₂. The mixture was stirred overnight and monitored by TLC upon

completion. Upon completion, the solvent was concentrated in vacuo.

Boc-protected thioamide was puriﬁed by ﬂash column chromatog-

raphy using an ethyl acetate/methylene chloride or ethyl acetate/

hexane gradient on silica gel.

EtO-Thiazole-Ala-NMeBoc (15b). EtO-Thiazole-Ala-NMeBoc was

synthesized following the General Thiazole Synthesis procedure,

utilizing 797 mg (3.65 mmol, 1.0 equiv) of thioamide-Ala-NMeBoc

(14b) and 2.92 g (29.2 mmol, 8.0 equiv) of potassium bicarbonate

dissolved in 36.5 mL of 1,2-dimethoxyethane. A 1.38 mL amount of

ethyl bromopyruvate (11.0 mmol, 3.0 equiv) was added dropwise (0.1

mL/min) to yield the thiazoline intermediate. The thiazoline was

dehydrated using 2.66 mL (32.9 mmol, 9.0 equiv) of pyridine, 2.03 mL

(14.6 mmol, 4.0 equiv) of triﬂuoroacetic anhydride, and 1.02 mL (7.30

mmol, 2.0 equiv) of triethylamine in 36.5 mL of 1,2-dimethoxyethane

to aﬀord the desired thiazole (826 mg, 72% yield) as a light yellow oil.

General Thiazole Synthesis (Modiﬁed Hantzsch). Thiazole syn-

thesis reaction was carried out under N₂with anhydrous 1,2-

dimethoxyethane. KHCO₃(8.0 equiv) was added to the dry ﬂask

containing peptidyl thioamide (1.0 equiv). Anhydrous 1,2-dimethoxy-

ethane (0.1 M) was added to the reaction, and it was stirred at room

temperature for 15 min. α-Bromo ketone residue (3.0 equiv) was

added (0.1 mL/min), and the reaction mixture was stirred overnight.

Upon reaction completion, conﬁrmed by TLC, the desired thiazoline

intermediate was concentrated in vacuo, redissolved in ethyl acetate,

extracted with brine, dried over sodium sulfate, ﬁltered, and

concentrated in vacuo. The crude thiazoline intermediate was

redissolved in 1,2-dimethoxyethane and stirred at 0 °C for 15 min.

Next, pyridine (9.0 equiv) was added (0.1 mL/min) to a solution of

thiazoline in 1,2-dimethoxyethane (0.05 M) at 0 °C under N₂for the

dehydration of the thiazoline to yield the desired thiazole. The reaction

was stirred for 15 min and then TFAA (4.0 equiv) was added to the

reaction mixture (0.1 mL/min). After 3 h, triethylamine (TEA) (2.0

equiv) was added to the reaction mixture (0.1 mL/min), and the

reaction was stirred at room temperature for an additional 2−3 h or

until complete as determined by TLC. Upon completion, the crude

reaction was washed with pH 1 hydrochloric acid solution (100 mL ×

2), saturated sodium bicarbonate solution (100 mL × 10), and ﬁnally

1

R_f: 0.78 (hexanes/ethyl acetate 1:1). H NMR (300 MHz, CDCl₃): δ

1.37−1.44 (t, 3H, OCH₂CH₃); 1.49 (s, 9H, C(CH₃)₃); 1.67−1.73 (d,

J = 7.2 Hz, 3H, CHCH₃); 2.79 (s, 3H, NCH₃); 4.36−4.46 (q, 2H,

OCH₂CH₃); 5.40−5.80 (m, 1H, 1αH); 8.13 (s, 1H, SCH). ¹³C NMR

(75 MHz, CDCl₃): δ 14.7, 17.1, 28.6, 29.4, 61.5, 80.9, 128.2, 147.2,

155.5, 161.5, 173.8. HRMS (ESI-TOF): M + Na⁺, found 337.1187

C₁₄H₂₂N₂O₄S requires 337.1198.

EtO-Thiazole-Ala-NMeH (15b-1). EtO-Thiazole-Ala-NMeH was

synthesized following the General Amine Deprotection procedure.

The amine was taken on to the next reaction without further

puriﬁcation or characterization. (571 mg, quantitative yield) as a light

brown oil.

EtO-Thiazole-Ala-NMe-Val-NHBoc (17c). EtO-Thiazole-Ala-NMe-

Val-NHBoc was synthesized following the General Peptide Synthesis

procedure, utilizing 512 mg (2.39 mmol, 1.0 equiv) of amine EtO-

Thiazole-Ala-NMeH (15b-1), 571 mg (2.63 mmol, 1.1 equiv) of acid

HO-Val-NHBoc (16a), 844 mg (2.63 mmol, 1.1 equiv) of TBTU, and

1.67 mL (9.56 mmol, 4 equiv) of DIPEA dissolved in 23.9 mL of

methylene chloride under N₂. The crude product underwent a ﬁnal

10605

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

puriﬁcation via column chromatography on silica gel (hexanes/ethyl

acetate 19:1 to 3:1) to aﬀord the desired peptide (562 mg, 57% yield)

as a yellow oil. R_f: 0.55 (hexanes/ethyl acetate 1:1). H NMR (300

•Compound 2. Compound 2 was synthesized following the

Macrocyclization Procedure, utilizing 196 mg (0.264 mmol, 1.0

equiv) of HO-Thiazole-Ala-NMe-Val-Pro-Oxazole-Thiazole-Pro-Leu-

NH₂(29-2b), 68.0 mg (0.210 mmol, 0.8 equiv) of TBTU, 80.0 mg

(0.210 mmol, 0.8 equiv) of HATU, 63.0 mg (0.210 mmol, 0.8 equiv),

and 370 μL (2.11 mmol, 8 equiv) of DIPEA in 377 mL of methylene

chloride. The crude product underwent an initial puriﬁcation via

column chromatography on silica gel (ethyl acetate/methanol 9:1),

and the resulting semipure residue was subjected to reversed-phase

HPLC puriﬁcation to aﬀord a 3.6:1 ratio of compound 2-A (8.9 mg)

and compound 2-B (7.6 mg) in a 37.6% yield. LC/MS (ESI): m/z

calcd for C₃₄H₄₄N₈O₆S₂(M + H⁺) = 724.28, found 725.00. HRMS

(ESI-TOF): M + Na⁺, found 747.2723 C₃₄H₄₄N₈O₆S₂requires

747.2706.

1

MHz, CDCl₃): δ 0.95−1.00 (d, J = 6.7 Hz, 3H, CHCH(CH₃)₂); 1.00−

1.06 (d, J = 6.7 Hz, 3H, CHCH(CH₃)₂); 1.39−1.46 (t, 3H,

OCH₂CH₃); 1.48 (s, 9H, C(CH₃)₃); 1.70−1.76 (d, J = 7.3 Hz, 3H,

CHCH₃); 1.95−2.12 (m, 1H, CHCH(CH₃)₂); 2.99 (s, 1H, NCH₃);

4.38−4.48 (q, 2H, OCH₂CH₃); 5.28−5.40 (m, 1H, NH); 6.15−6.24

(m, 1H, 1αH); 8.15 (s, 1H, SCH). ¹³C NMR (75 MHz, CDCl₃): δ

14.4, 16.0, 17.2, 19.7, 28.4, 30.3, 31.2, 50.8, 55.4, 61.5, 79.7, 128.2,

147.0, 156.0, 161.3, 171.4, 172.7.

EtO-Thiazole-Ala-NMe-Val-NH₂(17c-1), Fragment A. EtO-Thia-

zole-Ala-NMe-Val-NH₂was synthesized following the General Amine

Deprotection procedure. The amine was taken on to the next reaction

without further puriﬁcation or characterization (426 mg, quantitative

yield) as a light brown oil.

1

•Compound 2-A. H NMR (308 K, 600 MHz, CDCl₃): δ 0.97−

1.06 (m, 6H, CHCH(CH₃)₂); 0.96−1.04 (m, 6H, CH(CH₃)₂); 1.58−

1.62 (br, 3H, CHCH₃); 1.61−1.68 (m, 1H, CHCH₂CH(CH₃)₂);

1.72−1.78 and 1.84−1.94 (m, 2H, CHCH₂CH(CH₃)₂); 1.80 and

1.97−2.03 (m, 2H, CH₂γ Pro2); 2.01−2.06 (m, 2H, CH₂γ Pro1);

2.03−2.05 and 2.48−2.50 (m, 2H, CH₂β Pro1); 2.19−2.25 (m, 1H,

CHCH(CH₃)₂); 2.31−2.39 (m, 2H, CH₂β Pro2); 3.27 (s, 1H,

NCH₃); 3.69−3.83 (m, 4H, CH₂δ Pro); 4.71−4.75 (m, 1H, 1αH);

4.75−4.81 (m, 1H, 1αH); 5.02−5.09 (m, 1H, 1αH); 5.47−5.51 (m,

1H, 1αH); 5.89−5.94 (m, 1H, 1αH); 7.11−7.20 (m, 1H, NH); 7.48−

7.54 (m, 1H, NH); 7.55 (s, 1H, SCH); 8.04 (s, 1H, SCH); 8.08 (s, 1H,

OCH). ¹³C NMR (150 MHz, CDCl₃): δ 17.8, 18.1, 19.3, 20.9 (CH₂γ

Pro2), 22.5 (CH₂γ Pro1), 22.4, 23.4, 24.3, 29.5, 30.0 (CH₂β Pro1),

30.5, 30.6, 35.1 (CH₂β Pro2), 41.3, 46.7, 49.1, 51.3, 55.0, 59.8, 62.5,

122.2, 122.4, 137.9, 140.9, 142.7, 148.9, 154.0, 161.1, 170.8, 171.4,

172.3, 172.9, 173.8, 174.3. △βγ = (7.5. ppm; 14.2 ppm).

Experimental procedures and compound characterizations for 18−

28a-1 were performed as described in ref 3.

EtO-Thiazole-Ala-NMe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc

(29-2). EtO-Thiazole-Ala-NMe-Val-Pro-Oxazole-Thiazole-Pro-Leu-

NHBoc was synthesized following the General Peptide Synthesis

procedure, utilizing 506 mg (0.88 mmol, 1.0 equiv) of acid HO-Pro-

Oxazole-Thiazole-Pro-Leu-NHBoc (28a-1), 304 mg (0.97 mmol, 1.1

equiv) of amine EtO-Thiazole-Ala-NMe-Val-NH₂(17c-1), 225 mg

(0.70 mmol, 0.8 equiv) of TBTU, 266 mg (0.70 mmol, 0.8 equiv) of

HATU, and 1.22 mL (7.00 mmol, 8.0 equiv) of DIPEA dissolved in

9.00 mL of methylene chloride. The crude product underwent a ﬁnal

puriﬁcation via column chromatography on silica gel (ethyl acetate/

methanol 99:1) to aﬀord the desired peptide (294 mg, 38% yield). R_f:

1

0.30 (ethyl acetate/methanol 99:1). H NMR (300 MHz, CDCl₃): δ

1

•Compound 2-B. H NMR (318 K, 600 MHz, CDCl₃): δ 0.67−

0.64−0.74 (m, 3H, CHCH(CH₃)₂); 0.74−0.82 (m, 3H, CHCH-

(CH₃)₂); 0.85−0.91 (d, J = 6.7 Hz, 3H, CH(CH₃)₂); 0.91−0.96 (d, J =

6.7 Hz, 3H, CH(CH₃)₂); 1.26−1.34 (t, 3H, OCH₂CH₃); 1.35 (s, 9H,

C(CH₃)₃); 1.39−1.49 (m, 2H, CHCH₂CH(CH₃)₂); 1.57−1.64 (d, J =

8.7 Hz, 3H, CHCH₃); 1.65−1.78 (m, 1H, CHCH(CH₃)₂); 1.84−1.96

(m, 2H, CHCH₂CH₂CH₂); 1.87−2.05 (m, 1H, CHCH(CH₃)₂);

1.96−2.00 and 2.09−2.16 (m, 2H, CHCH₂CH₂CH₂); 2.00−2.09 (m,

2H, CHCH₂CH₂CH₂); 2.16−2.43 (m, 2H, CHCH₂CH₂CH₂); 2.79

and 2.91 (s, 1H, NCH₃); 3.59−3.85 and 3.98−4.16 (m, 4H,

CHCH₂CH₂CH₂); 4.24−4.36 (q, 2H, OCH₂CH₃); 4.41−4.53 (m,

1H, 1αH); 4.62−4.76 (m, 1H, 1αH); 5.23−5.36 (m, 1H, 1αH); 5.43−

5.52 (m, 1H, 1αH); 5.84−6.14 (m, 1H, 1αH); 7.31−7.43 (m, 1H,

NH); 7.86 and 8.07 (s, 1H, SCH); 8.04 (s, 1H, SCH); 8.21 and 8.26

(s, 1H, OCH). ¹³C NMR (75 MHz, CDCl₃): δ 14.4, 16.0, 17.5, 19.8,

21.8, 22.0, 23.5, 24.5, 24.6, 25.4, 27.8, 28.4, 30.3, 31.7, 42.1, 47.1, 49.1,

50.4, 50.8, 54.3, 58.8, 61.0, 61.4, 61.7, 79.6, 120.7, 121.4, 128.3, 137.9,

142.8, 143.3, 146.9, 155.7, 156.4,161.1, 170.8, 171.4, 172.0, 172.5,

172.8, 174.3. LC/MS (ESI): m/z calcd for C₄₁H₅₈N₈O₉S₂(M + H⁺) =

893.37, found 893.00. HRMS (ESI-TOF): M + H⁺, found 871.3846

C₄₁H₅₈N₈O₉S₂requires 871.3840.

0.94 (m, 6H, CHCH(CH₃)₂); 0.94−1.11 (m, 6H, CH(CH₃)₂); 1.62−

1.69 (m, 1H, CHCH₂CH(CH₃)₂); 1.64−1.69 and 1.79−1.86 (m, 3H,

CHCH₃); 1.65−1.72 and 1.93−1.96 (m, 2H, CHCH₂CH(CH₃)₂);

1.76−1.85 and 1.94−2.03 (m, 2H, CH₂γ Pro1); 2.07−2.14 (m, 2H,

CH₂γ Pro2); 2.12−2.19 and 2.28−2.42 (m, 2H, CH₂β Pro2); 2.18−

2.20 and 2.33−2.39 (m, 2H, CH₂β Pro1); 2.41−2.46 (m, 1H,

CHCH(CH₃)₂); 3.24 (s, 1H, NCH₃); 3.58−67 and 4.18−4.22 (m, 2H,

CH₂δ Pro1); 3.67−3.85 (m, 2H, CH₂δ Pro2); 4.76−4.80 and 4.88−

4.92 (m, 1H, 1αH); 4.83−4.90 (m, buried, 1H, 1αH); 5.03−5.16 (m,

1H, 1αH); 5.37−5.42 and 5.52−5.59 (m, 1H, 1αH); 5.78−5.84 (m,

1H, 1αH); 7.00−7.06 (m, 1H, NH); 7.41−7.45 (m, 1H, NH); 7.60 (s,

1H, SCH); 7.89 (s, 1H, SCH); 8.31 (s, 1H, OCH). ¹³C NMR (150

MHz, CDCl₃): δ 16.7, 18.1, 18.2, 19.6, 20.1, 20.7 (CH₂γ Pro1), 22.0,

23.4, 24.0 (CH₂γ Pro2), 24.3, 30.3 (CH₂β Pro1), 30.7, 34.9 (CH₂β

Pro2), 46.8, 47.3, 48.8, 51.4, 55.0, 59.6, 59.9, 123.7, 124.3, 125.7,

137.9, 142.7, 148.9, 154.0, 161.1, 170.8, 171.4, 172.3, 172.9, 173.8,

174.3. Δβγ (Pro1, Pro2) = (9.6 ppm; 10.9 ppm).

Compound 3. Experimental procedures and compound character-

izations for 12a−17a-1 were performed as described in ref 3.

MeO-Oxazole-Thiazole-Pro-NMe-Leu-NBoc (27b). MeO-Oxazole-

Thiazole-Pro-NMe-Leu-NBoc was synthesized following the General

Peptide Synthesis procedure, utilizing 114 mg (0.41 mmol, 1.0 equiv)

of amine MeO-Oxazole-Thiazole-Pro-NH (25-1), 110 mg (0.45

mmol, 1.1 equiv) of acid HO-NMe-Leu-NBoc (26b), 106 mg (0.33

mmol, 0.8 equiv) of TBTU, 125 mg (0.33 mmol, 0.8 equiv) of HATU,

and 0.43 mL (2.46 mmol, 6 equiv) of DIPEA dissolved in 4.5 mL of

methylene chloride. The crude product underwent a ﬁnal puriﬁcation

via column chromatography on silica gel (hexanes/ethyl acetate 17:3

to 1:1) to aﬀord the desired peptide (213 mg, 97% yield) as white

ﬂakes. R_f: 0.3 (hexanes/ethyl acetate 3:1). ¹H NMR (300 MHz,

CDCl₃): δ 0.80−0.93 (m, 6H, CHCH₂CH(CH₃)₂); 1.38 and 1.41 (s,

9H, C(CH₃)₃); 1.39−1.54 (m, 1H, CHCH₂CH(CH₃)₂); 1.43−1.67

(m, 1H, CHCH₂CH(CH₃)₂); 1.93−2.22 (m, 2H, CHCH₂CH₂CH₂);

2.14−2.45 (m, 2H, CHCH₂CH₂CH₂); 2.76 and 2.78 (s, 3H, NCH₃);

3.49−3.76 (m, 2H, CHCH₂CH₂CH₂); 3.85 (s, 3H, OCH₃); 4.73−4.82

and 4.97−5.07 (m, 1H, 1αH); 5.40−5.47 (dd, J = 7.46, 2.44 Hz, 1H,

1αH); 7.99 (s, 1H, SCH); 8.23 (s, 1H, OCH). ¹³C NMR (75

MHz,CDCl₃): δ 22.0, 23.0, 24.5, 24.8, 28.4, 29.8, 31.7, 37.8, 47.1, 52.2,

53.5, 55.0, 58.7, 79.9, 121.5, 134.2, 142.1, 143.7, 156.1, 157.4, 161.4,

HO-Thiazole-Ala-NMe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc

(29-2a). HO-Thiazole-Ala-NMe-Val-Pro-Oxazole-Thiazole-Pro-Leu-

NHBoc was synthesized following the General Acid Deprotection

procedure, utilizing 294 mg (0.338 mmol, 1.0 equiv) of EtO-Thiazole-

Ala-NMe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-2) and 113

mg (2.70 mmol, 8 equiv) of LiOH·H₂O in 4.00 mL of ethanol. The

acid was taken on to the next reaction without further puriﬁcation

(222 mg, 78% yield). LC/MS (ESI): m/z calcd for C₃₉H₅₄N₈O₉S₂(M

+ H⁺) = 843.35, found 843.00.

HO-Thiazole-Ala-NMe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NH₂

(29-2b). HO-Thiazole-Ala-NMe-Val-Pro-Oxazole-Thiazole-Pro-Leu-

NH₂was synthesized following the General Amine Deprotection

procedure, utilizing 222 mg (0.264 mmol, 1.0 equiv) of HO-Thiazole-

Ala-NMe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-2a), 57.4 μL

(0.53 mmol, 2.0 equiv) of anisole, and 0.54 mL of triﬂuoroacetic acid

in 2.10 mL of methylene chloride. HO-Thiazole-Ala-NMe-Val-Pro-

Oxazole-Thiazole-Pro-Leu-NH₂was taken on to the next reaction

without further puriﬁcation or characterization (196 mg, quantitative

yield). LC/MS (ESI): m/z calcd for C₃₄H₄₆N₈O₇S₂(M + H⁺) =

743.29, found 743.00.

10606

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

171.5, 174.5. HRMS (ESI-TOF): M + Na⁺, found 529.2082

C₂₄H₃₄N₄O₆S requires 529.2097.

29.5, 31.8, 37.8, 46.7, 47.1, 47.5, 49.5, 53.5, 55.0, 58.6, 58.8, 61.4, 61.6,

80.1, 121.0, 121.7, 127.3, 127.5, 137.5, 142.5, 143.5,146.9, 155.2, 156.2,

156.8, 164.3, 170.4, 170.9, 171.6, 174.1, 175.0. LC/MS (ESI): m/z

calcd for C₄₁H₅₈N₈O₉S₂(M + H⁺) = 871.38, found 871.00. HRMS

(ESI-TOF): M + Na⁺, found 893.3653 C₄₁H₅₈N₈O₉S₂requires

893.3666

HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-NMe-Leu-NHBoc

(29-3a). HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-NMe-Leu-

NHBoc was synthesized following the General Acid Deprotection

procedure, utilizing 400 mg (0.470 mmol, 1.0 equiv) of EtO-Thiazole-

Ala-Val-Pro-Oxazole-Thiazole-Pro-NMe-Leu-NHBoc (29-3) and 80

mg (1.877 mmol, 4 equiv) of LiOH·H₂O in 5.00 mL of ethanol. The

acid was taken on to the next reaction without further puriﬁcation

(274 mg, 71% yield). LC/MS (ESI): m/z calcd for C₃₉H₅₅N₈O₉S₂(M

+ H⁺) = 843.35, found 843.00

HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-NMe-Leu-NH₂

(29-3b). HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-NMe-Leu-

NH₂was synthesized following the General Amine Deprotection

procedure, utilizing 274 mg (0.334 mmol, 1.0 equiv) of HO-Thiazole-

Ala-Val-Pro-Oxazole-Thiazole-Pro-NMe-Leu-NHBoc (29−3a), 70.0

μL (0.67 mmol, 2.0 equiv) of anisole, and 0.70 mL of triﬂuoroacetic

acid in 2.60 mL of methylene chloride. HO-Thiazole-Ala-Val-Pro-

Oxazole-Thiazole-Pro-NMe-Leu-NH₂was taken on to the next

reaction without further puriﬁcation or characterization (241 mg,

quantitative yield). LC/MS (ESI): m/z calcd for C₃₄H₄₆N₈O₇S₂(M +

H⁺) = 743.30, found 743.00.

HO-Oxazole-Thiazole-Pro-NMe-Leu-NBoc (27b-1). HO-Oxazole-

Thiazole-NMe-Leu-NBoc was synthesized following the General Acid

Deprotection procedure, utilizing 213 mg (0.42 mmol, 1.0 equiv) of

MeO-Oxazole-Thiazole-Pro-NMe-Leu-NBoc (27b) and 52.9 mg (1.26

mmol, 3.0 equiv) of LiOH·H₂O in 4.2 mL of methanol. The acid was

taken on to the next reaction without further puriﬁcation (207 mg,

quantitative yield) as a clear oil.

MeO-Pro-Oxazole-Thiazole-Pro-NMe-Leu-NBoc (28b). MeO-Pro-

Oxazole-Thiazole-Pro-NMe-Leu-NBoc was synthesized following the

General Peptide Synthesis procedure, utilizing 207 mg (0.42 mmol, 1.0

equiv) of acid HO-Oxazole-Thiazole-Pro-NMe-Leu-NBoc (27b-1),

59.5 mg (0.46 mmol, 1.1 equiv) of amine MeO-Pro-NH, 109 mg (0.34

mmol, 0.8 equiv) of TBTU, 129 mg (0.34 mmol, 0.8 equiv) of HATU,

and 0.44 mL (2.52 mmol, 6.0 equiv) of DIPEA dissolved in 4.2 mL of

methylene chloride. The crude product underwent a ﬁnal puriﬁcation

via column chromatography on silica gel (hexanes/ethyl acetate 4:1 to

0:1) to aﬀord the desired peptide (265 mg, 96% yield). R_f: 0.45

1

(hexanes/ethyl acetate 0:1). H NMR (300 MHz, CDCl₃): δ 0.82−

0.92 (m, 6H, CHCH₂CH(CH₃)₂); 1.38 and 1.41 (s, 9H, C(CH₃)₃);

1.42−1.50 (m, 1H, CHCH₂CH(CH₃)₂); 1.44−1.64 (m, 2H,

CHCH₂CH); 1.77−1.89 (m, 2H, CHCH₂CH₂CH₂); 1.91−2.12 (m,

2H, CHCH₂CH₂CH₂); 1.89−1.96 and 2.13−2.18 (m, 2H,

CHCH₂CH₂CH₂); 2.10−2.39 (m, 2H, CHCH₂CH₂CH₂); 2.75 and

2.77 (s, 3H, NCH₃); 3.60−3.78 and 4.01−4.09 (m, 4H,

CHCH₂CH₂CH₂); 3.62 and 3.64 (s, 3H, OCH₃); 4.50−4.59 and

5.19−5.28 (m, 1H, 1αH); 4.72−4.82 and 4.95−5.06 (m, 1H, 1αH);

5.37−5.46 (m, 1H, 1αH); 7.78 and 7.88 (s, 1H, SCH); 8.17 and 8.19

(s, 1H, OCH). ¹³C NMR (75 MHz,CDCl₃): δ 21.8, 22.0, 22.9, 24.4,

27.9, 28.3, 29.1, 29.7, 31.6, 37.8, 46.7, 47.1, 47.6, 48.7, 52.3, 53.5, 54.9,

58.6, 59.9, 60.3, 60.7, 79.9, 120.6, 137.8, 142.8, 143.1, 155.1, 156.1,

160.2, 171.0, 173.4. HRMS (ESI-TOF): M + Na⁺, found 626.2606

C₂₉H₄₁N₅O₇S requires 626.2625

•Compound 3. Compound 3 was synthesized following the

Macrocyclization Procedure, utilizing 241 mg (0.334 mmol, 1.0

equiv) of HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-NMe-Leu-

NH₂(29-3b), 87.0 mg (0.270 mmol, 0.8 equiv) of TBTU, 103.0 mg

(0.270 mmol, 0.8 equiv) of HATU, 75.0 mg (0.270 mmol, 0.8 equiv)

of DMTMM, and 350 μL (2.00 mmol, 6 equiv) of DIPEA in 334 mL

of methylene chloride. The crude product was directly subjected to

reversed-phase HPLC puriﬁcation to aﬀord a 1:1.1 of compound 3-A

(66.5 mg), and compound 3-B (103.9 mg) in a 25.2% overall yield.

HRMS (ESI-TOF): M + Na⁺, found 747.2711 C₃₄H₄₄N₈O₆S₂requires

747.2723.

HO-Pro-Oxazole-Thiazole-Pro-NMe-Leu-NBoc (28b-1), Fragment

B. HO-Pro-Oxazole-Thiazole-Pro-NMe-Leu-NBoc was synthesized

following the General Acid Deprotection procedure, utilizing 265

mg (0.44 mmol, 1.0 equiv) of MeO-Pro-Oxazole-Thiazole-Pro-NMe-

Leu-NBoc (28b), 55 mg (1.32 mmol, 3.0 equiv) of LiOH·H₂O in 4.40

mL of methanol. The acid was taken on to the next reaction without

further puriﬁcation (259 mg, quantitative yield) as clear ﬂakes.

EtO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-NMe-Leu-NHBoc

(29-3). EtO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-NMe-Leu-

NHBoc was synthesized following the General Peptide Synthesis

procedure, utilizing 259 mg (0.44 mmol, 1.0 equiv) of acid HO-Pro-

Oxazole-Thiazole-Pro-NMe-Leu-NBoc (28b-1), 137 mg (0.49 mmol,

1.1 equiv) of amine EtO-Thiazole-Ala-Val-NH₂(17a-1), 112 mg (0.35

mmol, 0.8 equiv) of TBTU, 133 mg (0.35 mmol, 0.8 equiv) of HATU,

and 0.46 mL (2.64 mmol, 6.0 equiv) of DIPEA dissolved in 4.40 mL of

methylene chloride. The crude product underwent a ﬁnal puriﬁcation

via column chromatography on silica gel (ethyl acetate/methanol

99:1) to aﬀord the desired peptide (400 mg, 94% yield). R_f: 0.30 (ethyl

1

•Compound 3-A. H NMR (318 K, 600 MHz, CDCl₃): δ 0.92−

0.96 and 0.99−1.03 (m, 3H, CHCH(CH₃)₂); 0.99−1.02 and 1.04−

1.08 (m, 3H, CHCH(CH₃)₂); 0.91−1.05 (m, 6H, CHCH₂CH-

(CH₃)₂); 1.44−1.57 and 1.60−1.69 (m, 1H, CHCH₂CH(CH₃)₂);

1.53−1.58 and 1.64−1.68 and 1.76−1.81 (m, 3H, CHCH₃); 1.68−

1.74 and 1.81−1.88 (m, 2H, CHCH₂CH(CH₃)₂); 1.74−2.09 (m, 2H,

CHCH₂CH₂CH₂); 2.00−2.18 (m, 2H, CHCH₂CH₂CH₂); 2.08−2.14

and 2.24−2.41 and 2.43−2.49 (m, 2H, CHCH₂CH₂CH₂); 2.13−2.20

and 2.22−2.52 (m, 2H, CHCH₂CH₂CH₂); 2.35−2.56 (m, 1H,

CHCH(CH₃)₂); 3.09−3.16 and 3.21−3.34 (m, 3H, NCH₃); 3.61−

4.00 (m, 2H, CHCH₂CH₂CH₂); 3.84−3.97 and 4.08−4.13 (m, 2H,

CHCH₂CH₂CH₂); 4.31−4.38 and 4.54−4.59 (m, 1H, 1αH); 4.76−

4.84 and 5.16−5.24 (m, 1H, 1αH); 5.32−5.47 (m, 1H, 1αH); 5.47−

5.75 (m, 1H, 1αH); 5.67−5.81 (m, 1H, 1αH); 6.80−6.85 and 6.91−

6.96 and 7.00−7.07 (m, 1H, NH); 7.11−7.15 and 7.21−7.27 and

7.34−7.39 (m, 1H, NH); 7.65 and 7.74 and 7.92 (s, 1H, SCH); 7.49

and 7.73 and 8.01 (s, 1H, SCH); 7.95 and 8.20 and 8.33 (s, 1H,

OCH). ¹³C NMR (318 K, 150 MHz,CDCl₃): δ 16.8, 17.5, 19.5, 19.7,

20.8 (CH₂γ Pro2), 21.1, 21.4, 21.8, 22.6, 23.0, 24.5, 24.6, 25.0 (CH₂γ

Pro1), 25.8, 26.5, 29.2, 29.5, 29.8, 30.0, 31.2 (CH₂β Pro1), 31.7, 32.3,

32.8, 33.8, 34.6, 34.9, 35.3, 35.8 (CH₂β Pro2), 37.5, 46.2, 46.8, 47.1,

47.4, 47.9, 48.9, 49.2, 53.6, 54.3, 55.9, 58.2, 59.0, 59.1, 59.4, 60.9, 63.0,

63.3, 121.0, 121.2, 122.3, 127.6, 127.9, 128.6, 129.0, 142.0, 142.1,

144.2,146.9, 155.2, 156.2, 156.8, 162.4, 164.3, 170.4, 170.9, 171.6,

174.1, 175.0. △βγ(Pro1, Pro2) = (6.7 ppm; 15.0 ppm).

1

acetate/methanol 99:1). H NMR (300 MHz, CDCl₃): δ 0.74−0.81

and 0.84−0.92 (dd, J = 21.8, 6.3 Hz, 3H, CHCH(CH₃)₂); 0.82−0.97

(dd, J = 14.4, 6.3 Hz, 3H, CHCH(CH₃)₂); 0.92−1.02 (dd, J = 11.9, 6.3

Hz, 6H, CHCH₂CH(CH₃)₂); 1.34−1.44 (t, 3H, OCH₂CH₃); 1.49 (s,

9H, C(CH₃)₃); 1.49−1.60 (m, 1H, CHCH₂CH(CH₃)₂); 1.52−1.73

(m, 2H, CHCH₂CH(CH₃)₂); 1.62−1.72 (d, J = 7.0 Hz, 3H, CHCH₃);

1.98−2.05 (m, 2H, CHCH₂CH₂CH₂); 2.00−2.22 (m, 2H,

CHCH₂CH₂CH₂); 2.07−2.19 and 2.24−2.31 (m, 2H,

CHCH₂CH₂CH₂); 2.25−2.49 (m, 2H, CHCH₂CH₂CH₂); 2.28−2.38

(m, 1H, CHCH(CH₃)₂); 2.80−2.89 (m, 3H, NCH₃); 3.61−3.68 and

3.73−3.87 (m, 2H, CHCH₂CH₂CH₂); 4.08−4.29 (m, 2H,

CHCH₂CH₂CH₂); 4.29−4.39 (m, 1H, 1αH); 4.34−4.46 (q, 2H,

OCH₂CH₃); 4.72−4.82 and 5.30−5.37 (m, 1H, 1αH); 4.82−4.91 and

5.06−5.16 (m, 1H, 1αH); 5.14−5.24 and 5.35−5.48 (m, 1H, 1αH);

5.46−5.57 (m, 1H, 1αH); 7.09−7.18 (d, J =8.6 Hz, 1H, NH); 7.36−

7.42 (d, J =7.3 Hz, 1H, NH); 7.94 and 8.05 (s, 1H, SCH); 8.04 and

8.08 (s, 1H, SCH); 8.29 (s, 1H, OCH). ¹³C NMR (75 MHz,CDCl₃): δ

14.3, 17.5, 17.8, 19.5, 21.0, 22.1, 22.2, 23.0, 24.5, 24.7, 25.5, 27.8, 28.3,

1

•Compound 3-B. H NMR (318 K, 600 MHz, CDCl₃): δ 0.82−

0.87 (d, J = 6.61 Hz, 3H, CHCH(CH₃)₂); 0.94−0.99 (d, J = 6.61 Hz,

3H, CHCH(CH₃)₂); 0.90−1.00 (m, 6H, CHCH₂CH(CH₃)₂); 0.95−

1.01 (m, 3H, CHCH₃); 1.45−1.52 (m, 1H, CHCH₂CH(CH₃)₂);

1.57−1.63 and 1.81−1.87 (m, 2H, CHCH₂CH(CH₃)₂); 1.76−1.82

and 1.89−1.94 (m, 2H, CHCH₂CH₂CH₂); 1.85−1.91 and 2.25−2.32

(m, 2H, CHCH₂CH₂CH₂); 1.96−2.02 and 2.06−2.12 (m, 2H,

CHCH₂CH₂CH₂); 2.22−2.29 (m, 2H, CHCH₂CH₂CH₂); 2.78−2.85

10607

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

(m, 1H, CHCH(CH₃)₂); 3.09−3.14 (s, 3H, NCH₃); 3.77−3.84 (m,

2H, CHCH₂CH₂CH₂); 3.77−3.83 and 3.84−3.91 (m, 2H,

CHCH₂CH₂CH₂); 4.65−4.71 (m, 1H, 1αH); 4.72−4.78 (m, 1H,

1αH); 5.07−5.14 (m, 1H, 1αH); 5.49−5.54 (m, 1H, 1αH); 5.63−5.69

(m, 1H, 1αH); 6.23−6.33 (m, 1H, NH); 7.15−7.24 (m, 1H, NH);

7.46 (s, 1H, SCH); 8.00 (s, 1H, SCH); 8.35 (s, 1H, OCH). ¹³C NMR

(318 K, 150 MHz,CDCl₃): δ 16.4, 16.6, 19.5, 20.4 (CH₂γ Pro2), 22.5

(CH₂γ Pro1), 22.7, 23.0, 24.5, 28.4, 31.7 (CH₂β Pro1), 35.8 (CH₂β

Pro2), 37.4, 45.4, 46.2, 47.8, 53.9, 56.6, 58.7, 64.2, 123.0, 129.5, 136.7,

140.5, 143.1, 147.9, 157.8, 161.0, 167.7, 168.5, 170.3, 171.3, 171.5,

173.3, 173.6. Δβγ (Pro1, Pro2) = (9.2 ppm; 15.4 ppm).

Compound 4. EtO-Thiazole-Ala-Gly-NHBoc (17b). EtO-Thiazole-

Ala-Gly-NHBoc was synthesized following the General Peptide

Synthesis procedure, utilizing 200 mg (0.995 mmol, 1.0 equiv) of

amine EtO-Thiazole-Ala-NH₂(15a-1), 192 mg (1.10 mmol, 1.1 equiv)

of acid HO-Gly-NHBoc (16c), 320 mg (0.995 mmol, 1.0 equiv) of

TBTU, and 0.70 mL (3.99 mmol, 4 equiv) of DIPEA dissolved in 10

mL of methylene chloride under N₂. The reaction mixture was stirred

for 3 h and was monitored by TLC upon completion. The crude

product underwent a ﬁnal puriﬁcation via column chromatography on

silica gel (hexanes/ethyl acetate 1:1) to aﬀord the desired peptide (220

mg, 62% yield) as a yellow oil. R_f: 0.325 (hexanes/ethyl acetate 1:1).

¹H NMR (300 MHz, CDCl₃): δ 1.33−1.38 (t, 3H, OCH₂CH₃); 1.39

(s, 9H, C(CH₃)₃); 1.59−1.62 (d, 3H, CHCH₃); 3.84−3.86 (s, 2H,

CH₂); 4.32−4.39 (m, 2H, OCH₂CH₃); 5.34−5.44 (m, 1H, 1αH), 8.05

(s, 1H, SCH). ¹³C NMR (75 MHz, CDCl₃): δ 14.09, 21.00, 28.51,

44.19, 47.40, 61.94, 80.08, 126.39, 127.91, 147.03, 156.16, 161.31,

169.92, 173.44. HRMS (ESI-TOF): M + Na⁺, found 380.1247

C₁₅H₂₃N₃O₅S requires 380.1256.

EtO-Thiazole-Ala-Gly-NH₂(17b-1), Fragment A. EtO-Thiazole-

Ala-Gly-NH₂was synthesized following the General Amine Depro-

tection procedure, utilizing 220 mg (0.616 mmol, 1.0 equiv) of EtO-

Thiazole-Ala-Gly-NHBoc (17b), 0.14 mL (1.23 mmol, 2.0 equiv) of

anisole, and 1.5 mL of triﬂuoroacetic acid in 4.5 mL of methylene

chloride. EtO-Thiazole-Ala-Gly-NH₂was taken on to the next reaction

without further puriﬁcation or characterization (201 mg, quantitative

yield) as a light brown oil.

EtO-Thiazole-Ala-Gly-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-

4). EtO-Thiazole-Ala-Gly-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc was

synthesized following the General Peptide Synthesis procedure,

utilizing 300 mg (0.521 mmol, 1.0 equiv) of acid HO-Pro-Oxazole-

Thiazole-Pro-Leu-NHBoc (28a-1), 201 mg (0.782 mmol, 1.5 equiv)

of amine EtO-Thiazole-Ala-Gly-NH₂(17b-1), 167 mg (0.521 mmol,

1.0 equiv) of TBTU, 198 mg (0.521 mmol, 1.0 equiv) of HATU, and

0.73 mL (4.17 mmol, 8.0 equiv) of DIPEA dissolved in 5.2 mL of

methylene chloride under N₂. The reaction mixture was stirred for 3 h

and was monitored by TLC and LCMS upon completion. The crude

product underwent a ﬁnal puriﬁcation via column chromatography on

silica gel (ethyl acetate/methanol 19:1) to aﬀord the desired peptide

(390 mg, 94% yield) as yellow ﬂakes. R_f: 0.25 (ethyl acetate/methanol

9:1). ¹H NMR (300 MHz, CDCl₃): δ 0.86−0.99 (m, 6H,

CH₃CHCH₃); 1.37 (s, 9H, C(CH₃)₃); 1.48−1.56 (t, 2H,

OCH₂CH₃); 1.56−1.65 (m, 1H, CH₃CHCH₃); 1.65−1.82 (d, 3H,

CHCH₃); 1.99−2.09 (m, 2H, CHCH₂CH₂CH₂); 2.09−2.19 (m, 2H,

CHCH₂CH₂CH₂); 2.21−2.32 (m, 2H, CHCH₂CH₂CH₂); 2.33−2.46

(m, 2H, CHCH₂CH₂CH₂); 3.63−3.73 (m, 2H, CHCH₂CH₂CH₂);

3.78−3.87 (m, 2H, CHCH₂CH₂CH₂); 3.86−3.94 (d, 2H, CH₂);

4.07−4.19 (m, 3H, OCH₂CH₃); 4.42−4.52 (m, 1H, 1αH); 4.51−4.59

(m, 1H, 1αH); 5.28−5.34 (m, 1H, 1αH); 5.45−5.54 (m, 1H, 1αH);

7.86 (s, 1H, SCH); 7.99 (s, 1H, SCH); 8.32 (s, 1H, OCH). ¹³C NMR

(75 MHz, CDCl₃): δ 17.26, 18.42, 20.52, 21.83, 23.34, 24.59, 25.60,

28.29, 28.65, 31.68, 42.04, 47.06, 47.42, 49.50, 50.38, 58.80, 61.35,

61.98, 79.60, 120.90, 127.43, 127.56, 137.35, 143.49, 146.46, 155.68,

156.51, 160.84, 169.42, 171.38, 172.81, 174.36. HRMS (ESI-TOF): M

+ Na⁺, found 837.3029 C₃₇H₅₀N₈O₉S₂requires 837.3040.

mmol, 8 equiv) of LiOH·H₂O in 4.78 mL of ethanol. The acid was

taken on to the next reaction without further puriﬁcation (354 mg,

94% yield). LC/MS (ESI): m/z calcd for C₃₅H₄₆N₈O₉S₂(M + H⁺) =

786.28, found 787.00

HO-Thiazole-Ala-Gly-Pro-Oxazole-Thiazole-Pro-Leu-NH₂(29-4b).

HO-Thiazole-Ala-Gly-Pro-Oxazole-Thiazole-Pro-Leu-NH₂was syn-

thesized following the General Amine Deprotection procedure,

utilizing 354 mg (0.449 mmol, 1.0 equiv) of HO-Thiazole-Ala-Gly-

Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-4a), 97 μL (0.897 mmol,

2.0 equiv) of anisole, and 1.25 mL of triﬂuoroacetic acid in 3.75 mL of

methylene chloride. HO-Thiazole-Ala-Gly-Pro-Oxazole-Thiazole-Pro-

Leu-NH₂was taken on to the next reaction without further

puriﬁcation or characterization (353 mg, quantitative yield). LC/MS

(ESI): m/z calcd for C₃₀H₃₈N₈O₇S₂(M + H⁺) = 686.80, found 687.00

•Compound 4. Compound 4 was synthesized following the

Macrocyclization Procedure, utilizing 353 mg (0.514 mmol, 1.0

equiv) of HO-Thiazole-Ala-Gly-Pro-Oxazole-Thiazole-Pro-Leu-NH₂

(29-4b), 165 mg (0.514 mmol, 1.0 equiv) of TBTU, 235 mg (0.617

mmol, 1.0 equiv) of HATU, and 0.72 mL (4.11 mmol, 8 equiv) of

DIPEA in 147 mL of methylene chloride under N₂. The reaction was

monitored by LCMS upon completion. The crude product underwent

an initial puriﬁcation via column chromatography on silica gel (ethyl

acetate/methanol 9:1), and the resulting semipure residue was

subjected to reversed-phase HPLC puriﬁcation to aﬀord a 1:0.1:4.48

of compound 4-A (14.1 mg), compound 4-B (3.9 mg), and compound

4-C (52.3 mg) in a 66.6% overall yield. HRMS (ESI-TOF): M + Na⁺,

found 691.2094 C₃₀H₃₆N₈O₆S₂requires 691.2097.

1

•Compound 4-A. H NMR (298 K, 600 MHz, CDCl₃): δ 0.89−

0.99 (m, 6H, CH(CH₃)₂); 0.92−1.01 (d, 3H, CHCH₃); 1.53−1.59

(m, 1H, CHCH₂CH(CH₃)₂); 1.48−1.59 (m, 2H, CHCH₂CH); 1.84−

2.08 (m, 2H, CH₂γ Pro1); 1.96−2.06 (m, 2H, CH₂γ Pro2); 2.02−2.45

(m, 2H, CH₂β Pro1); 2.24−2.28 (m, 2H, CH₂β Pro2); 3.35−4.21 (m,

2H, CH₂δ Pro2); 3.83−3.88 (m, 2H, CH₂δ Pro1); 4.49−4.52 (m, 1H,

1αH); 4.75−4.81 (m, 1H, 1αH); 5.09−5.13 (m, 2H, CH₂); 5.54−5.46

(m, 1H, CHCH₃); 5.55−5.58 (m, 1H, CHCH₂CH); 7.96 (s, 1H,

SCH); 8.24 (s, 1H, SCH); 8.38 (s, 1H, OCH).¹³C NMR (150

MHz,CDCl₃): δ 16.0, 22.1, 22.15 (CH₂γ Pro2), 24.4, 25.65 (CH₂γ

Pro1), 29.64 (CH₂β Pro1), 31.56 (CH₂β Pro2), 41.4, 45.51,48.04,

49.76, 58.8, 60.45, 62.37, 63.74, 121.4, 122.5,125.63, 127.83, 139.17,

143.4, 148.47. Δβγ = (3.99 ppm; 9.41 ppm)

1

•Compound 4-B. H NMR (278 K, 600 MHz, CD₃CN): δ 0.87−

0.91 (d, 3H, CHCH₃); 0.91−0.97 (m, 6H, CH(CH₃)₂); 1.44−1.51

(m, 1H, CHCH₂CH(CH₃)₂); 1.60−1.77 (m, 2H, CHCH₂CH); 1.85−

1.97 (m, 2H, CH₂γ Pro1); 1.90−1.96 (m, 2H, CH₂γ Pro2); 2.08−2.18

(m, 2H, CH₂β Pro1); 1.90−2.33 (m, 2H, CH₂β Pro2); 3.56−3.65 (m,

2H, CH₂δ Pro2); 3.65−3.76 (m, 2H, CH₂δ Pro1); 4.34−4.39 (m, 1H,

1αH); 4.74−4.76 (m, 1H, 1αH); 5.07−5.09 (m, 2H, CH₂); 5.34−5.38

(m, 1H, CHCH₃); 5.47−5.55 (m, 1H, CHCH₂CH); 7.94 (s, 1H,

SCH); 8.01 (s, 1H, SCH); 8.47 (s, 1H, OCH). ¹³C NMR (278 K, 150

MHz, CD₃CN): δ 19.96 (CH₂γ Pro2), 21.28, 21.63 (CH₂γ Pro1),

22.74, 24.27, 31.25 (CH₂β Pro1), 35.23 (CH₂β Pro2), 40.87, 45.28,

46.71, 47.39, 58.73, 59.41, 62.03, 63.3, 122.13, 124.43, 125.54, 128.47,

140.58, 142.67, 143.3. Δβγ = (9.62 ppm; 15.27 ppm).

1

•Compound 4-C. H NMR (298 K, 600 MHz, CD₃CN): δ 0.87−

0.91 (d, 3H, CHCH₃); 0.89−97 (m, 6H, CH(C₃)₂); 1.49−1.57 (m,

1H, CHCH₂CH(CH₃)₂); 1.57−1.87 (m, 2H, CHCH₂CH); 1.81−2.04

(m, 2H, CH₂γ Pro1); 1.94−2.02 (m, 2H, CH₂γ Pro2); 1.97−2.41 (m,

2H, CH₂β Pro1); 2.13−2.33 (m, 2H, CH₂β Pro2); 3.77−3.84 (m, 2H,

CH₂δ Pro2); 3.78−3.91 (m, 2H, CH₂δ Pro1); 4.44−4.50(m, 1H,

1αH); 4.71−4.79 (m, 1H, 1αH); 5.04−5.14 (m, 2H, CH₂); 5.39−5.43

(m, 1H, CHCH₃); 5.49−5.54 (m, 1H, CHCH₂CH); 7.51 (s, 1H,

SCH); 7.92 (s, 1H, SCH); 7.72 (s, 1H, OCH). ¹³C NMR (298 K, 150

MHz, CD₃CN): δ 22.1 (CH₂γ Pro2), 22.21, 22.97, 23.45, 24.4, 25.5

(CH₂γ Pro1), 29.6 (CH₂β Pro1), 31.7 (CH₂β Pro2), 41.27, 45.51,

46.67, 48.11, 58.8, 60.38, 62.3, 63.67, 121.68, 122.63, 125.69, 139.3,

143.45. Δβγ = (4.1 ppm; 9.6 ppm).

HO-Thiazole-Ala-Gly-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-

4a). HO-Thiazole-Ala-Gly-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc

was synthesized following the General Acid Deprotection procedure,

utilizing 390 mg (0.478 mmol, 1.0 equiv) of EtO-Thiazole-Ala-Gly-

Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-4) and 161 mg (3.83

Compound 5. Experimental procedures and compound character-

izations for 12c−15c were performed as described.⁴⁰

EtO-Thiazole-Gly-NH₂(15c-1). EtO-Thiazole-Gly-NH₂was synthe-

sized following the General Amine Deprotection procedure. The

10608

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

amine was taken on to the next reaction without further puriﬁcation or

characterization (66.1 mg, quantitative yield) as a light brown oil.

EtO-Thiazole-Gly-Val-NHBoc (17d). EtO-Thiazole-Gly-Val-NHBoc

was synthesized following the General Peptide Synthesis procedure,

utilizing 66.1 mg (0.35 mmol, 1.0 equiv) of amine EtO-Thiazole-Gly-

NH₂(15c-1), 83.6 mg (0.39 mmol, 1.1 equiv) of acid HO-Val-NHBoc

(16a), 125 mg (0.39 mmol, 1.1 equiv) of TBTU, and 244 μL (1.40

mmol, 4 equiv) of DIPEA dissolved in 35.0 mL of methylene chloride

under N₂. The crude product underwent a ﬁnal puriﬁcation via column

chromatography on silica gel (hexanes/ethyl acetate 19:1 to 3:1) to

aﬀord the desired peptide (135 mg, 99% yield) as yellow oil. R_f: 0.33

Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-5a), 240 μL (0.218 mmol,

2.0 equiv) of anisole, and 0.20 mL of triﬂuoroacetic acid in 0.80 mL of

methylene chloride. HO-Thiazole-Gly-Val-Pro-Oxazole-Thiazole-Pro-

Leu-NH₂was taken on to the next reaction without further

puriﬁcation or characterization (78 mg, quantitative yield). LC/MS

(ESI): m/z calcd for C₃₂H₄₃N₈O₇S₂(M + H⁺) = 715.27, found 715.00.

•Compound 5. Compound 5 was synthesized following the

Macrocyclization Procedure, utilizing 78.0 mg (0.109 mmol, 1.0

equiv) of HO-Thiazole-Gly-Val-Pro-Oxazole-Thiazole-Pro-Leu-NH₂

(29-5b), 42.0 mg (0.131 mmol, 1.2 equiv) of TBTU, 50.0 mg

(0.131 mmol, 1.2 equiv) of HATU, and 120 μL (0.66 mmol, 6 equiv)

of DIPEA in 110 mL of methylene chloride. The crude product was

directly subjected to reversed-phase HPLC puriﬁcation to aﬀord a

2.4:1:2.9 of compound 5-A (3.1 mg), compound 5-B (4.7 mg), and

compound 5-C (4.2 mg) in a 28.6% overall yield. LC/MS (ESI): m/z

calcd for C₃₄H₄₆N₈O₇S₂(M + Na⁺+ 2H⁺) = 721.27, found 721.00.

HRMS (ESI-TOF): M + 2Na⁺+ H⁺, found 743.2390 C₃₂H₄₀N₈O₆S₂

requires 743.2386.

1

(hexanes/ethyl acetate 1:1). H NMR (300 MHz, CDCl₃): δ 0.91−

0.97 (d, J = 7.0 Hz, 3H, CHCH(CH₃)₂); 0.97−1.03 (d, J = 7.0 Hz, 3H,

CHCH(CH₃)₂); 1.43 (t, 3H, OCH₂CH₃); 1.45 (s, 9H, C(CH₃)₃);

2.16−2.33 (m, 1H, CHCH(CH₃)₂); 3.98−4.07 (m, 1H, αH); 4.39−

4.49 (q, 2H, OCH₂CH₃); 4.79−4.86 (d, J = 6.82 Hz, 2H, αH); 4.98−

5.16 (m, 1H, NH); 7.00−7.19 (m, 1H, NH); 8.15 (s, 1H, SCH). ¹³C

NMR (75 MHz, CDCl₃): δ 14.4, 18.1, 19.6, 28.4, 31.3, 41.2, 59.9, 61.4,

79.5, 128.3, 146.5, 156.4, 161.4, 165.8, 169.9, 173.1. HRMS (ESI-

TOF): M + H⁺, found 386.1738 C₁₇H₂₇N₃O₅S requires 386.1778.

EtO-Thiazole-Gly-Val-NH₂(17c-1), Fragment A. EtO-Thiazole-

Gly-Val-NH₂was synthesized following the General Amine Depro-

tection procedure. The amine was taken on to the next reaction

without further puriﬁcation or characterization (101 mg, quantitative

yield) as a light brown oil.

1

•Compound 5-A. H NMR (298 K, 600 MHz, CDCl₃): δ 0.87−

0.95 (m, 6H, CHCH(CH₃)₂); 0.82−0.90 and 0.94−1.00 (m, 6H,

CHCH₂CH(CH₃)₂); 1.63−1.67 (m, 1H, CHCH(CH₃)₂); 1.76−1.81

(m, 1H, CHCH₂CH(CH₃)₂); 1.80−1.88 (m, 2H, CHCH₂CH-

(CH₃)₂); 1.97−2.06 (m, 2H, CHCH₂CH₂CH₂); 2.01−2.08 (m, 2H,

CHCH₂CH₂CH₂); 2.21−2.25 and 2.36−2.41 (m, 2H,

CHCH₂CH₂CH₂); 2.30−2.38 (m, 2H, CHCH₂CH₂CH₂); 3.79−3.86

(m, 4H, CHCH₂CH₂CH₂); 3.98−4.03 (m, 2H, 2αH); 4.70−4.75 (m,

1H, 1αH); 5.10−5.14 (m, 1H, 1αH); 5.47−5.49 and 5.57−5.60 (m,

1H, 1αH); 5.52−5.56 and 5.63−5.69 (m, 1H, 1αH); 6.81−6.86 (m,

1H, NH); 7.13−7.18 (m, 1H, NH); 7.48 and 7.61 (s, 1H, SCH); 7.55

and 7.73 (s, 1H, SCH); 7.88 and 8.03 (s, 1H, OCH). ¹³C NMR (298

K, 150 MHz, CDCl₃): δ 10.9, 14.1, 19.6, 20.4 (CH₂γ Pro1), 21.2, 21.4,

22.1, 22.7, 23.3, 25.3, 27.2 (CH₂γ Pro2), 31.1, 34.0 (CH₂β Pro2), 35.1

(CH₂β Pro1), 39.4, 48.0, 48.7, 55.5, 59.4, 66.8, 119.6, 120.3, 128.8,

130.8, 138.4, 140.8, 141.0, 143.5, 146.9, 152.1, 155.9, 169.4, 171.3,

171.7, 173.0, 174.4, 175.1. Δβγ (Pro1, Pro2) = (14.7 ppm; 6.8 ppm).

EtO-Thiazole-Gly-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-

5). EtO-Thiazole-Gly-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc was

synthesized following the General Peptide Synthesis procedure,

utilizing 184 mg (0.32 mmol, 1.0 equiv) of acid HO-Pro-Oxazole-

Thiazole-Pro-Leu-NHBoc (28a-1), 101 mg (0.35 mmol, 1.1 equiv) of

amine EtO-Thiazole-Gly-Val-NH₂(17c-1), 83.5 mg (0.26 mmol, 0.8

equiv) of TBTU, 98.9 mg (0.26 mmol, 0.8 equiv) of HATU, and 0.45

mL (2.56 mmol, 8.0 equiv) of DIPEA dissolved in 3.20 mL of

methylene chloride. The crude product underwent a ﬁnal puriﬁcation

via column chromatography on silica gel (ethyl acetate/methanol

99:1) to aﬀord the desired peptide (224 mg, 84% yield). R_f: 0.30 (ethyl

1

•Compound 5-B. H NMR (308 K, 600 MHz, CDCl₃): δ 0.84−

1

acetate/methanol 99:1). H NMR (300 MHz, CDCl₃): δ 0.68−0.80

0.90 (m, 6H, CHCH(CH₃)₂); 0.92−1.04 (m, 6H, CHCH₂CH-

(CH₃)₂); 1.53−1.63 (m, 1H, CHCH₂CH(CH₃)₂); 1.64−1.66 (m, 1H,

CHCH(CH₃)₂); 1.64−1.69 and 1.97−2.04 (m, 2H, CHCH₂CH-

(CH₃)₂); 1.90−2.10 (m, 2H, CHCH₂CH₂CH₂); 2.07−2.12 and 2.47−

2.52 (m, 2H, CHCH₂CH₂CH₂); 2.06−2.10 and 2.32−2.38 (m, 2H,

CHCH₂CH₂CH₂); 2.13−2.22 and 2.38−2.48 (m, 2H,

CHCH₂CH₂CH₂); 3.65−3.69 (m, 2H, 2αH); 3.72−3.99 (m, 4H,

CHCH₂CH₂CH₂); 4.97−5.04 (m, 1H, 1αH); 5.08−5.18 (m, 1H,

1αH); 5.39−5.55 (m, 1H, 1αH); 5.40−5.45 and 5.56−5.60 (m, 1H,

1αH); 6.81−6.86 (m, 1H, NH); 7.12−7.17 (m, 1H, NH); 7.66 and

7.79 (s, 1H, SCH); 7.69 (s, 1H, SCH); 8.00 (s, 1H, OCH). ¹³C NMR

(308 K, 150 MHz, CDCl₃): δ 19.0, 20.6 (CH₂γ Pro2), 22.1, 22.6, 22.7,

23.3, 24.0 (CH₂γ Pro1), 24.4, 31.0, 31.7 (CH₂γ Pro1), 34.9 (CH₂β

Pro2), 41.4, 47.3, 48.7, 49.4, 57.6, 59.3, 59.4, 70.2, 119.7, 120.2, 135.9,

140.7, 142.2, 143.5, 152.1, 155.6, 170.3, 171.9, 172.1, 175.5, 175.6,

176.3. Δβγ (Pro1, Pro2) = (14.3 ppm; 7.7 ppm).

(d, J = 20.2, 6.2 Hz, 6H, CHCH(CH₃)₂); 0.81−0.96 (ddd, J = 29.1,

12.8, 6.2 Hz, 6H, CHCH₂CH(CH₃)₂); 1.26−1.34 (t, 3H, OCH₂CH₃);

1.36 (s, 9H, C(CH₃)₃); 1.34−1.53 (m, 2H, CHCH₂CH(CH₃)₂);

1.63−1.79 (m, 1H, CHCH(CH₃)₂); 1.86−1.97 (m, buried, 2H,

CHCH₂CH₂CH₂); 1.99−2.13 (m, 2H, CHCH₂CH₂CH₂); 1.93−2.03

and 2.07−2.15 (m, 2H, CHCH₂CH₂CH₂); 2.08−2.24 (m, 1H,

CH(CH₃)₂); 2.16−2.43 (m, 2H, CHCH₂CH₂CH₂); 3.58−3.86 and

3.99−4.14 (m, 4H, CHCH₂CH₂CH₂); 4.26−4.36 (q, 2H, OCH₂CH₃);

4.33−4.47 (m, 1H, 1αH); 4.41−4.53 (m, 1H, 1αH); 4.58−4.77 (m,

2H, 2αH); 4.63−4.73 and 5.10−5.19 (m, 1H, 1αH); 5.39−5.53 (m,

1H, 1αH); 6.94−7.02 (d, J = 8.6 Hz, 1H, NH); 7.35−7.43 (d, J = 8.6

Hz, 1H, NH); 7.87 and 7.96 (s, 1H, SCH); 7.96 and 8.01 (s, 1H,

SCH); 8.03−8.10 (m, 1H, NH); 8.18−8.24 (m, 1H, OCH). ¹³C NMR

(75 MHz, CDCl₃): δ 14.3, 17.8, 18.2, 19.4, 21.0, 21.7, 23.4, 24.5, 24.6,

25.5, 27.9, 28.3, 30.4, 31.7, 41.1, 42.0, 47.1, 49.4, 50.4, 58.5, 58.8, 60.3,

61.4, 62.1, 79.5, 120.9, 128.2, 137.6, 142.6, 143.4, 146.5, 155.8,

156.6,161.2, 169.4, 170.7, 171.7, 172.4, 172.9, 174.4. LC/MS (ESI):

m/z calcd for C₃₉H₅₄N₈O₉S₂(M + H⁺) = 843.35, found 843.00.

HRMS (ESI-TOF): M + Na⁺, found 865.3433 C₃₉H₅₄N₈O₉S₂requires

865.3353

HO-Thiazole-Gly-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-

5a). HO-Thiazole-Gly-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc

was synthesized following the General Acid Deprotection procedure,

utilizing 224 mg (0.266 mmol, 1.0 equiv) of EtO-Thiazole-Gly-Val-

Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-5) and 33.6 mg (0.80

mmol, 3 equiv) of LiOH·H₂O in 2.70 mL of ethanol. The acid was

taken on to the next reaction without further puriﬁcation (89.2 mg,

41% yield). LC/MS (ESI): m/z calcd for C₃₇H₅₀N₈O₉S₂(M + H⁺) =

815.27, found 815.00.

1

•Compound 5-C. H NMR (308 K, 600 MHz, CDCl₃): δ 0.82−

0.90 (m, 6H, CHCH(CH₃)₂); 0.94−1.13 (m, 6H, CHCH₂CH-

(CH₃)₂); 1.62−1.81 (m, 2H, CHCH₂CH(CH₃)₂); 1.63−1.66 (m, 1H,

CHCH(CH₃)₂); 1.70−1.87 (m, 1H, CHCH₂CH(CH₃)₂); 2.02−2.06

(m, 2H, CHCH₂CH₂CH₂); 2.11−2.26 (m, 2H, CHCH₂CH₂CH₂);

2.32−2.44 and 2.51−2.59 (m, 2H, CHCH₂CH₂CH₂); 2.32−2.38 and

2.40−2.45 (m, 2H, CHCH₂CH₂CH₂); 3.74−4.00 (m, 4H,

CHCH₂CH₂CH₂); 3.99−4.03 (m, 2H, 2αH); 4.87−4.93 (m, 1H,

1αH); 5.05−5.17 (m, 1H, 1αH); 5.46−5.54 (m, 1H, 1αH); 5.52−5.64

(m, 1H, 1αH); 6.81−6.86 (m, 1H, NH); 7.12−7.17 (m, 1H, NH);

7.94 (s, 1H, SCH); 7.96 (s, 1H, SCH); 8.23 and 8.24 (s, 1H, OCH).

¹³C NMR (308 K, 150 MHz, CDCl₃): δ 19.7, 20.5, 21.2, 21.8, 22.1,

23.2, 24.5 (CH₂γ Pro2), 24.7, 27.1 (CH₂γ Pro1), 31.0, 31.6 (CH₂γ

Pro2), 32.3 (CH₂β Pro2), 42.0, 47.3, 48.7, 50.1, 58.6, 58.8, 59.3, 59.5,

66.8, 120.5, 121.1, 136.9, 140.8, 142.5, 142.6, 143.6, 156.9, 165.2,

171.9, 172.1, 174.1, 174.9, 175.0. Δβγ (Pro1, Pro2) = (5.2 ppm; 7.1

ppm).

HO-Thiazole-Gly-Val-Pro-Oxazole-Thiazole-Pro-Leu-NH₂(29-5b).

HO-Thiazole-Gly-Val-Pro-Oxazole-Thiazole-Pro-Leu-NH₂was syn-

thesized following the General Amine Deprotection procedure,

utilizing 89.2 mg (0.109 mmol, 1.0 equiv) of HO-Thiazole-Gly-Val-

10609

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

Compound 6. MeO-Oxazole-Thiazole-Pro-Gly-NHBoc (27c).

MeO-Oxazole-Thiazole-Pro-Gly-NHBoc was synthesized following

the General Peptide Synthesis procedure, utilizing 110 mg (0.393

mmol, 1.0 equiv) of amine OMe-Oxazole-Thiazole-Pro-NH (25-1),

103 mg (0.59 mmol, 1.5 equiv) of acid HO-Gly-NHBoc (26c), 126

mg (0.393 mmol, 1.0 equiv) of TBTU, 149 mg (0.393 mmol, 1.0

equiv) of HATU, and 0.40 mL (1.57 mmol, 4 equiv) of DIPEA

dissolved in 4 mL of methylene chloride under N₂. The reaction

mixture was stirred for 3 h and was monitored by TLC upon

completion. The crude product underwent a ﬁnal puriﬁcation via

column chromatography on silica gel (hexanes/ethyl acetate 1:1 to

1:3) to aﬀord the desired peptide (151 mg, 88% yield) as yellow ﬂakes.

and LCMS upon completion. The crude product underwent a ﬁnal

puriﬁcation via column chromatography on silica gel (ethyl acetate/

methanol 19:1) to aﬀord the desired peptide (170 mg, 74% yield) as

1

yellow ﬂakes. R_f: 0.325 (ethyl acetate/methanol 9:1). H NMR (300

MHz, CD₃OD): δ 0.94−1.74 (m, 6H, CH₃CHCH₃); 1.46 (s, 9H,

C(CH₃)₃); 1.48−1.57 (t, 2H,CH₂); 1.61−1.68 (dd, 3H,CHCH₃);

1.45−1.7 (m, 2H, CHCH₂CH₂CH₂); 1.92−2.02 (m, 2H, CHCH₂

CH₂CH₂); 2.03−2.11 (m, 2H, CHCH₂CH₂CH₂); 2.21−2.33 (m, 2H,

CHCH₂CH₂CH₂); 2.33−2.49 (m, 2H, CHCH₂CH₂CH₂); 3.81−3.93

(m, 2H, CHCH₂CH₂CH₂); 4.04−4.16 (m, 2H, CHCH₂CH₂CH₂);

4.16−4.24 (t, 3H, OCH₂CH₃); 4.37−4.47 (m, 2H, OCH₂CH₃); 4.70−

4.79 (m, 1H, 1αH); 5.28−5.42 (dd, 1H, 1αH); 5.54−5.69 (m, 1H,

1αH); 8.24 (s, 1H, SCH); 8.33 (s, 1H, SCH); 8.44 (s, 1H, OCH). ¹³C

NMR (75 MHz, CD₃OD): 13.26, 18.57, 18.72, 19.68, 24.90, 27.38,

30.89, 31.79, 32.06, 42.53, 48.59, 49.17, 58.58, 58.81, 59.09, 61.26,

79.26, 121.75, 127.80, 128.33, 137.50, 142.71, 143.31, 146.20, 150.32,

157.01, 160.94, 161.48, 169.67, 171.81, 172.06, 172.31. LC/MS (ESI):

m/z calcd for C₃₆H₄₈N₈O₉S₂(M + H⁺) = 800.94, found 801.00.

HRMS (ESI-TOF): M + Na⁺, found 823.2874 C₃₆H₄₈N₈O₉S₂requires

823.2884.

HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-Gly-NHBoc (29-

6a). HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-Gly-NHBoc was

synthesized following the General Acid Deprotection procedure,

utilizing 170 mg (0.212 mmol, 1.0 equiv) of EtO-Thiazole-Ala-Val-

Pro-Oxazole-Thiazole-Pro-Gly-NHBoc (29-6) and 71 mg (1.69 mmol,

8 equiv) of LiOH·H₂O in 2.20 mL of ethanol. Upon completion, the

reaction was diluted with methylene chloride. The acid was taken on

to the next reaction without further puriﬁcation (160 mg, 97.5% yield).

LC/MS (ESI): m/z calcd for C₃₄H₄₄N₈O₉S₂(M + H⁺) = 772.89,

found 773.00.

1

R_f: 0.65 (hexanes/ethyl acetate 1:3). H NMR (300 MHz, CDCl₃): δ

1.29 (s, 9H, C(CH₃)₃); 1.97−2.13 (m, 2H, CHCH₂CH₂CH₂); 2.16−

2.29 (m, 2H, CHCH₂CH₂CH₂); 3.41−3.56 (m, 2H, CHCH₂

CH₂CH₂); 3.57−3.72 (t, 2H, CH₂); 3.80 (s, 3H, OCH₃); 5.28−5.49

(m, 1H, 1αH); 7.95 (s, 1H, SCH), 8.22 (s, 1H, OCH). ¹³C NMR (75

MHz, CDCl₃): 24.20, 28.01, 29.54, 31.72, 34.77, 43.12, 46.17, 46.81,

52.32, 58.78, 58.92, 79.67, 121.82, 122.03, 133.71, 141.69, 142.65,

143.87, 143.97, 155.98, 157.44, 157.75, 161.62, 168.77, 174.42. HRMS

(ESI-TOF): M + H⁺, found 437.1482 C₁₉H₂₄N₄O₆S requires

437.1450.

HO-Oxazole-Thiazole-Pro-Gly-NHBoc (27c-1). HO-Oxazole-Thia-

zole-Pro-Gly-NHBoc was synthesized following the General Acid

Deprotection procedure, utilizing 151 mg (0.345 mmol, 1.0 equiv) of

MeO-Oxazole-Thiazole-Pro-Gly-NHBoc (27c) and 116 mg (2.76

mmol, 8.0 equiv) of LiOH·H₂O in 3.5 mL of methanol. Upon

completion, the reaction was diluted with methylene chloride. The

acid was taken on to the next reaction without further puriﬁcation

(137 mg, 94% yield) as yellow ﬂakes.

HO-Thiazole-Ala-NMe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NH₂

(29-6b). HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-Gly-NH₂

was synthesized following the General Amine Deprotection procedure,

utilizing 160 mg (0.207 mmol, 1.0 equiv) of HO-Thiazole-Ala-Val-Pro-

Oxazole-Thiazole-Pro-Gly-NHBoc (29-6a), 0.05 mL (0.42 mmol, 2.0

equiv) of anisole, and 0.50 mL of triﬂuoroacetic acid in 2.50 mL of

methylene chloride. The reaction was completed in 1 h; the reaction

was concentrated in vacuo. HO-Thiazole-Ala-Val-Pro-Oxazole-Thia-

zole-Pro-Gly-NH₂was taken on to the next reaction without further

puriﬁcation or characterization (196 mg, quantitative yield). LC/MS

(ESI): m/z calcd for C₃₀H₃₈N₈O₆S₂(M + H⁺) = 670.80, found 671.00.

•Compound 6. Compound 6 was synthesized following the

Macrocyclization Procedure, utilizing 140 mg (0.208 mmol, 1.0

equiv) of HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-Gly-NH₂

(29-6b), 67.0 mg (0.208 mmol, 1.0 equiv) of TBTU, 79.0 mg

(0.208 mmol, 1.0 equiv) of HATU, 57.4 mg (0.208 mmol, 1.0 equiv)

of DMTMM, and 0.29 mL (1.66 mmol, 8 equiv) of DIPEA in 297 mL

of methylene chloride under N₂. The reaction was monitored by

LCMS upon completion. The crude product underwent an initial

puriﬁcation via column chromatography on silica gel (ethyl acetate/

methanol 9:1), and the resulting semipure residue was subjected to

reversed-phase HPLC puriﬁcation to aﬀord a 5.5:1 of compound 6-A

(18.4 mg) and compound 6-B (6.3 mg) in a 15% overall yield. LC/MS

(ESI): m/z calcd for C₂₉H₃₄N₈O₆S₂(M + H⁺) = 654.76, found 655.00

HRMS (ESI-TOF): M + H⁺found 655.2110 C₂₉H₃₄N₈O₆S₂requires

655.2121.

MeO-Pro-Oxazole-Thiazole-Pro-Gly-NHBoc (28c). MeO-Pro-Oxa-

zole-Thiazole-Pro-Gly-NHBoc was synthesized following the General

Peptide Synthesis procedure, utilizing 137 mg (0.324 mmol, 1.0 equiv)

of acid HO-Oxazole-Thiazole-Pro-Gly-NHBoc (27c-1), 63 mg (0.486

mmol, 1.5 equiv) of the amine MeO-Pro-NH, 123 mg (0.324 mmol,

1.0 equiv) HATU, and 0.45 mL (2.59 mmol, 4.0 equiv) of DIPEA

dissolved in 4 mL of methylene chloride under N₂. The reaction

mixture was stirred for 3 h and was monitored by TLC upon

completion. The crude product underwent a ﬁnal puriﬁcation via

column chromatography on silica gel (hexanes/ethyl acetate 1:3) to

aﬀord the desired peptide (157 mg, 91% yield) as yellow ﬂakes. R_f:

0.45 (hexanes/ethyl acetate 0:1). ¹H NMR (300 MHz, CDCl₃): δ 1.30

(s, 9H, C(CH₃)₃); 1.83−1.98 (m, 2H, CHCH₂CH₂CH₂); 2.01−2.12

(m, 2H, CHCH₂CH₂CH₂); 2.12−2.23 (m, 2H, CHCH₂CH₂CH₂);

2.23−2.34 (m, 2H, CHCH₂CH₂CH₂); 3.06−3.19 (m, 2H, CHCH₂

CH₂CH₂); 3.44−3.58 (m, 2H, CH₂); 3.64 (s, 3H, OCH₃); 3.88−3.95

(m, 2H, CHCH₂CH₂CH₂); 4.48−4,58 (m, 1H, 1αH); 5.18−5.30 (m,

1H, 1αH); 7.84 (s, 1H, SCH), 8.18 (s, 1H, OCH). ¹³C NMR (75

MHz, CDCl₃): 24.13, 25.09, 28.23, 29.54, 31.79, 43.34, 52.20, 52.31,

60.10, 60.79, 79.60, 120.90, 136.69, 142.93, 155.78, 156.21, 156.34,

156.64, 168.40, 168.50, 172.25, 173.31. HRMS (ESI-TOF): M + H⁺,

found 534.2015 C₂₄H₃₁N₅O₇S requires 534.1978.

HO-Pro-Oxazole-Thiazole-Pro-Gly-NHBoc (28c-1), Fragment B.

HO-Pro-Oxazole-Thiazole-Pro-Gly-NHBoc was synthesized following

General Acid Deprotection procedure, utilizing 157 mg (0.295 mmol,

1.0 equiv) of MeO-Pro-Oxazole-Thiazole-Pro-Gly-NHBoc (28c) and

151 mg (2.36 mmol, 8.0 equiv) of LiOH·H₂O in 2.95 mL of methanol.

Upon completion, the reaction was diluted with methylene chloride.

The acid was taken on to the next reaction without further puriﬁcation

(150 mg, 98% yield) as yellow ﬂakes.

EtO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-Gly-NHBoc (29-

6). EtO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-Gly-NHBoc was

synthesized following the General Peptide Synthesis procedure,

utilizing 150 mg (0.288 mmol, 1.0 equiv) of acid HO-Pro-Oxazole-

Thiazole-Pro-Gly-NHBoc (28c-1), 130 mg (0.433 mmol, 1.5 equiv) of

amine EtO-Thiazole-Ala-Val-NH₂(17a-1), 90 mg (0.288 mmol, 1.0

equiv) of TBTU, 109 mg (0.288 mmol, 1.0 equiv) of HATU, 79 mg

(0.288 mmol, 1.0 equiv) of DMTMM, and 0.4 mL (2.31 mmol, 8.0

equiv) of DIPEA dissolved in 5.8 mL of methylene chloride under N₂.

The reaction mixture was stirred for 3 h and was monitored by TLC

1

•Compound 6-A. H NMR (318 K, 600 MHz, CDCl₃): δ 0.86−

1.01 (m, 6H, CH(CH₃)₂); 1.7−1.78 (d, 3H, CHCH₃); 1.4−1.47 (m,

2H, CHCH); 1.97−2.07 (m, 2H, CH₂γ Pro1); 2.02−2.13 (m, 2H,

CH₂γ Pro2); 1.98−2.35 (m, 2H, CH₂β Pro2); 2.07−2.3 (m, 2H, CH₂β

Pro1); 3.83−3.92 (m, 2H, CH₂δ Pro2); 3.84−3.87 (m, 2H, CH₂δ

Pro1); 4.17−4.24 (m, 2H, CH₂); 4.72−4.78 (m, 1H, 1αH); 4.78−4.99

(m, 1H, 1αH); 5.37−5.56 (m, 1H, CHCH₃); 5.48−5.5 (m, 1H, 1αH);

7.52 (s, 1H, SCH); 7.94 (s, 1H, SCH); 8.38 (s, 1H, OCH). ¹³C NMR

(318 K, 150 MHz, CDCl₃): δ 17.61, 19.58, 22.86CH₂γ Pro2), 23.34,

26.41 (CH₂γ Pro1), 31.82 (CH₂β Pro1), 35.85 (CH₂β Pro2), 42.07,

47.8, 49.01, 59.39, 60.27, 63.86, 64.1, 120.17, 121.89, 122.67, 125.49,

127.6, 142.94, 143.3, 149.4. Δβγ = (5.41 ppm; 12.99 ppm).

1

•Compound 6-B. H NMR (318 K, 600 MHz, CD₃OD): δ 0.95−

1.04 (m, 6H, CH(CH₃)₂); 1.44−1.47 (d, 3H, CHCH₃); 1.35−1.39

10610

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

(m, 2H, CHCH); 1.87−2.0 (m, 2H, CH₂γ Pro1); 1.9−2.24 (m, 2H,

CH₂γ Pro2); 1.89−2.15 (m, 2H, CH₂β Pro1); 2.32−2.54 (m, 2H,

CH₂β Pro2); 3.7−3.79 (m, 2H, CH₂δ Pro2); 3.72−3.88 (m, 2H, CH₂δ

Pro1); 4.85−4.87 (m, 1H, 1αH); 5.07−5.37 (m, 2H, CH₂); 5.43−5.5

(m, 1H, CHCH₃); 5.48−5.50 (m, 1H, 1αH); 5.93−6.00 (m, 1H,

1αH); 8.1 (s, 1H, SCH); 8.44 (s, 1H, SCH); 8.74 (s, 1H, OCH). ¹³C

NMR (318 K, 150 MHz, CD₃OD): δ 17.53, 18.33, 19.22, 24.55 (CH₂γ

Pro2), 25.82 (CH₂γ Pro1), 31.51 (CH₂β Pro1), 32.11 (CH₂β Pro2),

42.3, 46.42, 46.81, 47.63, 59.22, 60.24, 60.56, 63.62, 120.87, 122.89,

129.05, 150.85. Δβγ = (5.69 ppm; 7.56 ppm).

0.80 (dd, J = 23.0, 6.9 Hz, 3H, CHCH(CH₃)₂); 0.65−0.71 and 0.77−

0.85 (dd, J = 38.3, 6.9 Hz, 3H, CHCH(CH₃)₂); 1.35 (s, 9H,

C(CH₃)₃); 1.35 (t, 3H, OCH₂CH₃); 1.97−2.14 (m, 1H,

CHCH(CH₃)₂); 3.11−3.49 (m, 2H, CHCH₂Ph); 3.82−4.00 (m,

1H, 1αH); 4.30−4.42 (q, 2H, OCH₂CH₃); 5.22−5.38 (m, 1H, NH);

5.51−5.64 (m, 1H, 1αH); 7.00−7.24 (m, 5H, Ph); 7.28−7.41 (m, 1H,

NH); 8.00 (s, 1H, SCH). ¹³C NMR (75 MHz, CDCl₃): δ 14.6, 17.8,

19.6, 28.7, 30.7, 38.9, 41.2, 52.6, 60.5, 62.0, 80.1, 127.2, 127.8, 128.9,

129.5, 136.5, 147.4, 156.1, 161.7, 171.9. HRMS (ESI-TOF): M + H⁺,

found 476.2207 C₂₄H₃₃N₃O₅S requires 476.2219.

EtO-Thiazole-Phe-Val-NH₂(17e-1), Fragment A. EtO-Thiazole-

Phe-Val-NH₂was synthesized following the General Amine Depro-

tection procedure. The amine was taken on to the next reaction

without further puriﬁcation or characterization (150 mg, quantitative

yield) as a clear oil.

Compound 7. Compound 12d is commercially available (CAS no.

51987-73-6).

NH₂-Phe-NHBoc (13d). NH₂-Phe-NHBoc was synthesized follow-

ing the General Amide Formation procedure, utilizing 285 mg (1.02

mmol, 1.0 equiv) of MeO-Phe-NHBoc (12d) dissolved in 5.0 mL of

ammonium hydroxide (25% in water) and 5.0 mL of methanol. The

resulting amide was taken on to the next reaction without further

puriﬁcation (270 mg, quantitative yield) as white crystals. R_f: 0.15

EtO-Thiazole-Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-

7). EtO-Thiazole-Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc was

synthesized following the General Peptide Synthesis procedure,

utilizing 212 mg (0.36 mmol, 1.0 equiv) of acid HO-Pro-Oxazole-

Thiazole-Pro-Leu-NHBoc (28a-1), 150 mg (0.40 mmol, 1.1 equiv) of

amine EtO-Thiazole-Phe-Val-NH₂(17e-1), 93.1 mg (0.29 mmol, 0.8

equiv) of TBTU, 110 mg (0.29 mmol, 0.8 equiv) of HATU, and 0.38

mL (2.16 mmol, 6.0 equiv) of DIPEA dissolved in 3.60 mL of

methylene chloride. The crude product underwent a ﬁnal puriﬁcation

via column chromatography on silica gel (ethyl acetate/methanol

99:1) to aﬀord the desired peptide (253 mg, 74% yield). R_f: 0.30 (ethyl

1

(hexanes/ethyl acetate 1:1). H NMR (300 MHz, CDCl₃): δ 1.43 (s,

9H, C(CH₃)₃); 2.88−3.25 (m, 2H, CHCH₂Ph); 4.29−4.52 (m, 1αH);

5.09−5.32 (m, 1H, NH); 5.72−5.97 (br, 1H, NH₂); 5.97−6.17 (br,

1H, NH₂); 7.13−7.46 (m, 5H, Ph). ¹³C NMR (75 MHz, CDCl₃): δ

28.7, 38.6, 55.8, 80.7, 127.2, 128.9, 129.5, 136.8, 155.8, 174.3.

Thioamide-Phe-NHBoc (14d). Thioamide-Phe-NHBoc was synthe-

sized following the General Thioamide Formation procedure, utilizing

270 mg (1.02 mmol, 1.0 equiv) of NH₂-Phe-NHBoc (13d) and 311

mg (0.77 mmol, 0.75 equiv) of Lawesson’s Reagent dissolved in 10.2

mL of 1,2-dimethoxyethane. The thioamide was puriﬁed via column

chromatography on silica gel (hexanes/ethyl acetate 19:1 to 9:1) to

aﬀord the desired thioamide (171 mg, 60% yield) as white crystals. R_f:

1

acetate/methanol 99:1). H NMR (300 MHz, CDCl₃): δ 0.57−0.65

(d, J = 6.91 Hz, 3H, CHCH(CH₃)₂); 0.73−0.84 (d, J = 6.91 Hz, 3H,

CHCH(CH₃)₂); 0.94−1.01 (d, J = 6.91 Hz, 3H, CHCH₂CH(CH₃)₂);

0.99−1.06 (d, J = 6.91 Hz, 3H, CHCH₂CH(CH₃)₂); 1.36−1.45 (t, 3H,

OCH₂CH₃); 1.45 (s, 9H, C(CH₃)₃); 1.44−1.58 (m, 2H, CHCH₂CH-

(CH₃)₂); 1.73−1.87 (m, 1H, CHCH₂CH(CH₃)₂); 1.95−2.09 (m, 2H,

CHCH₂CH₂CH₂); 2.00−2.20 (m, 2H, CHCH₂CH₂CH₂); 2.03−2.14

and 2.22−2.32 (m, 2H, CHCH₂CH₂CH₂); 2.17−2.28 (m, 1H,

CHCH(CH₃)₂); 2.12−2.17 and 2.25−2.53 (m, 2H,

CHCH₂CH₂CH₂); 3.18−3.37 and 3.41−3.59 (m, 2H, CHCH₂Ph);

3.64−3.93 (m, 2H, CHCH₂CH₂CH₂); 4.06−4.29 (m, 2H,

CHCH₂CH₂CH₂); 4.21−4.33 (m, 1H, 1αH); 4.36−4.49 (q, 2H,

OCH₂CH₃); 4.48−4.62 (m, 1H, 1αH); 4.69−4.79 and 5.21−5.27 (m,

1H, 1αH); 5.43−5.72 (m, 1H, 1αH); 5.48−5.62 (m, 1H, 1αH); 6.97−

7.30 (m, 5H, Ph); 7.35−7.44 (m, 1H, NH); 7.95 (s, 1H, SCH); 7.96−

7.98 (m, 1H, NH); 8.00 and 8.06 (s, 1H, SCH); 8.24 and 8.28 (s, 1H,

OCH); 8.29−8.31 (m, 1H, NH). ¹³C NMR (75 MHz,CDCl₃): δ 14.6,

17.2, 19.6, 21.9, 23.7, 24.9, 25.7, 28.7, 29.3, 29.7, 31.8, 40.7, 42.3, 47.1,

49.6, 50.4, 52.5, 58.9, 61.5, 79.8, 121.1, 126.9, 127.6, 128.6, 129.3,

136.7, 137.6, 143.5, 147.2, 155.8, 156.9,161.5, 162.1, 171.0, 171.3,

172.8, 174.7. LC/MS (ESI): m/z calcd for C₄₆H₆₀N₈O₉S₂(M + Na⁺)

= 955.38, found 955.00. HRMS (ESI-TOF): M + Na⁺, found 955.3806

C₄₆H₆₀N₈O₉S₂requires 955.3823.

1

0.6 (hexanes/ethyl acetate 1:1). H NMR (300 MHz, CDCl₃): δ 1.39

(s, 9H, C(CH₃)₃); 2.98−3.25 (m, 2H, CHCH₂Ph); 4.66−4.82 (m,

1αH); 5.46−5.62 (m, 1H, NH); 7.17−7.37 (m, 5H, Ph); 7.67−7.87

(br, 1H, NH₂); 7.87−8.09 (br, 1H, NH₂). ¹³C NMR (75 MHz,

CDCl₃): δ 28.7, 42.4, 61.4, 81.0, 127.2, 128.9, 129.5, 136.8, 155.8,

208.7.

EtO-Thiazole-Phe-NHBoc (15d). EtO-Thiazole-Phe-NHBoc was

synthesized following the General Thiazole Synthesis procedure,

utilizing 171 mg (0.61 mmol, 1.0 equiv) of Thioamide-Phe-NHBoc

(14d) and 489 mg (4.88 mmol, 8.0 equiv) of potassium bicarbonate

dissolved in 6.1 mL of 1,2-dimethoxyethane. A 0.23 mL amount of

ethyl bromopyruvate (1.83 mmol, 3.0 equiv) was added dropwise (0.1

mL/min) to yield the thiazoline intermediate. The thiazoline was

dehydrated using 0.44 mL (5.49 mmol, 9.0 equiv) of pyridine, 0.34 mL

(2.44 mmol, 4.0 equiv) of triﬂuoroacetic anhydride, and 0.17 mL (1.22

mmol, 2.0 equiv) of triethylamine in 6.1 mL of 1,2-dimethoxyethane to

aﬀord the desired thiazole (210 mg, 92% yield) as a clear oil. R_f: 0.55

1

(hexanes/ethyl acetate 1:1). H NMR (300 MHz, CDCl₃): δ 1.31 (s,

9H, C(CH₃)₃); 1.33 (t, 3H, OCH₂CH₃); 3.02−3.41 (m, 2H,

CHCH₂Ph); 4.25−4.42 (q, 2H, OCH₂CH₃); 5.09−5.31 (m, 1H,

1αH); 5.44−5.64 (m, 1H, NH); 6.99−7.24 (m, 5H, Ph); 7.99 (s, 1H,

SCH). ¹³C NMR (75 MHz, CDCl₃): δ 14.6, 28.4, 41.5, 54.1, 61.7,

80.4, 126.9, 127.2, 128.9, 129.5, 136.5, 147.4, 155.2, 161.7, 173.7.

HRMS (ESI-TOF): M + H⁺, found 377.1526 C₁₉H₂₄N₂O₄S requires

377.1535.

EtO-Thiazole-Phe-NH₂(15d-1). EtO-Thiazole-Phe-NH₂was syn-

thesized following the General Amine Deprotection procedure. The

amine was taken on to the next reaction without further puriﬁcation or

characterization (154 mg, quantitative yield) as a clear oil.

HO-Thiazole-Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-

7a). HO-Thiazole-Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc

was synthesized following the General Acid Deprotection procedure,

utilizing 253 mg (0.267 mmol, 1.0 equiv) of EtO-Thiazole-Phe-Val-

Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-7), and 89.6 mg (2.14

mmol, 8 equiv) of LiOH·H₂O in 2.67 mL ethanol. The acid was taken

on to the next reaction without further puriﬁcation (208 mg, 86%

yield). LC/MS (ESI): m/z calcd for C₃₉H₅₄N₈O₉S₂(M + H⁺) =

905.36, found 905.00.

HO-Thiazole-Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NH₂(29-

7b). HO-Thiazole-Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NH₂was

synthesized following the General Amine Deprotection procedure,

utilizing 208 mg (0.23 mmol, 1.0 equiv) of HO-Thiazole-Phe-Val-Pro-

Oxazole-Thiazole-Pro-Leu-NHBoc (29-7a), 50.0 μL (0.46 mmol, 2.0

equiv) of anisole, and 0.46 mL of triﬂuoroacetic acid in 1.84 mL of

methylene chloride. HO-Thiazole-Phe-Val-Pro-Oxazole-Thiazole-Pro-

Leu-NH₂was taken on to the next reaction without further

puriﬁcation or characterization (185 mg, quantitative yield). LC/MS

(ESI): m/z calcd for C₃₉H₄₈N₈O₇S₂(M + H⁺) = 805.31, found 805.00.

•Compound 7. Compound 7 was synthesized following the

Macrocyclization Procedure, utilizing 185 mg (0.230 mmol, 1.0

EtO-Thiazole-Phe-Val-NHBoc (17e). EtO-Thiazole-Phe-Val-

NHBoc was synthesized following the General Peptide Synthesis

procedure, utilizing 154 mg (0.56 mmol, 1.0 equiv) of amine EtO-

Thiazole-Phe-NH₂(15d-1), 133 mg (0.61 mmol, 1.1 equiv) of acid

HO-Val-NHBoc (16a), 144 mg (0.45 mmol, 0.8 equiv) of TBTU, 171

mg (0.45 mmol, 0.8 equiv) of HATU, and 0.59 mL (3.36 mmol, 6

equiv) of DIPEA dissolved in 5.6 mL of methylene chloride under N₂.

The crude product underwent a ﬁnal puriﬁcation via column

chromatography on silica gel (hexanes/ethyl acetate 1:0 to 7:3) to

aﬀord the desired peptide (190 mg, 72% yield) as a clear oil. R_f: 0.50

1

(hexanes/ethyl acetate 1:1). H NMR (300 MHz, CDCl₃): δ 0.65−

10611

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

equiv) of HO-Thiazole-Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NH₂

(29-7b), 59.0 mg (0.184 mmol, 0.8 equiv) of TBTU, 70.0 mg

(0.184 mmol, 0.8 equiv) of HATU, 51.0 mg (0.184 mmol, 0.8 equiv)

of DMTMM, and 240 μL (1.38 mmol, 6 equiv) of DIPEA in 230 mL

of methylene chloride. The crude product was directly subjected to

reversed-phase HPLC puriﬁcation to aﬀord compound 7 (12.9 mg) in

a 14.2% overall yield. LC/MS (ESI): m/z calcd for C₃₉H₄₆N₈O₆S₂(M

+ H⁺) = 787.31, found 787.00. HRMS (ESI-TOF): M + Na⁺, found

809.2864 C₃₉H₄₆N₈O₆S₂requires 809.2880.

9H, C(CH₃)₃); 1.40 (t, 3H, OCH₂CH₃); 3.13−3.41 (m, 2H,

CHCH₂Ph); 4.31−4.50 (q, 2H, OCH₂CH₃); 5.19−5.36 (m, 1H,

1αH); 5.36−5.54 (m, 1H, NH); 7.03−7.32 (m, 5H, Ph); 8.05 (s, 1H,

SCH). ¹³C NMR (75 MHz, CDCl₃): δ 14.6, 28.4, 41.8, 54.1, 61.7,

80.4, 126.9, 127.2, 128.9, 129.5, 136.5, 147.4, 155.2, 161.7, 173.7.

HRMS (ESI-TOF): M + H⁺, found 377.1525 C₁₉H₂₄N₂O₄S requires

377.1535.

EtO-Thiazole-^D-Phe-NH₂(15e-1). EtO-Thiazole-D-Phe-NH₂was

synthesized following the General Amine Deprotection procedure.

The amine was taken on to the next reaction without further

puriﬁcation or characterization (124 mg, quantitative yield) as a clear

oil.

•Compound 7. ¹H NMR (308 K, 600 MHz, CDCl₃): δ 0.28−0.34

(d, J = 5.9 Hz, 3H, CHCH(CH₃)₂); 0.83−0.91 (d, J = 5.9 Hz, 3H,

CHCH(CH₃)₂); 0.91−0.97 (d, J = 5.9 Hz, 3H, CHCH₂CH(CH₃)₂);

0.97−1.04 (d, J = 5.9 Hz, 3H, CHCH₂CH(CH₃)₂); 1.54−1.62 (m, 1H,

CH CH₂CH(CH₃)₂); 1.57−1.66 and 1.95−2.03 (m, 2H, CHCH₂CH-

(CH₃)₂); 1.77−1.85 and 1.94−2.02 (m, 2H, CHCH₂CH₂CH₂); 1.91−

2.00 and 2.07−2.15 (m, 2H, CHCH₂CH₂CH₂); 1.92−1.99 and 2.32−

2.41 (m, 2H, CHCH₂CH₂CH₂); 2.19−2.32 (m, 2H,

CHCH₂CH₂CH₂); 2.19−2.27 and 3.25−3.32 (m, 2H, CHCH₂Ph);

2.64−2.73 (m, 1H, CH(CH₃)₂); 3.66−3.74 and 3.93−4.01 (m, 2H,

CHCH₂CH₂CH₂); 3.80−3.88 and 3.90−3.97 (m, 2H,

CHCH₂CH₂CH₂); 4.63−4.70 (m, 1H, 1αH); 4.69−4.76 (m, 1H,

1αH); 5.09−5.17 (m, 1H, 1αH); 5.34−5.43 (m, 1H, 1αH); 5.47−5.53

(m, 1H, 1αH); 5.99−6.06 (d, J = 11.0 Hz, 1H, NH); 6.89−6.97 and

7.16−7.33 (m, 5H, Ph); 6.99−7.05 (d, J = 11.0 Hz, 1H, NH); 7.10−

7.17 (d, J = 8.8 Hz, 1H, NH); 7.46 (s, 1H, SCH); 8.02 (s, 1H, SCH);

8.49 (s, 1H, OCH). ¹³C NMR (150 MHz, CDCl₃): δ 15.1, 19.5, 20.1

(CH₂γ Pro2), 22.0, 22.3 (CH₂γ Pro1), 23.3, 24.2, 28.5, 31.7 (CH₂β

Pro1), 35.4 (CH₂β Pro2), 36.8, 41.3, 46.6, 47.5, 48.9, 51.4, 57.0, 58.8,

64.1, 122.3, 125.8, 126.9, 128.6, 128.7, 137.2, 140.4, 143.4, 146.8,

157.4, 159.9, 162.0, 169.0, 170.4, 170.9, 172.7, 173.3 Δβγ (Pro1, Pro2)

= (9.4 ppm; 15.3 ppm).

EtO-Thiazole-^D-Phe-Val-NHBoc (17f). EtO-Thiazole-D-Phe-Val-

NHBoc was synthesized following the General Peptide Synthesis

procedure, utilizing 124 mg (0.45 mmol, 1.0 equiv) of amine EtO-

Thiazole-^D-Phe-NH₂(15e-1), 109 mg (0.50 mmol, 1.1 equiv) of acid

HO-Val-NHBoc (16a), 116 mg (0.36 mmol, 0.8 equiv) of TBTU, 137

mg (0.36 mmol, 0.8 equiv) of HATU, and 0.47 mL (2.70 mmol, 6

equiv) of DIPEA dissolved in 4.5 mL of methylene chloride under N₂.

The crude product underwent a ﬁnal puriﬁcation via column

chromatography on silica gel (hexanes/ethyl acetate 19:1 to 2:1) to

aﬀord the desired peptide (157 mg, 73% yield) as a clear oil. R_f: 0.48

1

(hexanes/ethyl acetate 1:1). H NMR (300 MHz, CDCl₃): δ 0.66−

0.80 (dd, J = 18.6, 6.5 Hz, 3H, CHCH(CH₃)₂); 0.66−0.74 and 0.79−

0.86 (dd, J = 31.5, 6.9 Hz, 3H, CHCH(CH₃)₂); 1.38 (s, 9H,

C(CH₃)₃); 1.37 (t, 3H, OCH₂CH₃); 1.86−2.14 (m, 1H,

CHCH(CH₃)₂); 3.13−3.49 (m, 2H, CHCH₂Ph); 3.84−3.98 (m,

1H, 1αH); 4.32−4.43 (q, 2H, OCH₂CH₃); 5.11−5.21 (d, J = 9.0 Hz,

1H, NH); 5.22−5.32 (d, J = 9.0 Hz, 1H, NH); 5.53−5.64 (m, 1H,

1αH); 7.04−7.28 (m, 5H, Ph); 8.00 (s, 1H, SCH). ¹³C NMR (75

MHz, CDCl₃): δ 14.6, 17.8, 19.6, 28.7, 30.7, 38.9, 41.2, 52.6, 60.2,

61.7, 80.1, 127.2, 127.8, 129.2, 129.5, 136.5, 147.4, 156.1, 161.4, 166.1,

171.9. HRMS (ESI-TOF): M + Na⁺, found 498.2024 C₂₄H₃₃N₃O₅S

requires 498.2039.

Compound 8. Compound 12e is commercially available (CAS no.

77119-84-7).

NH₂-D-Phe-NHBoc (13e). NH₂-D-Phe-NHBoc was synthesized

following the General Amide Formation procedure, utilizing 360 mg

(1.29 mmol, 1.0 equiv) of MeO-^D-Phe-NHBoc (12e) dissolved in 6.5

mL of ammonium hydroxide (25% in water) and 6.5 mL of methanol.

The resulting amide was taken on to the next reaction without further

puriﬁcation (341 mg, quantitative yield) as a white powder (rotamers

2:1). R_f: 0.15 (hexanes/ethyl acetate 1:1). ¹H NMR (300 MHz,

CDCl₃): δ 1.43 (s, 9H, C(CH₃)₃); 2.88−3.25 (m, 2H, CHCH₂Ph);

4.27−4.56 (m, 1αH); 5.11−5.35 (m, 1H, NH); 5.74−5.99 (br, 1H,

NH₂); 5.99−6.19 (br, 1H, NH₂); 7.11−7.43 (m, 5H, Ph). ¹³C NMR

(75 MHz, CDCl₃): δ 28.7, 38.9, 55.8, 80.7, 127.2, 128.9, 129.5, 136.8,

155.8, 174.3.

Thioamide-^D-Phe-NHBoc (14e). Thioamide-D-Phe-NHBoc was

synthesized following the General Thioamide Formation procedure,

utilizing 341 mg (1.29 mmol, 1.0 equiv) of NH₂-D-Phe-NHBoc (13e)

and 392 mg (0.97 mmol, 0.75 equiv) of Lawesson’s Reagent dissolved

in 12.9 mL of 1,2-dimethoxyethane. The thioamide was puriﬁed via

column chromatography on silica gel (hexanes/ethyl acetate 19:1 to

9:1) to aﬀord the desired thioamide (225 mg, 62% yield) as white

crystals. R_f: 0.6 (hexanes/ethyl acetate 1:1). ¹H NMR (300 MHz,

CDCl₃): δ 1.39 (s, 9H, C(CH₃)₃); 2.98−3.25 (m, 2H, CHCH₂Ph);

4.66−4.82 (m, 1αH); 5.46−5.62 (m, 1H, NH); 7.17−7.37 (m, 5H,

Ph); 7.75−7.95 (br, 1H, NH₂); 7.95−8.18 (br, 1H, NH₂). ¹³C NMR

(75 MHz, CDCl₃): δ 28.7, 42.4, 61.4, 81.0, 127.2, 128.9, 129.5, 136.8,

155.8, 208.7.

EtO-Thiazole-^D-Phe-Val-NH₂(17f-1), Fragment 1. EtO-Thiazole-

D-Phe-Val-NH₂was synthesized following the General Amine

Deprotection procedure. The amine was taken on to the next reaction

without further puriﬁcation or characterization (124 mg, quantitative

yield) as a light brown oil.

EtO-Thiazole-^D-Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc

(29-8). EtO-Thiazole-^D-Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-

NHBoc was synthesized following the General Peptide Synthesis

procedure, utilizing 177 mg (0.30 mmol, 1.0 equiv) of acid HO-Pro-

Oxazole-Thiazole-Pro-Leu-NHBoc (28a-1), 124 mg (0.33 mmol, 1.1

equiv) of amine EtO-Thiazole-^D-Phe-Val-NH₂(17f-1), 77.0 mg (0.24

mmol, 0.8 equiv) of TBTU, 91.2 mg (0.24 mmol, 0.8 equiv) of HATU,

and 0.42 mL (2.40 mmol, 8.0 equiv) of DIPEA dissolved in 3.00 mL of

methylene chloride. The crude product underwent a ﬁnal puriﬁcation

via column chromatography on silica gel (ethyl acetate/methanol

99:1) to aﬀord the desired peptide (208 mg, 73% yield). R_f: 0.30 (ethyl

1

acetate/methanol 99:1). H NMR (300 MHz, CDCl₃): δ 0.57−0.63

and 0.68−0.76 (d, J = 6.91 Hz, 3H, CHCH(CH₃)₂); 0.51−0.64 and

0.75−0.80 (m, 3H, CHCH(CH₃)₂); 0.91−0.99 (d, J = 6.91 Hz, 3H,

CHCH₂CH(CH₃)₂); 0.96−1.04 (3H, CHCH₂CH(CH₃)₂); 1.34−1.43

(t, 3H, OCH₂CH₃); 1.42 (s, 9H, C(CH₃)₃); 1.41−1.57 (m, 2H,

CHCH₂CH(CH₃)₂); 1.71−1.84 (m, 1H, CHCH₂CH(CH₃)₂); 1.92−

2.07 (m, 2H, CHCH₂CH₂CH₂); 1.97−2.19 (m, 2H,

CHCH₂CH₂CH₂); 2.02−2.12 and 2.18−2.28 (m, 2H,

CHCH₂CH₂CH₂); 2.12−2.25 (m, 1H, CHCH(CH₃)₂); 2.10−2.15

and 2.20−2.51 (m, 2H, CHCH₂CH₂CH₂); 3.18−3.35 and 3.40−3.57

(m, 2H, CHCH₂Ph); 3.64−3.91 (m, 2H, CHCH₂CH₂CH₂); 4.07−

4.25 (m, 2H, CHCH₂CH₂CH₂); 4.22−4.34 (m, 1H, 1αH); 4.33−4.46

(q, 2H, OCH₂CH₃); 4.47−4.60 (m, 1H, 1αH); 4.67−4.77 and 5.21−

5.33 (m, 1H, 1αH); 5.42−5.69 (m, 1H, 1αH); 5.45−5.60 (m, 1H,

1αH); 6.97−7.30 (m, 5H, Ph); 7.35−7.44 (m, 1H, NH); 7.91 and 7.93

(s, 1H, SCH); 7.96−7.98 (m, 1H, NH); 7.97 and 8.03 (s, 1H, SCH);

8.22 and 8.28 (s, 1H, OCH); 8.29−8.31 (m, 1H, NH). ¹³C NMR (75

MHz,CDCl₃): δ 14.4, 17.1, 19.4, 21.7, 23.4, 24.6, 25.6, 28.3, 29.7, 31.8,

40.7, 42.2, 47.1, 49.5, 50.4, 52.5, 52.8, 58.2, 58.8, 61.4, 79.7, 120.9,

127.0, 127.6, 128.6, 129.3, 136.7, 137.8, 142.7, 143.6, 147.0, 155.8,

EtO-Thiazole-^D-Phe-NHBoc (15e). EtO-Thiazole-D-Phe-NHBoc

was synthesized following the General Thiazole Synthesis procedure,

utilizing 225 mg (0.80 mmol, 1.0 equiv) of Thioamide-^D-Phe-NHBoc

(14e) and 644 mg (6.43 mmol, 8.0 equiv) of potassium bicarbonate

dissolved in 8.0 mL of 1,2-dimethoxyethane. A 0.30 mL amount of

ethyl bromopyruvate (2.40 mmol, 3.0 equiv) was added dropwise (0.1

mL/min) to yield the thiazoline intermediate. The thiazoline was

dehydrated using 0.58 mL (7.20 mmol, 9.0 equiv) of pyridine, 0.44 mL

(3.20 mmol, 4.0 equiv) of triﬂuoroacetic anhydride, and 0.22 mL (1.60

mmol, 2.0 equiv) of triethylamine in 8.0 mL of 1,2-dimethoxyethane to

aﬀord the desired thiazole (169 mg, 56% yield) as a clear oil. R_f: 0.55

1

(hexanes/ethyl acetate 1:1). H NMR (300 MHz, CDCl₃): δ 1.38 (s,

10612

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

156.6,161.4, 171.0, 171.3, 172.5, 172.8, 174.4. LC/MS (ESI): m/z

calcd for C₄₆H₆₀N₈O₉S₂(M + Na⁺) = 955.38, found 955.00. HRMS

(ESI-TOF): M + Na⁺, found 955.3811 C₄₆H₆₀N₈O₉S₂requires

955.3823.

HO-Thiazole-^D-Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc

(29-8a). HO-Thiazole-^D-Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-

NHBoc was synthesized following the General Acid Deprotection

procedure, utilizing 208 mg (0.22 mmol, 1.0 equiv) of EtO-Thiazole-^D-

Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-8) and 73.8 mg

(1.76 mmol, 8 equiv) of LiOH·H₂O in 2.20 mL of ethanol. The acid

was taken on to the next reaction without further puriﬁcation (199 mg,

quantitative yield). LC/MS (ESI): m/z calcd for C₄₄H₅₆N₈O₉S₂(M +

H⁺) = 905.36, found 905.00.

HO-Thiazole-^D-Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NH₂(29-

8b). HO-Thiazole-^D-Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NH₂was

synthesized following the General Amine Deprotection procedure,

utilizing 199 mg (0.22 mmol, 1.0 equiv) of HO-Thiazole-^D-Phe-Val-

Pro-Oxazole-Thiazole-Pro-Leu-NHBoc (29-8a), 47.8 μL (0.44 mmol,

2.0 equiv) of anisole, and 0.44 mL of triﬂuoroacetic acid in 1.76 mL of

methylene chloride. HO-Thiazole-^D-Phe-Val-Pro-Oxazole-Thiazole-

Pro-Leu-NH₂was taken on to the next reaction without further

puriﬁcation or characterization (177 mg, quantitative yield). LC/MS

(ESI): m/z calcd for C₃₉H₄₈N₈O₇S₂(M + H⁺) = 805.31, found 805.00.

•Compound 8. Compound 8 was synthesized following the

Macrocyclization Procedure, utilizing 177 mg (0.220 mmol, 1.0

equiv) of HO-Thiazole-^D-Phe-Val-Pro-Oxazole-Thiazole-Pro-Leu-NH₂

(29-8b), 58.0 mg (0.180 mmol, 0.8 equiv) of TBTU, 69.0 mg (0.180

mmol, 0.8 equiv) of HATU, 50.0 mg (0.180 mmol, 0.8 equiv) of

DMTMM, and 230 μL (1.32 mmol, 8 equiv) of DIPEA in 220 mL of

methylene chloride. The crude product was directly subjected to

reversed-phase HPLC puriﬁcation to aﬀord a compound 8 (22.1 mg)

in a 19.8% yield. HRMS (ESI-TOF): M + Na⁺, found 809.2865

C₃₉H₄₆N₈O₆S₂requires 809.2880.

CHCH₂CH₂CH₂); 3.93 (s, 3H, OCH₃); 4.69−4.84 (m, 1H, 1αH);

5.10−5.23 (m, 1H, 1αH); 7.29−7.39 (m, 2H, CHCHCH); 7.39−7.49

(m, 3H, CHCHCH); 8.08 (s, 1H, SCH), 8.29 (s, 1H, OCH). ¹³C

NMR (75 MHz, CDCl₃): δ 24.53, 28.30, 34.15, 37.96, 47.35, 52.32,

59.56, 60.50, 79.97, 115.63, 122.07, 127.39, 128.82, 128.89, 129.33,

129.47, 136.45, 141.82, 143.75, 155.39, 155.49, 157.91, 161.48. HRMS

(ESI-TOF): M + H⁺, found 527.1949 C₂₆H₃₀N₄O₆S requires

527.1920.

HO-Oxazole-Thiazole-Pro-^D-Phe-NHBoc (27d-1). HO-Oxazole-

Thiazole-Pro-^D-Phe-NHBoc was synthesized following the General

Acid Deprotection procedure, utilizing 199 mg (0.378 mmol, 1.0

equiv) of MeO-Oxazole-Thiazole-Pro-^L-Phe-NHBoc (27d) and 127

mg (3.03 mmol, 8.0 equiv) of LiOH·H₂O in 5 mL of methanol. The

acid was taken on to the next reaction without further puriﬁcation

(188 mg, 97% yield) as yellow ﬂakes.

MeO-Pro-Oxazole-Thiazole-Pro-^L-Phe-NHBoc (28d). MeO-Pro-

Oxazole-Thiazole-Pro-^D-Phe-NHBoc was synthesized following the

General Peptide Synthesis procedure, utilizing 188 mg (0.368 mmol,

1.0 equiv) of acid HO-Oxazole-Thiazole-Pro-^L-Phe-NHBoc (27d-1),

71 mg (0.552 mmol, 1.5 equiv) of the amine MeO-Pro-NH, 140 mg

(0.368 mmol, 1.0 equiv) of HATU, and 0.51 mL (2.94 mmol, 8.0

equiv) of DIPEA dissolved in 4 mL of methylene chloride under N₂.

The reaction mixture was stirred for 3 h and was monitored by TLC

upon completion. The crude product underwent a ﬁnal puriﬁcation via

column chromatography on silica gel (hexanes/ethyl acetate 1:3) to

aﬀord the desired peptide (220 mg, 96% yield) as yellow ﬂakes. R_f: 0.3

1

(hexanes/ethyl acetate 0:1). H NMR (300 MHz, CDCl₃): δ 1.22 (s,

9H, C(CH₃)₃); 1.71−1.80 (m, 2H, CHCH₂CH₂CH₂); 1.91−2.07 (m,

2H, CHCH₂CH₂CH₂); 2.13−2.26 (m, 2H, CHCH₂CH₂CH₂); 2.81−

2.94 (m, 2H, CHCH₂CH₂CH₂); 3.35−3.45 (t, 2H, CHCH₂C); 3.57−

3.69 (m, 2H, CHCH₂CH₂CH₂); 3.54 (s, 3H, OCH₃); 3.87−3.9 (m,

2H, CHCH₂CH₂CH₂); 4.39−4.51 (m, 1H, 1αH); 5.10−5.19 (m, 1H,

1αH); 5.43−5.54 (m, 1H, 1αH); 7.03−7.09 (m, 2H, CHCHCH);

7.09−7.17 (m, 3H, CHCHCH); 7.71 (s, 1H, SCH); 7.81 (o, 1H,

OCH). ¹³C NMR (75 MHz, CDCl₃): δ 24.35, 25.21, 29.12, 34.50,

38.71, 48.59, 48.68, 51.94, 61.11, 60.60, 79.31, 126.99, 128.33, 128.57,

129.27, 129.34, 136.23, 142.97, 143.47, 155.06, 156.54, 165.39, 171.00,

173.22, 173.48. HRMS (ESI-TOF): M + H⁺found 624.2477

C₃₁H₃₇N₅O₇S requires 624.2447.

HO-Pro-Oxazole-Thiazole-Pro-^L-Phe-NHBoc (28d-1), Fragment B.

HO-Pro-Oxazole-Thiazole-Pro-^D-Phe-NHBoc was synthesized follow-

ing General Acid Deprotection procedure, utilizing 220 mg (0.353

mmol, 1.0 equiv) of MeO-Pro-Oxazole-Thiazole-Pro-^L-Phe-NHBoc

(28d) and 118 mg (2.82 mmol, 8.0 equiv) of LiOH·H₂O in 4 mL of

methanol. The acid was taken on to the next reaction without further

puriﬁcation (204 mg, 95% yield) as yellow ﬂakes.

•Compound 8. ¹H NMR (318 K, 600 MHz, CDCl₃): δ 0.31−0.37

(d, J = 8.2 Hz, 3H, CHCH(CH₃)₂); 0.86−0.91 (d, J = 8.2 Hz, 3H,

CHCH(CH₃)₂); 0.93−0.99 (d, J = 8.2 Hz, 3H, CHCH₂CH(CH₃)₂);

0.99−1.04 (d, J = 8.2 Hz, 3H, CHCH₂CH(CH₃)₂); 1.57−1.63 (m, 1H,

CH CH₂CH(CH₃)₂); 1.58−1.65 and 1.97−2.04 (m, 2H, CHCH₂CH-

(CH₃)₂); 1.79−1.85 and 1.95−2.02 (m, 2H, CHCH₂CH₂CH₂); 1.93−

2.00 and 2.07−2.13 (m, 2H, CHCH₂CH₂CH₂); 1.94−2.00 and 2.33−

2.41 (m, 2H, CHCH₂CH₂CH₂); 2.21−2.32 (m, 2H,

CHCH₂CH₂CH₂); 2.21−2.28 and 3.25−3.31 (m, 2H, CHCH₂Ph);

2.66−2.72 (m, 1H, CH(CH₃)₂); 3.67−3.73 and 3.95−4.02 (m, 2H,

CHCH₂CH₂CH₂); 3.82−3.88 and 3.90−3.97 (m, 2H,

CHCH₂CH₂CH₂); 4.65−4.71 (m, 1H, 1αH); 4.72−4.77 (m, 1H,

1αH); 5.11−5.17 (m, 1H, 1αH); 5.36−5.43 (m, 1H, 1αH); 5.48−5.54

(m, 1H, 1αH); 5.99−6.06 (d, J = 10.6 Hz, 1H, NH); 6.91−6.97 and

7.16−7.23 and 7.26−7.33 (m, 5H, Ph); 6.99−7.04 (d, J = 10.6 Hz, 1H,

NH); 7.09−7.15 (d, J = 10.6 Hz, 1H, NH); 7.47 (s, 1H, SCH); 8.00

(s, 1H, SCH); 8.49 (s, 1H, OCH). ¹³C NMR (318 K, 150 MHz,

CDCl₃): δ 15.1, 19.5, 20.1 (CH₂γ Pro2), 22.0, 22.3 (CH₂γ Pro1), 23.3,

24.2, 28.5, 31.7 (CH₂β Pro1), 35.5 (CH₂β Pro2), 36.8, 41.3, 46.6, 47.5,

48.9, 51.4, 57.0, 58.8, 64.1, 122.3, 125.8, 126.9, 128.6, 128.7, 137.2,

140.4, 143.4, 146.8, 157.4, 159.9, 162.0, 169.0, 170.4, 170.9, 172.7,

173.3. Δβγ (Pro1, Pro2) = (9.4 ppm; 15.4 ppm).

EtO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-^L-Phe-NHBoc

(29-9). EtO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-^D-Phe-

NHBoc was synthesized following the General Peptide Synthesis

procedure, utilizing 204 mg (0.335 mmol, 1.0 equiv) of acid HO-Pro-

Oxazole-Thiazole-Pro-^L-Phe-NHBoc (28d-1), 150 mg (0.503 mmol,

1.5 equiv) of amine EtO-Thiazole-Ala-Val-NH₂(17a-1), 86 mg (0.268

mmol, 0.8 equiv) of TBTU, 102 mg (0.268 mmol, 0.8 equiv) of

HATU, 0.47 mL (2.68 mmol, 8.0 equiv) of DIPEA dissolved in 4 mL

of methylene chloride under N₂. The reaction mixture was stirred for 3

h and was monitored by TLC upon completion. The crude product

underwent a ﬁnal puriﬁcation via column chromatography on silica gel

(ethyl acetate/methanol 5:1) to aﬀord the desired peptide (266 mg,

Compound 9. MeO-Oxazole-Thiazole-Pro-Phe-NHBoc (27d).

MeO-Oxazole-Thiazole-Pro-^L-Phe-NHBoc was synthesized following

the General Peptide Synthesis procedure, utilizing 125 mg (0.45

mmol, 1.0 equiv) of amine OMe-Oxazole-Thiazole-Pro-NH (25-1),

179 mg (0.675 mmol, 1.5 equiv) of acid HO-^L-Phe-NHBoc (26d), 144

mg (0.45 mmol, 1.0 equiv) of TBTU, 171 mg (0.45 mmol, 1.0 equiv)

of HATU, and 0.63 mL (3.6 mmol, 4 equiv) of DIPEA dissolved in 4.5

mL of methylene chloride under N₂. The reaction mixture was stirred

for 3 h and was monitored by TLC upon completion. The crude

product underwent a ﬁnal puriﬁcation via column chromatography on

silica gel (hexanes/ethyl acetate 1:1 to 1:3) to aﬀord the desired

peptide (199 mg, 84% yield) as yellow ﬂakes. R_f: 0.58 (hexanes/ethyl

1

89% yield) as yellow ﬂakes. R_f: 0.3 (ethyl acetate/methanol 5:1). H

NMR (300 MHz, CDCl₃): δ 0.76−0.95 (dd, 6H, CH₃CHCH₃); 1.37

(s, 9H, C(CH₃)₃); 1.20−1.27 (dd, 3H,CHCH₃); 1.53−1.69 (t,

2H,OCH₂CH₃); 1.99−2.05 (m, 2H, CHCH₂CH₂CH₂); 2.05−2.14

(m, 2H, CHCH₂CH₂CH₂); 2.14−2.27 (m, 2H, CHCH₂CH₂CH₂);

2.23−2.48 (m, 2H, CHCH₂CH₂CH₂); 2.54−2.78 (m, 2H,

CHCH(CH₃)₂); 2.79−3.15 (m, 2H, CHCH₂CH₂CH₂); 3.22−3.44

(m, 2H, CHCH₂C); 3.58−3.92 (m, 2H, CHCH₂CH₂CH₂); 4.09−4.22

(m, 1H, 1αH); 4.29−4.43 (t, 3H, OCH₂CH₃); 4.6−4.84 (m, 1H,

1αH); 5.17−5.35 (m, 1H, 1αH); 5.39−5.44 (m, 1H, 1αH); 5.44−5.54

(m, 1H, 1αH); 7.04−7.19 (m, 2H, CHCHCH); 7.19−7.29 (m, 3H,

CHCHCH); 7.92 (s, 1H, SCH); 8.05 (s, 1H, SCH); 8.28 (s, 1H,

1

acetate 1:3). H NMR (300 MHz, CDCl₃): δ 1.35 (s, 9H, C(CH₃)₃);

2.15−2.52 (m, 2H, CHCH₂CH₂CH₂); 2.87−2.99 (m, 2H, CHCH₂

CH₂CH₂); 3.15−3.25 (m, 2H, CHCH₂C); 3.28−3.48 (m, 2H,

10613

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

OCH). ¹³C NMR (75 MHz, CDCl₃): δ 14.18, 19.51, 21.54, 24.53,

24.92, 28.29, 29.64, 31.51, 39.21, 47.49, 49.41, 53.86, 58.30, 58.59,

60.36, 61.69, 126.82, 127.46, 128.53, 128.77, 129.35, 129.43, 136.24,

143.53, 146.90, 154.35, 155.18, 156.72, 161.33, 161.55, 170.88, 172.60,

173.45. LC/MS (ESI): m/z calcd for C₄₃H₅₄N₈O₉S₂(M + Na⁺) =

913.00, found 913.00. HRMS (ESI-TOF): M + H⁺found 891.3527

C₄₃H₅₄N₈O₉S₂requires 891.3533.

HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-^L-Phe-NHBoc

(29-9a). HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-^D-Phe-

NHBoc was synthesized following the General Acid Deprotection

procedure, utilizing 266 mg (0.299 mmol, 1.0 equiv) of EtO-Thiazole-

Ala-Val-Pro-Oxazole-Thiazole-Pro-^L-Phe-NHBoc (29-9) and 100 mg

(2.39 mmol, 8 equiv) of LiOH·H₂O in 3 mL of ethanol. Upon

completion, the reaction was diluted with methylene chloride. The

acid was taken on to the next reaction without further puriﬁcation

(240 mg, 93% yield). LC/MS (ESI): m/z calcd for C₄₁H₅₀N₈O₉S₂(M

+ H⁺) = 863.01, found 863.00.

HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-^L-Phe-NH₂(29-

9b). HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-^D-Phe-NH₂was

synthesized following the General Amine Deprotection procedure,

utilizing 240 mg (0.278 mmol, 1.0 equiv) of HO-Thiazole-Ala-Val-Pro-

Oxazole-Thiazole-Pro-^L-Phe-NHBoc (29-9a), 0.06 mL (0.556 mmol,

2.0 equiv) of anisole, and 0.75 mL of triﬂuoroacetic acid in 2.25 mL of

methylene chloride. The reaction was completed in 1 h; the reaction

was concentrated in vacuo. HO-Thiazole-Ala-Val-Pro-Oxazole-Thia-

zole-Pro-^L-Phe-NH₂was taken on to the next reaction without further

puriﬁcation or characterization (221 mg, quantitative yield). LC/MS

(ESI): m/z calcd for C₃₆H₄₂N₈O₇S₂(M + H⁺) = 762.90, found 762.00.

•Compound 9. Compound 9 was synthesized following the

Macrocyclization Procedure, utilizing 221 mg (0.29 mmol, 1.0

equiv) of HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-^L-Phe-NH₂

(29-9b), 75.0 mg (0.232 mmol, 0.8 equiv) of TBTU, 89 mg (0.232

mmol, 0.8 equiv) of HATU, 69 mg (0.184 mmol, 1.0 equiv) of

DEPBT, and 1.21 mL (6.96 mmol, 24 equiv) of DIPEA in 434 mL of

methylene chloride under N₂. The reaction was monitored by TLC

and LCMS upon completion. The crude product underwent an initial

puriﬁcation via column chromatography on silica gel (ethyl acetate/

methanol 9:1), and the resulting semipure residue was subjected to

reversed-phase HPLC puriﬁcation to aﬀord compound 9 (1.1 mg) in

35.9% yield. LC/MS (ESI): m/z calcd for C₃₆H₄₀N₈O₆S₂(M + H⁺) =

744.88, found 745.00. HRMS (ESI-TOF): M + Na⁺, found 767.2398

C₃₆H₄₀N₈O₆S₂requires 767.2410.

2.48 (m, 2H, CHCH₂CH₂CH₂); 2.85−3.13 (m, 2H,

CHCH₂C);3.26−3.81 (m, 2H, CHCH₂CH₂CH₂); 3.57−3.72 (t,

2H, CH₂); 3.82 (s, 3H, OCH₃); 4.35−4.81 (m, 1H, 1αH); 5.37−5.59

(m, 1H, 1αH); 7.09−7.19 (m, 2H, CHCHCH); 7.19−7.29 (m, 3H,

CHCHCH); 8.07 (s, 1H, SCH), 8.29 (s, 1H, OCH). ¹³C NMR (75

MHz, CDCl₃): 23.80, 28.28, 31.60, 39.32, 46.72, 53.13, 53.89, 58.78,

79.88, 127.13, 128.55, 128.79, 129.32, 129.42, 134.18, 136.19, 141.94,

143.71, 155.35, 157.85, 161.52, 171.59, 173.16, 174.16. HRMS (ESI-

TOF): M + H⁺found 527.1959 C₂₆H₃₀N₄O₆S requires 527.1920.

HO-Oxazole-Thiazole-Pro-^D-Phe-NHBoc (27e-1). HO-Oxazole-

Thiazole-Pro-^D-Phe-NHBoc was synthesized following the General

Acid Deprotection procedure, utilizing 132 mg (0.251 mmol, 1.0

equiv) of MeO-Oxazole-Thiazole-Pro-Gly-NHBoc (27e) and 84 mg

(2.01 mmol, 8.0 equiv) of LiOH·H₂O in 2.5 mL of methanol. The acid

was taken on to the next reaction without further puriﬁcation (122 mg,

95% yield) as yellow ﬂakes.

MeO-Pro-Oxazole-Thiazole-Pro-^D-Phe-NHBoc (28e). MeO-Pro-

Oxazole-Thiazole-Pro-^D-Phe-NHBoc was synthesized following the

General Peptide Synthesis procedure, utilizing 122 mg (0.238 mmol,

1.0 equiv) of acid HO-Oxazole-Thiazole-Pro-^D-Phe-NHBoc (27e-1),

46 mg (0.357 mmol, 1.5 equiv) of the amine MeO-Pro-NH, 91 mg

(0.238 mmol, 1.0 equiv) of HATU, and 0.33 mL (1.91 mmol, 8.0

equiv) of DIPEA dissolved in 3 mL of methylene chloride under N₂.

The reaction mixture was stirred for 3 h and was monitored by TLC

upon completion. The crude product underwent a ﬁnal puriﬁcation via

column chromatography on silica gel (hexanes/ethyl acetate 1:3) to

aﬀord the desired peptide (121 mg, 82% yield) as yellow ﬂakes. R_f: 0.3

1

(hexanes/ethyl acetate 0:1). H NMR (300 MHz, CDCl₃): δ 1.37 (s,

9H, C(CH₃)₃); 1.45−1.7 (m, 2H, CHCH₂CH₂CH₂); 1.76−2.08 (m,

2H, CHCH₂CH₂CH₂); 2.18−2.43 (m, 2H, CHCH₂CH₂CH₂); 2.54−

2.7 (m, 2H, CHCH₂CH₂CH₂); 2.82−3.08 (m, 2H, CHCH₂

CH₂CH₂); 3.19−3.44 (m, 2H, CHCH₂CH₂CH₂); 3.69 (s, 3H,

OCH₃); 4.49−4.76 (m, 1H, 1αH); 5.17−5.35 (m, 1H, 1αH); 5.36−

5.62 (m, 1H, 1αH); 6.97−7.14 (m, 2H, CHCHCH); 7.14−7.27 (m,

3H, CHCHCH); 7.83 (s, 1H, SCH), 8.24 (s, 1H, OCH). ¹³C NMR

(150 MHz, CDCl₃): 24.53, 25.23, 28.61, 29.61, 31.65, 39.27, 47.66,

48.78, 52.24, 58.72, 59.86, 60.77, 79.74, 120.92, 127.66, 128.49,

128.74, 129.32, 129.40, 142.52, 143.28, 155.32, 156.49, 160.31, 171.03,

171.58, 173.93. HRMS (ESI-TOF): M + H⁺found 624.2486

C₃₁H₃₇N₅O₇S requires 624.2447.

HO-Pro-Oxazole-Thiazole-Pro-^D-Phe-NHBoc (28e-1), Fragment B.

HO-Pro-Oxazole-Thiazole-Pro-^D-Phe-NHBoc was synthesized follow-

ing General Acid Deprotection procedure, utilizing 121 mg (0.195

mmol, 1.0 equiv) of MeO-Pro-Oxazole-Thiazole-Pro-^D-Phe-NHBoc

(28e) and 65 mg (1.56 mmol, 8.0 equiv) of LiOH·H₂O in 2 mL of

methanol. The acid was taken on to the next reaction without further

puriﬁcation (113 mg, 95% yield) as yellow ﬂakes.

•Compound 9. ¹H NMR (288 K, 600 MHz, CDCl₃): δ 0.85−0.99

(m, 6H, CH(CH₃)₂); 1.22−1.29 (m, 2H, CHCH); 1.25−1.36 (d, 3H,

CHCH₃); 1.59−1.64 (m, 2H, CH₂γ Pro2); 2.02−2.15 (m, 2H, CH₂γ

Pro1); 1.88−2.26 (m, 2H, CH₂β Pro2); 2.31−2.38 (m, 2H, CH₂β

Pro1); 3.01−3.12 (m, 2H, CHCH₂C); 3.73−3.81 (m, 2H, CH₂δ

Pro2); 3.8−3.9 (m, 2H, CH₂δ Pro1); 4.70−4.76 (m, 1H, 1αH); 4.76−

4.82 (m, 1H, 1αH); 5.08−5.10 (m, 1H, CHCH₃); 5.22−5.28 (m, 1H,

1αH); 5.30−5.36 (m, 1H, 1αH); 7.18−7.32 (m, 3H, CHCHCH);

7.42−7.44 (m, 2H, CHCCH); 7.79 (s, 1H, SCH); 7.91 (s, 1H, SCH);

8.39 (s, 1H, OCH). ¹³C NMR (288 K, 150 MHz, CDCl₃): δ 19.8,

22.82, 25.49 (CH₂γ Pro2), 27.33 (CH₂γ Pro1), 31.42 (CH₂β Pro1),

35.85 (CH₂β Pro2), 37.24, 42.67, 45.78, 46.6, 48.01, 52.48, 56.77,

58.82, 64.16, 114.69, 116.46, 125.62, 126.47, 126.58, 126.96, 128.24,

129.95, 143.4. Δβγ = (4.7 ppm; 10.36 ppm).

EtO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-^D-Phe-NHBoc

(29-10). EtO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-^D-Phe-

NHBoc was synthesized following the General Peptide Synthesis

procedure, utilizing 113 mg (0.185 mmol, 1.0 equiv) of acid HO-Pro-

Oxazole-Thiazole-Pro-^D-Phe-NHBoc (28e-1), 83 mg (0.278 mmol,

1.5 equiv) of amine EtO-Thiazole-Ala-Val-NH₂(17a-1), 59 mg (0.185

mmol, 1.0 equiv) of TBTU, 70 mg (0.185 mmol, 1.0 equiv) of HATU,

51 mg (0.185 mmol, 1.0 equiv) of DMTMM, and 0.26 mL (1.48

mmol, 8.0 equiv) of DIPEA dissolved in 2 mL of methylene chloride

under N₂. The reaction mixture was stirred for 3 h and was monitored

by TLC upon completion. The crude product underwent a ﬁnal

puriﬁcation via column chromatography on silica gel (ethyl acetate/

methanol 19:1) to aﬀord the desired peptide (158 mg, 96% yield) as

Compound 10. MeO-Oxazole-Thiazole-Pro-^D-Phe-NHBoc (27e).

MeO-Oxazole-Thiazole-Pro-^D-Phe-NHBoc was synthesized following

the General Peptide Synthesis procedure, utilizing 75 mg (0.267

mmol, 1.0 equiv) of amine OMe-Oxazole-Thiazole-Pro-NH (25-1),

106 mg (0.401 mmol, 1.5 equiv) of acid HO-^D-Phe-NHBoc (26e), 86

mg (0.267 mmol, 1.0 equiv) of TBTU, 102 mg (0.267 mmol, 1.0

equiv) of HATU, and 0.37 mL (2.14 mmol, 8.0 equiv) of DIPEA

dissolved in 2.7 mL of methylene chloride under N₂. The reaction

mixture was stirred for 3 h and was monitored by TLC upon

completion. The crude product underwent a ﬁnal puriﬁcation via

column chromatography on silica gel (hexanes/ethyl acetate 1:1 to

1:3) to aﬀord the desired peptide (132 mg, 94% yield) as yellow ﬂakes.

1

yellow ﬂakes. R_f: 0.325 (ethyl acetate/methanol 9:1). H NMR (300

MHz, CDCl₃): δ 0.84−1.02 (dd, 6H, CH₃CHCH₃); 1.19−1.27 (t,

2H,OCH₂CH₃); 1.38 (s, 9H, C(CH₃)₃); 1.64−1.68 (dd, 3H,CHCH₃);

1.85−2.01 (m, 2H, CHCH₂CH₂CH₂); 2.12−2.24 (m, 2H, CHCH₂

CH₂CH₂); 2.25−2.36 (m, 2H, CHCH₂CH₂CH₂); 2.83−3.09 (m, 2H,

CHCH₂CH₂CH₂); 3.11−3.24 (m, 2H, CHCH₂CH); 3.59−3.76 (m,

2H, CHCH₂CH₂CH₂); 4.03−4.14 (m, 2H, CHCH₂CH₂CH₂); 4.27−

4.42 (t, 3H, OCH₂CH₃); 4.43−4.6 (m, 1H, 1αH); 4.62−4.79 (m, 1H,

1αH); 5.06−5.29 (dd, 1H, 1αH); 5.31−5.54 (m, 1H, 1αH); 7.06−

7.18 (m, 2H, CHCHCH); 7.18−7.28 (m, 3H, CHCHCH); 7.91 (s,

1

R_f: 0.58 (hexanes/ethyl acetate 1:3). H NMR (300 MHz, CDCl₃): δ

1.4 (s, 9H, C(CH₃)₃); 1.8−2.14 (m, 2H, CHCH₂CH₂CH₂); 2.18−

10614

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

1H, SCH); 8.05 (s, 1H, SCH); 8.25 (s, 1H, OCH). ¹³C NMR (75

MHz, CDCl₃): δ 17.00, 17.50, 18.49, 19.49, 21.03, 23.76, 29.64, 31.58,

32.17, 43.41, 46.74, 47.69, 49.48, 58.65, 60.38, 61.38, 61.68, 79.83,

115.58, 116.93, 127.17, 127.55, 128.56, 128.79, 129.40, 129.48, 136.18,

146.79, 155.30, 161.37, 171.06, 171.66, 173.16, 174.45. LC/MS (ESI):

m/z calcd for C₄₃H₅₄N₈O₉S₂(M + H⁺) = 891.07, found 891.00.

HRMS (ESI-TOF): M + H⁺found 891.3527 C₄₃H₅₄N₈O₉S₂requires

891.3533.

HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-^D-Phe-NHBoc

(29-10a). HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-^D-Phe-

NHBoc was synthesized following the General Acid Deprotection

procedure, utilizing 158 mg (0.177 mmol, 1.0 equiv) of EtO-Thiazole-

Ala-Val-Pro-Oxazole-Thiazole-Pro-^D-Phe-NHBoc (29-10) and 59 mg

(1.42 mmol, 8 equiv) of LiOH·H₂O in 2.00 mL ethanol. Upon

completion, the reaction was diluted with methylene chloride. The

acid was taken on to the next reaction without further puriﬁcation

(150 mg, 98% yield). LC/MS (ESI): m/z calcd for C₄₁H₅₀N₈O₉S₂(M

+ H⁺) = 863.01, found 863.00.

Twitching Motility Assays. Subsurface stab twitching motility assays

were performed as previously described.³Brieﬂy, P. aeruginosa PAO1

was stab-inoculated through a 1% agar LANS plate containing no

compound (untreated control) or the indicated San B analogues (50

μM). S. aureus was inoculated through a 1% agar MH plate and served

as a negative control for the assay. Plates were incubated for 48 h at 37

°C. The zone of motility was measured and recorded (in mm²).

In Vitro Antimicrobial Assays. For bacterial cytotoxicity assays,

overnight cultures of E. coli or S. aureus were grown in MH broth at 37

°C with shaking (250 rpm). On the next day, cultures were diluted

1:100 in fresh MH broth, and bacteria were grown statically in the

presence of 50 μM San B analogues for 5 h at 37 °C. Either gentamicin

or vancomycin-treated cells (50 μM; Sigma-Aldrich) were the positive

controls for the assays, and DMSO-treated cells (1% v/v; Sigma-

Aldrich) were the negative controls. Microbial viability was assessed

using the BacTiter-Glo microbial assay (Promega, Madison, WI)

following the manufacturer’s instructions. Relative ﬂuorescence units

were measured using a MPL4 Orion microplate luminometer

(Berthold Technologies).

HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-^D-Phe-NH₂(29-

10b). HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-^D-Phe-NH₂

was synthesized following the General Amine Deprotection procedure,

utilizing 150 mg (0.174 mmol, 1.0 equiv) of HO-Thiazole-Ala-Val-Pro-

Oxazole-Thiazole-Pro-^D-Phe-NHBoc (29-10a), 0.04 mL (0.347 mmol,

2.0 equiv) of anisole, and 0.50 mL of triﬂuoroacetic acid in 2.50 mL of

methylene chloride. The reaction was completed in 1 h and

concentrated in vacuo. HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-

Pro-^D-Phe-NH₂was taken on to the next reaction without further

puriﬁcation or characterization (140 mg, quantitative yield). LC/MS

(ESI): m/z calcd for C₃₆H₄₂N₈O₇S₂(M + H⁺) = 762.90, found 763.00.

•Compound 10. Compound 10 was synthesized following the

Macrocyclization Procedure, utilizing 140 mg (0.184 mmol, 1.0 equiv)

of HO-Thiazole-Ala-Val-Pro-Oxazole-Thiazole-Pro-^D-Phe-NH₂(29-

10b), 59.0 mg (0.184 mmol, 1.0 equiv) of TBTU, 69.9 mg (0.184

mmol, 1.0 equiv) of HATU, 50.8 mg (0.184 mmol, 1.0 equiv) of

DMTMM, and 0.26 mL (1.47 mmol, 8 equiv) of DIPEA in 263 mL of

methylene chloride under N₂. The reaction was monitored by TLC

and LCMS upon completion. The crude product underwent an initial

puriﬁcation via column chromatography on silica gel (ethyl acetate/

methanol 9:1), and the resulting semipure residue was subjected to

reversed-phase HPLC puriﬁcation to aﬀord a compound 10 (33.3 mg)

in a 18.3% yield. LC/MS (ESI): m/z calcd for C₃₆H₄₀N₈O₆S₂(M +

H⁺) = 744.88, found 745.00. HRMS (ESI-TOF): MH⁺found

745.2590 C₃₆H₄₀N₈O₆S₂requires 745.2590.

ASSOCIATED CONTENT

* Supporting Information

NMR, mass spectral, and bacterial cytotoxicity data for

compounds. This material is available free of charge via the

Internet at http://pubs.acs.org.

■

S

AUTHOR INFORMATION

Corresponding Author

*Tel: +61-4-1672-8896; fax:+61-4-9385-6141; e-mail: s.

mcalpine@unsw.edu.au.

■

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank the University of New South Wales Sydney for

support of H.W., D.R.M., and S.R.M., and the Frasch

foundation (658-HF07).

REFERENCES

■

(1) Pelae

́

z, F. Biochem. Pharmacol. 2006, 71, 981−990.

•Compound 10. ¹H NMR (288 K, 600 MHz, CDCl₃): δ 0.77−0.92

(m, 6H, CH(CH₃)₂); 1.32−1.35 (d, 3H, CHCH₃); 1.32−1.45 (m, 2H,

CHCH); 1.90−1.98 (m, 2H, CH₂γ Pro2); 2.05−2.13 (m, 2H, CH₂γ

Pro1); 2.13−2.29 (m, 2H, CH₂β Pro1); 2.22−2.29 (m, 2H, CH₂β

Pro2); 3.02−3.13 (m, 2H, CHCH₂C); 3.77−3.81 (m, 2H, CH₂δ

Pro2); 4.13−4.23 (m, 2H, CH₂δ Pro1); 4.36−4.42 (m, 1H, 1αH);

4.39−4.77 (m, 1H, 1αH); 5.31−5.36 (m, 1H, CHCH₃); 5.21−5.33

(m, 1H, 1αH); 5.33−5.39 (m, 1H, 1αH); 7.11−7.25 (m, 2H,

CHCCH); 7.26−7.31 (m, 3H, CHCHCH); 7.73 (s, 1H, SCH); 8.08

(s, 1H, SCH); 8.31 (s, 1H, OCH). ¹³C NMR (288 K, 150 MHz,

CDCl₃): δ 19.39, 20.4, 23.86 (CH₂γ Pro2), 25.53 (CH₂γ Pro1), 27.01

(CH₂β Pro1), 28.93, 31.89 (CH₂β Pro2), 39.24, 46.52, 47.48, 58.65,

58.81, 59.29, 61.54, 62.35, 121.3, 127.22, 127.4, 128.65, 129.42, 129.5,

143.5. Δβγ = (2.38 ppm; 8.03 ppm).

Experimental Procedures: Biological Methods. Strains,

Media, Antibiotics, and Cell Culture. Pseudomonas aeruginosa strain

PAO1 (ATCC 15692), as well as clinical isolates of enterotoxigenic

Escherichia coli (Type O88) and penicillin-resistant Staphylococcus

aureus (SA), were kind gifts from Dr. Suhelen Egan at the School of

Biotechnology and Biomolecular Sciences, University of New South

Wales. E. coli was propagated on Luria−Bertani (LB) medium with

1.5% agar (10 g/L tryptone; 5 g/L yeast extract; 10 g/L sodium

chloride; 15 g/L agar). P. aeruginosa was propagated on LB medium

lacking sodium chloride (LANS; 10 g/L tryptone; 5 g/L yeast extract;

15 g/L agar). S. aureus was propagated on Mueller−Hinton (MH)

medium (Carl Roth GmbH, Karlsruhe, Germany) containing 1.5%

agar.

(2) Butler, M. S.; Buss, A. D. Biochem. Pharmacol. 2006, 71, 919−

929.

(3) Singh, E.; Ramsey, D. M.; McAlpine, S. R. Org. Lett. 2012, 14,

1198−1201.

(4) Dalisay, D. S.; Rogers, W. W.; Edison, A. S.; Molinski, T. F. J. Nat.

Prod. 2009, 72, 732−738.

́

(5) Angel, V. Molluscan Res. 2002, 22, 289−301.

(6) Matsunaga, S.; Fusetani, N.; Hashimoto, K.; Koseki, K.; Noma,

M. J. Am. Chem. Soc. 1986, 108, 847−849.

(7) Maddock, J.; Pattenden, G.; Wight, P. G. J. Comput.-Aided Mol.

Des. 1993, 7, 573−586.

́

(8) Riego, E.; Hernandez, D.; Albericio, F.; Alvarez, M. Synthesis

2005, 1907, 1922.

(9) Baumann, M.; Bazendale, I. R.; Ley, S. V.; Nikbin, N. Beilstein J.

Org. Chem. 2011, 7, 422−495.

(10) Duchler, M. J. Drug Target. 2012, 20, 389−400.

(11) Boger, D. L.; Lee, J. K. J. Org. Chem. 2000, 65, 5996−6000.

(12) Dang, Q.; Yan, L.; Cashion, D. K.; Kasibhatla, S. R.; Jiang, T.;

Taplin, F.; Jacintho, J. D.; Li, H.; Sun, Z.; Fan, Y.; DaRe, J.; Tian, F.; Li,

W.; Gibson, T.; Lemus, R.; van Poelje, P. D.; Potter, S. C.; Erion, M.

D. J. Med. Chem. 2011, 54, 153−165.

(13) Rzuczek, S. G.; Pilch, D. S.; Liu, A.; LaVoie, E. J.; Rice, J. E. J.

Med. Chem. 2010, 53, 3632−3644.

(14) Kehraus, S.; Konig, G. M.; Wright, A. D.; Woerheide, G. J. Org.

̈

Chem. 2002, 67, 4989−4992.

(15) Roy, R. S.; Kelleher, N. L.; Milne, J. C.; Walsh, C. T. Chem. Biol.

1999, 6, 305−318.

10615

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

The Journal of Organic Chemistry

Article

(16) Takeuchi, Y.; Marshall, G. R. J. Am. Chem. Soc. 1998, 120,

5363−5372.

(17) Tan, L. T.; Williamson, R. T.; Gerwick, W. H.; Watts, K. S.;

McGough, K.; Jacobs, R. J. Org. Chem. 2000, 65, 419−425.

(18) Siemion, I. Z.; Wieland, T.; Pook, K.-H. Angew. Chem., Int. Ed.

1975, 14, 702−703.

(19) Deng, S.; Taunton, J. J. Am. Chem. Soc. 2002, 124, 916−917.

(20) Pan, P. S.; Vasko, R. C.; Lapera, S. A.; Johnson, V. A.; Sellers, R.

P.; Lin, C.-C.; Pan, C.-M.; Davis, M. R.; Ardi, V. C.; McAlpine, S. R.

Bio. Org. Med. Chem. 2009, 17, 5806−5825.

(21) Otrubova, K.; Lushington, G. H.; Vander Velde, D.; McGuire,

K. L.; McAlpine, S. R. J. Med. Chem. 2008, 51, 530−544.

(22) Otrubova, K.; McGuire, K. L.; McAlpine, S. R. J. Med. Chem.

2007, 50, 1999−2002.

(23) Rodriguez, R. A.; Pan, P.-S.; Pan, C.-M.; Ravula, S.; Lapera, S.

A.; Singh, E. K.; Styers, T. J.; Brown, J. D.; Cajica, J.; Parry, E.;

Otrubova, K.; McAlpine, S. R. J. Org. Chem. 2007, 72, 1980−2002.

(24) Heller, M.; Sukopp, M.; Tsomaia, N.; John, M.; Mierke, D. F.;

Reif, B.; Kessler, H. J. Am. Chem. Soc. 2006, 128, 13806−13814.

(25) Chatterjee, J.; Mierke, D. F.; Kessler, H. J. Am. Chem. Soc. 2006,

128, 15164−15172.

(26) Davis, M. R.; Singh, E. K.; Wahyudi, H.; Alexander, L. D.;

Kunicki, J.; Nazarova, L. A.; Fairweather, K. A.; Giltrap, A. M.; Jolliffe,

K. A.; McAlpine, S. R. Tetrahedron 2012, 68, 1029−1051.

(27) Shaw, P. E. EMBO Rep. 2002, 3, 521−526.

(28) Rajakumar, P.; Selvam, S.; Shanmugaiah, V.; Mathivanan, N.

Bioorg. Med. Chem. Lett. 2007, 17, 5270−5273.

(29) Cai, Y.; Chai, D.; Wang, R.; Bai, N.; Liang, B.-B.; Liu, Y. J.

Antimicrob. Chemother. 2011, 66, 968−978.

(30) Svarstad, H.; Bugge, H. C.; Dhillion, S. S. Biodivers. Conserv.

2000, 9, 1521−1541.

(31) Sagan, S.; Karoyan, P.; Lequin, O.; Chassaing, G.; Lavielle, S.

Curr. Med. Chem. 2004, 11, 2799−2822.

(32) Baynham, P. J.; Ramsey, D. M.; Gvozdyev, B. V.; Cordonnier, E.

M.; Wozniak, D. J. J. Bacteriol. 2006, 188, 132−140.

́

(33) Tugyi, R.; Uray, K.; Ivan, D.; Fellinger, E.; Perkins, A.; Hudecz,

F. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 413−418.

(34) Chatterjee, J.; Gilon, C.; Hoffman, A.; Kessler, H. Acc. Chem.

Res. 2008, 41, 1331−1342.

(35) Haubner, R.; Gratias, R.; Diefenbach, B.; Goodman, S. L.;

Jonczyk, A.; Kessler, H. J. Am. Chem. Soc. 1996, 118, 7461−7472.

(36) Aumailley, M.; Gurrath, M.; Muller, G.; Calvete, J.; Timpl, R.;

̈

Kessler, H. FEBS Lett. 1991, 291, 50−54.

(37) Mas-Moruno, C.; Rechenmacher, F.; Kessler, H. Anti-Cancer

Agents Med. Chem. 2010, 10, 753.

(38) Styers, T. J.; Rodriguez, R. A.; Pan, P.-S.; McAlpine, S. R.

Tetrahedron Lett. 2006, 47, 515−517.

(39) Rasmussen, L.; White, E. L.; Pathak, A.; Ayala, J. C.; Wang, H.;

Wu, J.-H.; Benitez, J. A.; Silva, A. J. Antimicrob. Agents Chemother.

2011, 55, 4134−4143.

(40) Moody, C. J.; Bagley, M. C. J. Chem. Soc., Perkin Trans. 1 1998,

601−608.

10616

dx.doi.org/10.1021/jo3017499 | J. Org. Chem. 2012, 77, 10596−10616

Article Doi

Synthesis, structurea-activity analysis, and biological evaluation of sanguinamide B analogues

DOI: 10.1021/jo3017499

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jo3017499

Full text of DOI:10.1021/jo3017499

Products guided by the article

R&D Labs maybe for 1414375-91-9

Relevant to this article

Hot Product