Copper(I)-Mediated Denitrogenative Macrocyclization for the Synthesis of Cyclic α3β-Tetrapeptide Analogues

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Accepted Article

Title: Cu(І)-Mediated Denitrogenative Macrocyclization for the

Synthesis of Cyclic α3β-Tetrapeptide Analogs

Authors: Chun-Chi Chen, Sheng-Fu Wang, Yung-Yu Su, Yuya A. Lin,

and Po-Chiao Lin

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To be cited as: Chem. Asian J. 10.1002/asia.201700339

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Cu(І)-Mediated Denitrogenative Macrocyclization for the

Synthesis of Cyclic α₃β-Tetrapeptide Analogs

Chun-Chi Chen, Sheng-Fu Wang, Yung-Yu Su, Yuya A. Lin and Po-Chiao Lin*^[a]

Dedication ((optional))

Abstract: A copper(I)-mediated denitrogenative reaction has been

successfully developed for the preparation of cyclic tetrapeptides.

The key reactive intermediate, ketenimine, triggers theintramolecular

cyclization via the attack of the terminal amine group generating an

internal β-amino acid with an amidine linkage. The chemistry

developed here provides a new synthetic route for the preparation of

cyclic α₃β-tetrapeptide analogs which contain important biological

properties and rich structural information for conformational studies.

With the success of this Cu(I)-catalyzed macrocyclization, two

histone deacetylase (HDAC) inhibitor analogs consist of cyclic α₃β-

tetrapeptide framework have been successfully synthesized.

Ring size is crucial for a successful head-to-tail peptide

macrocyclization, cyclization of peptides containing more than

seven amino acids is generally feasible. However, synthesis of

the small-to-medium sized cyclic peptides can be obstructed by

the trans-amide bonds that go against the ring-like conformation

required for cyclization.^[7]To overcome this limitation, cyclization

of long peptides are known to accelerate by the formation of

intra-peptide hydrogen bonds and transient β-sheet structure.

Moreover, the introduction of a cis-amide bond in the middle of

peptide chain may provide a suitable geometry for cyclization.

To this end, proline/pseudo-proline as well as modified

heterocyclic amino acids have been incorporated in the

sequence to induce the cisoid conformation facilitating the

subsequent peptide cyclization.^[8]Furthermore, the incorporation

of C-terminal D-amino acids^[9]and N-methyl amino acids^[10]could

also exert turn-inducing effects creating an appropriate

stereochemical configuration of the peptide backbones in

subsequent cyclization. The use of metal ions provides another

Introduction

Discovery and characterization of bioactive macrocycles have

inspired chemists in the field of medicinal chemistry. Cyclic

peptides are of particular significance owing to the remarkable

capacity for functional fine-tuning.^[1]Compared to linear peptides,

peptide cycles retain the variability in amino acid residues with

additional tuning in ring size, and can significantly resist

degradation by exo- and endoproteases enabling the practical

use of cyclic peptides as therapeutic agents.^[2]Cyclic peptides,

particularly cyclic tetrapeptides (CTPs), are important model

ligands acting as reverse turn analogs for protein specific

recognition.^[3]Reverse turns are loop-shaped motifs that

contribute toward the structural stability of proteins by

connecting residues of α-helices and β-strands.^[4]The structural

rigidity and protein surface location of reverse turns make these

ideal sites for receptor recognition. Many natural cyclic peptides

have been characterized to contain biological activities toward

multiple unrelated classes of receptors which might be ascribed

to the the idea that reverse-turn motifs could be ligands for more

non-covalent,

auxiliary-based

strategy

which

may

conformationally preorganize a peptide for macrocyclization.

Metals ions such as Pd(II), Ni(II), Cu(II),^[11]lithium,^[12]sodium ,^[13]

and Ag(I)^[14](can you put Ag(I) earlier together with other

transition metals? I don’t want to mess up the reference so I

didn’t correct here) all have been reported to promote peptide

macrocyclization by forming complexes with the peptides of

interest.

In addition, cis/trans isomerization of amide bonds usually

makes CTPs stay in a conformationally dynamic state in

aqueous solution.^[15]The ring strain of 12-membered cyclic

structures leads to a marked increase of cis-amide population

and therefore induces distortion of amide bond geometry.^{[4, 16]}

The lowered barrier of cis/trans amide isomerization can result in

conformational heterogeneity of CTPs. Incorporation of a β-

amino acid in the tetrapeptide sequence results in a less

strained 13-membered cyclic transition state and decreased

amide isomerization allowing CTPs to be more easily

synthesized with conformational homogeneity.^[17]α₃β CTPs were

found to be more resistant to hydrolysis. Yudin et al. have

reported a chemical strategy for constructing α₃β CTPs by

transformation of aziridine group to β-amino acids.^[18]The crucial

N-acyl aziridine group facilitates the macrocyclization and the

subsequent ring-opening reaction with nucleophiles to obtain

site-specific β-amino acid incorporated CTPs.^[19]Moreover, C-

linked carbohydrate-β³-amino acids have been incorporated in

the synthesis of CTPs which exhibit stable β- or γ -

structures.^[20]Recently, Wong et al. have reported a new

approach to synthesize 15-membered CTPs from a proline

motif by reductive cleavage using samarium(II) iodide (SmI₂).^[21]

than one receptor.^[5]Therefore, the synthesis of

a

conformationally diverse library of CTPs and their analogs is

certainly of great significance for discovery of biological active

peptides.

Accordingly, the development of facile synthetic strategies

of cyclic peptides is in high demand for rapid compound

generation with various ring sizes and side chain functionalities.^[6]

[a]

C.-C. Chen, S.-F. Wang, Y.-Y. Su, Y. A. Lin, Prof. P.-C. Lin*

Department of Chemistry

Nation Sun Yat-sen University

70 Lienhai Rd., Kaohsiung 80424 (Taiwan)

E-mail: pclin@mail.nsysu.edu.tw

Ghadiri et al. reported

containing CTPs as potent and selective inhibitors of Histone

deacetylase (HDAC).^[22]HDACs are Zn²⁺-dependent enzymes

a series of β-amino acids

Supporting information for this article is given via a link at the end of

the document.

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which catalyze the removal of acetyl groups from ε -N-

acetyllysine of histones. The acetylation and deacetylation of

lysine modulate the packing of chromatin complex and thereby

modulate gene transcription.^[23]The development of HDAC

inhibitors promises an avenue to gaining further understanding

of epigenetic regulation as well as treatment of cancers. The

proposed 13-membered α₃β CTPs have backbone similar to

azumamides, naturally occurring HDAC inhibitors containing a

single β³-amino acid,^[24]and side chains through rational design

inspired by a fungal metabolite apicidin A.^[5c]The scaffolds for

inhibition of HDAC isolated from HeLa have been optimized by

systematically screening the chirality of the amino acids and the

dihydropyrimidin-4-ones as the exclusive products. The resulting

dihydropyrimidin-4-ones could then be converted into β- and β³-

amino acid analogs via nucleophilic attack. This Cu(I)-catalyzed

strategy provided an important route to prepare β-amino acids

with well-defined stereochemistry that was preserved from the

starting α-amino acids. The Cu(I) catalyst was first involved in

the formation of sulfonyl triazole intermediate and subsequently

participate in the equilibrium with α-imino diazo specie. Through

a Wolff-type rearrangement, the α-imino diazo intermediate can

be converted into a highly reactive ketenimine intermediate

which have been used in many synthetic applications.^[27]Notably,

the linear sp hybrid center of the ketenimine intermediate is

highly reactive towards nucleophiles even with considerable

steric hindrance. Therefore, the introduction of ketenimine as a

key intermediate in the peptide cyclization should be of great

significance to improve cyclization efficiency and facilitate the

expansion of structural diversity.

position of the β-amino acid.^[22]

A variety of side chain

functionalities was also investigated by elaborate design.The

synthesis of one-bead-one-compound combinatorial libraries^[25]

of α₃β-CTPs could further allow the discovery of potent HDAC

ligands using a convenient screening platform. In this research,

a Cu(I)-catalyzed denitrogenative annulations is proposed to

enable macrocyclization of tetrapeptides through the formation

of β-amino acid linkages.

Results and Discussion

Accordingly, a linear α₂βα₂pentapeptide composed of four L-α-

glycine and a β-glycine was first selected as a model to evaluate

the applicability of this newly developed chemistry in the

preparation of β-amino acid-containing linear or cyclic peptides.

The general principle of this strategy is illustrated in Figure 1b in

which the ketenimine intermediate is subjected to nucleophilic

addition by the N-terminus of another peptide fragment. The

desired linear α₂βα₂pentapeptide would be obtained with an

amidine linkage. To synthesize the peptide precursors,

asillustrated in Scheme 1, Boc-protected glycine 1 was coupled

with propargylamine to provide compound 2 (93%). After

deprotection, the amino compound 3 was further coupled with

Boc-protected glycine 1 to furnish dipeptide 4 (85%). On the

other hand, Boc-protected glycine methyl ester 5 was prepared

by treating 1 with methyl iodide and the isolated yield was 97%.

After Boc deprotection, amino compound 6 was coupled with 1

using HOBt and EDC to obtain Boc-protected dipeptide 7 (95%).

Subsequently, the treatment of trifluoroacetic acid (TFA) led to

complete removal of the Boc group to provide the nucleophilic

amino dipeptide 8.

The ketenimine-directed coupling reaction was initiated by

activation of the terminal alkyne in compound 4. In the presence

of CuI catalyst, potassium carbonate (K₂CO₃) and tosyl azide,

the ketenimine intermediate immediately reacted with dipeptide

8 to give the desired pentapeptide 9. The details of reaction

conditions have been carefully studied and summarized in Table

1. First, it was found that a catalytic amount of copper iodide (0.1

equiv.) in dichloromethane (DCM) with K₂CO₃(2 equiv.) can

enable successful coupling between 4 and 8 to obtain the

desired α₂βα₂pentapeptide 9 though in a low yield of 32% (entry

1). To optimize the reaction efficiency, the amount of CuI, the

equivalent of K₂CO₃, and the reaction solvents have been

carefully screened. The fact that excess K₂CO₃(5 equiv.) cannot

improve the yield may be due to the decomposition of tosyl

azide in the presence of excess base. The use of acetonitrile

(ACN) as reaction solvent would retard the reaction while using

Figure 1. (a) Reported strategy for the preparation of β-amino acid analogs. (b)

the proposed method in this work to synthesize β-amino acid containing

peptides.

This strategy is inspired by our previous work on the preparation

of β- and β³- amino acids directly from the corresponding α-

amino acids.^[26]As shown in Figure 1a, N-propargyl benzamide

(i), directly transformed from α-amino acids, reacts with tosyl

azide in the presence of catalytic amount of CuI and K₂CO₃to

generate a highly reactive ketenimine intermediate (ii) which

immediately undergoes cyclization to form 4-sulfanimido-1,3-

oxazines (iii). A ring opening/closing process leads to a rapid

equilibrium that ultimately furnishes the more stable

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Scheme 1. Synthetic pathway of linear α₂βα₂-tetrapeptide precursors.

and a β-homoleucine as the third amino acid. The key Cu(I)-

catalyzed denitrogentaive reaction triggers

a

head-to-tail

cyclization and thereby connects residues 3 and 4 with a β-

linked amidine. With traditional solution-phase peptide synthesis,

linear tetrapeptide 14 was synthesized by a coupling reaction of

dipeptide 11 and 12. An unusual amino acid analog, 5-

methylhex-1-yn-3-amine (blue moiety in 11), can be prepared

from α-leucine by reported procedure.^[26]The target structure is a

HDAC inhibitor analog which usually carries a critical Zn²⁺-

coordinating amino acid side chains such as epoxyketone,

ethylketone, amide, or carboxylic acid.^{[22b, 28]}The modification of

Zn-coordinating functionality can significantly affect the binding

affinity of CTPs to HDACs.^[28]For this purpose, an O-allyl group

has been introduced to the side chains of CTP analog as CTP 2

demonstrating facile structural expansion of the Zn-coordinating

motif.

N,N-dimethylformamide (DMF) with two equivalent K₂CO₃can

improve the isolated yield to 40% (entry 5 and 6, respectively).

Accordingly, an attempt using co-solvent system consists of

DMF and DCM in the ratio of 1:1 successfully increased the

isolated yield up to 48% (entry 8). Under this optimal condition, a

short αβα tripeptide 10 which prepared from 2 and 6 can be

obtained with the isolated yield up to 85% (entry 9).

Figure 2. Retrosynthesis of CTPs via Cu(I)-catalyzed denitrogenative

cyclization.

Table 1. Condition optimization of synthesis linear α_n-β-α_npeptides.

The synthesis of dipeptide building block 11 initiated with the

conversion of Boc-L-Leu to the corresponding Weinreb amide

(Scheme 2). The obtained 16 was then reduced using lithium

aluminum hydride (LAH) to furnish aldehyde 17. Subsequently,

the aldehyde was subjected to

a

homologation using

Bestmann−Ohira reagent to produce Boc-protected amino

alkynes (compound 18, 60% over two steps). Deprotection of

the Boc group afforded the free amine (compound 19,

quantitative yield), which was then set up for coupling with Boc-

Trp-OH to give the dipeptide building block 20. Finally,

deprotection of the Boc protecting group led to dipeptide building

block 11 containing a free amine along with a terminal alkyne

group for further coupling reactions.

In scheme 3, glycine and O-allyl substituted L-serine were

coupled with L-alanine to construct the other dipeptide building

blocks 12 and 13. To prepare O-allyl substituted serine, L-serine

methyl ester hydrochloride was protected with triphenylmethyl

group afforded N-trityl-L-serine methyl ester which was then

subjected to O-allylation. After the removal of trityl group,

To apply this strategy in the synthesis of CTPs, two α₃β amidine

analogs of apicidin A, CTP 1 and 2, were selected as targets.

The retrosynthesis is illustrated in Figure 2. The target CTP 1

are composed of three α-amino acids including glycine (amino

acid 1), tryptophan (amino acid 2) and alanine (amino acid 4),

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Scheme 2. Synthesis of Building Block Dipeptide 11

byproduct was determined as 1 to 1 using coupling reagents

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

hexafluorophosph-ate (HBTU) and triethylamine (TEA). To

suppress undesired side reactions, different coupling reagents

and bases have been evaluated in the coupling reaction, and

were summarized in Table S1. Finally, a mild condition using

HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-

b]pyridin-ium 3-oxid hexafluorophosphate), HOAt (1-Hydroxy-7-

azabenzotriazole) and 2,4,6-trimethylpyridine (TMP) present the

optimized condition with improved ratio of 25 and undesired

byproduct as 5 to 1. Furthermore, 25 was separated from the

undesired byproduct by column chromatography to further

improve the purity of 25 up to the ratio of 15/1. After the removal

of Boc protecting group by TFA, linear tetrapeptide precursor 14

and 15 were collected ready for the next peptide cyclization.

Scheme 4. Synthesis of CTP Precursors Linear Tetrapeptides 14 and 15

Scheme 3. Synthesis of Building Block Dipeptide 12 and 13

Table 2. Optimization of Cu(I)-catalyzed Dinitrogenative Macrocyclization of

Tetrapeptides.

compound 21 was ready for the use in the synthesis of dipeptide

13.^[29]Glycine 6 and O-allyl serine 21 were then reacted with

Boc-Ala-OH to obtain protected dipeptides 22 (99%) and 23

(84%), respectively. Subsequently, basic hydrolysis of the ester

groups with diluted sodium hydroxide furnished the

corresponding dipeptides 12 (90%) and 13 (92%) exposing the

carboxylic acid group for next amide bond formation.

O

H

N

O

NH

O

HN

TsN

NH

Cu(I), TsN₃, Base

solvent, r.t.

H

N

NH₂

O

H

O

N

H

14

CTP 1

Entry

Catalyst (eq.)

Base

Solvent (M)

Concentration (mM) Yield (%)

Then, the dipeptide 11 was coupled with 12 and 13 to form the

desired linear tetrapeptide precursors 24 (75%) and 25 (75%).

(Scheme 4) High levels of epimerization were observed under

general coupling conditions in the case of the tetrapeptide 25.

According to previous reports, epimerization occurred during

solid phase peptide synthesis could produce as high as 80% of

the unnatural epimer for glycoylated serine-containing

peptides.^[30]In contrast, less than 1% epimerization was

observed for glycosylated threonine derivatives.^[31]The

epimerization occurrence was considered during the coupling

processes by activation and cyclization, then abstraction of the

α-hydrogen to form the other epimer (Scheme S1). (Data are

shown in supporting information) Apparently, adopting O-allyl

substituted serine 21 as second amino acids results in a quick

abstraction of the α-hydrogen as a ratio of 25 and undesired

K₂CO₃

TEA

1

2

CuI (0.2)

CuI (0.5)

CuI (0.2)

CuCl (0.2)

CuI (0.5)

CuI (1.0)

ACN

10

5

19

20

ACN

3

THF

12

TEA

4

ACN

solvent^[a]

trace

11

K₂CO₃

5

6^[b]

7^[c]

8

DCM

ACN

14

co-solvent^[d]

22

9

5

32

[a] THF, DCM, Dioxane, DMF. [b] reflux. [c] 50^oC. [d] DCM / DMF = 1:1

The cyclization of peptides is expected to be very difficult

compared to the linear peptide coupling reaction. Therefore, the

condition of peptide cyclization should be optimized for the

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synthesis of the desired CTPs. (Table 2) Poor solubility of the

linear tetrapeptide in less polar solvents (entry 5) causes poor

cyclization. Heating the reaction mixture (entry 6 and 7) led

toundesired decomposition of tosyl azide and thereby

considerably reduce the cyclization efficiency. In the presence of

excess base or using organic base like triethylamine instead of

K₂CO₃showed no significant improvement either. To improve

the substrate solubility, a DCM and DMF co-solvent system

along with the increase of CuI (0.5 equiv.) successfully raise the

cyclization yield to 22% (entry 8). Finally, the optimal reaction

condition was found by increasing the amount of CuI to 1

equivalent while keeping K₂CO₃(2 equiv.) and DCM/DMF

solvent system as before, and the desired CTP 1 can be

obtained with an isolated yield up to 32%. When the optimized

conditions were applied in the synthesis of CTP 2, the desired

CTP 2 can be isolated with a yield of 35%, as shown in Scheme

5.

Considering the reaction mechanism, two possible pathways

may involve in the CTP cyclization. The ketenimine intermediate

I was generated when linear tetrapeptide 15 was treated with

tosyl azide in the presence of CuI and K₂CO₃(Scheme 6). The

reactive ketenimine intermediate can then be attacked directly

by N-terminal amino group forming the desired CTP 2 as shown

in path (a). However, according to our reported mechanism in

the synthesis of β-amino acids,^[26]the ketenimine intermediate I

may first trigger the formation of dihydropyrimidin-4-one

(Intermediate II, path b). Next, the N-terminal amine initiates an

acyl substitution to open the ring to obtain the isomeric product

CTP 2’. To characterize the precise structure of the obtained

product, several 2D NMR experiments have been performed

1

and carefully analyzed. H-¹H correlation spectroscopy (COSY)

and ¹H-¹³C heteronuclear single quantum correlation (HSQC)

can allow correct assignment of most protons and carbons in

CTP 2. (Data included in supporting information) To determine

the correct linkage of β-amino acids, heteronuclear multiple

bond correlation (HMBC) experiment can provide critical

information. Carbon 26 of the amidine bond (numbered in CTP 2,

Figure 3) was found at δ167.1 and showed a correlation with

protons at carbon 25 which are separated by two bonds, as

shown in Figure S1. Furthermore, carbon 26 also gave a

correlation with the proton on carbon 1 in the HMBC spectrum

(Figure 3) which supports the structural arrangement of CTP 2.

In the case of CTP 2’, the correlation of the amidine carbon with

corresponding methylene protons separated by four bonds are

rarely observed. Therefore, the correct structure of CTP product

can be determined as CTP 2. The ring strain of reaction

intermediates in two pathways may be the explanation for the

preference of pathway (a) in which the nucleophilic attack may

Scheme 5. Synthesis of CTP 2

Scheme 6. Two Possible Pathways for CTP Cyclization.

occur in

a

13-membered ring structure. However, the

nucleophilic acyl substitution depicted in pathway (b) has to

overcome a larger ring strain of 11-membered ring intermediate

making it a less favored pathway.

O

H

O

H

N

O

N

O

HN ¹

NH

H₂N

.

NH

13

26

TsN

25

N

H

TsN

O

N

O

H

N

H

CTP 2

(a)

NH

O

NH

O

H

N

NH₂

N

NH₂

H

O

.

(b)

NTs

15

Intermediate I

(b)

O

H

O

H

N

O

HN

NH

H₂N

NH

11

O

TsN

N

O

NTs

N

H

N

H

N

Intermediate II

CTP 2'

H

Figure 3. Partial spectrum of HMBC experiment in CTP 2.

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General Experimental Procedures:

Conclusions

Synthesis of 2-((tert-Butoxycarbonyl)amino)acetic acid (1)^[34]

In summary, we have developed a novel synthetic method for

the preparation of cyclic α₃β-tetrapeptides as histone

deacetylases (HDACs) inhibitor analogs via copper(I)- catalyzed

formation of ketenimine intermediate, which immediately

undergoes intramolecular cyclization by nucleophilic attack to

form the corresponding tosyl-amidine structure. The cyclization

afforded two forms of the cyclic beta peptide analogs with

adequate yields. To the best of our knowledge, this is the first

reported method that enables the direct formation of α₃β CTP

analogs via head-to-tail cyclizations. Further, various allyl group

transformation could allow the structural diversity of the cyclic

α₃β-tetrapeptides analogs to be explored. Due to the stability of

amidines, the corresponding transformations to amides or other

functional groups were rarely addressed. There has been a

successful example of convertin amidine to amide using

concentrated HCl.^[33]However, the development of milder

alternatives to furnish native CTPs is ongoing in our group.

Furthermore, tosyl amidine analogs of CTPs are new and have

never been studied. Therefore, with the synthetic method

developed in this research, a variety of CTPs analogs could be

synthesized and carefully used in the screening for biological

activities. We believe that this method is an attractive option for

the construction of cyclic α₃β-tetrapeptides as histone

deacetylase (HDAC) inhibitors analogs.

To a stirred solution of sodium hydroxide (1600 mg, 40 mmol) in water

(54 mL) was added glycine (2000 mg, 26.66 mmol) and di-tert-butyl

dicarbonate (7.35 mL, 32 mmol) at rt, and reacted for 16 h under nitrogen

atmosphere. After completion of the reaction monitored by TLC, the pH

was adjusted to 2-3 by 1N HCl, and then extracted with ethyl acetate (50

mL x 2). The combined the organic layers were dried over with MgSO₄,

filtered and concentrated under reduced pressure. The desired product

(1) was afforded (4483 mg, 96%) as a white solid. mp 87-90 C; ¹H NMR

(300 MHz, CDCl₃) δ 5.02 (br, 1H), 3.96 (s, 2H), 1.46 (s, 9H); ¹³C NMR

(75 MHz, CDCl₃) δ 174.9, 156.2, 80.7, 42.5, 28.5; IR (KBr) ν 3354, 2980,

1729, 1714, 1251 cm⁻¹

; HRMS (ESI-TOF) calcd for C₇H₁₃NO₄Na

[M+Na]⁺198.0742, found 198.0737.

Synthesis of tert-Butyl (2-oxo-2-(prop-2-yn-1-ylamino)ethyl)carba-

mate (2)^[35]

To a stirred solution of Boc-Gly-OH (1) (2000 mg, 11.42 mmol) in N,N-

dimethylformamide (40 mL) was added N-(3-Dimethylaminopropyl)-N’-

ethylcarbodiimide hydrochloride (EDC·HCl) (4376 mg, 22.83 mmol), and

1-Hydroxybenzotriazole hydrate (HOBt) (3085 mg, 22.83 mmol) at rt and

allowed to stir for 20 min under nitrogen atmosphere. Next,

propargylamine (0.8 mL, 13.7 mmol) and triethylamine (4.8 mL, 34.26

mmol) were added and reacted at rt for 9 h under nitrogen atmosphere.

After completion of the reaction monitored by TLC, the reaction was

quenched by water (10 mL). The solvent was removed and the resulting

residue was extracted with ethyl acetate (30 mL x 2), 1N HCl (30 mL),

and aqueous sodium bicarbonate solution (30 mL). The combined the

organic layers were dried over with MgSO₄, filtered and concentrated

under reduced pressure to afford crude. The crude residue was purified

by silica gel column chromatography using 10% ethyl acetate in hexane

as a solvent system to afford the desired product (2) (2263 mg, 93%) as

a white solid. mp 116-118 C; ¹H NMR (300 MHz, MeOD) δ 3.98 (d, J =

1.6 Hz, 2H), 3.70 (s, 2H), 2.56 (s, 1H), 1.45 (s, 9H); ¹³C NMR (75 MHz,

MeOD) δ 172.4, 158.6, 80.9, 80.6, 72.3, 44.6, 29.5, 28.8; IR (KBr) ν 3307,

2980, 2124, 1668, 1251 cm⁻¹; HRMS (ESI-TOF) calcd for C₁₀H₁₆N₂O₃Na

[M+Na]⁺235.1053, found 235.1054.

Experimental Section

General Considerations. All reactions were performed under

an atmosphere of nitrogen and the workups were carried out in

air. All the solvents used for the condition optimization were

dried using reported procedures.^[32]Especially, N,N-

Dimethylformamide should be freshly prepared. Unless noted,

all materials were purchased from commercial suppliers and

used as received. Tosyl azide was prepared in house using

conventional procedure. Cuprisorb resin was purchased from

Synthesis of 2-Amino-N-(prop-2-yn-1-yl)acetamide (3)^[36]

1

To

a

stirred

solution

of

tert-Butyl

(2-oxo-2-(prop-2-yn-1-

Seachem Laboratory and dried in high vacuum before used. H

TM

&

¹³C NMR spectra were recorded on Bruker Ultrasheild

300

ylamino)ethyl)carbamate (2) (2000 mg, 9.42 mmol) in dichloromethane

(31 mL) was added trifluoroacetic acid (5 mL) and the resultant mixture

was stirred at rt for 60 min under nitrogen atmosphere. After completion

of the reaction monitored by TLC, solvent was removed under reduced

pressure and the resulting residue was extracted with water (20 mL) and

dichloromethane (20 mL). The combined aqueous layers were

concentrated under reduced pressure to afford the desired product (3)

(1130 mg, quant.) as a colorless liquid. ¹H NMR (300 MHz, MeOD) δ

4.03 (s, 2H), 3.67 (s, 2H), 2.64 (s, 1H); ¹³C NMR (75 MHz, MeOD) δ

167.1, 80.1, 72.9 ,41.6, 29.7; IR (KBr) ν 3325, 2978, 2191, 1748, 1713,

1223 cm⁻¹; HRMS (ESI-TOF) calcd for C₅H₉N₂O [M+H]⁺113.0715, found

113.0717.

& 75 MHz spectrometer and Varian UNITY INOVA 500 & 125

MHz spectrometer respectively. NMR data are reported as

follows: chemical shift, multiplicity (s = singlet, d = doublet, t =

triplet, q = quartet, m = multiplet), coupling constants (Hz).

1

Solvent residual peaks calibrations: for H NMR: CDCl₃: 7.2600

ppm, CD₃OD: 3.3100 ppm. For ¹³C NMR: CDCl₃: 77.23 ppm,

CD₃OD: 49.15 ppm. Melting Points of the products were

measured in open capillary tubes using Fargo Melting Point

Apparatus MP-2D. Infra-Red spectra were recorded using

Perkin Elmer 100 FTIR Spectrometer. High Resolution Mass

Spectra (HRMS) were performed on an Electronspray Ionization

Time-of-Flight (ESI-TOF), Fast Atom Bombardment (FAB), and

Electron Ionization (EI) mass spectrometer. Nominal and exact

m/z values are reported in Daltons. Flash chromatography was

performed using silica gel (43-60 m, Merck).

Synthesis of tert-Butyl (2-oxo-2-((2-oxo-2-(prop-2-yn-1-ylamino)-

ethyl)amino)ethyl)carbamate (4)^[37]

To a stirred solution of Boc-Gly-OH (1) (1577 mg, 9.0 mmol) in N,N-

dimethylformamide (20 mL) was added N-(3-Dimethylaminopropyl)-N’-

ethylcarbodiimide hydrochloride (EDC·HCl) (2588 mg, 13.5 mmol), and

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1-Hydroxybenzotriazole hydrate (HOBt) (1824 mg, 13.5 mmol) at rt and

allowed to stir for 20 min under nitrogen atmosphere. Next, 2-amino-N-

(prop-2-yn-1-yl)acetamide (3) (1211 mg, 10.8 mmol) and triethylamine

(3.10 mL, 22.5 mmol) were added and stirring continued at rt for 10 h

under nitrogen atmosphere. After completion of the reaction monitored by

TLC, the reaction was quenched by water (5 mL), the solvent was

removed under reduced pressure. The resulting residue was extracted

with ethyl acetate (30 mL x 2), 1N HCl (30 mL), and aqueous sodium

bicarbonate solution (30 mL). The combined the organic layers were

dried over with MgSO₄, filtered and concentrated under reduced pressure

to afford crude. The crude residue was purified by silica gel column

chromatography using 20% ethyl acetate in hexane as a solvent system

to afford the desired product (4) (2060 mg, 85%) as a white solid. mp

147-150 C; ¹H NMR (300 MHz, MeOD) δ 3.98 (d, J = 2.5 Hz, 2H), 3.87

(s, 2H), 3.73 (s, 2H), 2.56 (t, J = 2.4 Hz, 1H), 1.46 (s, 9H); ¹³C NMR (75

MHz, MeOD) δ 173.3, 171.4, 158.9, 81.2, 80.5, 72.4, 45.1, 43.4, 29.6,

28.9; IR (KBr) ν 3307, 2980, 2121, 1680, 1245 cm⁻¹; HRMS (ESI-TOF)

calcd for C₁₂H₁₉N₃O₄Na [M+Na]⁺292.1268, found 292.1268.

sodium bicarbonate solution (30 mL). The combined the organic layers

were dried over with MgSO₄, filtered and concentrated under reduced

pressure to afford crude. The crude residue was purified by silica gel

column chromatography using 10% ethyl acetate in hexane as a solvent

system to afford the desired product (7) (2328 mg, 95%) as a colorless

liquid. ¹H NMR (300 MHz, MeOD) δ 3.96 (s, 2H), 3.75 (s, 2H), 3.72 (s,

3H), 1.45 (s, 9H); ¹³C NMR (75 MHz, MeOD) δ 173.2, 171.9, 158.5, 80.9,

52.8, 44.6, 41.9, 28.8; IR (KBr) ν 2979, 1748, 1696, 1215 cm⁻¹; HRMS

(ESI-TOF) calcd for C₁₀H₁₈N₂O₅Na [M+Na]⁺269.1108, found 269.1109.

Synthesis of Methyl 2-(2-aminoacetamido)acetate (8)^[41]

To a stirred solution of methyl (tert-Butoxycarbonyl)glycylglycinate (7)

(2000 mg, 8.12 mmol) in dichloromethane (27 mL) was added

trifluoroacetic acid (5 mL) and the resultant mixture was stirred at rt for 40

min under nitrogen atmosphere. After completion of the reaction

monitored by TLC, the solvent was removed and then extracted with

water (20 mL) and dichloromethane (20 mL). The combined the aqueous

layers were concentrated under reduced pressure. The desired product

was afforded (8) (1242 mg, quant.) as a white solid. mp 134-137 C; ¹H

NMR (300 MHz, MeOD) δ 4.03 (s, 2H), 3.76-3.72 (m, 5H); ¹³C NMR (75

MHz, MeOD) δ 171.7, 168.0, 52.9, 42.0, 41.5; IR (KBr) ν 3244, 2963,

1743, 1680, 1203 cm⁻¹; HRMS (ESI-TOF) calcd for C₅H₁₁N₂O₃[M+H]⁺

147.0770, found 147.0768

Synthesis of Methyl 2-((tert-butoxycarbonyl)amino)acetate (5)^[38]

To a stirred solution of Boc-Gly-OH (1) (2000 mg, 11.42 mmol) in N,N-

dimethylformamide (18 mL) was added potassium carbonate (3160 mg,

22.84 mmol). The solution of iodomethane (6480 mg, 45.69 mmol) in

N,N-dimethylformamide (20 mL) was added dropwise by addition funnel.

The reaction was allowed to stir at rt for 12 h under nitrogen atmosphere.

After completion of the reaction monitored by TLC, the solvent was

removed under reduced pressure and then extracted with water (15 mL)

and ethyl acetate (30 mL x 2). The combined organic layers were dried

over with MgSO₄, filtered and concentrated under reduced pressure. The

desired product was afforded (5) (2100 mg, 97%) as a colorless liquid. ¹H

NMR (300 MHz, CDCl₃) δ 4.99 (br, 1H), 3.92 (d, J = 5.3 Hz, 2H), 3.75 (s,

3H), 1.45 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.0, 155.9, 80.2, 52.4,

42.5, 28.5; IR (KBr) ν 2979, 1748, 1714, 1211 cm⁻¹; HRMS (EI) calcd for

C₈H₁₅NO₄Na [M+Na]⁺212.0893, found 212.0896.

Synthesis of Methyl 2,2-dimethyl-4,7,10,17-tetraoxo-14-(tosylimino)-

3-oxa-5,8,11,15,18-pentaazaicosan-20-oate (9)

To a stirred solution of methyl glycylglycinate (8) (200 mg, 1.37 mmol) in

cosolvent (N,N-dimethylformamide (6 mL) and dichloromethane (6 mL))

was

added

tert-butyl

(2-oxo-2-((2-oxo-2-(prop-2-yn-1-

ylamino)ethyl)amino)ethyl)carbamate (4) (300 mg, 1.11 mmol),

potassium carbonate (308 mg, 2.23 mmol), tosyl azide (243 mg, 1.24

mmol) followed by copper iodide (106 mg, 0.56 mmol) and the resultant

mixture was stirred at rt for 2 h under nitrogen atmosphere. After

completion of the reaction monitored by TLC, the reaction mixture was

treated with cuprisorb resin (800 mg) for 30 min to remove the copper

Synthesis of Methyl 2-aminoacetate (6)^[39]

traces, filtered through

a pad of Celite, washed with excess

To a stirred solution of methyl (tert-Butoxycarbonyl)glycinate (5) (2000

mg, 10.58 mmol) in dichloromethane (26 mL) was added trifluoroacetic

acid (4 mL) and the resultant mixture was stirred at rt for 30 min under

nitrogen atmosphere. After completion of the reaction monitored by TLC,

the solvent was removed and then extracted with water (20 mL) and

dichloromethane (20 mL). The combined aqueous layers were

concentrated under reduced pressure. The desired product was afforded

(6) (988 mg, quant.) as a white solid. mp 162-165 C; ¹H NMR (300 MHz,

MeOD) δ 3.84 (s, 3H), 3.83 (s, 2H); ¹³C NMR (75 MHz, MeOD) δ 169.1,

53.6, 41.0; IR (KBr) ν 3417, 2962, 1754, 1184 cm⁻¹; HRMS (EI) calcd for

C₃H₇NO₂[M]⁺89.0477, found 89.0474.

dichloromethane (10 mL) and combined filtrate was concentrated under

reduced pressure. Then filtrate dissolved with ethyl acetate (50 mL) and

extracted with water (50 mL), combined the organic layers were dried

over with MgSO₄, filtered and concentrated under reduced pressure to

afford the crude. The crude residue was purified by silica gel column

chromatography using 50 % ethyl acetate in hexane as a solvent system

to afford the desired product (9) (315 mg, 48%) as a yellow liquid. ¹H

NMR (500 MHz, MeOD) δ 7.75 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.1 Hz,

2H), 3.98 (s, 2H), 3.89 (s, 2H), 3.86 (s, 2H), 3.75 (s, 2H), 3.71 (s, 3H),

3.61 (t, J = 6.4 Hz, 2H), 2.97 (t, J = 6.4 Hz, 2H), 2.41 (s, 3H), 1.45 (s,

9H) ; ¹³C NMR (125 MHz, MeOD) δ 173.5, 172.2, 171.8, 171.4, 169.1,

158.8, 144.1, 142.2, 130.5, 127.4, 81.1, 52.8, 45.8, 45.0, 43.8, 42.0, 38.4,

34.9, 28.9, 21.6; IR (KBr) ν 3302, 2933, 1748, 1668, 1273 cm⁻¹; HRMS

(ESI-TOF) calcd for C₂₄H₃₆N₆O₉SNa [M+Na]⁺607.2162, found 607.2153.

Synthesis of Methyl 2-(2-((tert-butoxycarbonyl)amino)acetamido)-

acetate (7)^[40]

Synthesis of Methyl 2,2-dimethyl-4,7-dioxo-11-(tosylimino)-3-oxa-

5,8,12-triazatetradecan-14-oate (10)

To a stirred solution of Boc-Gly-OH (1) (2102 mg, 12 mmol) in N,N-

dimethylformamide (35 mL) was added N-(3-Dimethylaminopropyl)-N’-

ethylcarbodiimide hydrochloride (EDC·HCl) (4379 mg, 22.84 mmol), and

1-Hydroxybenzotriazole hydrate (HOBt) (3086 mg, 22.84 mmol). The

reaction was stirred for 20 min at rt under nitrogen atmosphere. Next,

methyl glycinate (6) (892 mg, 10 mmol) and triethylamine (4.20 mL, 30

mmol) were added and stirring continued at rt for 9 h under nitrogen

atmosphere. After completion of the reaction monitored by TLC, the

reaction was quenched by water (5 mL), solvent was removed and then

extracted with ethyl acetate (30 mL x 2), 1N HCl (30 mL), and aqueous

To a stirred solution of Methyl glycinate (6) (302 mg, 3.39 mmol) in

cosolvent (N,N-dimethylformamide (17 mL) and dichloromethane (17

mL))

was

added

tert-Butyl

(2-oxo-2-(prop-2-yn-1-

ylamino)ethyl)carbamate (2) (720 mg, 3.39 mmol), potassium carbonate

(938 mg, 6.79 mmol), tosyl azide (736 mg, 3.73 mmol) followed by

copper iodide (323 mg, 1.70 mmol) and the resultant mixture was stirred

at rt for 1 h under nitrogen atmosphere. After completion of the reaction

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monitored by TLC, the reaction mixture was treated with cuprisorb resin

(2000 mg) for 30 min to remove the copper traces, filtered through a pad

of Celite, washed with excess dichloromethane (20 mL) and the

combined filtrate was concentrated under reduced pressure. Then the

filtrate was dissolved with ethyl acetate (80 mL) and extracted with water

(80 mL). The combined the organic layers were dried over with MgSO₄,

filtered and concentrated under reduced pressure to afford the crude.

The crude residue was purified by silica gel column chromatography

using 50 % ethyl acetate in hexane as a solvent system to afford the

desired product (10) (1357 mg, 85%) as a yellow solid. mp 56-58 C; ¹H

NMR (300 MHz, MeOD) δ 7.73 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.2 Hz,

2H), 4.00 (s, 2H), 3.69 (s, 2H), 3.64 (s, 3H), 3.59 (t, J = 6.7 Hz, 2H), 2.97

(t, J = 6.7 Hz, 2H), 2.41 (s, 3H), 1.45 (s, 9H); ¹³C NMR (75 MHz, MeOD)

δ 173.0, 171.2, 168.9, 158.7, 144.2, 142.1, 130.5, 127.4, 81.0, 52.9, 44.9,

12

h

under nitrogen atmosphere. After completion of the reaction

monitored by TLC, the reaction was quenched by water (10 mL),

concentrated under reduced pressure to remove solvent and extracted

with ethyl acetate (20 mL x 2) and water (20 mL). The combined organic

layers were dried over with MgSO₄, filtered and concentrated under

reduced pressure to afford the crude. The crude residue was purified by

silica gel column chromatography using 10% ethyl acetate in hexane as

a solvent system to afford the desired product (18) (553 mg, 60%) as a

colorless liquid. ¹H NMR (300 MHz, CDCl₃) δ 4.62 (br, 1H), 4.43 (br, 1H),

2.25 (s, 1H), 1.89-1.73 (m, 1H), 1.57-1.48 (m, 2H), 1.45 (s, 9H), 0.97-

0.89 (m, 6H); ¹³C NMR (75 MHz, CDCl₃) δ 155.0, 84.1, 80.1, 70.9, 45.4,

41.5, 28.6, 25.2, 22.9, 22.1; IR (KBr) ν 3314, 2934, 2872, 2117, 1705,

1699, 1368, 1249, 1171 cm⁻¹; HRMS (ESI-TOF) calcd for C₁₂H₂₁NO₂Na

[M+Na]⁺234.1470, found 234.1467.

44.3, 38.5, 34.6, 28.8, 21.5; IR (KBr) ν 3300, 2932, 1747, 1275 cm⁻¹

;

HRMS (ESI) calcd for C₂₀H₃₀N₄O₇SNa [M+Na]⁺493.1733, found

493.1742.

Synthesis of (S)-5-Methylhex-1-yn-3-amine (19)^[43]

To a stirred solution of tert-Butyl (S)-(5-methylhex-1-yn-3-yl)carbamate

(18) (800 mg, 3.79 mmol) in dichloromethane (15 mL) was added

trifluoroacetic acid (3 mL) and the resultant mixture was stirred at rt for 30

min under nitrogen atmosphere. After completion of the reaction

monitored by TLC, the solvent was removed under reduced pressure,

then extracted with water (20 mL) and dichloromethane (20 mL). The

combined aqueous layers were concentrated under reduced pressure.

The desired product was afforded (19) (445 mg, quant.) as a colorless

liquid. ¹H NMR (300 MHz, CDCl₃) δ 4.02-3.92 (m, 1H), 2.53 (d, J = 1.8

Hz, 1H), 1.95-1.82 (m, 1H), 1.82-1.69 (m, 1H), 1.67-1.54 (m, 1H), 1.02-

0.85 (m, 6H); ¹³C NMR (75 MHz, CDCl₃) δ 78.0, 76.4, 42.2, 42.2, 25.1,

23.0, 21.1; IR (KBr) ν 3312, 3257, 2964, 2127, 1672, 1530, 1378, 1203,

1142 cm⁻¹; HRMS (ESI-TOF) calcd for C₇H₁₄N [M+H]⁺112.1126, found

112.1124.

Synthesis of (S)-tert-Butyl (1-(methoxy(methyl)amino)-4-methyl-1-

oxopentan-2-yl)carbamate (16)^[42]

To

a stirred solution of Boc-L-Leu-OH (2313 mg, 10 mmol) in

dichloromethane (45 mL) was added N-(3-Dimethylaminopropyl)-N’-

ethylcarbodiimide hydrochloride (EDC·HCl) (2876 mg, 15 mmol), and 1-

Hydroxybenzotriazole hydrate (HOBt) (2027 mg, 15 mmol). The reaction

was stirred at rt for 20 min under nitrogen atmosphere. Next, N,O-

Dimethylhydroxylamine hydrochloride (1171 mg, 12 mmol) and

triethylamine (4.18 mL, 30 mmol) were added and stirring continued at rt

for 12 h under nitrogen atmosphere. After completion of the reaction

monitored by TLC, the reaction was quenched by water (10 mL), the

solvent was removed and then extracted with ethyl acetate (30 mL x 2),

1N HCl (30 mL), and aqueous sodium hydrogen carbonate solution (30

mL). The combined organic layers were dried over with MgSO₄, filtered

and concentrated under reduced pressure to afford crude. The crude

residue was purified by silica gel column chromatography using 50%

ethyl acetate in hexane as a solvent system to afford the desired product

(16) (2632 mg, 96%) as a white solid. mp 87-90 C; ¹H NMR (300 MHz,

CDCl₃) δ 5.04 (br, 1H), 4.72 (br, 1H), 3.78 (s, 3H), 3.20 (s, 3H), 1.78-1.65

(m, 1H), 1.52-1.32 (m, 11H), 0.99-0.91 (m, 6H); ¹³C NMR (75 MHz,

CDCl₃) δ174.2, 155.9, 79.7, 61.8, 49.2, 42.3, 32.4, 28.6, 25.0, 23.6, 21.8;

IR (KBr) ν 3328, 2980, 1756, 1668, 1368, 1212, 1176 cm⁻¹; HRMS (FAB)

calcd for C₁₃H₂₇N₂O₄[M+H]⁺275.1971, found 275.1977.

Synthesis of tert-Butyl ((S)-3-(1H-indol-3-yl)-1-(((S)-5-methylhex-1-

yn-3-yl)-amino)-1-oxopropan-2-yl)carbamate (20)

To a stirred solution of Boc-L-Trp-OH (1016 mg, 3.34 mmol) in N,N-

dimethylformamide (11 mL) was added N,N,N’,N’-Tetramethyl-O-(1H-

benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (1900 mg, 5.01

mmol). The reaction was stirred for 20 min at rt. Then the solution (8 mL)

of (S)-5-methylhex-1-yn-3-amine (300 mg, 2.7 mmol) in N,N-

dimethylformamide and triethylamine (0.94 mL, 2.70 mmol) were added

and stirring continued at rt for 9 h under nitrogen atmosphere. After

completion of the reaction monitored by TLC, water (5 mL) was added to

quench the reaction and solvent was removed. The resulting residue was

dissolved with ethyl acetate (30 mL), extracted with 1N HCl (30 mL), and

brine (30 mL x 2). The combined organic layers were dried over with

MgSO₄, filtered and concentrated under reduced pressure to afford the

crude. The crude residue was purified by silica gel column

chromatography using 10% ethyl acetate in hexane as a solvent system

to afford the desired product (20) (1073 mg, quant.) as a yellow liquid. ¹H

NMR (300 MHz, CDCl₃) δ 8.11 (br, 1H), 7.69 (d, J = 7.7 Hz, 1H), 7.36 (d,

J = 8.0 Hz, 1H), 7.28-7.10 (m, 3H), 7.07 (br, 1H), 5.88 (br, 1H), 4.70 (dd,

J = 15.0, 7.5 Hz, 1H), 4.46-4.39 (m, 1H), 3.36-3.10 (m, 2H), 2.16 (d, J =

1.7 Hz, 1H), 1.75-1.60 (m, 1H), 1.43 (s, 9H), 1.41-1.32 (m 2H), 0.92-0.82

(m 6H); ¹³C NMR (75 MHz, CDCl₃) δ 170.8, 155.7, 136.5, 127.6, 123.6,

122.6, 120.1, 119.2, 111.4, 110.8, 83.3 ,80.4, 71.1 55.3, 44.8, 39.9, 28.5,

25.1, 22.8, 22.1; IR (KBr) ν 3308, 2959, 2872, 1695, 1661, 1506 1368,

1248, 1168 cm⁻¹; HRMS (ESI-TOF) calcd for C₂₃H₃₁N₃O₃Na [M+Na]⁺

420.2263 found 420.2256.

Synthesis of (S)-tert-Butyl (5-methylhex-1-yn-3-yl)carbamate (18)^[42]

A stirred solution of tert-Butyl (S)-(1-(methoxy(methyl)amino)-4-methyl-1-

oxopentan-2-yl)carbamate (16) (1200 mg, 4.37 mmol) in tetrahydrofuran

(15 mL) was cooled to 0 ℃ using a ice bath. Lithium aluminium hydride

(61 mg x 3, 4.81 mmol) was added and stirring continued for 20 min at

0

℃ under nitrogen atmosphere. After completion of the reaction

monitored by TLC, water (6 mL) and 1N HCl (12 mL) were added to

quench reaction and then extracted with ethyl acetate (20 mL x 2). The

combined the organic layers were dried over with MgSO₄, filtered and

concentrated under reduced pressure. The tert-Butyl (S)-(4-methyl-1-

oxopentan-2-yl)carbamate (17) crude residue was afforded (flask A).

Compound (17) was directly used without further purification. Next, to

another flask (flask B) containing

a stirred solution of Dimethyl-2-

oxopropylphosponate (1090 mg, 6.56 mmol) in acetonitrile (11 mL) was

added tosyl azide (1294 mg, 6.56 mmol) and potassium carbonate (1209

mg, 8.75 mmol). The reaction was stirred at rt for 2 h under nitrogen

atmosphere, when completion of the reaction was observed by TLC.

Then the crude residue (17) in flask A was dissolved with methanol (5

mL). The completed reaction mixture (flask B) was added into the flask A

by addition funnel at 0 C with ice bath. Next, the reaction reacted at rt for

Synthesis of (S)-2-Amino-3-(1H-indol-3-yl)-N-((S)-5-methylhex-1-yn-

3-yl) propanamide (11)

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To

a

stirred solution of tert-Butyl ((S)-3-(1H-indol-3-yl)-1-(((S)-5-

monitored by TLC, the reaction was concentrated under reduced

pressure to remove the solvent then extracted with water (10 mL) and

dichloromethane (20 mL). The combined aqueous layers and

concentrated under reduced pressure. The desired product was afforded

(21) (1165 mg, quant.) as a colorless liquid. ¹H NMR (300 MHz, CDCl₃) δ

5.90-5.73 (m, 1H), 5.30-5.16 (m, 2H), 4.28-4.22 (m, 1H), 4.08-3.92 (m,

2H), 3.92-3.85 (m, 2H), 3.83 (s, 3H); ¹³C NMR (75MHz, CDCl₃) δ 168.1,

133.3, 118.8, 72.6, 66.5, 53.8, 53.7; IR (KBr) ν 3398, 2923, 2855, 1755,

1679, 1532 1442, 1245, 1203, 1135 cm⁻¹; HRMS (ESI-TOF) calcd for C_7-

H₁₄NO₃[M+H]⁺160.0974, found 160.0973.

methylhex-1-yn-3-yl)amino)-1-oxopropan-2-yl)carbamate (20) (1600 mg,

4.02 mmol) in dichloromethane (20 mL) was added trifluoroacetic acid (4

mL) and the resultant mixture was stirred for 60 min at rt under nitrogen

atmosphere. After completion of the reaction monitored by TLC, solvent

was removed and then the pH was adjusted to 8-9 by 1N NaOH.

Following extraction with ethyl acetate (20 mL), the combined organic

layers were dried over with MgSO₄, filtered and concentrated under

reduced pressure to afford crude. The crude residue was purified by

silica gel column chromatography using 10% methanol in

dichloromethane as a solvent system to afford the desired product (11)

(1220 mg, quant.) as a yellow liquid. ¹H NMR (300 MHz, MeOD) δ 7.68

(d, J = 7.7 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.22-7.04 (m, 3H), 4.69 (td, J

= 7.8, 1.8 Hz, 1H), 3.99 (t, J = 6.6 Hz, 1H), 3.38-3.16 (m, 2H), 2.68 (d, J =

2.2 Hz, 1H), 1.84-1.71 (m, 1H), 1.63-1.42 (m, 2H), 0.93 (d, J = 6.6 Hz,

6H) ; ¹³C NMR (75 MHz, MeOD) δ 170.6, 138.4, 128.6 125.8, 122.9,

120.4, 119.4, 112.6, 108.3, 83.8, 72.7, 55.3, 45.5, 41.0, 29.4, 26.2, 23.0,

22.4; IR (KBr) ν 3404, 3395, 2876, 2109, 1705, 1685, 1528, 1207, 1139

cm⁻¹; HRMS (EI) calcd for C₁₈H₂₂N₃O [M-H]⁺296.1763, found 296.1769.

Synthesis of (S)-Methyl 2-(2-((tert-butoxycarbonyl)amino)propan-

amido)acetate (22)^[47]

To a stirred solution of (tert-Butoxycarbonyl)-L-alanine (1667 mg, 8.81

mmol) in N,N-dimethylformamide (35 mL) was added N,N,N’,N’-

Tetramethyl-O-(1H-benzotriazol-1-yl)uronium

hexafluorophosphate

(HBTU) (5010 mg, 13.21 mmol) and reacted for 20 min at rt under

nitrogen atmosphere. Then the solution (8 mL) of methyl glycinate (6)

(941 mg, 10.57 mmol) dissolved in N,N-dimethylformamide and N,N-

diisopropylethylamine (3.1 mL, 17.62 mmol) were added and the stirring

was continued for 9 h at rt under nitrogen atmosphere. After completion

of the reaction monitored by TLC, water (5 mL) was added to quench the

reaction and concentrated under reduced pressure. The resulting residue

was dissolved with ethyl acetate (30 mL), extracted with 1N HCl (30 mL),

and brine (30 mL x 2). The combined organic layers were dried over with

MgSO₄, filtered and concentrated under reduced pressure to afford the

crude. The crude residue was purified by silica gel column

chromatography using 10% ethyl acetate in hexane as a solvent system

to afford the desired product (22) (2275 mg, 99%) as a colorless liquid.

¹H NMR (300 MHz, MeOD) δ 4.11 (br, 1H), 3.94 (dd, J = 25.7, 17.6 Hz,

2H), 3.72 (s, 3H), 1.45 (s, 9H), 1.33 (d, J = 7.2 Hz, 3H); ¹³C NMR (75

MHz, MeOD) δ 176.6, 171.8, 157.8, 80.8, 52.7, 51.7, 42.0 28.8, 18.5; IR

(KBr) ν 3323, 2981, 1755, 1696, 1369, 1250, 1168 cm⁻¹; HRMS (ESI-

TOF) calcd for C₁₁H₂₅N₂O₅Na [M+Na]⁺283.1270, found 283.1263.

Synthesis of (S)-Methyl 3-(allyloxy)-2-aminopropanoate (21)^[44-46]

To a stirred solution of L-serine methyl ester hydrochloride (1560 mg,

10.0 mmol) in dichloromethane (13.3 mL) was added triethylamine (1.67

mL, 12.0 mmol) at 0 ℃ with ice bath. When the stirred solution became

clear. Then the solution of trityl chloride (3070 mg, 11.0 mmol) in

dichloromethane (20 mL) was added dropwise by addition funnel and the

reaction was reacted 2 h under nitrogen atmosphere. After completion of

the reaction monitored by TLC, solvent was removed partially. Following

direct wet-loading the crude residue was purified by silica gel column

chromatography using 3% methanol in dichloromethane as a solvent

system to afford the desired product (S)-methyl 3-hydroxy-2-

(tritylamino)propanoate (21-1) (2890 mg, 80%) as a white solid. mp 148-

151 C; ¹H NMR (300 MHz, CDCl₃) δ 7.52-7.43 (m 6H), 7.31-7.24 (m,

6H), 7.24-7.15 (m, 3H), 3.77-3.64 (m, 1H), 3.62-3.51 (m, 2H), 3.30 (s,

3H), 2.98 (br, 1H), 2.27 (br, 1H); ¹³C NMR (75 MHz, CDCl₃) δ 174.1,

145.8, 129.0, 128.1 126.8, 71.2, 65.2, 58.0, 52.2; IR (KBr) ν 3445, 3958,

Synthesis of (S)-Methyl 3-(allyloxy)-2-((S)-2-((tert-butoxycarbonyl)-

amino)propan amido)propanoate (23)

2951, 1773, 1596, 1490, 1205 cm⁻¹

C₂₃H₂₃NO₃Na [M+Na]⁺384.1576, found 384.1575.

; HRMS (ESI-TOF) calcd for

To a stirred solution of (tert-Butoxycarbonyl)-L-alanine (1500 mg, 7.93

mmol) in N,N-dimethylformamide (16 mL) was added N,N,N’,N’-

To a stirred solution of sodium hydride (284 mg, 11.83 mmol) in N,N-

dimethylformamide (15 mL) at 0 ℃ (ice bath) was added allyl bromide

(1.5 mL, 17.36 mmol) into the mixture. Then the solution (10 mL) of (S)-

Methyl 3-hydroxy-2-(tritylamino)propanoate (21-1) (2850 mg, 7.89 mmol)

dissolved in N,N-dimethylformamide was added dropwise by addition

funnel. The reaction was reacted 1 h under nitrogen atmosphere. After

completion of the reaction monitored by TLC, aqueous sodium

bicarbonate solution (10 mL) was added to quench the reaction.

Following extraction with brine (30 mL) and ether (30 mL x 2), the

combined organic layers were dried over with MgSO₄, filtered and

concentrated under reduced pressure. The desired product was afforded

(S)-Methyl 3-(allyloxy)-2-(tritylamino)propanoate (21-2) (2910 mg, 92%)

as a colorless liquid. ¹H NMR (300 MHz, CDCl₃) δ 7.55-7.46 (m 6H),

7.31-7.23 (m, 6H), 7.23-7.13 (m 3H), 5.95-5.77 (m, 1H), 5.32-5.12 (m,

2H), 4.04-3.90 (m ,2H), 3.81-3.72 (m 1H), 3.61-3.43 (m, 2H), 3.22 (s, 3H),

2.77 (br, 1H); ¹³C NMR (75 MHz, CDCl₃) δ 174.5, 146.1, 134.6, 129.0,

128.0, 126.6, 117.1, 72.9, 72.2, 71.1, 56.6, 51.9; IR (KBr) ν 3321, 3058,

2949, 2924, 1737, 1646, 1596, 1491, 1328, 1205 cm⁻¹; HRMS (ESI-TOF)

calcd for C₂₆H₂₈NO₃[M+H]⁺402.2069, found 402.2072.

Tetramethyl-O-(1H-benzotriazol-1-yl)uronium

hexafluorophosphate

(HBTU) (4511 mg, 11.90 mmol). The reaction was reacted 20 mins at rt

under nitrogen atmosphere. Then the solution (8 mL) of methyl glycinate

(1147 mg, 7.21 mmol) dissolved in N,N-dimethylformamide and N,N-

diisopropylethylamine (2.5 mL, 14.41 mmol) were added and the stirring

was continued for 9 h at rt under nitrogen atmosphere. After completion

of the reaction monitored by TLC, water (5 mL) was added to quench the

reaction and concentrated under reduced pressure. The resultant residue

was dissolved with ethyl acetate (30 mL), extracted with 1N HCl (30 mL),

and brine (30 mL x 2). The combined organic layers were dried over with

MgSO₄, filtered and concentrated under reduced pressure to afford the

crude. The crude residue was purified by silica gel column

chromatography using 10% ethyl acetate in hexane as a solvent system

to afford the desired product (23) (2005 mg, 84%) as a colorless liquid.

¹H NMR (300 MHz, CDCl₃) δ 6.79 (br, 1H), 5.91-5.74 (m, 1H), 5.29-5.14

(m 2H), 5.04 (br, 1H), 4.76-4.65 (m, 1H), 4.21 (br, 1H), 4.04-3.91 (m, 2H),

3.91-3.84 (m, 1H), 3.76 (s, 3H), 3.68-3.61 (m, 1H), 1.45 (s, 9H), 1.38 (d,

J = 7.1 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 172.8, 170.6, 155.4, 134.1,

117.6, 80.1, 72.3, 69.6, 52.7, 52.7, 50.2, 28.4, 18.7; IR (KBr) ν 3319,

2980, 1748, 1668, 1516, 1368, 1248, 1168 cm⁻¹; HRMS (ESI-TOF) calcd

for C₁₅H₂₆N₂O₆Na [M+Na]⁺353.1689, found 353.1691.

To a stirred solution of (S)-Methyl 3-(allyloxy)-2-(tritylamino)propanoate

(21-2) (2900 mg, 7.22 mmol) in dichloromethane (20 mL) was added

trifluoroacetic acid (4 mL) and the resultant mixture was stirred for 30 min

at rt under nitrogen atmosphere. After completion of the reaction

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Synthesis of (S)-2-(2-((tert-Butoxycarbonyl)amino)propanamido)-

3.31-3.30 (m, 2H), 2.57 (d, J = 2.2 Hz, 1H), 1.80-1.63 (m, 1H), 1.54-1.42

(m, 1H), 1.42 (s, 9H), 1.27 (d, J = 7.1 Hz, 3H), 0.93-0.85 (m, 6H); ¹³C

NMR (75 MHz, MeOD) δ 176.7, 172.9, 171.4, 158.0, 138.2, 129.0, 124.8,

122.5, 120.0, 119.5, 112.4, 110.8, 84.1, 81.0, 72.1, 55.5, 52.1, 45.4, 43.9,

40.8, 28.9, 26.1 23.0, 22.5, 18.2; IR (KBr) ν 3286, 2957 2108, 1655, 1509,

1247, 1164 cm⁻¹; HRMS (ESI-TOF) calcd for C₂₈H₃₉N₅O₅Na [M+Na]⁺

548.2849, found 548.2854.

acetic acid (12)^[48]

To a stirred solution of methyl (tert-Butoxycarbonyl)-L-alanylglycinate

(22) (1500 mg, 5.76 mmol) in methanol (20 mL) was added 1N NaOH (10

mL) and the resultant mixture was stirred for 8 h at rt under nitrogen

atmosphere. After completion of the reaction monitored by TLC, the

reaction was concentrated under reduced pressure to remove methanol,

and then extracted with dichloromethane (30 mL). The aqueous layers

were collected and the pH was adjusted to 2-3 by 2N HCl. Following

extraction with dichloromethane (20 mL x 2), the combined organic layers

were dried over with MgSO₄, filtered and concentrated under reduced

pressure. The desired product was afforded (12) (1280 mg, 90%) as a

colorless liquid. ¹H NMR (300 MHz, MeOD) δ 4.11 (br, 1H), 3.98-3.84 (m,

2H), 1.44 (s, 9H), 1.33 (d, J = 7.2 Hz, 3H), ¹³C NMR (75 MHz, MeOD) δ

176.4, 172.9, 157.8, 80.8, 51.7, 41.9, 28.8, 18.6; IR (KBr) ν 3324, 2981,

Synthesis of tert-Butyl ((S)-1-(((S)-1-(((S)-3-(1H-indol-3-yl)-1-(((S)-5-

methylhex-1-yn-3-yl)amino)-1-oxopropan-2-yl)amino)-3-(allyloxy)-1-

oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (25)

To

a

stirred

solution

of

(S)-3-(Allyloxy)-2-((S)-2-((tert-

butoxycarbonyl)amino) propanamido) propanoic acid (13) (1600 mg, 5.06

mmol) in N,N-dimethylformamide (10 mL) was added N,N,N’,N’-

Tetramethyl-O-(1H-benzotriazol-1-yl)uronium

hexafluorophosphate

1667, 1257, 1369, 1251, 1176 cm⁻¹

; HRMS (ESI-TOF) calcd for

(HBTU) (2410 mg, 6.07 mmol) and reacted for 20 min at rt under nitrogen

atmosphere. Then the solution (6 mL) of (S)-2-Amino-3-(1H- indol-3-yl)-

N-((S)-5-methylhex-1-yn-3-yl)propenamide (11) (1360 mg, 4.60 mmol) in

N,N-dimethylformamide and N,N-diisopropylethylamine (1.6 mL, 9.21

mmol) was added and allowed to stir for further 4 h at rt under nitrogen

atmosphere. After completion of the reaction monitored by TLC, water (5

mL) was added to quench the reaction and the reaction was

concentrated under reduced pressure. The resulting residue was

dissolved with ethyl acetate (30 mL), extracted with 1N HCl (30 mL), and

brine (30 mL x 2). The combined organic layers were dried over with

MgSO₄, filtered and concentrated under reduced pressure to afford the

crude. The crude residue was purified by silica gel column

chromatography using 20% ethyl acetate in hexane as a solvent system

to afford the desired product (25) (2055 mg, 75%) as a yellow liquid. ¹H

NMR (300 MHz, MeOD) δ 7.58 (d, J = 7.7 Hz, 1H), 7.32 (d, J = 8.1 Hz,

1H), 7.15-6.90 (m, 3H), 5.92-5.74 (m, 1H), 5.29-5.10 (m, 2H), 4.72-4.60

(m, 2H), 4.58-4.48 (m, 1H), 4.46-4.34 (m, 1H), 3.99-3.93 (m, 2H), 3.76-

3.57 (m, 2H), 3.29-3.16 (m, 2H), 2.59 (d, J = 2.3 Hz, 1H), 1.75-1.63 (m,

1H), 1.51-1.43 (m 2H), 1.38 (s, 9H), 1.19 (d, J = 7.2 Hz, 3H), 0.89 (dd, J

= 6.6, 1.9 Hz, 6H); ¹³C NMR (75 MHz, MeOD) δ 176.4, 172.6, 172.0,

138.2, 135.7, 129.0, 125.0, 124.7, 122.6, 120.0, 119.6, 118.0, 112.4,

110.7, 84.1, 81.1, 73.4, 72.2, 70.1, 55.4, 52.1, 45.5, 40.8, 30.8, 28.8,

28.7, 26.1, 22.9, 22.5, 17.9; IR (KBr) ν 3286, 2957, 2108, 1655, 1509,

1247, 1164 cm⁻¹; HRMS (ESI-TOF) calcd for C₃₃H₄₅N₅O₆Na [M+Na]⁺

618.3268, found 618.3275.

C₁₀H₁₈N₂O₅Na [M+Na]⁺269.1113, found 269.1117.

Synthesis of (S)-3-(Allyloxy)-2-((S)-2-((tert-butoxycarbonyl)amino)-

propanamido) propanoic acid (13)

To

a

stirred solution of (S)-Methyl 3-(allyloxy)-2-((S)-2-((tert-

butoxycarbonyl) amino)propanamido)propanoate (23) (1900 mg, 5.75

mmol) in methanol (20 mL) was added 1N NaOH (8 mL) and the

resultant mixture was stirred for 8 h at rt under nitrogen atmosphere.

After completion of the reaction monitored by TLC, the reaction was

concentrated under reduced pressure to remove methanol, and then

extracted with dichloromethane (30 mL). The aqueous layers were

collected and the pH was adjusted to 2-3 by 2N HCl. Following extraction

with dichloromethane (20 mL x 2), the combined organic layers were

dried over with MgSO₄, filtered and concentrated under reduced pressure.

The desired product was afforded (13) (1676 mg, 92%) as a colorless

liquid. ¹H NMR (300 MHz, CDCl₃) δ 7.06 (br, 1H), 5.91-5.74 (m, 1H),

5.29-5.14 (m, 2H), 4.78-4.65 (m, 1H), 4.27 (br, 1H), 3.99 (d, J = 5.7 Hz,

2H), 3.95-3.87 (m, 1H), 3.73-3.63 (m, 1H), 1.43 (s, 9H), 1.37 (d, J = 7.1

Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 173.5, 172.6, 155.9, 134.0, 118.2,

80.8, 72.6, 69.3, 52.8, 50.5, 28.5, 18.6; IR (KBr) ν 3320, 2980, 1751,

1669, 1520, 1367, 1248, 1210 cm⁻¹

; HRMS (ESI-TOF) calcd for

C₁₄H₂₄N₂O₆Na [M+Na]⁺339.1532, found 339.1533.

Synthesis of tert-Butyl ((S)-1-((2-(((S)-3-(1H-indol-3-yl)-1-(((S)-5-

methylhex-1-yn-3-yl)amino)-1-oxopropan-2-yl)amino)-2-oxoethyl)-

amino)-1-oxopropan-2-yl)carbamate (24)

Synthesis of (S)-2-(2-((S)-2-Aminopropanamido)acetamido)-3-(1H-

indol-3-yl)-N-((S)-5-methylhex-1-yn-3-yl)propenamide (14)

To a stirred solution of tert-Butyl ((S)-1-((2-(((S)-3-(1H-Indol-3-yl)-1-(((S)-

5-methylhex-1-yn-3-yl)amino)-1-oxopropan-2-yl)amino)-2-

To a stirred solution of (tert-Butoxycarbonyl)-L-alanylglycine (12) (1200

mg, 4.87 mmol) in N,N-dimethylformamide (10 mL) was added N,N,N’,N’-

oxoethyl)amino)-1-oxopropan-2-yl)carbamate (24) (1587 mg, 3.02 mmol)

in dichloromethane (15 mL) was added trifluoroacetic acid (5 mL) and the

resultant mixture was stirred at rt for 60 min under nitrogen atmosphere.

After completion of the reaction monitored by TLC, the reaction was

concentrated under reduced pressure then the pH was adjusted to 8-9 by

1N NaOH, and concentrated under reduced pressure to afford the crude.

The crude residue was purified by silica gel column chromatography

using 10% methanol in dichloromethane as a solvent system to afford the

desired product (14) (1295 mg, quant.) as a yellow solid. mp 131-133 C;

¹H NMR (300 MHz, MeOD) δ 7.61 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 8.1 Hz,

1H), 7.14-6.97 (m, 3H), 4.71-4.59 (m, 2H), 3.92-3.76 (m, 2H), 3.52 (dd, J

= 13.7, 6.9 Hz, 1H), 3.30-3.10 (m, 2H), 2.60 (d, J = 2.3Hz, 1H), 1.80-1.62

(m, 1H), 1.57-1.40 (m, 1H), 1.27 (d, J = 7.0 Hz, 3H), 0.94-0.85 (m, 6H);

¹³C NMR (75 MHz, MeOD) δ 178.6, 172.8, 171.3, 138.1, 129.0, 125.0,

122.6, 120.1, 119.5, 112.4, 110.5, 84.2, 72.2, 55.5, 51.4, 45.4, 43.7, 40.8,

29.0, 26.1, 23.0, 22.5, 20.9; IR (KBr) ν 3402, 2962, 2117, 1680, 1532

Tetramethyl-O-(1H-benzotriazol-1-yl)uronium

hexafluorophosphate

(HBTU) (2215 mg, 5.84 mmol) and reacted 20 min at rt. Then the

solution (6 mL) of (S)-2-Amino-3-(1H-indol-3-yl)-N-((S)-5-methylhex-1-yn-

3-yl)propenamide (11) (1310 mg, 4.43 mmol) in N,N-dimethylformamide

and N,N-diisopropylethylamine (1.5 mL, 8.86 mmol) was added and

stirring was continued for 5 h at rt under nitrogen atmosphere. After

completion of the reaction monitored by TLC, water (5 mL) was added to

quench the reaction and the reaction was concentrated under reduced

pressure. The resulting residue was dissolved with ethyl acetate (30 mL),

extracted with 1N HCl (30 mL), and brine (30 mL x 2). The combined

organic layers were dried over with MgSO₄, filtered and concentrated

under reduced pressure to afford the crude. The crude residue was

purified by silica gel column chromatography using 20% ethyl acetate in

hexane as a solvent system to afford the desired product (24) (1750 mg,

75%) as a yellow solid. mp 203-206 C; ¹H NMR (300 MHz, MeOD) δ

7.59 (d, J = 7.6 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.12-6.97 (m, 3H),

4.69-4.59 (m, 2H), 4.02 (dd, J = 14.4, 7.1 Hz, 1H), 3.89-3.73 (m, 2H),

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1440, 1206 cm⁻¹

426.2505, found 426.2515.

;

HRMS (ESI-TOF) calcd for C₂₃H₃₂N₅O₃[M+H]⁺

Synthesis of N-((3S,6S,9S,13S)-3-((1H-Indol-3-yl)methyl)-6-((allyl-

oxy)methyl)-13-isobutyl-9-methyl-2,5,8-trioxo-1,4,7,10-tetraaza-

cyclotridecan-11-ylidene)-4-methylbenzenesulfonamide (CTP2)

Synthesis of (S)-N-((S)-3-(1H-Indol-3-yl)-1-(((S)-5-methylhex-1-yn-3-

yl)amino)-1-oxo-propan-2-yl)-3-(allyloxy)-2-((S)-2-aminopropan-

amido)propenamide (15)

To a stirred solution of (S)-N-((S)-3-(1H-Indol-3-yl)-1-(((S)-5-methylhex-1-

yn-3-yl)amino)-1-oxopropan-2-yl)-3-(allyloxy)-2-((S)-2-

aminopropanamido)propenamide (15) (89 mg, 0.18 mmol) in cosolvent

(N,N-dimethylformamide (18 mL) and dichloromethane (18 mL)) was

added potassium carbonate (50 mg, 0.36 mmol), tosyl azide (53 mg, 0.27

mmol) followed by copper iodide (34 mg, 0.18 mmol) under nitrogen

atmosphere, and the resultant mixture was stirred at rt for 1.5 h. After

completion of the reaction monitored by TLC, the reaction mixture was

treated with cuprisorb resin (100 mg) for 30 min to remove the copper

To a stirred solution of tert-Butyl ((S)-1-(((S)-1-(((S)-3-(1H-indol-3-yl)-1-

(((S)-5-methylhex-1-yn-3-yl)amino)-1-oxopropan-2-yl)amino)-3-(allyloxy)-

1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (25) (1200 mg,

2.01 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (4

mL) and the resultant mixture was stirred at rt for 60 min under nitrogen

atmosphere. After completion of the reaction monitored by TLC, the

reaction was concentrated under reduced pressure then the pH was

adjusted to 8-9 by 1N NaOH, and concentrated under reduced pressure

to afford the crude. The crude residue was purified by silica gel column

chromatography using 10% methanol in dichloromethane as a solvent

system to afford the desired product (15) (1007 mg, quant.) as a white

solid. mp 214-217 C; ¹H NMR (300 MHz, MeOD) δ 7.60 (d, J = 7.7 Hz,

1H), 7.32 (d, J = 8.1 Hz, 1H), 7.13-6.98 (m, 3H), 5.83-5.65 (m, 1H), 5.30-

5.14 (m, 2H), 4.69-4.60 (m, 2H), 4.43 (t, J = 5.2 Hz, 1H), 4.05-3.91 (m,

2H), 3.72-3.64 (m, 1H), 3.64-3.56 (m, 1H), 3.34 (q, J = 6.9 Hz, 1H), 3.30-

3.15 (m, 2H), 2.61 (d, J = 2.3 Hz, 1H), 1.78-1.58 (m, 1H), 1.55-1.35 (m,

2H), 1.11 (d, J = 6.9 Hz, 3H), 0.88 (d, J = 6.5 Hz, 6H); ¹³C NMR (75 MHz,

MeOD) δ 178.7, 172.5, 171.8, 138.1, 135.7, 129.0, 125.1, 122.6, 120.1,

119.6, 118.0, 112.5, 110.2, 84.1, 73.4, 72.3, 70.3, 55.4, 55.2, 51.3, 45.4,

40.8, 28.7, 26.1, 23.0, 22.5, 21.1; IR (KBr) ν 3286, 3056, 2961, 2917,

1509, 1247, 1164 cm⁻¹; HRMS (ESI-TOF) calcd for C₂₇H₃₈N₅O₄[M+H]⁺

496.2924, found 496.2919.

traces, filtered through

a pad of Celite, washed with excess

dichloromethane (10 mL) and the combined filtrate was concentrated

under reduced pressure. Then the filtrate was dissolved with ethyl

acetate (30 mL) and extracted with water (30 mL). The combined organic

layers were dried over with MgSO₄, filtered and concentrated under

reduced pressure to afford the crude. The crude residue was purified by

silica gel column chromatography using

5

%

methanol in

dichloromethane as a solvent system to afford the desired product CTP2

(41 mg, 35%) as a yellow liquid. ¹H NMR (500 MHz, MeOD) δ 7.72 (d, J

= 8.2 Hz, 2H), 7.59 (d, J = 7.8 Hz, 1H), 7.36-7.29 (m, 3H), 7.11-6.97 (m,

3H), 5.97-5.85 (m, 1H), 5.32-5.13 (m, 2H), 4.75-4.66 (m, 3H), 4.05-3.94

(m, 3H), 3.83-3.78 (m, 1H), 3.64-3.58 (m, 1H), 3.36-3.32 (m, 1H), 3.22-

3.15 (m, 1H), 3.07-3.01 (m, 1H), 2.61 (t, J = 12.9 Hz, 1H), 2.40 (s, 3H),

1.43-1.35 (m, 1H), 1.28 (d, J = 6.7 Hz, 3H), 1.03-0.85 (m, 2H), 0.69-0.62

(m, 6H); ¹³C NMR (125 MHz, MeOD) δ 174.3, 173.8, 171.7, 167.1, 144.2,

142.0, 138.2, 136.0, 130.7, 128.7, 127.2, 124.5, 122.6, 120.0, 119.5,

117.8, 112.4, 110.7, 73.5, 68.1, 56.5, 53.5, 51.8, 49.9, 45.3, 39.9, 28.5,

25.7, 23.1, 22.6, 21.6, 16.3; IR (KBr) ν 3296, 3061, 2955, 2929, 1652,

1548, 1457 cm⁻¹; HRMS (ESI-TOF) calcd for C₃₄H₄₄N₆O₆SNa [M+Na]⁺

687.2941, found 687.2939.

Synthesis of N-((3S,9S,13S)-3-((1H-Indol-3-yl)methyl)-13-isobutyl-9-

methyl-2,5,8-trioxo-1,4,7, 10-tetraazacyclotridecan-11-ylidene)-4-me-

thylbenzenesulfonamide (CTP 1)

To a stirred solution of (S)-2-(2-((S)-2-Aminopropanamido)acetamido)-3-

(1H-indol-3-yl)-N-((S)-5-methylhex-1-yn-3-yl)propanamide (14) (50 mg,

0.12 mmol) in cosolvent (N,N-dimethylformamide (12 mL) and

dichloromethane (12 mL)) was added potassium carbonate (32 mg, 0.24

mmol), tosyl azide (35 mg, 0.18 mmol) followed by copper iodide (23 mg,

0.12 mmol) under nitrogen atmosphere, and the resultant mixture was

stirred at rt for 1.5 h. After completion of the reaction monitored by TLC,

the reaction mixture was treated with cuprisorb resin (80 mg) for 30 min

to remove the copper traces, filtered through a pad of Celite, washed with

excess dichloromethane (10 mL) and the combined filtrate was

concentrated under reduced pressure. Then the filtrate was dissolved

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

combined organic layers were dried over with MgSO₄, filtered and

concentrated under reduced pressure to afford the crude. The crude

residue was purified by silica gel column chromatography using 5 %

methanol in dichloromethane as a solvent system to afford the desired

product CTP1 (22 mg, 32%) as a yellow liquid. ¹H NMR (500 MHz,

MeOD) δ 7.71 (d, J = 8.2 Hz, 2H), 7.60 (d, J = 7.9 Hz, 1H), 7.35-7.29 (m,

3H), 7.12-6.98(m, 3H), 4.76-4.68 (m, 2H), 4.36(d, J = 14.2 Hz, 1H), 4.12-

4.04 (m, 1H), 3.38-3.32 (m, 1H), 3.28 (d, J = 14.2Hz, 1H), 3.19-3.12 (m,

1H), 3.09-3.03 (m 1H), 2.56(t, J = 12.9Hz, 1H), 2.40 (s, 3H), 1.45-1.38 (m,

1H), 1.30 (d, J = 6.9Hz, 3H), 1.03-0.96 (m, 1H), 0.94-0.85 (m, 1H), 0.71-

0.65 (m, 6H); ¹³C NMR (125 MHz, MeOD) δ 174.5, 173.7, 172.0, 167.1,

144.2, 142.0, 138.2, 130.7, 128.7, 127.2, 124.4, 122.6, 120.0, 119.5,

112.4, 110.6, 56.8, 51.9, 49.7, 45.6, 45.3, 40.0, 28.6, 25.7, 23.1 22.7,

21.6, 16.3; IR (KBr) ν 3333, 3083, 2980, 2936, 1747, 1683, 1680, 1540,

Acknowledgements

This work is supported by grants from Ministry of Science and

Technology (MOST104-2738-M-110-001-, MOST104-2113-M-

110-001- and MOST104-2627-M-007-003-) and National Sun

Yat-sen University (04B39131 and 04B39132).

Keywords: Macrocyclization •β-amino acid •Cyclic Tetrapeptide

•HDAC inhibitor analogs •ketenimines

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Chun-Chi Chen, Sheng-Fu Wang, Yung-

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Cu(І)-Mediated Denitrogenative

Macrocyclization for the Synthesis of

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Cyclic α₃β-Tetrapeptide Analogs

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Copper(I)-Mediated Denitrogenative Macrocyclization for the Synthesis of Cyclic α3β-Tetrapeptide Analogues

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