A combinatorial approach towards pharmaceutically relevant cyclic peptides - Chapter 4: Synthesis of 1,5-connected triazole-containing cyclic pseudotetrapeptides

A combinatorial approach towards pharmaceutically relevant cyclic peptides

Springer, J.

Publication date

2008

Link to publication

Citation for published version (APA):

Springer, J. (2008). A combinatorial approach towards pharmaceutically relevant cyclic

peptides.

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4.1 Introduction

Although the use of the copper-catalyzed 1,3-dipolar cycloaddition reaction between

an azide and an alkyne providing 1,4-disubstituted triazoles has received much attention in the

last years,

1-4

the use of cycloaddition reactions between azides and alkynes leading to

1,5-disubstituted triazoles is still limited. While the 1,4-1,5-disubstituted triazole has been shown to

be a good mimic for a trans amide bond (Chapter 3),

5,6

the 1,5-substituted triazole has been

speculated as a cis amide bond surrogate.

7

Introduction of these moieties in cyclic peptides

locks the backbone in a specific conformation and may subsequently lead to enhanced

receptor selectivities and activities.

Scheme 4.1 Synthesis of 1,2,3-triazoles.

N N N R 1 R2 NN N R1 NNN R1 R2 _R2 Δ [Ru] 1,4-isomer 1,5-isomer 1 1 4 4 5 5 NN N R1 R2 1,5-isomer 1 4 5

The 1,5-disubstituted triazole can be traditionally accessed via a classical Huisgen 1,3-dipolar

cycloaddition reaction (Scheme 4.1),

8

generally leading to a mixture of both isomers of the

triazoles. Using tethered azides and alkynes

9-12

1,5-disubstituted triazoles are obtained

exclusively, induced by the formation of a six-membered ring. For example, azidoacetamide 1

could efficiently be closed to the 1,5-disubstituted triazole-containing cyclic dipeptide 2 in

71% yield, simply by heating in toluene (Scheme 4.2).

9

The cyclic peptide 2 could be

subsequently opened by treatment with concentrated HCl and immediate protection of the

amine, to give 1,5-connected containing amino acid 3. This 1,5-disubstituted

triazole-containing amino acid 3 was incorporated in peptoid oligomers. In solution, these peptoids

resulted in the formation of hairpin structures around the 1,5-substituted triazole, indicating a

β-turn around the triazole linkage. This confirmed the ability of the 1,5-disubstituted triazole

linkage in mimicking a cisoid-like amide bond, as these bonds also often result in the

formation hairpin-like structures and

β-turns in peptides.

Scheme 4.2 Synthesis of 1,5-substituted triazoles by an intramolecular thermal cycloaddition

reaction using a tethered azide/alkyne.

HN O N3 C6H5CH3 ,Δ 71% HN N O N N 1) HCl, H2O 80 οC 2) Fmoc-OSu dioxane, K2CO3(aq) 65% (over 2 steps) FmocHN N O N N HO 1 2 3

However, in this example only the use of unsubstituted alkynes and azides was shown and the

reactions required harsh reaction conditions. In a different approach, the 1,5-disubstituted

1,2,3-triazoles could also be accessed by the reaction of bromomagnesium acetylides to

azides,

13

from which the intermediates could be trapped by different electrophiles. Although

the original process

14

was optimized in terms of yield and substrate scope, the use was still

limited because of the sensitive substrates and intermediates. In a similar fashion,

trimethylsilylacetylenes reacted with organoazides to exclusively furnish 1,5-substituted

triazoles.

15

Analogous to the copper-catalyzed cycloaddition between azides and alkynes leading to the

selective formation of 1,4 substituted triazoles, a catalytic version of the reaction between

azides and alkynes selectively leading to 1,5-substituted triazoles was investigated. In 2005

Zhang et al. first published a ruthenium-catalyzed version of this reaction (Scheme 4.3).

16

After an initial screening of some ruthenium catalysts like Ru(OAc)

2

(PPh

3

)

2

and

CpRu(PPh

3

)

2

Cl (entry 1 and 2), the reaction between benzyl azide 4 and phenylacetylene 5 in

the presence of the catalyst Cp

*

Ru(PPh

3

)

2

Cl (entry 3) in benzene at 80

ο

C led to the exclusive

formation of the 1,5-disubstituted triazole 6 in a good yield.

Scheme 4.3 Ruthenium-catalyzed cycloaddition reaction between an azide and an alkyne.

Ru Cl N H R1 R2 N N Ru Cl N R2 N N R1 Ru Cl N N N R2 R1 Ru Cl L L or + N N N R1 R2 N3 [Ru] N N N N N N + 1 Ru(OAc)2(PPh3)2 - 100% 2 CpRu(PPh3)2Cl 85% 15% 3 Cp*Ru(PPh3)2Cl 100% -+ 4 5 6a 6b A B C D E

The ruthenium-catalyzed reaction proved to be suited not only for terminal alkynes, but also

for internal alkynes,

17

providing 1,4,5-trisubstituted triazoles. This suggests a different

mechanism compared to the copper-catalyzed version

18

in which the first step in the catalytic

cycle is the formation of an end-on coordinated copper-acetylide species. The complete

mechanism of the ruthenium-catalyzed reaction is still under investigation, but a hypothesis

was made by Zhang,

16

based on the cyclotrimerization process of alkynes.

19,20

That is,

oxidative coupling of an alkyne and an azide on the ruthenium catalyst might initially give a

ruthenacycle (Scheme 4.3, with B more likely then C), which undergoes reductive elimination

releasing the triazole product E.

The examples shown by Zhang only describe simple azides and alkynes. Recently, this

reaction has been used in pseudopeptide synthesis, aiming at a cis-prolyl peptide bond

derivative.

7

The moiety Xaa

−ψ(1,5-triazole)−Ala could be a Xaa−cis−Pro dipeptide isostere,

retaining the stereochemistry, a similar hybridization and a similar number of non-hydrogen

atoms. Several ruthenium-catalyzed cycloaddition reactions were employed with different

amino acid derivatives containing azides and alkynes (Table 4.1). The amino alkynes 7a-g

and azido esters 8a-g (entry 1-7) provided the 1,5-disubstituted triazole-containing

pseudodipeptides 9a-g in moderate to high yield. Some of the resulting triazole-containing

pseudodipeptides were incorporated into the enzyme Bovine pancreatic nuclease replacing a

parent type VI

β-turn. The resulting semi-synthetic enzymes were evaluated for their activity

and, indeed, all of the proteins retained full catalytic activity. In addition, CD-spectroscopy

showed no alteration of the secondary structure of the semi-synthetic enzyme. The catalytic

values of the proteins were independent of the Xaa-residue, whereas in the wild-type enzyme

the propensity of cis-trans isomerization of the amide bond between Xaa and Pro (influenced

by the nature of the Xaa-residue) strongly affected the catalytic activity of the protein.

Table 4.1 Synthesis of 1,5-disubstituted triazole-containing dipeptides.

N H R PG1 ₊ N3 OPG2 O CH3 Cp*_RuCl(COD) 4 mol% N N HN PG1 OPG2 N R CH3 O 7 8 9

entry R PG1 PG2 solvent temp yield(%)

1 H Boc tBu toluene rt 45

2 CH2CONH2(Trt) Boc tBu dioxane 60 οC 62

3 CH2CONH2(Trt) Boc Bn dioxane 60 οC 55

4 CH3 Boc tBu toluene rt 67

5 CH2Ph Boc tBu toluene rt 90

6 CH(CH3)2 Boc tBu toluene rt 92

7 CH(CH3)2 Fmoc tBu toluene rt 91

Although it would be very interesting to investigate the influence of this turn-inducing

element on the properties of cyclic peptides, the 1,5-connected 1,2,3-triazoles have not yet

been applied in the synthesis of cyclic peptides. Therefore, we envisioned a 1,5-connected

1,2,3-triazole incorporated into a cyclic tetrapeptide (Scheme 4.4).

Cyclo-[Pro

−Val−Pro−Tyr]

21

10 was chosen as the model compound, as previously several analogues

were made containing 1,4-disubstituted triazoles (also, Chapter 3).

22,23

In target molecule

cyclo-[Pro

−ψ(triazole)−Gly−Pro−Tyr] 11 the parent amide bond between proline and valine

was replaced by a 1,5-disubstituted triazole and the valine residue was replaced by simple

glycine. In cyclo-[Tyr

−Pro−Gly−ψ(triazole)−Gly] 12 the secondary amide bond between

valine and proline was replaced by a 1,5-disubstituted triazole, with two glycine residues at

the sides. Finally, in cyclo-[Tyr

−Pro−Val−ψ(triazole)−Gly] 13 the same secondary amide

bond between valine and proline was replaced by a 1,5-disubstituted triazole, retaining the

parent valine adjacent to the triazole linkage.

Scheme 4.4 1,5-Disubstituted 1,2,3-triazole-containing cyclic tetrapeptides derived from

cyclo-[Pro

−Val−Pro−Tyr] 10.

N NH N HN O O O O Me Me HO cyclo-[Pro_{−Val−Pro−Tyr]} 10 N N N HN O O O HO cyclo-[Pro−Ψ(triazole)−Gly−Pro−Tyr] 11 N NH N HN O O O Me Me HO cyclo-[Tyr_{−Pro−Val−Ψ(triazole)−Gly]} 13 N NH N HN O O O HO cyclo-[Tyr_{−Pro−Gly−Ψ(triazole)−Gly]} 12 N N N N N N

The ruthenium-catalyzed cycloaddition reaction is not suited for difficult ring closure-type

reactions, because this procedure has to be performed at a high concentration (~0.5-1.0 M).

Therefore, the final cyclization step was planned via a classical lactamization to obtain the

cyclic lactams 14 (Scheme 4.5). This is preferably done opposite to the 1,5-substituted

triazole, as this should facilitate the cyclization because of the proposed cisoid character of

this bond. This positions the mutually reactive end-groups in close proximity. The linear

precursors 15 can be obtained from the two dipeptide fragments bearing the alkyne 16 and the

azide 17 by the key ruthenium-catalyzed cycloaddition reaction. These dipeptide fragments

should be easily accessible from the corresponding amino acids and amino acid derivatives by

well-known transformations.

Scheme 4.5 Strategy for the synthesis of 1,5-connected 1,2,3-triazole-containing cyclic

peptides.

NH N HN HN O R2 O O R1 R3 N N R4 H2N H N O R2 R1 N N N R4 N H O CO2H R3 PGHN H N O R2 R1 N3 R4 N H O R3 OPG O + 14 15 16 17

4.2

Towards the synthesis of cyclo-[Pro

−ψ

−ψ

−ψ

−ψ(triazole)−−−−Gly−−−−Pro−−−−Tyr] 11

The synthesis of the required alkyne dipeptide fragment N-Boc

−Tyr(OBn)−Pro−≡ (19)

started from known N-Boc

−Pro−≡ (18, see Section 3.7).

24,25

Deprotection by treatment with

TFA and subsequent coupling

26,27

with commercially available N-Boc

−Tyr(OBn)−OH

provided the desired dipeptide N-Boc

−Tyr(OBn)−Pro−≡ (19) in 97% yield over two steps.

Scheme 4.6 Synthesis of alkyne-containing dipeptide fragment 19.

BocHN N O BnO N Boc _{1) TFA, CH} 2Cl2 2) N-Boc−Tyr(OBn)−OH EDCI, HOBt 97% 19 18

The azide-containing dipeptide fragment 21 was made in one step from azido glycine 20

28,29

by coupling with H

−Pro−OtBu in 94% using EDCI and HOBt (Scheme 4.7). Although the use

of small molecular weight azides is reported to be hazardous,

30,31

so far, in our hands azido

glycine proved to be relatively safe to handle, although precaution has to be taken.

Scheme 4.7 Synthesis of azide-containing dipeptide fragment 21.

N₃ N O CO2tBu H−Pro−OtBu EDCI, HOBt 94% N3 O OH 20 21

With the two dipeptide fragments 19 and 21 in hand, the stage was set for the

ruthenium-catalyzed cycloaddition reaction. Coupling of the two peptide fragments 19 and 21 was

performed by reaction of both fragments in benzene at a concentration of 1 M with the

ruthenium catalyst at elevated temperatures (Scheme 4.8). This cleanly afforded the

1,5-disubstituted triazole-containing linear tetrapeptide 22 in 99% yield. The reaction was

relatively fast and only took one hour to go to completion. The reaction was performed on a 2

g scale with a relatively low catalyst loading (0.01 mol%).

Scheme 4.8 Ruthenium-catalyzed cycloaddition reaction between the dipeptide fragments 19

and 21.

BocHN N O N N N N O CO_2tBu BocHN N O N₃ N O CO_2tBu BnO BnO + Cp*Ru(PPh3)2Cl 2mol% C6H6, 1M 99% 19 21 22

Simultaneous deprotection of both the C- and N-termini of the linear precursor 22 by

treatment with TFA afforded the linear precursor as a TFA salt, ready for lactamization

towards the desired cyclic peptide 11. However, all attempts to afford the monocyclic product

failed. On the contrary, by lactamization with HATU and DIPEA considerable amounts of the

dimeric cyclic product 23 were obtained (25% yield). Coupling conditions using EDCI/HOBt,

PyBOP/DIPEA or PyBOP/DIPEA with the use of a syringe pump all provided only dimeric

products, without a trace of the monomeric cyclic pseudopeptide.

Scheme 4.9 Macrolactamization of linear precursor 22.

BocHN N O N N N N O CO2tBu N N N HN O O O BnO N N BnO 1) TFA, CH2Cl2 98% 2) HATU,DIPEA CH2Cl2 N N N NH O O O OBn N N 22 N N N O O N N N H O OBn 23 25% 11 0%

Re-evaluation of the design of the target molecule 11 led to the conclusion that the termini of

the linear precursor are probably not in close proximity (Scheme 4.10, B), preventing the

formation of a peptide bond between the C- and N-termini of the linear precursor. In fact,

three turn-inducing elements have been incorporated, two prolines and the 1,5-connected

triazole, increasing the helical pitch of the linear peptide too far. On the contrary, this favours

the coupling with a second linear peptide, leading to the formation of dimeric products C.

Scheme 4.10 Proposed overcrossing of the linear precursor leading to the formation of

dimers.

H2N COOH NH2 HOOC HN C O HN C NH C O O A B C

4.4 Synthesis

of

cyclo-[Tyr

−−−−Pro−−−−Gly−ψ

−ψ

−ψ(triazole)−−−−Gly] 12

−ψ

As the previous strategy only led to the formation of dimers, a new strategy was set

up. In this case, the tertiary amide bond of one of the proline units was replaced by the

1,5-disubstituted triazole, leading to the target molecule cyclo-[Tyr

−Pro−Gly−ψ(triazole)−Gly]

12. The linear precursor for the final lactamization would have alternating trans-cis-trans

amide bonds, thus ultimately favouring the formation of monomeric cyclic products. For

simplicity reasons, glycine was chosen instead of the valine residue to validate this new

strategy. We expected that replacement of the valine by a glycine would not effect the

biological activity, as only the tyrosine residue is conserved in tyrosinase inhibitors. The

linear precursor for 12 required the synthesis of two new dipeptide fragments containing the

azide and the alkyne. The C-terminal tert-butyl protective group from the initial strategy was

replaced by a methyl ester, because of the commercial availability of H-Tyr(OBn)-OMe.

However, this ester cannot be deprotected simultaneously with the N-Boc protective group

under acidic conditions and a two step deprotection has to be employed to provide the

unprotected linear precursor from the protected tetrapeptide precursor.

Scheme 4.11 Synthesis of azide dipeptide fragment.

N3 H N O 1) H−Tyr(OBn)−OMe EDCI, HOBt 2) NaN₃, DMF, _Δ 52% (2 steps) Cl O OH 24 25 CO₂Me OBn

The synthesis of required the azide-containing dipeptide fragment started from

α-chloroacetic

acid 24 (Scheme 4.11). EDCI mediated coupling with H

−Tyr(OBn)−OMe and subsequent

nucleophilic replacement of the chloride by an azide by treatment with NaN

3

in DMF

32

provided the dipeptide N

3

−Gly−Tyr(OBn)−OMe 25 in 52% yield over two steps. A second

route can also be employed by coupling of azido glycine 20 to H

−Tyr(OBn)−OMe to provide

the dipeptide fragment, but in the former route the use of potentially explosive azido glycine

is avoided.

Scheme 4.12 Synthesis of 1,5-connected triazole-containing cyclic peptide 12.

BocN H N O N N N N H O OMe O + RuCp*Cl(PPh3)2 5mol% C₆H₆, 1M 62% BocHN OBn 26 BocHN N N N N H O CO₂Me OBn 1) TFA, CH2Cl2 2) N-Boc−Pro−OH EDCI, HOBt 86% over two steps

1) LiOH, THF 2) TFA, CH2Cl2 HN H N O N N N N H O CO2H OBn TFA DPPA NaHCO3, DMF

87% over three steps N NH O N N N HN O O OR 30 R = Bn H2, Pd/C 99% 27 28 29 N3 H N O 25 CO2Me OBn 12 R = H

A stepwise procedure was chosen to assemble the required linear precursor, but addition of a

dipeptide fragment to the azide-containing dipeptide fragment 25 should also be possible.

Thus, coupling of N-Boc protected propargylamine 26 to N

3

−Gly−Tyr(OBn)−OMe 25 using

the ruthenium catalyst RuCp

*

Cl(PPh

3

)

2

cleanly provided the 1,5-disubstituted

triazole-containing tripeptide 27 in moderate yield of 62% (Scheme 4.12). Deprotection of the N-Boc

terminal group by treatment with TFA in CH

2

Cl

2

and subsequent coupling with

N-Boc

−Pro−OH using EDCI and HOBt gave the protected linear tetrapeptide 28 in a good 86%

yield over two steps. Cleavage of the protective groups, first by saponification with lithium

hydroxide to liberate the C-terminus and subsequent treatment with TFA in CH

2

Cl

2

to liberate

the N-terminus gave the linear tetrapeptide 29 as its TFA salt.

Now the final macrolactamization of the linear precursor should provide the desired cyclic

product. Several condition for macrolactamization of linear peptides to cyclic product have

been described using a wide range of coupling conditions.

33-35

One of the oldest and still most

successful coupling reagents, diphenyl phosphoryl azide (DPPA), was picked as the method

of choice. Solid NaHCO

3

was chosen as the base for the neutralization of the TFA-salt of the

linear precursor. Thus, macrolactamization using the coupling reagent DPPA and NaHCO

3

as

the base under high dilution (6.10

-3

) in DMF gave the desired monocyclic lactam 30 in a good

yield of 87% over three steps. Final cleavage of the benzyl protective group by hydrogenation

provided the target compound 12 in quantitative yield.

4.3 Synthesis

of

cyclo-[Tyr

−−−−Pro−−−−Val−ψ

−ψ

−ψ

−ψ(triazole)−−−−Gly] 13

Synthesis of the dipeptide fragment N-Boc

−Pro−Val−≡ 36 started from

N-Boc

−Val−OH 31 (Scheme 4.13). Although the synthesis of enantiopure N-Boc−Val−≡ 32 has

been reported earlier

36

starting from 31 via the aldehyde 34 using the Corey-Fuchs

transformation of the aldehyde to the alkyne, our initial attempts towards enantiopure alkyne

failed.

Scheme 4.13 Synthesis of alkyne-containing dipeptide fragment 36.

BocHN 1) TFA, CH2Cl2 2) _{N-Boc−Pro−OH} EDCI, HOBt 18% 35 36 Me Me N H Me Me O NBoc BocHN 34 Me Me BocHN 32 Me Me BocHN 31 Me Me O OH OH O H 1) pyridine 2) NaBH4, MeOH 92% (2 seps) N N N F F F (COCl)2 DMSO, DIPEA 88% BocHN Me Me O N OMe Me LiAlH4 Et2O 68% K2CO3 37, MeOH 56% 33 Me P O O N2 OMeOMe 37

Following the general route for the transformation of the carboxylic acid to the alkyne, similar

to proline (Section 3.7), starting from N-Boc

−Val−OH 31, the acid moiety was first reduced

to the alcohol 32 in 92% yield over two steps, followed by oxidation via a Swern reaction

providing the aldehyde 34 in 88% yield.

37-39

The aldehyde 34 could also be synthesized from

the commercially available Weinreb amide 33 by reduction with lithium aluminiumhydride.

40-42

Transformation of the aldehyde 34 to the alkyne using the Bestmann-Ohira reagent 37

43-45

provided the amino alkyne 35 in moderate yield of 56% (Scheme 4.13). The [

α]26

_{D measured}

for N-Boc

−Val−≡ 35 proved to be only -9.2, while -45 has been reported

36

for this compound.

Apparently, extensive racemization induced by methoxide during the reaction had occurred.

Nevertheless, by removing the N-Boc protective group by treatment with TFA in CH

2

Cl

2

and

subsequent coupling with N-Boc

−Pro−OH the desired dipeptide 36 could be obtained, from

which the undesired diastereomer could be removed by column chromatography to obtain the

desired dipeptide in 18% overall.

Scheme 4.14 Ruthenium-catalyzed cycloaddition reaction with dipeptide fragment 36 and

alkyne 35.

BocN H N O N N N N H O CO2Me N3 H N O + Cp*Ru(PPh3)2Cl 5mol% C₆H₆, 1M 36 25 37 CO2Me OBn N H Me Me O NBoc Me Me OBn Cp*Ru(PPh3)2Cl 5mol% C6H6, 1M 50% BocHN N N N N H O CO2Me Me Me OBn 38 N3 H N O 25 CO2Me OBn + BocHN 35 Me Me

No product was obtained from the ruthenium-catalyzed cycloaddition reaction between the

two dipeptide fragments 25 and 36 (Scheme 4.14). Fortunately, reaction of the N-Boc

−Val−≡

35 with the azido dipeptide 25 did provide the 1,5-disubstituted product 38 in a moderate 50%

yield as a mixture of diasteromers, due to the use of racemic 35.

Scheme 4.15 Synthesis of valine-alkyne by an enantioselective copper-catalyzed propargylic

amination reaction.

BocHN Me Me HN Me Me AcO Me Me MeO ligand 41/CuI DIPEA o-anisidine MeOH, 25 οC _{40 76%, 83 %ee} 1) PhI(OAc)2 2) (Boc)2O

69% over two steps

N O N N O Me Me ligand ₄₁ 39 35

A stereoselective route to the target N-Boc

−Val−≡ (35) was recently discovered by Detz in

our group. (Scheme 4.15).

46

Starting from propynyl acetate 39 propargyl amine 40 was

synthesized by a copper-catalyzed propargylic amination using o-anisidine and copper-pybox

complex. The propargylic amine 40 was obtained both in a good yield of 76% and

enantiomeric excess of 83%. The o-anisidine group was removed by treatment with

PhI(OAc)

247

and the amine was N-Boc protected in an overall yield of 69%. Evaluation of the

optical rotation of the N-Boc protected propargyl amine ([

α]

D

= -51.6) and comparison with

literature

36

revealed the proper S-configuration and enantiopurity.

With N-Boc

−Val−≡ 35 in hand, the linear precursor was elongated comparable to the other

linear precursors (Scheme 4.16). Thus, coupling of N-Boc

−Val−≡ 35 to the azido-containing

dipeptide fragment 25 by the ruthenium catalyzed cycloaddition reaction cleanly provided the

1,5-disubstituted triazole containing tripeptide 38, albeit in moderate yield of 51%. Removal

of the N-Boc terminal group by treatment with TFA in CH

2

Cl

2

and subsequent coupling with

N-Boc

−Pro−OH using EDCI and HOBt gave the protected linear tetrapeptide 42 in 89% over

two steps.

Scheme 4.16 Synthesis of 1,5-connected triazole-containing cyclic peptide 13.

BocN H N O N N N N H O CO2Me + RuCp*Cl(PPh3)2 5mol% C₆H₆, 1M 51% BocHN OBn 35 BocHN N N N N H O CO2Me OBn 1) TFA, CH2Cl2 2) N-Boc−Pro−OH EDCI, HOBt 89% over 2 steps 1) LiOH, THF 2) TFA, CH2Cl2 HN H N O N N N N H O CO2H OBn TFA DPPA NaHCO3, DMF 81% over 3 steps N NH O N N N HN O O OR 44 R = Bn H2, Pd/C 99% 38 42 43 N3 H N O 25 CO2Me OBn 13 R = H Me Me Me Me Me Me Me Me Me Me

Cleavage of the protective groups, first by saponification with lithium hydroxide and

subsequent treatment with TFA in CH

2

Cl

2

gave the linear tetrapeptide 43 as its TFA salt.

DPPA mediated macrolactamization using sodium bicarbonate as the base under high dilution

in DMF gave the monocyclic lactam 44 in good yield of 81% over three steps, similar to the

previous target 30.

Figure 4.1

1

H NMR of 44 at rt (top) and 100

ο

C (bottom) in DMSO-d6.

Interestingly, the

1

H NMR spectrum of 44 showed broad signals (Figure 4.1). This indicates

the presence of multiple conformations of the cyclic peptide at room temperature. Indeed, the

signals of the

1

H NMR sharpened upon heating of the sample to 100

ο

C in DMSO-d6.

Final cleavage of the benzyl protective group by hydrogenolysis provided the target

compound 13 in quantitative yield. The

1

H spectrum also gave broad peaks, indicating

multiple conformations.

4.5 Conclusions

In conclusion, new routes have been developed for the incorporation of 1,5-connected

1,2,3-triazoles in cyclic tetrapeptides. Two new cyclic tetrapeptides have been synthesized,

based on the natural tyrosinase inhibitor cyclo-[Pro

−Val−Pro−Tyr]. A ruthenium-catalyzed

cycloaddition reaction was employed for the synthesis of 1,5-connected triazoles using the

appropriate amino acid-derived building blocks. The linear precursors with the incorporated

1,5-connected triazoles were cyclized using traditional lactamization strategies under high

dilution. Investigation of the place of the 1,5-connected triazole led to the conclusion, that

replacement can only be done on a cisoid amide bond, like the parent proline amide bond.

Replacement of a parent transoid amide bond by the 1,5-connected triazole led in this

example to an overturned linear precursor, resulting in the formation of dimers on attempted

cyclization.

4.6 Acknowledgments

Dr. M. Vitale, R. Drost and M. Buit are kindly acknowledged for their contributions to

this chapter. Drs. R. Detz and Z. Abiri are kindly acknowledged for providing the enantiopure

N-Boc

−Val−≡ 35.

4.7 Experimental

section

For general experimental details, see Section 2.9. The synthesis of compounds 18, 19 has been described in Section 3.16.

Azidoacetic acid (20). Bromoacetic acid (1 equiv) and a saturated aqueous solution of sodium

azide (2 equiv) were stirred at 0 οC for 24 hours. The mixture was acidified with aqueous hydrochloric acid to pH 5. The product 20 was obtained by extraction with Et2O and careful evaporation of the

solvents in vacuo. IR (neat) 2113, 1729, 1419, 1220 cm-1

. 1H NMR (400 MHz, CDCl3) δ 10.74 (bs 1 H), 3.99 (s,

2 H) ppm. 13C (100 MHz, CDCl3) δ 174.4, 49.9 ppm.

N3−−−−Gly−−−−Pro−−−−OtBu (21). To a solution of N3−Gly−OH 20 (1.53 g, 15.2 mmol, 1.3 equiv) in

CH2Cl2 (40 mL) were added HOBt (2.05 g, 15.2 mmol, 1.3 equiv) and EDCI (2.90 g, 15.2

mmol, 1.3 equiv) and the mixture was stirred at room temperature for 30 minutes. H−Pro−OtBu (2.00 g, 11.7 mmol, 1 equiv) in CH2Cl2 (10 mL) was added and the mixture was stirred overnight

at room temperature. The solution was diluted with CHCl3 (80 mL). The organic layer was washed with water, a

1 M aqueous solution of hydrochloric acid, a aqueous solution of saturated sodium bicarbonate and brine. The organic layer was dried over MgSO4, filtered and concentrated in vacuo. The product was purified by flash

column chromatography [silica gel, ethyl acetate/petroleum ether, boiling range 40-65 οC, 3:7] to obtain the product 21 (2.8 g, 94%). IR (neat) ν 2979, 2106, 1736, 1664, 1377, 1282, 1154 cm-1

. 1H NMR (400 MHz, CDCl3) δ 4.33 (m, 1 H), 3.81 (m, 2 H), 3.45 (m, 2 H), 1.95 (m, 4 H), 1.37 (s, 9 H) ppm. 13C (100 MHz, CDCl3) δ

170.7, 170.4, 165.6, 81.2, 59.6, 59.4, 50.6, 46.6, 46.0, 31.2, 28.7, 27.6, 24.4, 22.0 ppm. HRMS (FAB) calc. for C11H19N4O3 [MH+] 255.1459, found 255.1451.

N-Boc−−−−Tyr(OBn)−−−−Pro−−−−ψψψψ(triazole)−−−−Gly−−−−Pro−−−−OtBu (22). In a sealed tube were added N3−Gly−Pro−OtBu 21 (0.225 g, 0.5 mmol, 1

equiv), N-Boc−Tyr(OBn)−Pro−≡ 19 (0.172 g, 0.6 mmol, 1.2 equiv) and the catalyst Cp*Ru(PPh3)2Cl in benzene (1 mL) and the mixture

was stirred at 80 οC for one hour. Solvents were removed in vacuo and the product was purified by flash column chromatography [silica gel, CH2Cl2/MeOH, 98:2] to obtain the

product 22 (0.350 g, 99%) as a white solid. IR (neat) ν 2977, 1735, 1708, 1672, 1643, 1510, 1444, 1241, 1155 cm-1. 1H NMR (400 MHz, C6D6) as a mixture of rotamers δ 7.85 (t, 0.3 H), 7.43-7.17 (m,10 H), 6.95 (d, J = 8.0 Hz, 2 H), 6.88 (d, J = 8.0 Hz, 1 H), 5.92 (AB, Jab = 16.0 Hz, 1 H), 5.82 (d, J = 8.0 Hz, 1 H), 5.54 (AB, Jab = 16.0 Hz, 1 H), 5.13 (m, 1 H), 4.97 (AB, Jab = 12.0 Hz, 1 H), 4.90 (AB, Jab = 12.0 Hz, 1 H), 4.74 (m, 1 H), 4.35 (t, J = 6.0 Hz, 1 H), 3.25 (m, 1 H), 3.10 (m, 1 H), 2.94 (m, 1 H), 2.86 (m, 2 H), 2.61 (m, 1 H), 2.09 (m, 1 H), 1.81 (m, 1 H), 1.51 (m, 23 H) ppm. 13C (100 MHz, CDCl3) δ 170.9, 170.2, 164.4, 157.8, 154.9, 140.0, 137.4, 131.3, 130.9, 130.5, 128.6, 128.1, 127.6, 127.4, 114.8, 80.2, 78.7, 69.4, 59.6, 53.4, 50.6, 50.2, 46.2, 45.6, 38.4, 31.8, 28.4, N3 N O _OtBu O BocHN N O N N N N O OtBu O BnO N3 O OH

28.1, 27.5, 27.4, 24.5, 24.2 ppm. RP-HPLC: Rt 5.82 min (λ = 254). HRMS (FAB) calc. for C11H20NO2 [MH+]

198.1496, found 198.1496.

Cyclo-[Pro−−−−Tyr(OBn)−−−−Pro−−−−ψψψ(triazole)−−−−Gly−−−−Pro−−−− ψ

Tyr(OBn)−−−−Pro−−−−ψψψ(triazole)−−−−Gly] (23). Linear peptide 22 ψ

(0.190 g, 0.25 mmol, 1 equiv) was dissolved in CH2Cl2 and

DIPEA (0.15 mL, 0.75 mmol, 3 equiv) and HATU (0.130 g, 0.27 mmol, 1.1 equiv) were added. The reaction mixture was stirred overnight at room temperature. The reaction was quenched with MeOH (5 mL) and stirred for 30 minutes. Solvents were removed in vacuo. Crude was redissolved in CH2Cl2 and washed with a 1 M aqueous solution of citric acid and a saturated aqueous solution of sodium

bicarbonate, dried over Na2SO4 and concentrated in vacuo. The product was purified by flash column

chromatography [silica gel, CH2Cl2/MeOH, 90:1] to provide the product 23 (0.042 g, 25%). IR (neat) ν x cm-1. 1

H NMR (400 MHz, CDCl3) δ 7.65 (s, 1 H), 7.42-7.20 (m, 6 H), 6.98 (s, 4 H), 5.68 (m, 1 H), 5.44 (m, 1 H), 5.31

(m, 1 H), 5.18 (m, 1 H), 4.81 (m, 2 H), 3.38 (m, 2 H), 3.02 (m, 2 H), 2.68 (m, 2 H), 1.93 (m, 1 H), 1.76 (m, 2 H), 1.24 (m, 2 H) ppm. 13C (100 MHz, CDCl3) δ 170.3, 166,6, 165,6, 157.9, 151.0, 139.6, 137.2, 132.2, 130.5,

128.4, 120.8, 114.7, 69.6, 61.3, 51.2, 51.0, 46.0, 45.7, 36.6, 35.4, 30.6, 29.8, 29.2, 24.0, 24.0 ppm. MS (MALDI-TOF) calc. for C58H65N12O8 [MH+] 1057.51, found 1057.54. RP-HPLC: Rt 5.43 min (λ = 220).

N3−−−−Gly−−−−Tyr(OBn)−−−−OMe (25). H2N−Tyr(OBn)−OMe (1.38 g, 4.29 mmol, 1 equiv)

and α-chloroacetic acid (0.446 g, 4.72 mmol, 1.1 equiv) were dissolved in dry CH2Cl2

(20 mL). To this mixture DIPEA (3 mL, 17.15 mmol, 4 equiv) and HOBt (0.984 g, 6.43 mmol, 1.5 equiv) were added. EDCI (1.23 g, 6.43 mmol, 1.5 equiv) was added at 0 οC. The mixture was stirred for one hour at 0 οC, allowed to warm to room temperature and stirred overnight at room temperature. The mixture was diluted with ethyl acetate (100 mL) and water (100 mL). The water layer was extracted with ethyl acetate (2 × 50 mL). The combined organic layer was washed with a 1 M solution of potassium hydrogensulphate (2 × 150 mL), a saturated solution of sodium bicarbonate (2 × 150 mL) and brine (150 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The crude mixture was dissolved

in DMF (40 mL) and sodium azide (0.809 g, 12.4 mmol, 5 equiv) was added. The mixture was stirred at 90οC overnight. The mixture was diluted with ethyl acetate (250 mL) and water (200 mL). The water layer was extracted with ethyl acetate (100 mL). The combined organic layer was washed with a saturated solution of sodium bicarbonate (3 × 200 mL), water (200 mL) and brine (200 mL). The organic layer was dried over Na2SO4

and concentrated in vacuo. The product 25 (0.827 g, 52%) was obtained as yellow solid. IR (neat) ν 3297, 3034, 2952, 2109, 1744, 1672, 1512, 1243, 1012 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.39-7.35 (m, 5 H), 7.04 (d, J =

8.8 Hz, 2 H), 6.94 (d, J = 8.4 Hz, 2 H), 6.68 (d, J = 7.6 Hz, 1 H), 5.07 (s, 2 H), 4.87 (m, 1 H), 3.99 (m, 2 H), 3.76 (s, 3 H), 3.11 (m, 2 H) ppm. 13C (100 MHz, CDCl3) δ 171.5, 166.3, 158.0, 136.8, 130.2, 128.6, 128.0, 127.6,

127.5, 115.0, 77.3, 70.0, 53.1, 52.5, 37.0 ppm. HRMS (FAB) calc. for C19H21N4O4 [MH+] 369.1565, found

369.1565.

tert-Butyl prop-2-ynylcarbamate (26). According to literature procedure. IR (neat) ν 3305,

N N N HN O O O BnO N N N N N NH O O O OBn N N N3 H N O OMe O OBn BocHN

2970, 1701, 1518, 1368, 1280, 1168 cm-1. 1H NMR (400 MHz, CDCl3) δ 4.97 (bs, 1 H), 3.93 (s, 2 H), 2.23 (t, J

= 2.5 Hz, 1 H), 1.40 (s, 9 H) ppm. 13C (100 MHz, CDCl3) δ 155.4, 80.2, 80.0, 71.2, 30.4, 28.4 ppm. HRMS

(FAB) calc. for C8H14NO2 [MH+] 156.1026, found 156.1019.

N-Boc−−−−Gly−ψ−ψ−ψ−ψ(triazole)−−−−Gly−−−−Tyr(OBn)−−−−OMe (27). A sealed tube was charged with N-Boc propargylamine 26 (0.127 g, 0.82 mmol, 1 equiv),

N3−Gly−Tyr(OBn)−OMe 25 (0.300 g, 0.85 mmol, 1 equiv) and

Cp*Ru(PPh3)2Cl (0.032 g, 0.04 mmol, 0.05 equiv) in benzene (4 mL). The

mixture was heated at 79 οC overnight. Solvents were evaporated in vacuo and the product was purified by flash column chromatography [silica gel, CH2Cl2/MeOH, 99:1] to afford the product 27 (0.265 g, 62%) as a colourless

oil. IR (neat) ν 3315, 2978, 1743, 1693, 1512, 1244 cm-1

. 1H NMR (400 MHz, CDCl3) δ 7.59 (s, 1 H), 7.46-7.35

(m, 5 H), 6.95 (d, J = 8.8 Hz, 2 H), 6.88 (d, J = 8.4 Hz, 2 H), 6.55 (bs, 1 H), 5.31 (s, 2 H), 5.07 (d, J = 2.8 Hz, 1 H), 5.04 (s, 2 H), 4.80 (m, 1 H), 4.29 (m, 1 H), 3.73 (s, 3 H), 3.07 and 2.97 (AB part of ABX, Jab = 14.0 Hz, Jax =

6.8 Hz, Jbx = 5.2 Hz), 1.45 (s, 9 H) ppm. 13C (100 MHz, CDCl3) δ 171.5, 165.0, 157.9, 136.9, 135.8, 133.8,

130.1, 128.7, 128.0, 127.5, 127.4, 115.0, 69.9, 53.5, 52.5, 50.8, 36.6, 32.9, 28.4 ppm. HRMS (FAB) calc. for C27H34N5O6 [MH+] 524.2511, found 524.2507. LC-MS (EI) Rt 7.72 min (λ = 254), calc. for C27H34N5O6 [MH+]

m/z 524.3, found 524.2.

N-Boc−−−−Pro−−−−Gly−ψ−ψ−ψ−ψ(triazole)−−−−Gly−−−−Tyr(OBn)−−−−OMe (28). N-Boc−Gly−ψ(triazole)−Gly−Tyr(OBn)−OMe 27 (0.200 g, 0.38 mmol, 1 equiv) was dissolved in TFA/CH2Cl2 (20 mL, 1:1). The mixture was

stirred at room temperature for four hours. Solvents were evaporated

in vacuo. The crude was dissolved in CH2Cl2 (5 mL). N-Boc−Pro−OH (0.163 g, 0.76 mmol, 2 equiv) was added

together with DIPEA (0.262 mL, 1.52 mmol, 4 equiv), HOBt (0.116 g, 0.76 mmol, 2 equiv) and EDCI (0.145 g, 0.76 mmol, 2 equiv). The reaction mixture was stirred overnight at room temperature. The mixture was diluted with ethyl acetate (100 mL) and water (100 mL). The water layer was extracted with ethyl acetate (2 × 50 mL). The combined organic layer was washed with a 1 M solution of potassium hydrogensulphate (2 × 150 mL), a saturated solution of sodium bicarbonate (2 × 150 mL) and brine (150 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The product was purified by flash column chromatography [silica gel,

CH2Cl2/MeOH, 95:5] to afford the product 28 (0.202 g, 86%) as a colourless oil. IR (neat) ν 3296, 2978, 1746,

1693, 1513, 1393, 1025 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.82 (bs, 1 H), 7.55 (s, 1 H), 7.45-7.33 (m, 5 H),

7.03 (m, 2 H), 6.90 (d, J = 8.4 Hz, 2 H), 5.12 (m, 2 H), 5.05 (s, 2 H), 4.75 (m, 1 H), 4.56 (m, 1 H), 4.16 (m, 2 H), 3.77 (s, 3 H), 3.41 (m, 2 H), 3.04 (m, 2 H), 2.01 (m, 4 H), 1.47 (s, 9 H) ppm. 13C (100 MHz, CDCl3) δ 173.3,

173.1, 171.3, 166.3, 158.0, 136.9, 133.1, 130.2, 130.0, 128.6, 128.0, 127.5, 115.0, 70.0, 59.5, 52.5, 50.9, 47.1, 36.3, 33.3, 30.0, 28.4, 24.4 ppm. HRMS (FAB) calc. for C32H41N6O7 [MH+] 621.3038, found 621.3041.

Cyclo-[Tyr(OBn)−−−−Pro−−−−Gly−−−−ψψψ(triazole)−−−−Gly] (30). The linear protected precursor 28ψ

(0.150 g, 0.24 mmol, 1 equiv) was dissolved in THF (1.8 mL). MeOH (0.6 mL) was added together with water (0.6 mL). LiOH (0.031 g, 0.72 mmol, 3 equiv) was added in one portion and the mixture was stirred at room temperature for three hours. The solution was poured into an aqueous 5% solution of ammonium dihydrogensulphate (15 mL) and 1 M solution of hydrochloric acid (0.5 mL). The water layer was saturated with sodium chloride. The water layer was extracted with ethyl acetate (3 × 25 mL). The organic layer was dried over

BocHN N N N N H O OMe O OBn BocN H N O N N N N H O OMe O OBn N NH O N N N HN O O OBn

Na2SO4 and concentrated in vacuo. The acid was dissolved in TFA/CH2Cl2 (2 mL, 1:1). The reaction mixture

was stirred at room temperature for two hours. Solvents were evaporated in vacuo. The crude was co evaporated with CHCl3 (2 ×) and n-heptane (2 ×). The crude was dissolved in DMF (20 mL). Solid NaHCO3 (0.150 g, 1.8

mmol, 15 equiv) was added. At -15 οC DPPA (0.035 mL, 0.16 mmol, 1.3 equiv) was added. The mixture was stirred for 96 hours with slow warming to room temperature. The DMF was evaporated. CHCl3 (25 mL) was

added and the organic layer was washed with an aqueous 5% solution of ammonium dihydrogensulphate (20 mL) saturated with sodium chloride, an aqueous 10% solution of potassium carbonate (20 mL) saturated with sodium chloride and brine (20 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The

product was purified by flash column chromatography [silica gel, CH2Cl2/MeOH, 90:10] to afford the product 30

(0.051 g, 87%) as an amorphous solid. 1H NMR (400 MHz, CDCl3/MeOD, 9:1) δ 7.54 (s, 1 H), 7.36-7.13 (m, 5

H), 7.09 (d, J = 8.0 Hz, 2 H), 6.81 (d, J = 8.0 Hz, 2 H), 5.02 (AB, J = 16.0 Hz, 1 H), 4.96 (s, 2 H), 4.92 (AB, J = 16.0 Hz, 1 H), 4.73 (m, 1 H), 4.60 (AB, J = 17.2 Hz, 1 H), 4.14 (d, J = 8.0 Hz, 1 H), 4.02 (AB, J = 17.0 Hz, 1 H), 3.30 (m, 2 H), 3.16 (m, 1 H), 2.67 (m, 1 H), 2.09 (m, 1 H), 1.92 (m, 3 H) ppm. 13C (100 MHz, CDCl3) δ

172.5, 168.3, 165.4, 157.4, 136.9, 133.8, 130.6, 129.4, 128.4, 127.8, 127.3, 114.5, 69.8, 59.2, 52.7, 49.8, 47.4, 36.5, 31.7, 30.7, 21.4 ppm. HRMS (FAB) calc. for C26H29N6O4 [MH+] 489.2255, found 489.2257. LC-MS (EI)

Rt 6.18 min (λ = 254), calc. for C26H29N6O4 [MH+] m/z 489.2, found 489.3.

Cyclo-[Tyr−−−−Pro−−−−Gly−−−−ψψψψ(triazole)−−−−Gly] (12). Benzyl protected cyclic peptide 30 (0.025 g,

0.051 mmol, 1 equiv) was dissolved in ethyl acetate/iPrOH (10 mL, 1:1). Pd/C (0.013 g) was added and the resulting mixture was subjected to a three-cycle of vacuum and H2 and

was stirred at room temperature under a H2 balloon overnight, The catalyst was removed by

filtration over a pad of Celite and the filtrate was concentrated in vacuo to afford the cyclic peptide 12 (0.020 g, 99%) as an off-white solid. 1H NMR (400 MHz, MeOD) δ 8.80 (bm,1 H), 8.17 (bm,1 H), 7.66 (s, 1 H), 7.10 (d, J = 8.4 Hz, 1 H), 6.95 (d, J = 8.4 Hz, 1 H), 6.87 (d,

J = 8.4 Hz, 1 H), 6.62 (d, J = 8.4 Hz, 1 H), 5.04 (s, 1 H), 4.89 (m, 2 H), 4.63 (m, 2 H), 4.17 (m, 1 H), 4.04 (AB, J

= 14.0 Hz, 1 H), 3.40 (m, 3 H), 3.05 (m, 1 H), 2.02 (m, 1 H), 1.79 (m, 3 H) ppm. 13C (100 MHz, DMSO-d6) δ 171.5, 164.7, 156.7, 155.6, 137.2, 134.6, 130.3, 128.4, 127.8, 114.8, 114.2, 69.1, 58.8, 52.1, 49.7, 47.1, 31.6, 30.1, 21.2 ppm. HRMS (FAB) calc. for C19H22N6O4 [MH+] 399.1783, found 399.1782.

(S)-tert-Butyl 1-hydroxy-3-methylbutan-2-yl carbamate (32). N-Boc−Val−OH (5.00 g, 23.0

mmol, 1 equiv) was dissolved in CH2Cl2 (80 mL) and the solution was cooled to -10 οC. Pyridine

(1.9 mL, 23.0 mmol, 1 equiv) and cyanuric fluoride (6.22 g, 46.1 mmol, 2 equiv) were added and the reaction mixture was stirred at -10 οC for one hour. The reaction mixture was partitioned between CH2Cl2

(500 mL) and cold water (200 mL). The aqueous layer was extracted with CH2Cl2 (2 × 150 mL). Combined

organic layer was washed with water (2 × 100 mL), dried over Na2SO4 and concentrated in vacuo. The residue

was then dissolved in MeOH (60 mL) and NaBH4 (3.50 g, 92.1 mmol, 4 equiv) was slowly added. The reaction

mixture was stirred overnight, after which additional NaBH4 (1.75 g, 46.1 mmol, 2 equiv) was slowly added and

the reaction mixture was stirred at room temperature for three days. Reaction mixture was neutralized with 1 M H2SO4 (30 mL). The solvent was removed in vacuo and the aqueous layer was diluted with water (50 mL).

Aqueous layer was extracted with EtOAc (3 × 50 mL) and the combined organic layer was with water (2 × 30 mL), dried over Na2SO4, filtered and the solvents were evaporated. Product was obtained by flash column

chromatography [silica gel, ethyl acetate/petroleum ether, boiling range 40-65 οC, 1:9] to yield the desired product 32 (4.32 g, 92%) as a white solid. IR (neat) ν 3352, 2972, 1693, 1513, 1391, 1367, 1248, 1173, 1078,

N NH O N N N HN O O OH BocHN Me Me OH

1048 cm-1. 1H NMR (400 MHz, CDCl3) δ 4.69 (s, 1 H), 3.68 (m, 2 H), 3.45 (s, 1 H), 2.40 (s, 1 H), 1.85 (m, 1 H),

1.47 (s, 1 H), 0.96 (m, 6 H) ppm.

tert-Butyl 3-methyl-1-oxobutan-2-yl carbamate (34). Oxalyl chloride (12.8 mL, 25.5 mmol, 1.2 equiv) was dissolved in CH2Cl2 (65 mL). The mixture was cooled to -60 οC. DMSO (3.7 g, 46.8

mmol, 2.2 equiv) in CH2Cl2 (10 mL) was added drop wise over ten minutes. The reaction mixture

was stirred for ten minutes after which N-Boc−Valinol 32 (4.32 g, 21.3 mmol, 1 equiv) in CH2Cl2 (50 mL) was

added drop wise over 15 minutes. After stirring for 30 minutes, DIPEA (14.0 mL, 85.1 mmol, 4 equiv) was added over 15 minutes. The reaction mixture was stirred at -60 οC for 30 minutes before being allowed to warm to room temperature. The reaction mixture was washed with 5% aqueous hydrochloric acid solution (3 × 100 mL). The combined aqueous layer was extracted with CH2Cl2 (50 mL). Combined organic layer was washed

with water (3 × 50 mL) and brine (50 mL), dried over Na2SO4, filtered and concentrated in vacuo to yield the

product 34 (3.78 g, 88%)as yellow oil which was used without further purification. 1H NMR (400 MHz, CDCl3)

δ 9.66 (s, 1 H), 5.09 (m, 1 H), 4.26 (m, 1 H), 2.30 (m, 1 H), 1.47 (s, 9 H), 1.05 (d, J = 6.9 Hz, 3 H), 0.96 (d, J = 7.0 Hz, 3 H) ppm.

tert-Butyl 3-methyl-1-oxobutan-2-yl carbamate (34). LiAlH4 (0.29 g, 7.7 mmol, 8 equiv) was

added at -23 οC to a stirred solution of N-Boc−Val−N(OCH3)CH3 (0.25 g, 0.96 mmol, 1 equiv) in

Et2O (10 mL). The mixture was stirred for two hours at -23 οC. The reaction mixture was

quenched with a 1 M solution of potassium hydrogensulphate (10 mL) and diluted with Et2O (50 mL). The

aqueous layer was extracted with Et2O (3 × 50 mL). The combined organic layer was washed with an aqueous

solution of 10% hydrochloric acid (3 × 20 mL), an aqueous solution of saturated sodium bicarbonate (3 × 20 mL), brine (3 × 20 mL) and dried over MgSO4, filtered and concentrated in vacuo to yield the product 34 (0.131

g, 68%) as a yellow oil. Analytic data, see above.

tert-Butyl 4-methylpent-1-yn-3-yl carbamate (35). N-Boc−Pro−H 34 (3.78 g, 18.8 mmol, 1 equiv) was dissolved in MeOH (20 mL). The solution was cooled to 0 οC and potassium carbonate (5.2 g, 37.6 mmol, 2 equiv) and phosphonate 51 (4.33 g, 22.6 mmol, 1.2 equiv) were added. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was quenched with water (200 mL), diluted with Et2O (50 mL). The aqueous layer was extracted with Et2O (3 × 200

mL). The combined organic layer was washed with saturated aqueous sodium bicarbonate solution (50 mL), brine (50 mL) and dried over Na2SO4. Solvents were evaporated in vacuo to yield the crude product, which was

purified through flash column chromatography [silica gel, ethyl acetate/petroleum ether, boiling range 40-65 οC, 1:9] yielding the desired compound 35 (2.08 g, 56%) as a white solid. [α]26_D –9.23 (c 0.75 g/100 mL, CH2Cl2).

IR (neat) ν 2976, 2876,2812, 1694, 1503, 1367, 1219, 1172 cm-1

. 1H NMR (400 MHz, CDCl3) δ 4.75 (m, 1 H),

4.34 (m, 1 H), 2.27 (d, J = 2.4 Hz), 1.92 (m, 1 H), 1.47 (s, 9 H), 1.01 (d, J = 6.8 Hz, 6 H) ppm. 13C (100 MHz, CDCl3) δ 157.5, 81.2, 71.7, 48.6, 32.9, 28.3, 18.7, 17.5 ppm. HRMS (FAB) calc. for C11H20NO2 [MH+]

198.1496, found 198.1496.

tert-Butyl 4-methylpent-1-yn-3-yl carbamate (35). According to literature to obtain the product as a white solid. [α]26_D –51.6 (c 1.0 g/100 mL, CH2Cl2). Analytical data similar as above.

BocHN Me Me H O BocHN Me Me O H BocHN Me Me BocHN Me Me

(S)-tert-Butyl 2-((S)-4-methylpent-1-yn-3-ylcarbamoyl)pyrrolidine-1-carboxylate (36).

N-Boc−Val−≡ 35 (0.52 g, 2.64 mmol, 1 equiv, mixture of enantiomers) was dissolved in a mixture of CH2Cl2 (5 mL) and TFA (5 mL). The reaction mixture was stirred for two hours at

room temperature. Solvents were evaporated in vacuo to yield the TFA-salt. The TFA-salt was added to CH2Cl2 (50 mL) and DIPEA (0.44 mL, 2.64 mmol, 1 equiv). The mixture was stirred for ten

minutes at room temperature. N-Boc−Pro−OH (0.55 g, 2.77 mmol, 1.05 equiv), HOBt (0.36 g, 2.64 mmol, 1 equiv) and EDCI (0.41 g, 2.64 mmol, 1 equiv) were added. After stirring overnight at room temperature the mixture was diluted with CHCl3 (90 mL) and washed with water (100 mL), an aqueous solution of saturated

sodium bicarbonate (100 mL), a 1 M solution of potassium hydrogensulphate (100 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The product was purified by flash column

chromatography [silica gel, ethyl acetate/petroleum ether, boiling range 40-65 οC, 1:9] to yield the desired product 36 (0.135 g, 18%, pure diastereoisomer) as a white solid. IR (neat) ν 2973, 1699, 1665, 1537, 1399, 1219, 1166 cm-1. 1H NMR (400 MHz, CDCl3) as a mixture of rotamers δ 7.47 (s, 0.5 H), 6.25 (s, 0.5 H), 4.65 (s,

1 H), 4.27 (m, 1 H), 3.40 (m, 2 H), 2.46 (s, 1 H), 2.16 (s, 1 H), 1.98 (m, 3 H), 1.49 (s, 9 H), 0.98 (m, 6 H) ppm.

13

C (100 MHz, CDCl3) δ 47.1, 46.8, 31.0, 28.4, 27.1, 18.6, 17.7 ppm. HRMS (FAB) calc. for C16H26N2O3 [MH+]

295.2013, found 295.2020.

N-Boc−−−−Val−ψ−ψ−ψ(triazole)−−−−Gly−−−−Tyr(OBn)−−−−OMe (38). A sealed tube was −ψ charged with N-Boc−Val−≡ 35 (0.099 g, 0.5 mmol, 1 equiv),

N3−Gly−Tyr(OBn)−OMe 25 (0.184 g, 0.5 mmol, 1 equiv) and

Cp*Ru(PPh3)2Cl (0.040 g, 0.05 mmol, 0.1 equiv) in benzene (2.5 mL). The

mixture was heated at 79 οC overnight. Solvents were evaporated in vacuo and the product was purified by flash column chromatography [silica gel, CH2Cl2/MeOH, 99:1] to afford the product 38 (0.143 g, 50%) as a colourless

oil. IR (neat) ν 3300, 2973, 1744, 1700, 1611, 1511, 1454, 1367, 1242, 1220, 1174, 1012 cm-1

. 1H NMR (400 MHz, CDCl3) δ 7.69 (m, 1 H), 7.55 (s, 1 H), 7.43 (m, 5 H), 6.96 (d, J = 8.8 Hz, 2 H), 6.88 (d, J = 8.8 Hz, 2 H),

6.50 (bs, 1 H), 5.21 (AB, J = 16.0 Hz, 1 H), 5.14 (m, 1 H), 5.04 (s, 2 H), 5.02 (m, 1 H), 4.78 (X part of ABX system, Jax = 6.7 Hz, Jbx = 5.3 Hz, 1 H), 4.63 (m, 1 H), 4.30 (m, 2 H), 3.71 (s, 3 H), 3.03 and 2.97 (AB part of

ABX, Jab = 14.0 Hz, Jax = 6.7 Hz, Jbx = 5.3 Hz, 2 H), 2.09 (m, 1 H), 1.41 (s, 9 H), 0.98 (d, J = 6.8 Hz, 3 H), 0.89

(d, J = 6.4 Hz, 3 H) ppm. 13C (100 MHz, CDCl3) δ 171.5, 169.4, 164.9, 161.2, 158.0, 155.4, 139.2, 136.9, 130.2,

128.6, 128.0, 127.5, 127.4, 115.2, 103.4, 70.0, 59.9, 53.4, 52.6, 52.2, 51.1, 45.3, 36.7, 29.7, 28.3, 27.7 ppm. HRMS (FAB) calc. for C30H40N5O6 [MH+] 566.2980, found 566.2976.

N-Boc−−−−Pro−−−−Val−ψ−ψ−ψ−ψ(triazole)−−−−Gly−−−−Tyr(OBn)−−−−OMe (42). N-Boc−Val−ψ(triazole)−Gly−Tyr(OBn)−OMe 38 (0.143 g, 0.25 mmol, 1 equiv) was dissolved in TFA/CH2Cl2 (12 mL, 1:1). The mixture was

stirred at room temperature for four hours. Solvents were evaporated

in vacuo. The crude was dissolved in CH2Cl2 (5 mL). N-Boc−Pro−OH

(0.108 g, 0.5 mmol, 2 equiv) was added together with DIPEA (0.174 mL, 1 mmol, 4 equiv), HOBt (0.077 g, 0.5 mmol, 2 equiv) and EDCI (0.096 g, 0.5 mmol, 2 equiv). The reaction mixture was stirred overnight at room temperature. The mixture was diluted with ethyl acetate (100 mL) and water (100 mL). The water layer was extracted with ethyl acetate (2 × 50 mL). The combined organic layer was washed with a 1 M solution of potassium hydrogensulphate (2 × 150 mL), a saturated solution of sodium bicarbonate (2 × 150 mL) and brine (150 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The product was purified by

BocHN N N N N H O OMe O Me Me OBn BocN H N O N N N N H O OMe O OBn Me Me N H Me Me O NBoc

flash column chromatography [silica gel, CH2Cl2/MeOH, 95:5] to afford the product 42 (0.147 g, 89%) as a

colourless oil. IR (neat) ν 3296, 2978, 1746, 1693, 1513, 1393, 1025 cm-1

. 1H NMR (400 MHz, CDCl3) δ 7.81

(bs, 1 H), 7.60 (s, 1 H), 7.49-7.31 (m, 5 H), 6.96 (d, J = 8.0 Hz, 2 H), 6.88 (d, J = 8.0 Hz, 2 H), 5.21 (m, 1 H) 5.10 (m, 1 H), 5.05 (s, 2 H), 4.76 (m, 2 H), 4.22 (m, 1 H), 3.72 (s, 3 H), 3.40 (m, 2 H), 3.07 and 3.01 (AB part of ABX, Jab = 14.0 Hz, Jax = 7.2 Hz, Jbx = 4.8 Hz, 2 H), 2.03 (m, 1 H), 2.11 (m, 1 H), 1.90 (m, 4 H), 1.49 (s, 9 H),

1.01 (d, J = 6.4 Hz, 3 H), 0.90 (d, J = 6.4 Hz, 3 H) ppm. 13C (100 MHz, CDCl3) δ 172.0, 172.0, 171.5, 165.2,

157.9, 138.8, 136.9, 130.8, 130.1, 128.7, 128.5, 127.9, 127.4, 114.9, 69.9, 68.1, 59.4, 53.8, 52.4, 51.1, 49.4, 47.1, 38.7, 36.4, 31.9, 28.4, 28.3, 27.1, 24.6, 19.6 ppm. HRMS (FAB) calc. for C35H47N6O7 [MH+] 663.3508, found

663.3509.

Cyclo-[Tyr(OBn)−−−−Pro−−−−Val−−−−ψψψ(triazole)−−−−Gly] (44). The linear protected precursor 42ψ

(0.130 g, 0.2 mmol, 1 equiv) was dissolved in THF (2 mL). MeOH (0.5 mL) was added together with water (0.5 mL). LiOH (0.025 g, 0.6 mmol, 3 equiv) was added in one portion and the mixture was stirred at room temperature for three hours. The solution was poured into an aqueous 5% solution of ammonium dihydrogensulphate (15 mL) and 1 M solution of hydrochloric acid (0.5 mL). The water layer was saturated with sodium chloride. The water layer was extracted with ethyl acetate (3 × 25 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The acid was dissolved in TFA/CH2Cl2 (2 mL, 1:1). The reaction mixture

was stirred at room temperature for two hours. Solvents were evaporated in vacuo. The crude was co evaporated with CHCl3 (2 ×) and n-heptane (2 ×). The crude was dissolved in DMF (20 mL). Solid NaHCO3 (0.124 g, 1.47

mmol, 15 equiv) was added. At -15 οC DPPA (0.027 mL, 0.13 mmol, 1.3 equiv) was added. The mixture was stirred for 96 hours with slow warming to room temperature. The DMF was evaporated. CHCl3 (25 mL) was

added and the organic layer was washed with an aqueous 5% solution of ammonium dihydrogensulphate (20 mL) saturated with sodium chloride, an aqueous 10% solution of potassium carbonate (20 mL) saturated with sodium chloride and brine (20 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The

product was purified by flash column chromatography [silica gel, CH2Cl2/MeOH, 98:2] to afford the product 44

(0.042 g, 81%) as an amorphous solid. IR (neat) ν 3306, 2961, 1671, 1644, 1510, 1212, 1025 cm-1

. 1H NMR (400 MHz, DMSO-d6, 100 οC) δ 7.70 (s, 1 H), 7.45-7.24 (m, 7 H), 7.10 (d, J = 8.6 Hz, 2 H), 6.90 (d, J = 8.6 Hz,

2 H), 5.07 (s, 2 H), 4.99 (AB, J = 17.1 Hz, 1 H), 4.94 (m, 1 H), 4.60 (AB, J = 17.0 Hz, 1 H), 4.59 (m, 1 H), 4.31 (d, J = 7.8 Hz, 1 H), 3.50 (m, 1 H), 3.34 (m, 1 H), 3.08 (m, 1 H), 2.66 (m, 1 H), 2.32 (m, 1 H), 2.05 (m, 2 H), 1.88 (m, 1 H), 1.66 (m, 1 H), 0.98 (d, J = 6.5 Hz, 3 H), 0.96 (d, J = 6.6 Hz, 3 H) ppm. HRMS (FAB) calc. for C29H35N6O4 [MH+] 531.2722, found 531.2727.

Cyclo-[Tyr−−−−Pro−−−−Val−−−−ψψψψ(triazole)−−−−Gly] (13). Benzyl protected cyclic peptide x (0.030 g,

0.057 mmol, 1 equiv) was dissolved in CH2Cl2/MeOH (10 mL, 9:1). Pd/C (0.015 g) was

added and the resulting mixture was subjected to a three-cycle of vacuum and H2 and was

stirred at room temperature under a H2 balloon overnight, The catalyst was removed by

filtration over a pad of Celite and the filtrate was concentrated in vacuo to afford the cyclic peptide 13 (0.025 g, 99%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) broad spectrum δ 9.20 (bm, 1 H), 8.78 (bm, 1 H), 8.33 (s, 1 H), 7.70-6.63 (m, 4 H), 5.10-3.91 (m, 6 H), 3.17 (m, 2 H), 3.02 (m, 2 H), 2.25 (m, 1 H), 2.10-1.50 (m, 4 H), 1.25 (m, 3 H), 0.87 (m, 3 H) ppm. 13C (100 MHz, DMSO-d6) δ 170.3, 167.0, 164.5, 155.6, 131.7, 130.0, 128.7, 128.4, 115.1, 114.8, 67.4, 40.2, 39.8, 38.1, 29.8, 28.4, 23.2, 21.6, 13.9, 11.2 ppm. HRMS (FAB) calc. for C22H29N6O4 [MH+] 441.2252, found 441.2250.

N NH O N N N HN O O OBn Me Me N NH O N N N HN O O OH Me Me

4.8

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