Aliphatic chain-containing macrocycles as diazonamide A analogs

DOI 10.1007/s10593-020-02704-6

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

1

Viktorija Vitkovska , Rimants Zogota , Toms Kalnins ,

1

Diana Zelencova , Edgars Suna *

1

Latvian Institute of Organic Synthesis,

2

1 Aizkraukles St., Riga LV-1006, Latvia; e-mail: edgars@osi.lv

Published in Khimiya Geterotsiklicheskikh Soedinenii,

020, 56(5), 586–602

Submitted December 30, 2019

Accepted after revision April 3, 2020

2

Aliphatic alkyl chain-containing 12–14-membered macrocycles have been designed as structural analogs of antimitotic natural product

diazonamide A. Macrocycles were synthesized from 5-bromooxazole in 7 to 9 linear steps using Ru-catalyzed ring-closing metathesis as

the key transformation. Heat effect of binding to ,-tubulin tetramer (T4-RB3 complex) has been measured for the synthesized macro-

cycles by isothermal titration calorimetry method.

Keywords: diazonamide A, anticancer agents, macrocycles, ring-closing metathesis.

Chemotherapeutics are the most efficient means for the

treatment of metastatic tumors. However, a majority of

cancer chemotherapeutic agents cause side effects such as

nausea, vomiting, diarrhea, hair loss, and pain. These side

effects result from undesired cytotoxicity toward normal

1

cells. A notable exception among cancer chemotherapeutic

agents is marine metabolite diazonamide A (1) (Fig. 1),

which does not cause systemic toxicity typical for other

antimitotic drugs, while exerting nanomolar cytotoxicity

2

against a range of human tumor cell lines. The greatly

reduced systemic toxicity renders diazonamide A (1) a

highly attractive cancer chemotherapeutic agent. Unfortu-

nately, highly complex structure of the natural product 1 is

an important hurdle for its use in the cancer treatment. The

decrease of the structural complexity of diazonamide A (1)

without affecting the anticancer activity is possible, as was

demonstrated by the development of anticancer agent DZ-2384

3

(

2) (Fig. 1). It has been shown that both natural product 1

2,3

and synthetic analog 2 are microtubule-targeting agents.

The structurally simplified diazonamide A analog 2

lacks CD subunit and heteroaromatic macrocycle as compared

to the parent natural product 1 (Fig. 1). Considering further

structural modifications, the substitution of the difficult to

synthesize tetracyclic subunit EFGH by less complex linker

is highly attractive from the synthetic viewpoint. To verify

Figure 1. Antimitotic diazonamide A (1) and the structurally

simplified synthetic analog 2.

0

586

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

Figure 2. Structure of diazonamide A analogs 3–6.

the importance of the tetracyclic subunit EFGH for the

binding of cytotoxic agents 1, 2 to tubulin, a series of

diazonamide analogs 3–6 that feature the replacement of

the EFGH subunit by aliphatic chain of various length were

designed and synthesized (Fig. 2). Binding energy of the

synthesized analogs 3–6 with tubulin has been measured by

isothermal titration calorimetry (ITC) assay.

The retrosynthetic analysis of diazonamide A analogs 3–6 is

depicted in Figure 3. We envisioned that macrocycles 7

could be constructed from the parent bisalkene inter-

mediates 8 using the ring-closing metathesis (RCM) as the

key step. The allyl moieties in the key intermediates 8

2

(

X = NR ) could be installed by amide coupling with chiral

unsaturated amino acids and by the C–N bond formation in

the S Ar-type reaction of bromooxazole 10 and allyl

N

amines. In addition, the allyl moiety could be also attached

to the oxazole subunit in the key intermediate 8

Figure 3. Retrosynthetic analysis of macrocycles 4–6.

(

X = C(OH)Ar) by the nucleophilic addition of allyl metal

species to ketone 11. The latter is accessible by regio-

4

selective lithiation of bioxazole 12, followed by the in situ

boronate and vinyl bromide to afford ester 17C. The

hydrolysis of the ester moiety afforded acid 16C in good

addition of lithiated species to aryl ketones.

6

The synthesis of macrocycles 4–6 commenced by the

preparation of bromooxazole 10 and amino acids 16B and

yield. The synthesis of amino acid 16B required the

preparation of organozinc species from iodide 18. The

intermediate N-Boc-protected organozinc species were

cross-coupled with allyl bromide in the presence of

1

6C using literature methods (Scheme 1). Thus, bromo-

oxazole 10 was synthesized from the commercially available

Cbz-protected (S)-tert-leucine 13 and 2-aminomalononitrile

tosylate 14, followed by amine-bromide exchange in

7

stoichiometric amounts of copper bromide. The formed

aminohexenoate 17B was converted into corresponding

5

8

compound 15 under the Sandmeyer reaction conditions.

acid 16B by saponification with LiOH (Scheme 1).

The synthesis of unsaturated amino acid 16C started with

the formation of ester 17A from the corresponding acid

Macrocycles 3–4 were prepared starting from oxazole

10 (Scheme 2). The allylamine moiety was installed in

bromooxazole 10 or its N-deprotected derivative by

1

6A. Subsequent reaction sequence included the hydro-

boration of ester 17A with 9-BBN, which was followed by

Suzuki cross coupling between the in situ generated

S Ar-type nucleophilic substitution of bromide in the

N

9

presence of a general base such as NEt . For the less

3

5

87

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

Scheme 1. Synthesis of oxazole building block 10 and amino acids 16B,C

nucleophilic aniline, the generation of the corresponding

potassium anilide by KHMDS was required to effect the

desired nucleophilic substitution of bromide in compound

macrocycle 7aA to 63% isolated yield (entry 5). It should

be noted that macrocycle 7aA was formed as a 2:3 mixture

of E:Z isomers that was anticipated for a medium-sized

(12-membered) macrocycles.¹

4,15

1

0. The cleavage of N-Cbz protection was accomplished by

HBr solution, and higher yields were achieved if morpholine

was added to trap the formed benzyl bromide and to avoid

the alkylation side reactions. The amide bond formation

with N-Boc-protected amino acids 16A–C gave the

corresponding dienes 8aA–C,bA. N-Phenyl diene 8cA was

prepared by the initial amide coupling of intermediate 19

with amino acid 16A, followed by regioselective allylation

of the most nucleophilic nitrogen in the deprotonated

compound 20 (Scheme 2).

Table 1. Screening of conditions for the RCM*

Next, the reaction conditions for the RCM step were

optimized using diene 8aA as a model substrate (0.1 M

1

0

solution in CH

2

Cl ) at 50°C (Table 1). Initial attempts to

2

effect the formation of the desired macrocycle 7aA using

catalyst Ru1 were not successful: incomplete conversion

of the starting diene 8aA was observed, and macrocycle

11

7

aA was not formed (entry 1). The lack of the RCM

product and the incomplete conversion of diene 8aA

pointed to a low catalytic activity of Ru1 that resulted in

slow reaction and concomitant decomposition of the

starting material under the tested conditions. Catalyst Ru2

1

2

was also inefficient (entry 2), however it was shown that

the catalytic activity of complex Ru2 could be increased by

introduction of a strong electron-withdrawing group in the

Entry

Conditions

Conversion**, %

Yield**, %

1

3

1

2

3

4

Ru1, 50°C, 22 h

Ru2, 50°C, 22 h

Ru3, 50°C, 22 h

Ru4, 50°C, 22 h

Ru4, 50°C, 72 h

30

100

61

0

2

-isopropoxystyrene ligand. Indeed, the nitro-substituted

catalyst Ru3 and the corresponding sulfonamide Ru4 afforded

9 and 41%, respectively, of the desired macrocycle 7aA

3

39

41

together with ca. 20% of unidentified side products with

the balance corresponding to 61–63% conversion (entries 3

and 4). We speculated that the metathesis-polymerization

side reaction might account for the formation of the side

products. Indeed, twofold increase of the dilution and

prolonged reaction time has helped to increase the yield of

63

4

5

***

83

76 (63* )

*

2 2

0.1 M solution of diene 8aA in CH Cl at 50°C.

* Determined by UPLC-MS assay.

** 0.05 M solution.

4

* Isolated yield.

5

88

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

Scheme 2. Synthesis of analogs 3 and 4aA–C,bA,cA

5

89

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

Scheme 3. Synthesis of analog 5

With the optimized cyclization conditions in hand,

dienes 8aA–C,bA,cA were subjected to RCM in the

presence of catalyst Ru4 (5 mol %) (Scheme 2). Isolation

of the RCM products was accomplished only for diene 8aA

to afford unsaturated macrocycles (E)-21 and (Z)-21 as

individual isomers after column chromatography. The other

macrocyclization products were obtained as mixtures of

E- and Z-isomers and directly converted into saturated

macrocycles 7aA–C,bA,cA under Pd-catalyzed hydrogena-

tion conditions. The end-game of the synthesis involved

N-Boc deprotection, followed by amide bond formation

with (S)-2-hydroxy-3-methylbutanoic acid to afford

unsaturated macrocycles (E)-3 and (Z)-3 as well as their

saturated analogs 4aA–C,bA,cA (Scheme 2).

of the lithiated intermediate to 3,5-difluorobenzaldehyde to

form alcohol 25 as a 1:1 mixture of diastereomers (Scheme 4).

17

Subsequent oxidation with Dess–Martin periodinane

provided ketone 26, which further reacted with allyl-

magnesium bromide to afford tertiary alcohol 27 (1:1

mixture of diastereomers). The cleavage of both N-Boc and

OTBS protecting groups under acidic conditions was followed

by the amide bond formation using acid 16A to give diene

28. The resulting diene was subjected to Ru-catalyzed

RCM under the optimized conditions (Table 1, entry 5).

The unsaturated macrocyclization product was formed as a

2:1 E:Z mixture of isomers in high (82%) yield. The

mixture of isomers was hydrogenated to form macrocycle

29. At this point, the stage was set for the N-deprotection/

amide bond formation sequence. However, the cleavage of

N-Boc protecting group in macrocycle 29 by trifluoroacetic

acid resulted in the concomitant trifluoroacetoxylation of

the tertiary alcohol-derived transient carbocation. After some

experimentation, it was found that the ester side product

can be hydrolyzed back to the tertiary alcohol by aqueous

LiOH. Subsequent amide bond formation with (S)-2-hydroxy-

3-methylbutanoic acid furnished macrocycle 6 as a mixture

of diastereomers 6A and 6B. Individual diastereomers were

obtained after separation by chromatography, however, the

configuration of the quaternary stereogenic center could

not be assigned for the individual diastereomers 6A,B

(Scheme 4).

Bioxazole-containing analog 5 was prepared by analogy

to the synthesis of macrocycles 3, 4 (Scheme 3). Thus, the

4

N-Boc deprotection of compound 22 was followed by the

nucleophilic substitution of bromide with N-methylallyl-

amine. Subsequent amide coupling with amino acid 16A

afforded diene 23. Disappointingly, the optimized RCM

conditions (Table 1, entry 5) turned out to be unsuitable for

the RCM of diene 23, as the formation of the desired

macrocycle was not observed. We hypothesized that the

chelation of catalytically active Ru species by substrate

may be responsible for the inhibition of the catalytic cycle.

It has been demonstrated that the addition of certain Lewis

acids such as Ti(IV) species helps to avoid the deactivation

16

of the Ru catalyst. Indeed, the addition of stoichiometric

amounts of Ti(Oi-Pr) improved the reaction rate and

The heat effect of binding (dissociation constants,

4

K values) between all synthesized diazonamide A analogs

d

allowed for the formation of the unsaturated macrocycle,

which was converted into the saturated analog 24 by

Pd-catalyzed hydrogenation. The end-game of the synthesis

involved reduction of the ester moiety, N-deprotection, and

amide bond formation sequence to give macrocycle 5

3–6 and α,β-tubulin tetramer (T4-RB3 complex) has been

measured by isothermal titration calorimetry (ITC) method

(Table 2). No heat effect of the binding has been observed

for most of the synthesized analogs (Table 2, entries

1–4, 7, 8). Low micromolar binding was observed for

diazonamide A analogs 4aC,bA and both diastereomers of

compound 6 (entries 5, 6, 9, and 10, respectively).

However, the measured heat effects of binding are 2 orders

(Scheme 3).

The synthesis of macrocycle 6 was started by regio-

4

selective lithiation of bioxazole 12, followed by the addition

5

90

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

Scheme 4. Synthesis of analogs 6A and 6B

of magnitude lower than that of DZ-2384 (2) (K

d

0.05 μM).

binding of these antimitotic agents to the α,β-tubulin

tetramer.

Hence, the ITC results provided strong evidence that the

presence of the tetracyclic subunit GHFE in the natural

product 1 and the synthetic derivative 2 is important for the

A series of structurally simplified macrocyclic analogs

of marine metabolite diazonamide A and synthetic anti-

mitotic agent DZ-2384 have been synthesized in 7 to 9 linear

steps from chiral, enantiomerically pure 5-bromooxazole

building block. Ruthenium-catalyzed ring-closing meta-

thesis was employed to construct 12–14-membered

macrocycles. The addition of stoichiometric amounts of

Table 2. Heat effect of binding between compounds 3–6 and

α,β-tubulin tetramer (T4-RB3 complex) measured by ITC method

Entry

Compound

(E)-3

(Z)-3

4aA

4aB

4aC

4bA

4cA

5

d

K , μM*

1

2

3

4

5

6

7

8

9

No heat observed

2.52 ± 0.83**

3.38

No heat observed

4.10

Ti(i-OPr) has helped to avoid the chelation of catalytically

4

active Ru species by bioxazole subunit-containing RCM

substrates. The starting chiral 5-bromooxazole building

block was prepared from (S)-tert-leucine in a straight-

forward two-step synthesis. Several of the synthesized

diazonamide analogs showed weak (low micromolar)

binding with α,β-tubulin tetramer (T4-RB3 complex), and

the measured heat effects were considerably lower than that

of DZ-2384. These data provided an evidence that the

presence of the tetracyclic subunit GHFE in diazonamide A

and its synthetic derivative DZ-2384 is important for the

6A

1

0

6B

1.52

*

Single ITC experiment.

* Duplicate ITC run.

5

91

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

binding to the α,β-tubulin tetramer. We believe that this

and concentrated under reduced pressure. The residue was

purified by column chromatography on silica gel using

gradient elution from pure hexane to 40% EtOAc in hexane.

General method IV. Catalyst Ru4 (0.05 equiv) and diene

finding will have implications in the design of diazonamide A

analogs as antimitotic agents.

Experimental

8

aA–C,bA,cA, 28 (1 equiv) were weighed in an oven-dried

1

13

H and C NMR spectra were recorded on a Bruker

ACE glass pressure tube, and anhydrous CH Cl (50 ml/mmol)

2

Avance Neo spectrometer with a 5-mm Double-Resonance

Broadband CryoProbe Prodigy probe (400 and 101 MHz,

was added. The resulting green solution was stirred at 60°C

for 30–48 h whereupon the color gradually changed to dark-

brown. The reaction mixture was cooled to room tempe-

rature and concentrated under reduced pressure.

respectively) using TMS or the residual solvent peaks as

1

internal reference (CDCl

3

: 7.26 ppm for H nuclei and

13

1

7

7.2 ppm for C nuclei; DMSO-d

6

: 2.50 ppm for H nuclei

General method V. 10% Pd on carbon (0.01 equiv) was

added to a solution of macrocyclic alkene (from General

method III or IV) in EtOAc (15 ml/mmol) under argon

atmosphere. The argon atmosphere was replaced by

hydrogen atmosphere and the black suspension was stirred

for 18–24 h at room temperature. Then the suspension was

filtered through a pad of Celite®, and the filtrate was

concentrated under reduced pressure. The residue was

purified by reversed-phase column chromatography on

1

3

1

and 39.5 ppm or C nuclei; CD OD: 3.31 ppm for H

3

1

3

13

nuclei and 49.0 ppm for C nuclei). Assignments in

C

NMR spectra of compounds 20, (Z)-3, 4bA and 6B were

1

13

1

13

made based on the COSY, H– C HSQC, and H– C

HMBC experiments. High-resolution mass spectra were

recorded on a Waters Synapt G2-Si TOF MS instrument

using ESI technique. Specific rotation was recorded on a

Kruss P3000 instrument. Analytical thin-layer chromato-

graphy (TLC) was performed on precoated silica gel F-254

plates (Merck).

silica gel using gradient elution from pure H

MeCN in H O.

General method VI. CF

added to a solution of macrocycle 7aA–C,bA,cA, (Z)-21,

or (E)-21 (1 equiv) in CH Cl (10 ml/mmol) at 0°C. The

2

O to 95%

2

All chemicals were used as obtained from commercial

sources and all reactions were performed under argon

atmosphere in an oven-dried (120°C) glassware, unless

3

CO H (50 equiv) was dropwise

2

noted otherwise. Anhydrous PhMe, Et

CH Cl

solvent through activated alumina columns.

General method I. Allylamine (2 equiv), followed by

2

O, THF, and

resulting solution was warmed to room temperature and

stirred for 3–5 h, whereupon all volatiles were concentrated

under reduced pressure. The resulting crude amine was

used in the next step without further purification. Thus,

(S)-2-hydroxy-3-methylbutanoic acid (1.5 equiv), EDC·HCl

(3 equiv), and HOBt (3 equiv) were added to a solution of

2

were obtained by passing commercially available

NEt (3 equiv), was dropwise added to a solution of bromo-

3

oxazole or bromobioxazole (1 equiv) in DMF (6 ml/mmol)

at room temperature. The orange solution was stirred for 18–

the above crude amine in CH

2

Cl (15 ml/mmol). Then

2

4 h at room temperature or at 50°C. Then the reaction

DIPEA (6 equiv) was added dropwise and the resulting

yellow solution was stirred at room temperature for 12–18 h,

whereupon the mixture was washed with 1 M aqueous HCl

(10 ml/mmol) two times. The organic layers were washed

mixture was diluted with EtOAc (10 ml/mmol) and washed

with H O (10 ml/mmol) two times. The organic layers were

washed with brine, dried over anhydrous Na SO , filtered,

2

4

and concentrated under reduced pressure. The residue was

purified by column chromatography on silica gel using

gradient elution from pure hexane to 30% EtOAc in hexane

with brine, dried over anhydrous Na

2

SO , and concentrated

4

under reduced pressure. The residue was purified by

reversed-phase column chromatography on silica gel using

or from pure CH

Cl

2 2

to 10% MeOH in CH

Cl

2 2

.

2

gradient elution from pure H O to 95% MeCN in H O.

2

General method II. 33% HBr solution in AcOH (60 equiv)

was added dropwise to a solution of Cbz-protected

aminooxazole (1 equiv) in 1,4-dioxane (3 ml/mmol) at

room temperature. The yellow solution was stirred for

Benzyl N-[(1S)-1-(5-amino-4-cyano-1,3-oxazol-2-yl)-

2,2-dimethylpropyl]carbamate (15). EDC·HCl (16.3 g,

85.2 mmol, 1.2 equiv) was added to a solution of

(2S)-2-{([benzyloxy)carbonyl]amino}-3,3-dimethylbutanoic

5,18

1

0 min, then cooled to 0°C (crushed ice batch), and 2 M

acid (13)

(18.8 g, 71.0 mmol, 1 equiv) and amino-

aqueous Na

2

CO

3

solution was carefully added (Caution!

malononitrile p-toluenesulfonate (14) (19.8 g, 78.1 mmol,

1.1 equiv) in pyridine (200 ml). The resulting orange

solution was stirred for 24 h at room temperature, where-

upon pyridine was evaporated under reduced pressure. The

Intense gas evolution!) until the mixture medium reached

pH 7–9. The mixture was extracted with EtOAc (10 ml/mmol)

four times. The combined organic layers were washed with

brine, dried over anhydrous Na

2

SO

4

,

and filtered.

residue was dissolved in EtOAc and washed with H

2

O

Morpholine (neat, 10 equiv) was added, and after stirring

for 1 h at room temperature, all volatiles were removed

under reduced pressure.

three times. The organic layers were washed with brine,

dried over anhydrous Na

2

SO , filtered, and evaporated

4

under reduced pressure. The crude product was used in the

next reaction without additional purification. Yield 21.5 g

General method III. N-Boc-protected carboxylic acid 16

1.5 equiv) was added at room temperature to a solution of

20

(

(92%), brown amorphous solid, [α]

D

–41.4° (c 1.0,

), δ, ppm (J, Hz): 7.38–

7.32 (5H, m, H Ph); 5.34 (1H, d, J = 9.8, NH); 5.12–5.09

(2H, m, CH ); 4.81 (2H, s, NH ); 4.58 (1H, d, J = 9.8, CH);

1

amine 9a,b, 19, 22, 27 (1 equiv) and EDC·HCl (2 equiv) in

anhydrous pyridine (15 ml/mmol). The yellow solution was

stirred for 3–18 h at room temperature, then pyridine was

evaporated under reduced pressure. The residue was

dissolved in EtOAc (10 ml/mmol) and washed with 0.1 M

aqueous HCl solution two times. The organic layers were

MeOH). H NMR spectrum (CDCl

3

2

13

0.98 (9H, s, C(CH

3

)

3

). C NMR spectrum (DMSO-d ),

6

δ, ppm: 162.3; 156.7; 151.9; 137.3; 128.8; 128.7; 128.2;

115.9; 82.8; 66.1; 58.2; 35.0; 26.7. Found, m/z: 329.1614

+

washed with brine, dried over anhydrous Na

2

SO

4

, filtered,

[M+H] . C17

H

21

N

4

O . Calculated, m/z: 329.1614.

3

5

92

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

Benzyl N-[(1S)-1-(5-bromo-4-cyano-1,3-oxazol-2-yl)-

,2-dimethylpropyl]carbamate (10). tert-Butylnitrite (1.2 ml,

0.1 mmol, 1.1 equiv) was added to a suspension of CuBr

pure hexane to 10% EtOAc in hexane. Yield 716 mg

1

2

(70%), colorless oil. H NMR spectrum (CDCl

3

), δ, ppm

1

2

(J, Hz): 5.79 (1H, ddt, J = 16.9, J = 10.2, J = 6.5,

(

4.1 g, 18.3 mmol, 2 equiv) in MeCN (80 ml) at room

CH

=CH

2.00 (2H, m, CH

(1H, m, CH ); 1.63 (9H, s, C(CH

agreement with the literature reported.

Methyl (2S)-2-[(tert-butoxycarbonyl)amino]hept-

6-enoate (17C). Anhydrous K CO (2.7 g, 19.5 mmol,

2

=CH); 5.09–5.04 (1H, m, CH); 5.04–4.95 (2H, m,

); 4.30–4.28 (1H, m, NH); 3.74 (3H, s, OCH ); 2.19–

); 1.95–1.89 (1H, m, CH ); 1.78–1.56

). NMR data are in

temperature under argon atmosphere. The resulting solution

2

3

was stirred for 5 min whereupon a solution of amine 15

2

(

3.0 g, 9.1 mmol, 1 equiv) in MeCN (30 ml) was added

2

3 3

)

7

dropwise at room temperature. Gas evolution was observed!

The reaction mixture was stirred for 1 h at room tempe-

rature. Then Et

2

O and H

2

O were added and layers were

2

3

separated. The organic layer was washed with 1 M aqueous

3 equiv) and MeI (1.2 ml, 19.5 mmol, 3 equiv) were added

to a solution of (2S)-2-[(tert-butoxycarbonyl)amino]pent-

4-enoic acid (16A) (1.4 g, 6.5 mmol, 1 equiv) in anhydrous

DMF (30 ml) at room temperature. The yellow suspension

HCl solution (3×20 ml), then with brine, dried over

anhydrous Na

2

SO , filtered, and concentrated under

4

reduced pressure. The residue was purified by reversed-

phase column chromatography on silica gel using gradient

was stirred for 18 h, whereupon it was diluted with Et

(50 ml), washed with H O (2×50 ml), then with aqueous

10% CuSO solution (50 ml) and brine. The organic layer

was dried over anhydrous Na SO and concentrated under

2

O

elution from 40 to 85% MeCN in H

2

O to give product as a

2

brown oil, which was additionally purified by direct-phase

column chromatography on silica gel using gradient elution

from pure hexane to 30% EtOAc in hexane. Yield 1.6 g

4

2

4

reduced pressure to give methyl ester 17A as a yellow oil,

yield 1.4 g (96%), which was used in the next step without

additional purification.

A solution of 9-BBN in THF (0.5 M, 26 ml, 13.1 mmol,

2.5 equiv) was added to a solution of methyl ester 17A (1.2 g,

5.2 mmol, 1 equiv) in anhydrous THF (20 ml) at 0°C

(crushed ice). The mixture was warmed to room tempe-

rature, stirred for 3 h, and then quenched with aqueous 1 M

2

0

1

(

46%), colorless oil, [α]

D

–17.7° (c 1.0, CHCl

3

). H NMR

spectrum (CDCl

3

), δ, ppm (J, Hz): 7.40–7.29 (5H, m,

H Ph); 5.40 (1H, d, J = 9.7, NH); 5.10 (2H, s, CH

2

); 4.76

1

3

(

1H, d, J = 9.7, CH); 1.00 (9H, s, C(CH

CDCl ), δ, ppm: 166.0; 155.9; 135.8; 130.9; 128.6; 128.4;

28.2; 116.0; 110.8; 67.5; 58.1; 35.5; 26.1. Found, m/z:

3

)

3

). C NMR

3

1

+

3

92.0603 [M+H] . C17

Methyl (2S)-2-[(tert-butoxycarbonyl)amino]hex-5-enoate

17B). An oven-dried Schlenk flask (50 ml) was charged

H19BrN

3 3

O . Calculated, m/z: 392.0610.

Na

2

CO solution (18 ml, 18.3 mmol, 3.5 equiv) while

3

(

passing a stream of argon through the resulting solution.

Vinyl bromide (1 M solution in THF, 21 ml, 20.9 mmol,

4 equiv), followed by a degassed solution of boronate from

with Zn powder (2.2 g, 34.0 mmol, 4 equiv) and attached to

high vacuum system (0.1 Torr). The flask was heated with

heatgun and then cooled back to room temperature. The

heating/cooling sequence was repeated three times. Then

the flask was disconnected from the vacuum, filled with

above was added to a stirred suspension of Pd

2

(dba)

3

(96 mg, 0.1 mmol, 0.02 equiv) and tri-o-tolylphosphine

(159 mg, 0.5 mmol, 0.1 equiv) in THF at −78°C (dry ice/

argon, and I

The flask was heated again with the heatgun until

2

(533 mg, 2.1 mmol, 0.3 equiv) was added.

Me CO bath). The resulting mixture was warmed to room

2

temperature and stirred for 20 h, whereupon the dark-

brown solution was diluted with H O (50 ml) and extracted

with EtOAc (3×50 ml). The organic extracts were combined,

evaporation of I

2

has started (red steam appeared). The

2

flask was cooled to room temperature under argon

atmosphere, and anhydrous DMF (10 ml) was added

dropwise, whereupon color of the suspension slowly

washed with brine, dried over anhydrous Na

2

SO , and

4

concentrated under reduced pressure. The residue was

purified by column chromatography on silica gel using

gradient elution from pure hexanes to 10% EtOAc in

(

within 1 min) changed from dark-brown to colorless. The

resulting suspension was cooled to 0°C (crushed ice), and a

1

solution of methyl (2R)-2-[(tert-butoxycarbonyl)amino]-

hexane. Yield 1.1 g (81%), colorless oil. H NMR spectrum

3-iodopropanoate (18) (2.3 g, 7.0 mmol, 1 equiv) in

(CDCl

4.91 (3H, m, CH

(3H, s, OCH ); 2.11–2.01 (2H, m, CH

CH ); 1.70–1.60 (1H, m, CH ); 1.55–1.38 (11H, m, C(CH

CH

3

), δ, ppm (J, Hz): 5.83–5.68 (1H, m, CH=CH ); 5.05–

2

anhydrous DMF (7 ml) was added dropwise. The resulting

yellow suspension was warmed to room temperature and

stirred for 3 h, whereupon the stirring was stopped to let

the solid settle to the bottom. The supernatant containing

organozinc species (~0.5 M, 8.4 ml, 4.2 mmol, 1 equiv)

was then carefully transferred by a syringe to a well-stirred

suspension of CuBr (301 mg, 2.1 mmol, 0.5 equiv) and

allyl bromide (0.7 ml, 8.4 mmol, 2 equiv) in anhydrous

DMF (5 ml) at –15°C (NaCl–ice bath). After the addition

was completed, the cooling bath was removed and the

stirring was continued for 20 h. Then, EtOAc (25 ml) was

added to the reaction mixture and stirring was continued

2

=CH, CH); 4.30–4.28 (1H, m, NH); 3.73

3

2

); 1.90–1.75 (1H, m,

2

3

) ,

3

6

2

). NMR data are in agreement with the literature reported.

(2S)-2-[(tert-Butoxycarbonyl)amino]hex-5-enoic acid

(16B). A solution of LiOH (44 mg, 1.9 mmol, 1 equiv) in

H O (10 ml) was added to a solution of ester 17B (450 mg,

2

1.9 mmol, 1 equiv) in 1,4-dioxane (10 ml). The colorless

mixture was stirred at room temperature for 3 h and

concentrated under reduced pressure. The residue was

partitioned between H O (20 ml) and EtOAc (20 ml). The

2

aqueous phase was acidified with 1 M HCl to pH 4 and

extracted with EtOAc (2×20 ml). The combined organic

layers were washed with brine, dried over anhydrous

for 15 min. The mixture was washed with H

organic layer was washed with aqueous 1 M Na

2×20 ml), then with H O (20 ml) and brine. After drying

over anhydrous Na SO and concentration under reduced

2

O (20 ml), the

2

S

2

O

3

(

2

Na

424 mg (99%), colorless oil. H NMR (CDCl

(J, Hz): 7.89 (1H, br. s, OH); 5.76–5.68 (1H, m, CH=CH

5.03–4.89 (3H, m, CH =CH, CH); 4.27–4.23 (1H, m, NH);

2

SO , and concentrated under reduced pressure. Yield

4

1

2

4

3

), δ, ppm

);

pressure, the residue was purified by column

chromatography on silica gel using gradient elution from

2

5

93

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

2

1

.12–2.03 (2H, m, CH

2

); 1.92–1.84 (1H, m, CH

2

); 1.74–

5.86–5.68 (2H, m, 2CH=CH

CH=CH ); 5.19–5.10 (2H, m, CH=CH

NH); 4.84 (1H, d, J = 9.4, CH–oxazole); 4.16–4.08 (1H, m,

CHCH ); 4.05–3.90 (2H, m, NCH ); 3.11 (3H, s, CH );

2.57–2.43 (2H, m, CHCH ); 1.44 (9H, s, C(CH ); 0.95

), δ, ppm:

2

); 5.31–5.20 (2H, m,

.68 (1H, m, CH ); 1.39 (9H, s, C(CH

2

3

)

3

). NMR data are in

2

); 4.95 (1H, br. s,

8

agreement with the literature reported.

(

2S)-2-[(tert-Butoxycarbonyl)amino]hept-6-enoic acid

16C). A solution of LiOH (101 mg, 4.2 mmol, 1 equiv) in

O (25 ml) was added to a solution of ester 17C (1.08 g,

2

3

(

2

3 3

)

13

H

2

(9H, s, C(CH

3

)

3

). C NMR spectrum (CDCl

3

4

.2 mmol, 1 equiv) in 1,4-dioxane (25 ml). The colorless

171.1; 160.0; 155.7; 151.7; 133.0; 131.1; 119.2; 118.9; 116.0;

mixture was stirred at room temperature for 3 h and

evaporated under reduced pressure. The residue was

84.5; 80.6; 55.0; 53.9; 36.3; 35.8; 28.3; 26.1. Found, m/z:

+

446.2771 [M+H] . C23

H

36

N

5

O . Calculated, m/z: 446.2767.

4

partitioned between H

2

O (50 ml) and EtOAc (50 ml). The

tert-Butyl N-{(1S)-1-[((1S)-1-{4-cyano-5-[methyl(prop-

2-en-1-yl)amino]-1,3-oxazol-2-yl}-2,2-dimethylpropyl)-

carbamoyl]pent-4-en-1-yl}carbamate (8aB) was obtained

from crude amine 9a (250 mg, 1.0 mmol), acid 16B (231 mg,

1.0 mmol), and EDC·HCl (232 mg, 1.2 mmol) in anhydrous

pyridine following the general method III. Yield 253 mg

aqueous phase was acidified with 1 M HCl to pH 4 and

extracted with EtOAc (2×20 ml). The combined organic

layers were washed with brine, dried over anhydrous

Na

2

SO , and concentrated under reduced pressure. Yield

4

1

9

22 mg (90%), colorless oil. H NMR spectrum (CDCl

δ, ppm (J, Hz): 9.53 (1H, br. s, OH); 5.85–5.69 (1H, m,

CH=CH ); 5.06–4.93 (3H, m, CH =CH, CH); 4.34–4.30

); 1.95–1.76 (1H, m,

); 1.56–1.37 (11H, m, C(CH

). NMR data are in agreement with the literature

3

),

2

0

1

(55%), yellow oil, [α]

spectrum (CDCl

CHNH); 5.87–5.69 (2H, m, 2CH=CH

CH=CH ); 5.09–4.97 (2H, m, CH=CH ); 4.95 (1H, d, J = 7.4,

NHCOO); 4.84 (1H, d, J = 9.0, CH–oxazole); 4.07–3.89

(3H, m, CH –CH=, CHCO); 3.11 (3H, s, CH ); 2.17–2.06

(2H, m, CH ); 1.99–1.85 (1H, m, CH ); 1.76–1.63 (1H, m,

CH ); 1.44 (9H, s, C(CH ); 0.96 (9H, s, C(CH ).

D

–42.5° (c 1.0, CHCl

3

). H NMR

2

3

), δ, ppm (J, Hz): 6.80 (1H, d, J = 9.0,

(

1H, m, NH); 2.13–2.03 (2H, m, CH

2

); 5.32–5.18 (2H, m,

CH

2

); 1.73–1.59 (1H, m, CH

2

3

)

3

,

2

6

reported.

2

3

2-((1S)-1-Amino-2,2-dimethylpropyl)-5-[methyl(prop-

2

-en-1-yl)amino]-1,3-oxazole-4-carbonitrile (9a) was obtain-

2

3

)

3

)

3 3

13

ed in a two-step sequence from benzyl N-[(1S)-1-(5-bromo-

C NMR spectrum (CDCl ), δ, ppm: 171.6; 160.1; 155.7;

3

4-cyano-1,3-oxazol-2-yl)-2,2-dimethylpropyl]carbamate (10).

151.7; 137.1; 131.1; 118.9; 116.0; 115.9; 84.5; 80.4; 55.0;

Step 1. Benzyl N-[(1S)-1-{4-cyano-5-[methyl(prop-2-en-

54.2; 53.9; 36.4; 35.8; 30.7; 29.8; 28.3; 26.1. Found, m/z:

+

1

-yl)amino]-1,3-oxazol-2-yl}-2,2-dimethylpropyl]carbamate

460.2925 [M+H] . C24

H

38

N

5

O . Calculated, m/z: 460.2924.

4

was obtained from bromooxazole 10 (6.0 g, 15.2 mmol),

N-methylallylamine (2.8 ml, 30.8 mmol), and NEt (6.4 ml,

tert-Butyl N-{(1S)-1-[((1S)-1-{4-cyano-5-[methyl(prop-

2-en-1-yl)amino]-1,3-oxazol-2-yl}-2,2-dimethylpropyl)-

carbamoyl]hex-5-en-1-yl}carbamate (8aC) was obtained

from crude amine 9a (350 mg, 1.4 mmol), acid 16C (343 mg,

1.4 mmol), and EDC·HCl (324 mg, 1.7 mmol) in anhydrous

pyridine following the general method III. Yield 495 mg

3

4

6.0 mmol) in anhydrous DMF following the general

method I. The crude product was purified by column

chromatography on silica gel using gradient elution from

pure hexanes to 30% EtOAc in hexane to afford the subtitle

2

0

20

1

compound. Yield 4.80 g (82%), yellow oil. [α]

D

–30.6°

), δ, ppm (J, Hz):

.39–7.27 (5H, m, H Ph); 5.86–5.73 (1H, m, CH=CH );

.43 (1H, d, J = 9.8, CH(NH)); 5.31–5.20 (2H, m, CH =);

.10 (2H, s, CH O); 4.57 (1H, d, J = 9.8, NH(CH)); 3.97

); 3.10 (3H, s, CH ); 0.97

), δ, ppm: 160.0;

(74%), yellow oil, [α]

spectrum (CDCl

CHNH); 5.88–5.68 (2H, m, 2CH=CH

CH=CH ); 5.05–4.92 (2H, m, CH=CH

m, NHCOO); 4.84 (1H, d, J = 9.5, CH–oxazole); 4.07–3.88

(3H, m, CH –CH=, CHCO); 3.11 (3H, s, CH ); 2.11–2.00

(2H, m, CH ); 1.87–1.81 (1H, m, CH ); 1.71–1.55 (1H, m,

CH ); 1.49–1.42 (11H, m, C(CH ,CH ); 0.98 (9H, s, C(CH ).

D

–42.3° (c 1.0, CHCl

3

). H NMR

1

(

c 1.0, CHCl

3

). H NMR spectrum (CDCl

3

), δ, ppm (J, Hz): 6.78 (1H, d, J = 8.8,

7

5

2

); 5.31–5.20 (2H, m,

); 4.91–4.87 (1H,

2

(

2H, dq, J = 14.0, J = 5.7, NCH

2

3

2

3

1

3

(

9H, s, C(CH

3

)

3

). C NMR spectrum (CDCl

3

2

1

56.1; 152.1; 136.1; 131.1; 128.5; 128.2; 128.1; 118.9;

16.0; 84.5; 67.2; 57.4; 53.9; 36.3; 35.7; 26.1. Found, m/z:

2

3

)

3

2

)

3 3

13

C NMR spectrum (CDCl ), δ, ppm: 171.8; 160.2; 151.9;

3

+

383.2083 [M+H] . C21

H

27

4 3

N O . Calculated, m/z: 383.2092.

138.1; 131.3; 119.1; 116.1; 115.3; 84.7; 54.8; 55.1; 54.1; 36.5;

Step 2. 2-((1S)-1-Amino-2,2-dimethylpropyl)-5-[methyl-

prop-2-en-1-yl)amino]-1,3-oxazole-4-carbonitrile (9a)

was obtained from benzyl N-[(1S)-1-{4-cyano-5-[methyl-

prop-2-en-1-yl)amino]-1,3-oxazol-2-yl}-2,2-dimethylpropyl]-

36.0; 33.4; 32.2; 31.2; 28.5; 26.4; 26.3; 25.1. Found, m/z:

+

(

474.3086 [M+H] . C25

H

40

N

5

O . Calculated, m/z: 474.3080.

4

2-((1S)-1-Amino-2,2-dimethylpropyl)-5-[benzyl(prop-

2-en-1-yl)amino]-1,3-oxazole-4-carbonitrile (9b) was

obtained in a two-step sequence from benzyl N-[(1S)-1-(5-bromo-

4-cyano-1,3-oxazol-2-yl)-2,2-dimethylpropyl]carbamate (10).

Step 1. 2-((1S)-1-Amino-2,2-dimethylpropyl)-5-bromo-

1,3-oxazole-4-carbonitrile. 33% HBr solution in AcOH

(28 ml, 163 mmol, 40 equiv) was added dropwise to a

solution of bromooxazole 10 (1.6 g, 4.1 mmol, 1 equiv) in

1,4-dioxane (15 ml) at room temperature. The yellow

solution was stirred for 10 min, and cooled (0°C) 2 M

(

carbamate (step 1) (3.57 g, 9.3 mmol) and 33% HBr in

AcOH (96 ml, 560.1 mmol) in 1,4-dioxane, followed by

the addition of morpholine (8.1 ml, 93.3 mmol) by the

general method II. Yield 762 mg (33%), orange oil. The

crude amine 9a was used in the next step without

additional purification.

tert-Butyl N-{(1S)-1-[((1S)-1-{4-cyano-5-[methyl(prop-

-en-1-yl)amino]-1,3-oxazol-2-yl}-2,2-dimethylpropyl)-

2

carbamoyl]but-3-en-1-yl}carbamate (8aA) was obtained

from crude amine 9a (254 mg, 1.0 mmol), EDC·HCl (292 mg,

Na

2

CO

3

aqueous solution was added till the mixture

medium reached pH 7–9. The mixture was extracted with

EtOAc (4×50 ml). The organic layers were combined and

washed with 1 M HCl aqueous solution (2×50 ml).

2

.0 mmol), and acid 16A (330 mg, 1.5 mmol) in anhydrous

pyridine following the general method III. Yield 365 mg

2

0

1

(

80%), colorless oil, [α]

D

–33.5° (c 1.3, CDCl

3

). H NMR

Aqueous layers were combined, and solid Na

added till the medium reached pH 9. Aqueous layer was

2

CO was

3

spectrum (CDCl

3

), δ, ppm (J, Hz): 6.91 (1H, br. s, NH);

5

94

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

extracted with EtOAc (3×50 ml). The organic layers were

combined, washed with brine, dried over anhydrous Na SO

and evaporated under reduced pressure. The crude subtitle

product was obtained as orange oil, yield 700 mg (67%), and

used in the next reaction without additional purification.

Step 2. 2-((1S)-1-Amino-2,2-dimethylpropyl)-5-[benzyl-

Step 2. 2-((1S)-1-Amino-2,2-dimethylpropyl)-5-(phenyl-

2

4

,

amino)-1,3-oxazole-4-carbonitrile (19) was obtained from

benzyl N-{(1S)-1-[4-cyano-5-(phenylamino)-1,3-oxazol-2-yl]-

2,2-dimethylpropyl}carbamate (step 1) (1.05 g, 2.6 mmol),

33% HBr solution in AcOH (27 ml, 156 mmol), and

morpholine (2.3 ml, 26.0 mmol) in 1,4-dioxane following

the general method II. The crude amine 19 was used in the

next step without additional purification. Yield 390 mg

(56%), yellow oil.

(

prop-2-en-1-yl)amino]-1,3-oxazole-4-carbonitrile (9b)

was obtained from 2-[(1S)-1-amino-2,2-dimethylpropyl]-

-bromo-1,3-oxazole-4-carbonitrile (700 mg, 2.7 mmol),

N-allylbenzylamine (0.8 ml, 5.4 mmol), and NEt (1.1 ml,

5

3

8

.1 mmol) in anhydrous DMF 50°C (72 h) following the

general method I. Purification by column chromatography

on silica gel using gradient elution from pure hexane to

3

0% EtOAc in hexane afforded compound 9b. Yield 295 mg

34%), yellow oil. Compound 9b was used in the next

(

reaction without additional purification.

tert-Butyl N-{(1S)-1-[((1S)-1-{5-[benzyl(prop-2-en-1-

yl)amino]-4-cyano-1,3-oxazol-2-yl}-2,2-dimethylpropyl)-

carbamoyl]but-3-en-1-yl}carbamate (8bA) was obtained

from compound 9b (295 mg, 0.9 mmol), acid 16A (196 mg,

0

.9 mmol), and EDC·HCl (209 mg, 1.1 mmol) in anhydrous

pyridine following the general method III. Yield 310 mg

2

0

1

(

65%), yellow oil, [α]

D

–28.0° (c 1.0, CHCl

3

). H NMR

spectrum (CDCl

3

), δ, ppm (J, Hz): 7.40–7.20 (5H, m, H Ph);

6

5

4

3

1

.97–6.86 (1H, m, NH); 5.88–5.66 (2H, m, 2CH=CH

.23 (2H, m, CH=CH ); 5.21–5.09 (2H, m, CH=CH

.89 (1H, m, NH); 4.84 (1H, d, J = 9.5, CH–oxazole); 4.69–

.48 (2H, m, CH Ph); 4.17–4.06 (1H, m, CHCO); 4.05–

.87 (2H, m, CH –CH=); 2.59–2.40 (2H, m, CH –CH=);

.44 (9H, s, C(CH

), δ, ppm: 171.1; 159.9; 155.7; 151.8; 135.5;

33.0; 131.2; 128.9; 128.1; 127.7; 119.2; 155.7; 84.8; 80.6;

5.0; 53.9; 52.4; 51.1; 35.9; 29.7; 28.3; 26.1. Found, m/z:

2

); 5.32–

2

); 4.98–

2

1

3

)

3

); 0.92 (9H, s, C(CH ) ). C NMR

3

spectrum (CDCl

3

1

5

tert-Butyl [(1S)-1-({(1S)-1-[4-cyano-5-(phenylamino)-

1,3-oxazol-2-yl]-2,2-dimethylpropyl}carbamoyl)but-3-en-

1-yl]carbamate (20) was obtained from aminooxazole 19

(390 mg, 1.4 mmol), EDC·HCl (553 mg, 2.9 mmol), and

acid 16A (466 mg, 2.2 mmol) in anhydrous pyridine

following the general method III. Yield 585 mg (87%),

+

22.3083 [M+H] . C29

H

40

N

5

O

4

. Calculated, m/z: 522.3080.

2

-((1S)-1-Amino-2,2-dimethylpropyl)-5-(phenylamino)-

,3-oxazole-4-carbonitrile (19) was obtained in a two-step

1

sequence from benzyl N-[(1S)-1-(5-bromo-4-cyano-1,3-

oxazol-2-yl)-2,2-dimethylpropyl]carbamate (10).

2

0

1

Step 1. Benzyl N-{(1S)-1-[4-cyano-5-(phenylamino)-

,3-oxazol-2-yl]-2,2-dimethylpropyl}carbamate. Aniline

colorless oil, [α]

spectrum (CDCl

D

–28.0° (c 1.0, CHCl

3

). H NMR

1

3

), δ, ppm (J, Hz): 7.39–7.32 (2H, m, H Ph);

(

0.4 ml, 4.2 mmol, 2 equiv) was added dropwise to a

7.23–7.16 (2H, m, H Ph); 7.14–7.08 (1H, m, H Ph); 5.75

(1H, ddd, J = 17.3, J = 10.0, J = 7.2, CH=CH ); 5.20–5.10

(2H, m, CH=CH ); 4.94 (1H, d, J = 9.3, CH–t-Bu); 4.18–

4.11 (1H, m, CHNHCOO); 2.59–2.45 (2H, m, CH –CH=);

solution of bromooxazole 10 (815 mg, 2.1 mmol, 1 equiv)

2

in anhydrous THF (20 ml) at room temperature. Then the

2

orange solution was cooled to –78°C (dry ice/Me

2

CO) and

2

13

KHMDS (1 M solution in THF (7.3 ml, 7.3 mmol, 3.5 equiv))

was added dropwise. The resulting dark solution was

stirred for 30 min at –78°C and quenched by the addition of

1.45 (9H, s, C(CH

3

)

3

); 1.00 (9H, s, C(CH ) ). C NMR

3

spectrum (CDCl ), δ, ppm: 171.3; 170.3; 156.8; 153.7;

3

137.6; 133.1; 129.6; 124.1; 119.3; 118.4; 113.8; 89.3; 80.6;

+

aqueous saturated NH

4

Cl solution. The organic layer was

55.4; 53.9; 35.8; 28.3; 26.2. Found, m/z: 490.2426 [M+Na] .

diluted with EtOAc (50 ml), washed with aqueous 1 M HCl

C

25

H

33NaN

5

O . Calculated, m/z: 490.2430.

4

(

2×40 ml), brine, and dried over anhydrous Na

2

SO

4

.

tert-Butyl N-{(1S)-1-[((1S)-1-{4-cyano-5-[phenyl(prop-

Filtration and evaporation under reduced pressure afforded

2-en-1-yl)amino]-1,3-oxazol-2-yl}-2,2-dimethylpropyl)-

carbamoyl]but-3-en-1-yl}carbamate (8cA). 60% NaH in

mineral oil (116 mg, 2.9 mmol) was added to a solution of

phenylaminooxazole 20 (450 mg, 1.0 mmol) in anhydrous

DMF (10 ml) at 0°C. After the hydrogen evolution was

ceased, the resulting yellow suspension was stirred for 15 min,

whereupon allyl bromide (0.08 ml, 1.0 mmol) was added

dropwise. The resulting solution was stirred for 1 h at 0°C,

the subtitle compound as a yellow amorphous solid, yield

20

1

7

60 mg (90%), [α]

D

–7.6° (c 1.0, CHCl ). H NMR spectrum

3

(

CDCl ), δ, ppm (J, Hz): 7.50 (1H, br. s, NHPh); 7.40–7.27

3

7H, m, H Ph); 7.22–7.08 (3H, m, H Ph); 5.45 (1H, d,

J = 9.8, CH(NH)); 5.14–5.09 (2H, m, CH

J = 9.8, NH(CH)); 1.02 (9H, s, C(CH

2

); 4.69 (1H, d,

13

3

)

3

). C NMR

spectrum (CDCl

3

), δ, ppm: 156.9; 156.1; 154.0; 137.6;

1

8

C

36.0; 129.6; 128.6; 128.3; 128.2; 124.1; 118.3; 113.9;

quenched by the addition of H

EtOAc (3×50 ml). The combined organic extracts were

washed with brine, dried over anhydrous Na SO , and

2

O, and extracted with

+

9.2; 67.4; 57.8; 35.7; 26.2. Found, m/z: 405.1928 [M+H] .

. Calculated, m/z: 405.1927.

23

H

25

N

4

O

3

2

4

5

95

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

concentrated under reduced pressure. The residue was

purified by column chromatography on silica gel using

gradient elution from pure hexane to 30% EtOAc in hexane

to afford the title compound 8cA. Yield 185 mg (38%),

(CDCl

(1H, d, J = 7.6, CHC(CH

NHCOO); 3.84 (1H, t, J = 14.5, CH

3.00 (1H, dt, J = 14.5, J = 3.1, CH

CH ); 1.61–1.49 (1H, m, CH ); 1.48–1.36 (10H, m, CH

C(CH ); 1.33–1.21 (1H, m, CH ); 1.06 (9H, s, C(CH ).

3

), δ, ppm (J, Hz): 5.88 (1H, d, J = 7.6, NHCO); 5.51

); 4.59–4.48 (2H, m, CHCH

); 3.17 (3H, s, CH );

); 2.02–1.76 (3H, m,

3

)

3

2

,

2

3

2

20

1

yellow oil, [α]

CDCl

D

–36.0° (c 1.0, CHCl

3

). H NMR spectrum

2

,

(

3

), δ, ppm (J, Hz): 7.45–7.27 (3H, m, H Ph); 7.25–

3

)

3

2

)

3 3

13

7

.08 (2H, m, H Ph); 6.96–6.90 (1H, m, NH); 5.98–5.88

C NMR spectrum (CDCl ), δ, ppm: 172.9; 160.1; 155.3;

3

(

1H, m, CH=CH

2H, m, CH=CH

2

); 5.79–5.71 (1H, m, CH=CH

); 5.19–5.11 (2H, m, CH=CH

2

); 5.30–5.23

150.1; 116.4; 83.5; 79.9; 58.9; 53.4; 49.1; 36.4; 33.6; 31.0;

+

(

2

); 5.01–4.92

28.5; 26.7; 25.4. Found, m/z: 442.2415 [M+Na] .

(

1H, m, CHNHCOO); 4.86 (1H, d, J = 9.4, CH–oxazole);

C

21

H

33NaN

5

O . Calculated, m/z: 442.2430.

4

.40 (2H, ddt, J = 16.5, J = 6.8, J = 1.4, CH

.08 (1H, m, NH); 2.58–2.44 (2H, m, CH

); 0.95 (9H, s, C(CH

), δ, ppm: 171.3; 158.7; 155.8; 153.1;

41.6; 133.2; 132.1; 129.8; 127.5; 125.2; 119.4; 118.8; 114.0;

8.7; 80.7; 55.2 (2C); 54.0; 36.0; 35.9; 28.4; 26.3. Found, m/z:

2

–CH=); 4.18–

tert-Butyl N-((8S,11S)-11-tert-butyl-14-cyano-2-methyl-

4

2

–CH=); 1.45

9-oxo-15-oxa-2,10,13-triazabicyclo[10.2.1]pentadeca-

1(14),12-dien-8-yl)carbamate (7aB) was obtained in a

two-step sequence from amide 8aB (200 mg, 0.4 mmol)

and ruthenium metathesis catalyst Ru4 (16 mg, 0.02 mmol)

1

3

(

9H, s, C(CH

3

)

3

) ). C NMR

3

spectrum (CDCl

3

1

8

in anhydrous CH

2

Cl following the general method IV. The

2

+

5

08.2921 [M+H] . C28

tert-Butyl N-((4Z,7S,10S)-10-tert-butyl-13-cyano-

-methyl-8-oxo-14-oxa-2,9,12-triazabicyclo[9.2.1]tetradeca-

H

38

N

5

O

4

. Calculated, m/z: 508.2924.

crude mixture of E/Z-isomers in 2:1 ratio of the resulting

macrocycle was hydrogenated in the presence of 10% Pd

on carbon (46 mg, 0.04 mmol) following the general

method V. Yield in two steps 90 mg (48%), white amorphous

2

1

(13),4,11-trien-7-yl)carbamate (21), obtained as a mixture

of E/Z-isomers in 59:41 ratio. Compound 21 was synthe-

sized from amide 8aA (350 mg, 0.8 mmol) and ruthenium

metathesis catalyst Ru4 (29 mg, 0.04 mmol) in anhydrous

2

0

1

solid, [α]

(CDCl

D

–59.7° (c 1.0, CHCl

3

). H NMR spectrum

3

), δ, ppm (J, Hz): 6.17 (1H, d, J = 9.3, NHCO); 5.50

(1H, d, J = 7.5, NHCOO); 4.88 (1H, d, J = 9.3, CHC(CH

4.36–4.27 (1H, m, CHCH ); 3.63–3.56 (1H, m, CH ); 3.19–

3.07 (4H, m, CH , CH ); 1.94–1.83 (1H, m, CH ); 1.64–

1.48 (2H, m, CH ); 1.44–1.30 (13H, m, CH , C(CH );

1.15–1.03 (10H, m, CH ). C NMR spectrum

(CDCl ), δ, ppm: 171.7; 160.2; 155.3; 149.7; 116.7; 83.3;

3

) );

3

CH

the crude product by column chromatography on silica gel

using gradient elution from pure CH Cl to 5% MeOH in

2

Cl

2

following the general method IV. Purification of

2

3

2

)

3 3

13

CH

2

Cl

2

afforded product 21. Yield 220 mg (65%), yellow

2

, C(CH

)

3 3

amorphous solid. Individual isomers were obtained after

separation by silica gel column chromatography using

3

79.9; 55.7; 53.9; 49.5; 36.1; 33.8; 32.9; 28.5; 26.5; 25.8;

24.6; 20.8. Found, m/z: 456.2574 [M+Na] . C22

Calculated, m/z: 456.2587.

+

CH

2

Cl

2

–EtOAc, 9:1 as eluent.

H

35NaN

5 4

O .

Compound (Z)-21. Yield 121 mg (37%), white amorphous

2

0

1

solid, [α]

D

–294.8° (c 1.0, MeOH). H NMR spectrum

tert-Butyl N-((9S,12S)-12-tert-butyl-15-cyano-2-methyl-

10-oxo-16-oxa-2,11,14-triazabicyclo[11.2.1]hexadeca-

1(15),13-dien-9-yl)carbamate (7aC) was obtained in a

two-step sequence from amide 8aC (350 mg, 0.7 mmol)

and ruthenium metathesis catalyst Ru4 (27 mg, 0.04 mmol)

(

5

4

CDCl ), δ, ppm (J, Hz): 6.36 (1H, d, J = 6.9, NH–CO);

3

.64–5.53 (2H, m, NH, CH=); 5.43–5.33 (1H, m, CH=);

.44 (1H, d, J = 6.9, CH–C(CH ); 4.41–4.30 (1H, m,

); 4.15–4.05 (1H, m, CH ); 3.67–3.59 (1H, m,

); 3.23 (3H, s, CH ); 2.55–2.47 (1H, m, CH ); 2.39–

); 1.39 (9H, s, C(CH ); 1.03 (9H, s,

). C NMR spectrum (CDCl ), δ, ppm: 172.2;

)

3 3

NH–CH–CH

2

CH

2

3

2

in anhydrous CH

2

Cl following the general method IV. The

2

.28 (1H, m, CH

2

3

)

3

crude mixture of E/Z-isomers in 2:1 ratio of the resulting

macrocycle was hydrogenated in the presence of 10% Pd

on carbon (88 mg, 0.08 mmol) following the general

method V. Yield in two steps 161 mg (44%), white

13

C(CH

3

)

3

1

5

4

60.5; 155.2; 150.9; 130.4; 128.0; 115.9; 86.2; 80.0; 57.7;

7.0; 53.8; 39.3; 36.4; 33.5; 28.3; 26.5. Found, m/z:

+

20

1

18.2494 [M+H] . C21

H

32

N

5

O

4

. Calculated, m/z: 418.2454.

amorphous solid, [α]

spectrum (CDCl

D

–144.6° (c 1.0, MeOH). H NMR

Compound (E)-21. Yield 84 mg (26%), beige amorphous

3

), δ, ppm (J, Hz): 6.54 (1H, br. s, NHCO);

20

o

1

solid, [α]

D

–226.7 (c 0.6, MeOH). H NMR spectrum

5.25 (1H, br. s, NHCOO); 4.82 (1H, d, J = 9.2, CHC(CH

4.19–4.10 (1H, m, CHCH ); 3.68–3.58 (1H, m, CH ); 3.19–

3.11 (4H, m, CH , CH ); 1.90–1.76 (2H, m, CH ); 1.70–

1.58 (2H, m, CH ); 1.48–1.38 (11H, m, CH , C(CH );

1.38–1.28 (2H, m, CH ); 1.23–1.15 (2H, m, CH ); 1.03

), δ, ppm:

3

) );

3

(

CDCl

3

), δ, ppm (J, Hz): 5.96 (1H, d, J = 6.2, NHCO); 5.74

);

.59–5.52 (1H, m, CH=); 5.28 (1H, s, NH); 4.54–4.43 (3H,

, NH–CH–CH ); 3.23 (3H, s, CH ); 2.84 (1H, t,

J = 13.1, CH ); 2.60–2.52 (1H, m, CH ); 1.41 (9H, s,

C(CH ); 1.05 (9H, s, C(CH ). C NMR spectrum

CDCl ), δ, ppm: 171.6; 160.1; 155.0; 151.1; 128.2; 123.8;

15.8; 86.1; 80.0; 57.8; 52.6; 49.8; 37.8; 33.7; 31.1; 28.3; 26.5.

2

(

1H, t, J = 11.1, CH=); 5.65 (1H, d, J = 6.2, CH–C(CH

3

)

3

2

5

2

)

3 3

m, CH

2

3

2

13

2

(9H, s, C(CH

3

)

3

). C NMR spectrum (CDCl

3

1

3

)

3

)

3

171.4; 160.2; 155.6; 149.8; 116.4; 84.0; 80.1; 55.7; 54.0;

(

49.5; 35.8; 34.8; 31.3; 28.3; 26.3; 26.0; 25.2; 24.8; 24.4.

+

1

Found, m/z: 448.2924 [M+H] . C23

H N

38 5

O . Calculated, m/z:

4

+

Found, m/z: 418.2489 [M+H] . C21

H

32

N

5

O

4

. Calculated, m/z:

448.2924.

4

18.2441.

tert-Butyl N-((7S,10S)-2-benzyl-10-tert-butyl-13-cyano-

8-oxo-14-oxa-2,9,12-triazabicyclo[9.2.1]tetradeca-1(13),11-

dien-7-yl)carbamate (7bA) was obtained in a two-step

sequence from amide 8bA (280 mg, 0.54 mmol) and

ruthenium metathesis catalyst Ru4 (20 mg, 0.03 mmol) in

tert-Butyl N-((7S,10S)-10-tert-butyl-13-cyano-2-methyl-

8

-oxo-14-oxa-2,9,12-triazabicyclo[9.2.1]tetradeca-1(13),11-

dien-7-yl)carbamate (7aA) was obtained by the hydro-

genation of alkene (Z)-21 (88 mg, 0.2 mmol) in the

presence of 10% Pd on carbon (23 mg, 0.02 mmol)

following the general method V. Yield 85 mg (96%),

anhydrous CH

2

Cl following the general method IV. The

2

crude mixture of E/Z-isomers in 2:1 ratio of the resulting

20

1

yellow oil, [α]

D

–63.0° (c 1.0, CHCl

3

). H NMR spectrum

macrocycle was hydrogenated in the presence of 10% Pd

5

96

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

on carbon (57 mg, 0.05 mmol) following the general

method V. Yield in two steps 90 mg (34%), white amorphous

2

0

1

solid, [α]

D

–30.3° (c 1.0, CHCl

3

). H NMR spectrum

(

CDCl

3

), δ, ppm (J, Hz): 7.40–7.26 (5H, m, H Ph); 5.87

(

1H, d, J = 7.5, NHCO); 5.51 (1H, d, J = 7.5, CHC(CH

.81 (1H, d, J = 15.6, NHCOO); 4.60–4.43 (3H, m, CH

); 3.73 (1H, t, J = 15.6, CH ); 3.05 (1H, dt, J = 15.6,

J = 3.3, CH ); 2.05–1.96 (1H, m, CH ); 1.88–1.70 (2H, m,

CH ); 1.58–1.37 (11H, m, CH , C(CH ); 1.24–1.12 (1H,

m, CH ); 1.08 (9H, s, C(CH

), δ, ppm: 172.9; 160.1; 155.3; 150.0; 135.6; 129.1;

28.4; 127.9; 116.0; 83.7; 79.9; 58.8; 53.4; 52.3; 46.1;

3.7; 31.1; 28.5; 26.7; 25.8; 18.2. Found, m/z: 496.2921

3

)

3

);

4

2

Ph,

CHCH

2

3 3

)

1

3

2

3

) ). C NMR spectrum

3

(

CDCl

3

1

3

m, CH

J = 14.7, J = 2.8, CH

dddd, J = 13.1, J = 5.4, J = 5.4, J = 2.8, CH

ddd, J = 13.1, J = 11.4, J = 11.4, CH ); 2.07 (1H, sept d,

J = 6.9, J = 3.4, CH(CH ); 1.08 (9H, s, C(CH ); 0.99

(3H, d, J = 6.9, CH CH); 0.83 (3H, d, J = 6.9, CH CH).

2

); 3.84 (1H, d, J = 3.5, CHOH); 3.73 (1H, dq,

); 3.25 (3H, s, NCH ); 2.54 (1H,

); 2.28 (1H,

2

3

+

[

M+H] . C27

H

38

N

5

O

4

. Calculated, m/z: 496.2924.

2

tert-Butyl N-((7S,10S)-10-tert-butyl-13-cyano-8-oxo-

-phenyl-14-oxa-2,9,12-triazabicyclo[9.2.1]tetradeca-

(13),11-dien-7-yl)carbamate (7cA) was obtained in a

2

1

3

)

2

)

3 3

3

1

3

two-step sequence from amide 8cA (185 mg, 0.4 mmol)

and ruthenium metathesis catalyst Ru4 (14 mg, 0.02 mmol)

C NMR spectrum (CD OD), δ, ppm: 176.1; 174.2; 162.3;

3

153.3; 131.9; 128.8; 117.0; 86.1; 76.8; 59.7; 57.8; 52.5;

in anhydrous CH

Cl

2 2

following the general method IV. The

39.3; 37.1; 34.5; 33.0; 26.8; 19.5; 16.1. Found, m/z:

+

crude mixture of E/Z-isomers in 2:1 ratio of the resulting

macrocycle was hydrogenated in the presence of 10% Pd

on carbon (39 mg, 0.04 mmol) following the general

method V. Yield in two steps 82 mg (47%), white amorphous

418.2455 [M+H] . C21

H

32

N

5

O . Calculated, m/z: 418.2454.

4

(2S)-N-((4E,7S,10S)-10-tert-Butyl-13-cyano-2-methyl-

8-oxo-14-oxa-2,9,12-triazabicyclo[9.2.1]tetradeca-1(13),4,11-

trien-7-yl)-2-hydroxy-3-methylbutanamide ((E)-3) was

2

0

1

solid, [α]

D

–126.9° (c 0.6, CHCl

3

). H NMR spectrum

obtained from compound (E)-21 (83 mg, 0.2 mmol), CF

3

CO H

2

(

CDCl

3

), δ, ppm (J, Hz): 7.49–7.27 (5H, m, H Ph); 5.91

(0.76 ml, 9.9 mmol), (S)-2-hydroxy-3-methylbutanoic acid

(35 mg, 0.3 mmol), EDC·HCl (114 mg, 0.6 mmol), HOBt

(81 mg, 0.6 mmol), and DIPEA (0.21 ml, 1.2 mmol)

following the General method VI. Yield 48 mg (58%),

(

1H, d, J = 7.5, NHCO); 5.56 (1H, d, J = 7.5, CHC(CH

.62–4.57 (2H, m, CHCH , NHCOO); 4.12 (1H, t, J = 14.4,

); 3.51 (1H, dt, J = 14.4, J = 3.2, CH ); 2.13–2.01 (1H,

m, CH ); 1.98–1.85 (2H, m, CH ); 1.68–1.56 (1H, m, CH );

.43 (9H, s, C(CH ); 1.32–1.23 (1H, m, CH ); 1.19–1.06

10H, m, CH , C(CH ). C NMR spectrum (CDCl ),

δ, ppm: 172.9; 159.3; 155.2; 150.4; 139.9; 130.1; 128.7;

3

)

3

);

4

CH

2

0

2

beige amorphous solid, [α]

D

–190.7° (c 1.0, MeOH).

OD), δ, ppm (J, Hz): 5.71–5.57

(2H, m, 2CH=); 4.92 (1H, dd, J = 5.3, J = 2.4, CHCH );

4.59–4.49 (1H, m, CH ); 3.89 (1H, d, J = 3.3, CHOH); 3.38

(1H, s, CHC(CH ); 3.35 (1H, s, CH ); 3.25 (3H, s,

NCH ); 3.14–3.04 (1H, m, CH ); 2.58–2.47 (1H, m, CH );

2.18–2.07 (1H, m, CH(CH ); 1.09 (9H, s, C(CH ); 1.01

(3H, d, J = 6.9, CH CH); 0.85 (3H, d, J = 6.9, CH CH).

1

(

3

)

3

2

H NMR spectrum (CD

3

13

2

3

)

3

2

1

2

27.5; 113.6; 85.8; 79.8; 58.8; 53.3; 50.1; 33.6; 31.1; 28.4;

3

)

3

2

+

6.6; 26.2; 17.9. Found, m/z: 482.2759 [M+H] . C26

H

36

N

5

O

4

.

3

2

Calculated, m/z: 482.2767.

3

)

2

)

3 3

(

2S)-N-((4Z,7S,10S)-10-tert-Butyl-13-cyano-2-methyl-

3

13

8

-oxo-14-oxa-2,9,12-triazabicyclo[9.2.1]tetradeca-1(13),4,11-

C NMR spectrum (CD OD), δ, ppm: 174.6; 172.8; 160.8;

3

trien-7-yl)-2-hydroxy-3-methylbutanamide ((Z)-3) was

obtained from compound (Z)-21 (110 mg, 0.3 mmol),

151.8; 130.5; 127.4; 115.5; 84.7; 75.3; 58.3; 56.3; 51.0;

37.9; 35.7; 33.1; 31.5; 25.3; 18.1; 14.7. Found, m/z:

+

CF

3

CO

2

H (1.0 ml, 13.2 mmol), (S)-2-hydroxy-3-methyl-

418.2477 [M+H] . C21

H

32

N

5

O . Calculated, m/z: 418.2454.

4

butanoic acid (47 mg, 0.4 mmol), EDC·HCl (152 mg,

(2S)-N-((7S,10S)-10-tert-Butyl-13-cyano-2-methyl-8-oxo-

14-oxa-2,9,12-triazabicyclo[9.2.1]tetradeca-1(13),11-dien-

7-yl)-2-hydroxy-3-methylbutanamide (4aA) was obtained

0

(

.8 mmol), HOBt (107 mg, 0.8 mmol), and DIPEA

0.28 ml, 1.6 mmol) following the general method VI.

20

Yield 60 mg (54%), white amorphous solid, [α]

D

–264.1°

from compound 7aA (80 mg, 0.2 mmol), CF

3

CO H (0.73 ml,

2

1

(

c 0.7, CD

3

OD). H NMR spectrum (CD

3

OD), δ, ppm

9.5 mmol), (S)-2-hydroxy-3-methylbutanoic acid (34 mg,

0.3 mmol), EDC·HCl (108 mg, 0.6 mmol), HOBt (76 mg,

0.6 mmol), and DIPEA (0.2 ml, 1.1 mmol) following the

General method VI. Yield 39 mg (50%), white amorphous

J, Hz): 5.84–5.75 (1H, m, CH=); 5.42 (1H, dddd, J = 11.4,

J = 10.2, J = 2.8, J = 2.8, CH=); 4.80 (1H, dd, J = 11.4,

J = 5.4, CHCH

2

); 4.36 (1H, s, CHC(CH ) ); 4.14–4.05 (1H,

3

2

0

1

solid, [α]

CD

.86 (1H, m, CHCH

3H, s, NCH ); 3.09 (1H, dt, J = 14.7, J = 3.2, CH

.02 (2H, m, CH , CH(CH ); 2.00–1.86 (2H, m, CH

.50–1.37 (1H, m, CH ); 1.32–1.21 (1H, m, CH ); 1.09

); 1.05–1.01 (1H, m, CH ); 1.00 (3H, d, J = 6.9,

CH); 0.93–0.85 (1H, m, CH ); 0.83 (3H, d, J = 6.9,

D

–109.5° (c 1.0, MeOH). H NMR spectrum

(

3

OD), δ, ppm (J, Hz): 4.41 (1H, s, CHC(CH ) ); 3.95–

3

2

); 3.86 (1H, d, J = 3.2, CHOH); 3.18

); 2.16–

);

(

3

2

1

2

3

)

2

(

9H, s, C(CH

3

)

3

2

CH

3

2

13

3

CH). C NMR spectrum (CD OD), δ, ppm: 175.8;

3

1

3

4

74.7; 161.6; 152.5; 117.4; 83.0; 76.8; 60.7; 52.6; 49.8;

6.4; 34.4; 32.9; 31.4; 26.7; 26.3; 19.6; 19.1. Found, m/z:

+

20.2599 [M+H] . C21

H

34

N

5

O . Calculated, m/z: 420.2611.

4

5

97

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

(

2S)-N-((8S,11S)-11-tert-Butyl-14-cyano-2-methyl-9-oxo-

5-oxa-2,10,13-triazabicyclo[10.2.1]pentadeca-1(14),12-

dien-8-yl)-2-hydroxy-3-methylbutanamide (4aB) was

1

obtained from compound 7aB (90 mg, 0.2 mmol), CF

3

CO H

2

(

1.0 ml, 12.5 mmol), (S)-2-hydroxy-3-methylbutanoic acid

37 mg, 0.3 mmol), EDC·HCl (119 mg, 0.6 mmol), HOBt

84 mg, 0.6 mmol), and DIPEA (0.2 ml, 1.2 mmol)

following the general method VI. Yield 41 mg (46%),

2

0

white amorphous solid, [α]

D

–88.9° (c 1.1, MeOH).

OD), δ, ppm (J, Hz): 4.79 (1H, s,

); 4.59 (1H, t, J = 5.3, CHCH ); 3.88 (1H, d,

J = 3.3, CHOH); 3.58–3.48 (1H, m, CH ); 3.41–3.34 (1H,

); 2.15–2.02 (1H, m, CH(CH );

); 1.70–1.56 (2H, m, CH ); 1.49–

); 1.36–1.18 (2H, m, CH ); 1.15 (9H, s,

); 1.02 (3H, d, J = 6.9, CH CH); 0.86 (3H, d, J = 6.9,

1

H NMR spectrum (CD

CHC(CH

3

)

3

2

m, CH

2

); 3.19 (3H, s, NCH

.79–1.71 (2H, m, CH

.37 (2H, m, CH

3

)

3 2

1

2

C(CH

3

)

3

13

CH

3

CH). C NMR spectrum (CD OD), δ, ppm: 175.9;

3

1

3

73.7; 161.7; 151.5; 117.6; 83.4; 76.8; 57.6; 53.3; 50.3;

6.2; 34.6; 33.4; 33.0; 26.8; 26.7; 25.8; 21.9; 19.6; 16.1.

+

Found, m/z: 434.2772 [M+H] . C22

H

36

N

5

O . Calculated, m/z:

4

34.2767.

2S)-N-((9S,12S)-12-tert-Butyl-15-cyano-2-methyl-10-oxo-

(

1

6-oxa-2,11,14-triazabicyclo[11.2.1]hexadeca-1(15),13-

dien-9-yl)-2-hydroxy-3-methylbutanamide (4aC) was

obtained from compound 7aC (113 mg, 0.3 mmol),

C(CH

CH ); 0.74 (3H, d, J = 6.9, CH

(DMSO-d ), δ, ppm: 172.6; 172.3; 159.9; 150.7; 136.3;

3

)

3

); 0.90 (3H, d, J = 6.9, CH

3

CH); 0.88–0.79 (1H, m,

13

CF

3

CO

2

H (1.2 ml, 15.1 mmol), (S)-2-hydroxy-3-methyl-

2

3

CH). C NMR spectrum

butanoic acid (45 mg, 0.4 mmol), EDC·HCl (146 mg,

6

0

1

.8 mmol), HOBt (103 mg, 0.8 mmol), and DIPEA (0.3 ml,

.5 mmol) following the general method VI. Yield 56 mg

128.8; 127.7; 127.0; 116.2; 81.3; 74.9; 58.6; 51.2; 50.5; 46.3;

33.2; 31.2; 30.2; 26.1; 25.3; 19.2; 17.8; 15.8. Found, m/z:

2

0

+

(

49%), white amorphous solid, [α]

D

–183.4° (c 1.0,

OD), δ, ppm (J, Hz): 4.84

496.2921 [M+H] . C27

H

38

N

5

O . Calculated, m/z: 496.2924.

4

1

CHCl

3

). H NMR spectrum (CD

3

(2S)-N-((7S,10S)-10-tert-Butyl-13-cyano-8-oxo-2-phenyl-

14-oxa-2,9,12-triazabicyclo[9.2.1]tetradeca-1(13),11-dien-

7-yl)-2-hydroxy-3-methylbutanamide (4cA) was obtained

(

1H, s, CHC(CH ) ); 4.57 (1H, dd, J = 10.4, J = 4.8,

3

CHCH

2

); 3.86 (1H, d, J = 3.7, CHOH); 3.83–3.74 (1H, m,

); 3.34–3.25 (1H, m, CH ); 3.18 (3H, s, NCH ); 2.13–

.05 (1H, m, CH(CH ); 1.87–1.69 (3H, m, CH ); 1.69–

.61 (2H, m, CH ); 1.45–1.36 (2H, m, CH ); 1.34–1.23

); 1.19–1.10 (2H, m, CH ); 1.08 (9H, s,

); 1.01 (3H, d, J = 6.9, CH CH); 0.86 (3H, d, J = 6.9,

CH

2

3

from compound 7cA (80 mg, 0.2 mmol), CF

3

CO H (0.6 ml,

2

1

3

)

2

8.3 mmol), (S)-(+)-2-hydroxy-3-methylbutanoic acid

(30 mg, 0.3 mmol), EDC·HCl (96 mg, 0.5 mmol), HOBt

(68 mg, 0.5 mmol), and DIPEA (0.2 ml, 1.0 mmol) following

the general method VI. Yield 40 mg (50%), white amor-

2

(

1H, m, CH

2

C(CH

3

)

3

13

20

1

CH

3

CH). C NMR spectrum (CD

3

OD), δ, ppm: 175.9;

phous solid, [α]

spectrum (CD

D

–111.0° (c 1.0, MeOH). H NMR

1

3

1

73.6; 162.0; 151.6; 117.4; 84.2; 76.8; 57.1; 53.4; 50.5;

3

OD), δ, ppm (J, Hz): 7.55–7.46 (2H, m, H Ph);

6.1; 36.0; 33.3; 33.0; 27.3; 26.8; 26.2; 25.7; 25.4; 19.6;

7.44–7.37 (3H, m, H Ph); 4.97–4.90 (1H, m, CHC(CH

4.50 (1H, d, J = 6.7, CH ); 4.28–4.18 (1H, m, CHCH

3.90 (1H, d, J = 3.3, CHOH); 3.57 (1H, d, J = 15.0, CH

2.24–2.08 (2H, m, CH ); 2.08–1.96 (1H, m, CH

1.85 (1H, m, CH ); 1.58–1.46 (1H, m, CH(CH

1.24 (1H, m, CH ); 1.24–1.17 (1H, m, CH ); 1.14 (9H, s,

C(CH ); 1.03 (3H, d, J = 7.0, CH CH); 0.87 (3H, d, J = 7.0,

CH

3

)

3

2

);

+

6.2. Found, m/z: 448.2937 [M+H] . C23

H

38

N

5

O

4

. Calcu-

2

lated, m/z: 448.2924.

(

2S)-N-((7S,10S)-2-Benzyl-10-tert-butyl-13-cyano-8-oxo-

2

); 1.96–

1

7

4-oxa-2,9,12-triazabicyclo[9.2.1]tetradeca-1(13),11-dien-

2

3

) ); 1.36–

2

-yl)-2-hydroxy-3-methylbutanamide (4bA) was obtained

2

from compound 7bA (80 mg, 0.2 mmol), CF

3

CO

2

H (0.7 ml,

3

)

3

13

9

0

.7 mmol), (S)-2-hydroxy-3-methylbutanoic acid (29 mg,

3

CH). C NMR spectrum (CD OD), δ, ppm: 175.9;

3

.2 mmol), EDC·HCl (93 mg, 0.5 mmol), HOBt (66 mg,

175.0; 161.1; 153.0; 141.6; 131.1; 129.7; 128.8; 114.8;

.5 mmol), and DIPEA (0.2 ml, 1.0 mmol) following the

85.5; 76.8; 60.9; 52.7; 51.2; 34.6; 32.9; 31.6; 27.1; 26.8; 19.6;

+

general method VI. Yield 25 mg (31%), white amorphous

19.0; 16.2. Found, m/z: 482.2765 [M+H] . C26

Calculated, m/z: 482.2767.

H

36

N

5 4

O .

2

0

1

solid, [α]

D

–52.0° (c 1.0, DMSO-d

6

). H NMR spectrum

(

DMSO-d

6

), δ, ppm (J, Hz): 8.38 (1H, d, J = 6.7, NHCO);

Methyl 2-{2-[(1S)-1-((2S)-2-{[(tert-butoxy)carbonyl]-

amino}pent-4-enamido)-2,2-dimethylpropyl]-5-[methyl-

(prop-2-en-1-yl)amino]-1,3-oxazol-4-yl}-1,3-oxazole-

4-carboxylate (23) was synthesized in a three-step

sequence from bioxazole 22.

Step 1. Methyl 2-[2-((1S)-1-amino-2,2-dimethylpropyl)-

5-bromo-1,3-oxazol-4-yl]-1,3-oxazole-4-carboxylate.

33% HBr solution in AcOH (2.6 ml, 15.0 mmol, 20 equiv)

was added to a solution of bioxazole 22 (370 mg, 0.8 mmol,

7

.53 (1H, d, J = 7.6, NHCOO); 7.43–7.26 (5H, m, H Ph);

.60 (1H, d, J = 5.7, OH); 4.89–4.83 (1H, m, CHCH ); 4.67

Ph); 4.61 (1H, d, J = 16.8, CH Ph);

.37–4.27 (1H, m, CHC(CH ); 3.78–3.71 (1H, m, CH N);

.70 (1H, dd, J = 5.6, J = 3.2, CHOH); 3.06 (1H, d, J = 15.0,

); 2.00 (1H, sept d, J = 6.8, J = 3.2, CH(CH ); 1.99–

.91 (1H, m, CH ); 1.79–1.71 (2H, m, CH ); 1.32–1.20

1H, m, CH ); 1.19–1.09 (1H, m, CH ); 1.03 (9H, s,

5

2

(

1H, d, J = 17.4, CH

2

4

3

)

3

2

CH

2

)

3 2

1

(

2

5

98

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

1

equiv) in 1,4-dioxane (2 ml). The reaction mixture was

CH

1.55–1.38 (10H, m, CH

CH ); 1.08 (9H, s, C(CH

2

); 2.08–1.95 (2H, m, CH

, C(CH

); 1.03–0.91 (1H, m, CH

2

); 1.93–1.83 (1H, m, CH

); 1.29–1.18 (1H, m,

).

2

);

stirred at room temperature for 15 min. Then Et

2

O (20 ml)

2

)

3 3

was added to the reaction mixture and white precipitate

2

3

)

3

2

13

was formed. The resulting suspension was decanted, and

C NMR spectrum (CDCl ), δ, ppm: 172.9; 162.2; 158.1;

3

the precipitate was washed with Et

2

O (2×40 ml) and

155.3; 154.8; 150.3; 142.7; 133.5; 100.1; 79.9; 58.8; 53.4;

evaporated under reduced pressure to provide crude

oxazole as a yellow oil, yield 280 mg (85%), which was

used in the next reaction without additional purification.

Step 2. Methyl 2-{2-((1S)-1-amino-2,2-dimethylpropyl)-

52.1; 49.4; 37.0; 33.8; 31.5; 28.5; 26.8; 25.1; 18.0. Found, m/z:

+

520.2772 [M+H] . C25

H

38

N

5

O . Calculated, m/z: 520.2771.

7

(2S)-N-{(7S,10S)-10-tert-Butyl-13-[4-(hydroxymethyl)-

1,3-oxazol-2-yl]-2-methyl-8-oxo-14-oxa-2,9,12-triazabicyclo-

[9.2.1]tetradeca-1(13),11-dien-7-yl}-2-hydroxy-3-methyl-

5

-[methyl(prop-2-en-1-yl)amino]-1,3-oxazol-4-yl}-1,3-

oxazole-4-carboxylate was obtained from methyl 2-[2-((1S)-

-amino-2,2-dimethylpropyl)-5-bromo-1,3-oxazol-4-yl]-1,3-

oxazole-4-carboxylate (step 1) (280 mg, 0.8 mmol), N-methyl-

butanamide (5). CF

3

CH OH (0.1 ml) was added to a

2

1

solution of compound 24 (25 mg, 0.05 mmol, 1 equiv) in

anhydrous THF (1 ml). The flask was cooled to 0°C

allylamine (0.1 ml, 1.3 mmol), and NEt

3

(0.3 ml, 1.9 mmol) in

(crushed ice bath), and LiBH (5 mg, 0.2 mmol, 5 equiv)

4

anhydrous DMF at 50°C following the general method I.

was added in one portion. The resulting mixture was warmed

The crude product was purified by column chromatography

to room temperature and stirred for 20 h, whereupon it was

on silica gel using gradient elution from pure CH

2

Cl

2

to

quenched by addition of aqueous saturated NH Cl solution

4

1

0% MeOH in CH

2

Cl

2

to obtain product as a yellow oil,

and extracted with EtOAc (3×10 ml). The combined

organic extracts were washed with brine, dried over

yield 110 mg (40%), which was used in the next reaction

without additional purification.

anhydrous Na

2

SO , filtered, and concentrated under

4

Step 3. Methyl 2-{2-[(1S)-1-((2S)-2-{[(tert-butoxy)-

carbonyl]amino}pent-4-enamido)-2,2-dimethylpropyl]-

reduced pressure to afford crude alcohol as colorless oil,

yield 17 mg (72%), which was used in the next step

without additional purification.

5

-[methyl(prop-2-en-1-yl)amino]-1,3-oxazol-4-yl}-1,3-

oxazole-4-carboxylate (23) was obtained from methyl

The title compound 5 was obtained from the crude

alcohol (17 mg, 0.03 mmol), CF

2

-{2-((1S)-1-amino-2,2-dimethylpropyl)-5-[methyl(prop-

3

CO H (0.1 ml, 1.6 mmol),

2

-en-1-yl)amino]-1,3-oxazol-4-yl}-1,3-oxazole-4-carboxylate

(S)-2-hydroxy-3-methylbutanoic acid (8 mg, 0.07 mmol),

EDC·HCl (14 mg, 0.07 mmol), HOBt (15 mg, 0.08 mmol),

and DIPEA (0.03 ml, 0.4 mmol) following the general

method VI. Yield 10 mg (59%), white amorphous solid,

(

step 2) (110 mg, 0.3 mmol), acid 16A (102 mg, 0.5 mmol),

and EDC·HCl (121 mg, 0.6 mmol) in anhydrous pyridine

following the general method III. Yield 110 mg (64%),

2

0

1

20

1

yellow oil, [α]

CDCl

D

–48.0° (c 1.0, CHCl

3

). H NMR spectrum

[α]

δ, ppm (J, Hz): 7.75 (1H, s, H oxazole); 4.53 (2H, s,

CH OH); 4.51 (1H, s, CHC(CH ); 4.00–3.90 (1H, m,

CHCH ); 3.87 (1H, d, J = 3.3, CHOH); 3.17 (3H, s,

NCH ); 3.09 (1H, dt, J = 14.9, J = 3.3, CH ); 2.18–1.88

(4H, m, CH , CH(CH ); 1.47–1.35 (1H, m, CH ); 1.34–

1.21 (2H, m, CH ); 1.12 (9H, s, C(CH ); 1.05–0.93 (4H,

m, CH CH, CH ); 0.85 (3H, d, J = 6.8, CH

spectrum (CD OD), δ, ppm: 175.9; 174.6; 158.8; 155.6;

D

–154.8° (c 1.0, CHCl

3

). H NMR spectrum (CD OD),

3

(

3

), δ, ppm (J, Hz): 8.19 (1H, s, H oxazole); 6.90 (1H,

d, J = 9.6, NHCO); 5.94–5.66 (2H, m, 2CH=CH

2

); 5.26–

); 5.02–4.97 (1H, m, NHCOO);

.96 (1H, d, J = 9.6, CHC(CH ); 4.19–4.04 (3H, m,

, CH ); 3.89 (3H, s, OCH ); 3.10 (3H, s, NCH );

.55–2.40 (2H, m, CH ); 1.42 (9H, s, C(CH ); 0.98 (9H,

). C NMR spectrum (CDCl ), δ, ppm: 171.1;

2

)

3 3

5

4

.05 (4H, m, 2CH=CH

2

3

)

3

2

CHCH

2

3

2

3

)

2

3

)

3

2

)

3 3

1

3

13

s, C(CH

3

)

3

2

3

CH). C NMR

1

3

62.1; 157.7; 155.4; 152.8; 142.7; 133.9; 133.3; 132.9;

3

19.1; 118.4; 103.3; 80.4; 60.5; 55.3; 54.0; 52.1; 37.7;

152.8; 142.5; 135.6; 101.0; 76.8; 60.6; 57.3; 52.7; 50.0; 37.4;

+

6.2; 28.4; 26.4; 26.3. Found, m/z: 546.2932 [M+H] .

34.6; 32.9; 31.8; 26.9; 26.3; 19.6; 19.1; 16.1. Found, m/z:

+

C

27

H

40

N

5

O

7

. Calculated, m/z: 546.2928.

492.2818 [M+H] . C24

H

38

N

5

O . Calculated, m/z: 492.2822.

6

Methyl 2-((7S,10S)-7-{[(tert-butoxy)carbonyl]amino}-

0-tert-butyl-2-methyl-8-oxo-14-oxa-2,9,12-triazabicyclo-

9.2.1]tetradeca-1(13),11-dien-13-yl)-1,3-oxazole-4-carb-

tert-Butyl N-((1S)-1-{4-(4-{[(tert-butyldimethylsilyl)-

oxy]methyl}-1,3-oxazol-2-yl)-5-[(3,5-difluorophenyl)-

(hydroxy)methyl]-1,3-oxazol-2-yl}-2,2-dimethylpropyl)-

carbamate (25). n-BuLi (1.6 M solution in hexane, 1.17 ml,

1.88 mmol, 2.05 equiv) was added gradually (within 20 min) to

1

[

oxylate (24). Alkene 23 (90 mg, 0.2 mmol, 1 equiv) was

added to a solution of ruthenium metathesis catalyst Ru4

4

(

12 mg, 0.02 mmol, 0.1 equiv) in anhydrous CH

2

Cl

2

(100 ml).

(0.05 ml,

a solution of bioxazole (S)-12 (427 mg, 0.92 mmol, 1.0 equiv)

After stirring at 50°C for 24 h, neat Ti(Oi-Pr)

4

in anhydrous Et O (9 ml) at –78°C. The color of the

2

0

.2 mmol, 1 equiv) was added dropwise. The stirring was

solution slowly changed from yellow to dark-brown. After

10 min of the stirring at –78°C, a solution of 3,5-difluoro-

benzaldehyde (313 mg, 2.20 mmol, 2.4 equiv) in anhydrous

continued for 5 days at 60°C, whereupon the reaction

mixture was cooled to room temperature and concentrated

under reduced pressure. The crude unsaturated macrocycle

Et O (2 ml) was added dropwise. The resulting solution

2

(

as a mixture of E/Z-isomers) was hydrogenated in the

was stirred for 10 min at –78°C, slowly warmed to room

presence of 10% Pd on carbon (18 mg, 0.02 mmol) following

temperature, stirred for 30 min, and cooled back to 0°C.

the general method V to afford product 24 as a white

Aqueous saturated NH

the mixture was extracted with Et

organic extracts were washed with brine, dried over

4

Cl solution (20 ml) was added, and

2

0

amorphous solid, yield 33 mg (39%), [α]

D

–160.0° (c 1.0,

2

O (3×20 ml). Combined

1

CHCl

1H, s, H oxazole); 5.81 (1H, d, J = 7.5, NHCO); 5.51 (1H,

d, J = 7.5, CHC(CH ); 4.60 (1H, d, J = 15.4, NHCOO);

.59–4.53 (1H, m, CHCH ); 3.89 (3H, s, OCH ); 3.89–3.83

1H, m, CH ); 3.27 (3H, s, NCH ); 3.05–3.00 (1H, m,

3

). H NMR spectrum (CDCl

3

), δ, ppm (J, Hz): 8.17

(

anhydrous Na

2

SO , and concentrated under reduced

4

3

)

3

pressure. The residue was purified by column chromato-

graphy on silica gel using gradient elution from 5% EtOAc

in hexane to 50% EtOAc in hexane to afford alcohol 25 as

4

(

2

3

2

3

5

99

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

a mixture of diastereomers, yield 284 mg (51%), colorless

3-en-1-yl}carbamate (28) was obtained in a two-step

oil, which was used in the next reaction without additional

purification.

sequence from alcohol 27.

Step 1. 1-{2-((1S)-1-Amino-2,2-dimethylpropyl)-4-[4-

(hydroxymethyl)-1,3-oxazol-2-yl]-1,3-oxazol-5-yl}-1-(3,5-

difluorophenyl)but-3-en-1-ol. 4 M HCl solution in

1,4-dioxane (5.1 ml, 20.4 mmol, 50 equiv) was added to a

solution of allylic alcohol 27 (265 mg, 0.41 mmol, 1.0 equiv)

in 1,4-dioxane (9 ml). The resulting solution was stirred at

room temperature for 18 h and concentrated under reduced

pressure. The residue was dissolved in EtOAc (10 ml),

tert-Butyl N-{(1S)-1-[4-(4-{[(tert-butyldimethylsilyl)-

oxy]methyl}-1,3-oxazol-2-yl)-5-(3,5-difluorobenzoyl)-1,3-

oxazol-2-yl]-2,2-dimethylpropyl}carbamate (26). Dess–

Martin periodinane (290 mg, 0.68 mmol, 1.5 equiv) was

portionwise added to a solution of crude alcohol 25 (277 mg,

0

.46 mmol, 1.0 equiv) in anhydrous CH

2

Cl (9 ml). The

2

resulting white suspension was stirred at room temperature

for 1 h, whereupon all volatiles were removed under reduced

pressure. The residue was purified by flash chromato-

graphy using gradient elution from pure hexane to 60%

EtOAc in hexane. Yield 215 mg (78%), white amorphous

washed with aqueous saturated NaHCO

3

solution and

and concentrated

brine, then dried over anhydrous Na SO

2

4

under reduced pressure. The residue was purified by

column chromatography on silica gel using gradient elution

from 1% MeOH in EtOAc to 10% MeOH in EtOAc to

2

0

1

solid, [α]

CDCl

D

–42.0° (c 1.0, CHCl

3

). H NMR spectrum

(

3

), δ, ppm (J, Hz): 7.61 (1H, dd, J = 1.4, J = 1.4,

afford product as a mixture of diastereomers. Yield 123 mg

1

H Ar); 7.48–7.41 (2H, m, H Ar); 7.07 (1H, tt, J = 8.4,

(69%), yellow oil. H NMR spectrum (CDCl

3

), δ, ppm

J = 2.4, H Ar); 5.45 (1H, d, J = 9.8, NH); 4.84 (1H, d,

(J, Hz): 8.22 (1H, s, NH

6.95 (2H, m, H Ar); 6.71–6.63 (1H, m, H Ar); 5.85–5.71

(1H, m, CH=CH ); 5.18–5.05 (2H, m, CH=CH ); 4.63 (2H,

d, J = 1.1, CH OH); 3.86–3.84 (1H, m, CHNH ); 3.08–2.98

(1H, m, CH ); 2.93–2.82 (1H, m, CH ); 1.99–1.70 (3H,

br. s, 2OH, NH ); 1.05–1.00 (9H, m, C(CH

spectrum (CDCl

2

); 7.65–7.64 (1H, m, H Ar); 7.03–

J = 9.8, CHNH); 4.74 (2H, s, CH

2

OH); 1.43 (9H, s,

); 0.92 (9H, s, C(CH );

Si). C NMR spectrum (CDCl ), δ, ppm

OC(CH

3

)

3

); 1.05 (9H, s, C(CH

3

)

3

)

3

2

1

3

0

.10 (6H, s, 2CH

3

2

(

J, Hz): 179.3; 166.1; 163.0 (dd, J = 251.6, J = 11.8);

2

13

1

3

55.4; 153.8; 144.8; 144.2; 139.2 (t, J = 8.2); 136.7; 134.5;

12.9–112.5 (m); 109.1 (t, J = 25.2); 80.5; 59.2; 36.2;

2

3

) ). C NMR

3

), δ, ppm (J, Hz): 165.9; 162.9 (dd,

+

1.7; 28.4; 26.4; 26.0; –5.3. Found, m/z: 606.2810 [M+H] .

J = 248.7, J = 12.6); 161.7; 156.5; 155.4; 149.0 (dt, J = 7.9,

J = 3.4); 141.1; 135.7; 132.2; 124.7; 119.4; 108.7–108.3

C

30

H

42

F

2

N

3

O Si. Calculated, m/z: 606.2811.

6

tert-Butyl N-((1S)-1-{4-(4-{[(tert-butyldimethylsilyl)-

oxy]methyl}-1,3-oxazol-2-yl)-5-[1-(3,5-difluorophenyl)-

-hydroxybut-3-en-1-yl]-1,3-oxazol-2-yl}-2,2-dimethyl-

propyl)carbamate (27). Allylmagnesium bromide (1 M

solution in Et O, 0.88 ml, 0.88 mmol, 2.5 equiv) was drop-

wise added to a solution of ketone 26 (214 mg, 0.35 mmol,

.0 equiv) in anhydrous THF (5 ml) at –15°C. After stirring

at –15°C for 30 min, the yellow solution was warmed to 0°C,

(m); 103.1 (dt, J = 25.4, J = 3.6); 74.5; 59.5; 57.1; 45.9;

+

35.7; 26.4. Found, m/z: 434.1896 [M+H] . C22

Calculated, m/z: 434.1891.

H

26

F

2

N

3 4

O .

1

Step 2. tert-Butyl N-{(1S)-1-[((1S)-1-{5-[1-(3,5-difluoro-

phenyl)-1-hydroxybut-3-en-1-yl]-4-[4-(hydroxymethyl)-

1,3-oxazol-2-yl]-1,3-oxazol-2-yl}-2,2-dimethylpropyl)-

carbamoyl]but-3-en-1-yl}carbamate (28) was obtained

from 1-{2-((1S)-1-amino-2,2-dimethylpropyl)-4-[4-(hydroxy-

methyl)-1,3-oxazol-2-yl]-1,3-oxazol-5-yl}-1-(3,5-difluoro-

phenyl)but-3-en-1-ol (step 1) (110 mg, 0.25 mmol), EDC·HCl

(54 mg, 0.28 mmol), and acid 16A (55 mg, 0.25 mmol) in

anhydrous pyridine following the general method III.

Purification by column chromatography on silica gel using

gradient elution from 30% EtOAc in hexane to 70% EtOAc

in hexane afforded product 28 as a mixture of diastereo-

2

1

quenched with aqueous saturated NH Cl solution, and

4

extracted with EtOAc (3×15 ml). Combined organic

extracts were washed with brine, dried over anhydrous

Na

2

SO , and concentrated under reduced pressure. The

4

residue was purified by column chromatography on silica

gel using gradient elution from pure hexane to 50% EtOAc

in hexane to afford tertiary alcohol 27 as a mixture of

1

diastereomers. Yield 188 mg (82%), colorless oil H NMR

spectrum (CDCl

mers. Yield 114 mg (71%), yellow amorphous solid.

1

3

), δ, ppm (J, Hz): 8.27 (0.5H, s, NH); 8.24

H NMR spectrum (CDCl ), δ, ppm (J, Hz): 8.20 (1H, d,

3

(

0.5H, s, NH); 7.59–7.54 (1H, m, H Ar); 7.02–6.94 (2H, m,

J = 12.4, NH); 7.66–7.64 (1H, m, H Ar); 7.19–7.04 (1H, m,

H Ar); 6.69–6.62 (1H, m, H Ar); 5.86–5.70 (1H, m,

NH); 7.02–6.94 (2H, m, H Ar); 6.72–6.60 (1H, m, H Ar);

CH=CH

CH

2

); 5.37–5.27 (1H, m, OH); 5.18–5.02 (2H, m,

5.87–5.66 (2H, m, 2CH=CH ); 5.19–5.03 (5H, m,

2

); 4.78 (1H, d, J = 9.6, CHNH); 4.66–4.60 (2H, m,

2CH=CH

2

, CHNH); 5.02–4.92 (1H, br. s, OH); 4.66–4.62

OH); 4.20–4.13 (1H, m, CHNH); 3.04 (1H, dt,

); 2.88 (1H, td, J = 14.5, J = 6.7,

); 2.58–2.46 (2H, m, CH , OH); 2.00 (1H, t, J = 6.0,

); 1.44 (9H, s, OC(CH ); 1.03 (4.5H, s,

); 1.02 (4.5H, s, C(CH

2

OH); 3.03 (1H, dt, J = 14.7, J = 7.5 CH

2

); 2.93–2.79

(2H, m, CH

2

(

1H, m, CH

OC(CH ); 1.03 (4.5H, s, C(CH

.92 (4.5H, s, C(CH ); 0.91 (4.5H, s, C(CH

2

); 1.44 (4.5H, s, OC(CH

); 1.01 (4.5H, s, C(CH

); 0.12–0.08

Si). C NMR spectrum (CDCl ), δ, ppm

3

)

3

); 1.43 (4.5H, s,

J = 13.4, J = 6.4 CH

2

3

)

3

)

3

)

3

);

CH

C(CH

2

0

3

)

3

)

3

2

3 3

)

1

3

13

(

6H, m, 2CH

3

)

3

) ). C NMR spectrum

J, Hz): 166.5; 162.9 (dd, J = 250.6, J = 12.0); 156.1;

(CDCl ), δ, ppm (J, Hz): 163.0 (dd, J = 249.0, J = 12.6);

3

1

55.4; 149.3; 149.0; 141.8 (t, J = 8.0); 135.6; 132.2; 125.1;

19.2; 108.7–108.4 (m); 103.0 (t, J = 25.1); 80.3; 74.5;

162.0; 161.9; 161.7; 156.4; 155.7; 149.0 (t, J = 8.0); 141.1;

135.7; 133.2; 132.1; 125.0; 119.5; 108.7–108.3 (m); 103.1

5

8.1; 57.4; 46.0; 45.9; 35.8; 28.4; 26.4; 26.0; –5.2. Found, m/z:

(dd, J = 25.5, J = 8.0); 80.6; 74.5; 57.1; 45.9; 45.8; 35.8; 28.4;

+

6

48.3294 [M+H] .

C

33

H

48

F

2

N

3

O

6

Si. Calculated, m/z:

26.43; 26.40. Found, m/z: 631.2950 [M+H] . C32

H

41

F

2

N

4

O .

7

48.3280.

Calculated, m/z: 631.2943.

tert-Butyl N-{(1S)-1-[((1S)-1-{5-[1-(3,5-difluorophenyl)-

-hydroxybut-3-en-1-yl]-4-[4-(hydroxymethyl)-1,3-oxazol-

-yl]-1,3-oxazol-2-yl}-2,2-dimethylpropyl)carbamoyl]but-

tert-Butyl N-{(2S,5S)-2-tert-butyl-10-(3,5-difluorophenyl)-

10-hydroxy-12-[4-(hydroxymethyl)-1,3-oxazol-2-yl]-4-oxo-

14-oxa-3,13-diazabicyclo[9.2.1]tetradeca-1(13),11-dien-

1

2

6

00

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

5

-yl}carbamate (29) was synthesized from amide 28 (102 mg,

.16 mmol) and ruthenium metathesis catalyst Ru4 (11.9 mg,

.02 mmol) in anhydrous CH Cl following the general

crude amine (39 mg, 0.077 mmol, 1.0 equiv) in anhydrous

DMF (5 ml). The yellow solution was stirred at room

0

2

temperature for 18 h, whereupon H O was added, and the

2

method IV. The crude unsaturated macrocycle was obtained as

a mixture of E/Z-isomers in 2:1 ratio after purification by

column chromatography on silica gel using gradient elution

from 25% EtOAc in hexane to 100% EtOAc. Yield 80 mg

resulting mixture was extracted with EtOAc (3×10 ml).

Combined organic extracts were washed with brine, dried

over anhydrous Na

2

SO , and concentrated under reduced

4

pressure. The residue was purified by reversed-phase

(

82%), brown amorphous solid.

column chromatography on silica gel using gradient elution

A solution of the crude macrocycle (80 mg, 0.13 mmol,

from 30 to 50% MeCN in H

diastereomers.

2

O to afford individual

1

.0 equiv) in MeOH (10 ml) was stirred for 5 days under

H

2

pressure (5 atm) in the presence of 10% Pd on carbon

Diastereomer 6A. Yield 10.5 mg (23%), white amorphous

2

0

1

(

71 mg, 0.07 mmol, 0.5 equiv). The resulting black

solid, [α]

(CD

D

–49.7° (c 0.7, MeOH). H NMR spectrum

suspension was filtered through a pad of Celite®, and the

filtrate was concentrated under reduced pressure. The

residue was purified by column chromatography on silica

gel using gradient elution from 30% EtOAc in hexane to

3

OD), δ, ppm (J, Hz): 8.62 (1H, d, J = 7.2, NH); 7.73

(1H, s, H Ar); 6.96–6.86 (2H, m, H Ar); 6.78 (1H, tt, J = 8.8,

J = 2.3, H Ar); 4.99–4.92 (1H, m, CH); 4.74 (1H, dd,

J = 7.2, J = 2.8, CH); 4.37 (2H, s, CH

2

OH); 3.87 (1H, d,

CH); 2.57–2.40

CH, CH(CH );

); 1.22 (9H, s, C(CH ); 0.99 (3H,

CH); 0.82 (3H, d, J = 6.9, CH CH).

C NMR spectrum (CD OD), δ, ppm (J, Hz): 174.3;

5

0% EtOAc in hexane to afford product 29 as a mixture of

J = 3.4, CHOH); 2.96–2.87 (1H, m, CH

(1H, m, CH CH); 2.16–1.80 (3H, m, CH

1.34–1.26 (4H, m, 2CH

d, J = 6.9, CH

2

diastereomers. Yield 52 mg (65%), white amorphous solid.

2

)

3 2

1

H NMR spectrum (CDCl

3

), δ, ppm (J, Hz): 8.06 (0.67H, s,

2

)

3 3

NH); 7.65–7.62 (0.67H, m, H Ar); 7.61–7.59 (0.33H, m,

H Ar); 7.41 (0.33H, s, NH); 6.94–6.86 (1.34H, m, H Ar);

3

13

3

6

.85–6.79 (0.66H, m, H Ar); 6.69–6.60 (1H, m, H Ar);

.26–6.19 (0.33H, m, NHCH); 6.16–6.09 (0.67H, m,

173.4; 163.1 (dd, J = 248.0, J = 12.6); 156.2; 153.0; 142.9

(t, J = 8.1); 137.5; 137.0; 136.3; 126.5; 110.1–109.7 (m);

103.5 (t, J = 26.0); 76.5; 73.8; 59.5; 55.3; 51.3; 40.2; 32.9;

NHCH); 5.54–5.42 (1H, m, CH

2

OH); 5.18–5.11 (0.33H, m,

CHNH); 4.90 (0.67H, d, J = 8.0, CHNH); 4.62 (1.34H,

31.6; 29.6; 26.9; 22.7; 19.6; 18.2; 15.2. Found, m/z: 605.2789

+

br. s, CH

2

OH); 4.57–4.51 (0.67H, m, CHCH

0.66H, br. s, CH OH); 4.31–4.24 (0.33H, m, CHCH

.41–2.28 (1H, m, CH CH); 2.27–2.14 (2H, m, CH CH);

.00–1.72 (4H, m, 2CH , CH CH); 1.42 (2.97H, s,

); 1.41 (6.03H, s, OC(CH ); 1.33–1.26 (1H, m,

); 1.24 (2.97H, s, C(CH ); 1.17 (6.03H, s, C(CH ).

C NMR spectrum (CDCl ), δ, ppm (J, Hz): 173.2; 163.0

2

); 4.49

[M+H] . C30

H

39

F

2

N

4

O . Calculated, m/z: 605.2787.

7

(

2

);

Diastereomer 6B. Yield 16.1 mg (34%), white amorphous

20

1

2

solid, [α]

(CD

D

–27.6° (c 0.6, MeOH). H NMR spectrum

2

3

OD ), δ, ppm (J, Hz): 8.67 (1H, d, J = 7.2, NH); 7.90

(1H, s, H Ar); 7.06–6.94 (2H, m, H Ar); 6.81 (1H, tt, J = 8.8,

J = 2.3, H Ar); 4.90–4.86 (1H, m, CH); 4.83–4.79 (1H, m,

OC(CH

3

)

3

)

3

CH

2

3

)

3

)

3 3

1

3

CH); 4.54 (2H, s, CH

2.59–2.42 (1H, m, CH

2.15–2.05 (1H, m, CH(CH

CH ); 1.46–1.12 (12H, m, C(CH

J = 6.9, CH CH); 0.89–0.69 (3H, m, CH

spectrum (CD

2

OH); 3.87 (1H, d, J = 3.4, CHOH);

(

dd, J = 257.6, J = 12.6); 161.9; 161.2; 156.2; 155.3; 155.2;

2

CH); 2.27–2.15 (1H, m, CH

); 1.98–1.76 (3H, m, CH

, 2CH ); 0.99 (3H, d,

2

CH);

1

2

51.4 (t, J = 7.4); 141.2; 135.7; 125.2; 107.8–107.4 (m);

02.9 (t, J = 25.3); 74.1; 58.9; 57.1; 53.4; 38.9; 33.6; 30.8;

3

)

2

CH,

2

3

)

3

2

+

13

8.5; 26.9; 21.9; 14.3. Found, m/z: 605.2771 [M+H] .

3

CH). C NMR

C

30

(

H

39

F

2

N

4

O

7

. Calculated, m/z: 605.2787.

3

OD), δ, ppm (J, Hz): 174.4; 173.4; 164.4

2S)-N-{(2S,5S)-2-tert-Butyl-10-(3,5-difluorophenyl)-

(dd, J = 248.0, J = 12.6); 156.3; 153.0; 143.0 (t, J = 8.4);

1

5

0-hydroxy-12-[4-(hydroxymethyl)-1,3-oxazol-2-yl]-4-oxo-

4-oxa-3,13-diazabicyclo[9.2.1]tetradeca-1(13),11-dien-

-yl}-2-hydroxy-3-methylbutanamide (diastereomers 6A

137.6; 137.0; 136.3; 125.9; 108.7–108.3 (m); 102.2 (t,

CH3 1.09

5.34 HO

CH₃

H3C

4.40

and 6B). CF

3

CO H (0.4 ml, 5.27 mmol, 65 equiv) was

2

H⁴^.⁶⁸

N

1

H

.99

dropwise added to a solution of macrocycle 29 (49 mg,

0.72

^C^H3

8.67

HN

N

0

.081 mmol, 1.0 equiv) in anhydrous CH

2

Cl (3 ml) at 0°C.

2

0

.89 H3C

.70 H

5.58 HO

O

H8.08

H 7.14

H

7

.55

O

The yellow solution was warmed to room temperature and

stirred for 3 h, whereupon all volatiles were removed under

reduced pressure. The residue was dissolved in THF

O

H4.82

OH 7.89

F

N

3

H

O

H

(

0.5 ml) and MeOH (0.5 ml), and a solution of LiOH

H

1

.71 –1.62

.20 –2.12 1.14–1.05

H

10 mg, 0.40 mmol, 5 equiv) in H

2

O (0.5 ml) was added

1.78 –1.71

2

7.02 –6.97 F

2

.41 –2.35

.31 –1.25

.71 –1.62

dropwise. The resulting brown solution was stirred for 1 h

at room temperature, and all volatiles were concentrated

0.66–0.58

1

under reduced pressure. The residue was dissolved in Et

2

O

CH3

HO

H C

(

10 ml) and washed with 1 M aqueous HCl (2×10 ml),

3

2

6.2

54.8

3

3.1

8.5

HN 161.6

172.7

brine, dried over anhydrous Na

2

SO

4

, and concentrated

H

N

1

41.7

5

^N124.1

55.1

under reduced pressure. Crude amine was obtained as

white amorphous solid, yield 41 mg (100%), which was

used in the next reaction without additional purification.

19.1

CH3

15.7

136.4

1

H3C

O

31.2

H

N

O

F

153.8

73.7

OH

H

172.3

^O21.3

1

62.3

5

0.3

(S)-2-Hydroxy-3-methylbutanoic acid (9.6 mg, 0.081 mmol,

H

HO ⁷⁴^.⁸

29.4

151.5

1

.05 equiv), EDC·HCl (15.6 mg, 0.081 mmol, 1.05 equiv),

HOBt (11 mg, 0.081 mmol, 1.05 equiv), and DIPEA

40 µl, 0.23 mmol, 3.0 equiv) were added to a solution of

102.9

O

19.7

38.1

1

07.5 –107.2

161.4

(

6B

F

6

01

Chemistry of Heterocyclic Compounds 2020, 56(5), 586–602

J = 26.0); 75.5; 73.8; 59.3; 55.3; 51.3; 38.1; 33.0; 31.6;

This work was funded by ERDF (project No. 1.1.1.1/16/A/281

1

2

9.6; 25.6; 21.5; 19.9; 18.2; 14.7. H NMR spectrum

''Simplified Analogues of Diazonamide A as Anticancer

Agents'').

(

DMSO-d ), δ, ppm (J, Hz): 8.67 (1H, d, J = 7.3, NH); 8.08

6

1H, s, H oxazole); 7.89 (1H, d, J = 2.2, ArCOH); 7.55

1H, d, J = 7.7, NH); 7.14 (1H, tt, J = 9.0, J = 2.4, H Ar);

We thank Prof. K. Jaudzems for assistance with NMR

experiments and the ITC assay.

7

.02–6.97 (1H, m, H Ar); 5.58 (1H, d, J = 5.7,

References

(

CH

ddd, J = 7.9, J = 5.0, J = 2.8, CHCH

CH CH); 4.40 (1H, d, J = 5.7, CH

J = 5.7, J = 3.3, CHOH); 2.41–2.35 (1H, m, CH

.20–2.12 (1H, m, CH CH); 1.99 (1H, sept d, J = 6.9, J = 3.3,

CH(CH ); 1.78–1.71 (1H, m, CH COH); 1.71–1.62 (2H,

CH, CH ); 1.14–1.05 (10H, m, C(CH , CH ); 0.89

CH); 0.72 (3H, d, J = 6.9, CH CH),

),

3

)

2

CCHOH); 5.34 (1H, t, J = 5.7, CH

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2

OH); 4.82 (1H,

2

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(

3

)

3

2

2. Cruz-Monserrate, Z.; Vervoort, H. C.; Bai, R.; Newman, D. J.;

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. Wieczorek, M.; Tcherkezian, J.; Bernier, C.; Prota, A. E.;

Chaaban, S.; Rolland, Y.; Godbout, C.; Hancock, M. A.;

Arezzo, J. C.; Ocal, O.; Rocha, C.; Olieric, N.; Hall, A.; Ding, H.;

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3H, d, J = 6.9, CH

.66–0.58 (1H, m, CH

3

1

3

0

2

). C NMR spectrum (DMSO-d

6

δ, ppm (J, Hz): 172.7; 172.3; 162.3 (dd, J = 248.0,

J = 13.2); 161.6; 155.1; 153.8; 151.5 (t, J = 8.4); 141.7;

1

24.1; 107.5–107.2 (m); 102.9 (t, J = 26.0); 74.8; 73.7;

5

8.5; 54.8; 50.3; 38.1; 33.1; 31.2; 29.4; 26.2; 21.3; 19.7; 19.1;

2

016, 8(365), 365ra159.

+

1

5.7. Found, m/z: 605.2792 [M+H] . C30

H

39

F

2

N

4

O

7

.

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Compd. 2019, 56, 355. [Khim. Getrotsikl. Soedin. 2019, 56,

355.]

Calculated, m/z: 605.2787.

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dissolved in the ice-cold 20 mM phosphate (NaPi) 0.1 mM

GTP buffer (pH 6.5) just before the experiment and used

within 6 h. Prechilled RB3 (STMN4) protein prepared in

the same buffer was added to tubulin just before the

experiment. Tubulin:RB3 concentration ratio varied from

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. Mangold, S. L.; Grubbs, R. H. Chem. Sci. 2015, 6, 4561.

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. Shibata, K.; Yoshida, M.; Doi, T.; Takahashi, T. Tetrahedron

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:1 to 1:1. Protein sample was stored on ice before loading

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ITC cell was set up and equilibrated at 10°C. Afterward

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80 μl of ice-cold tubulin protein sample was loaded into

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The titration experiment consisted of 1 × 0.3 μl and

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2

1

997, 62, 9376.

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injection volume) was always removed prior the data

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1

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6

02

Article Doi

Aliphatic chain-containing macrocycles as diazonamide A analogs

DOI: 10.1007/s10593-020-02704-6

Source and publish data:

Authors:

Article abstract of DOI:10.1007/s10593-020-02704-6

Full text of DOI:10.1007/s10593-020-02704-6

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