Synthesis of pyrazolo[1,2-a]pyrazole-based peptide mimetics

Tetrahedron 69 (2013) 6648e6665

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Tetrahedron

journal homepage: w ww.elsevier.com/locate/tet

Synthesis of pyrazolo[1,2-a]pyrazole-based peptide mimetics

a

b

ꢀ

Ana Novak , Ana Testen , Jure Bezensek , Uros Groselj , Martina Hrast ,

Marta Kasunic , Stanislav Gobec , Branko Stanovnik ^c, Jurij Svete ^a

Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva 5, SI-1000 Ljubljana, Slovenia

b

a,

,c,

*

ꢀ ^a

a

ꢀ

b

ꢀ

Faculty of Pharmacy, University of Ljubljana, Askerceva 7, SI-1000 Ljubljana, Slovenia

^cCentre of Excellence EN-FIST, Dunajska 156, SI-1000 Ljubljana, Slovenia

a r t i c l e i n f o

a b s t r a c t

Article history:

The synthesis of U-shaped conformationally constrained analogues of peptides based on the 3-amino-2-

oxo-1,5-diazabicyclo[3.3.0]octane-8-carboxylic acid scaffold was developed. [3þ2] Cycloadditions of

(1Z,4R*,5R*)-1-arylmethylidene-4-benzyloxycarbonylamino-3-oxo-5-phenylpyrazolidin-1-ium-2-ides to

tert-butyl acrylate and tert-butyl methacrylate gave the corresponding racemic cycloadducts, in most

cases as mixtures of isomers, which were separated by preparative chromatography. Selective de-

protection of the C- and the N-terminal of these heterocyclic dipeptides followed by coupling with (S)-

alanine derivatives and chromatographic separation furnished the non-racemic tripeptides as target

compounds. The structures of racemic cycloadducts and non-racemic ﬁnal products were determined by

NMR spectroscopy and X-ray diffraction. The synthesized compounds were also evaluated for inhibition

Received 5 March 2013

Received in revised form 13 May 2013

Accepted 28 May 2013

Available online 4 June 2013

Dedicated to Professor Emeritus Volker

€

Jager, Institut fur Organische Chemie, Uni-

€

versitat Stuttgart, on the Occasion of his

70th Anniversary

of MurD ligase and D-alanine:D-alanine ligase.

Keywords:

Peptide mimics

Pyrazolo[1,2-a]pyrazoles

Biological activity

Stereoselectivity

Synthesis

1. Introduction

acylamino-3-pyrazolidinones as starting compounds, which in turn

are easily available by simple treatment of a,b-dehydro-a-amino

The synthesis of molecules that can mimic the structure and,

hence, the properties of peptides certainly represent an interesting

and important research topic in the ﬁelds of organic synthesis and

medicinal chemistry. Within this context, replacement of a di-

peptide motif in a given native (or natural) substrate with a U-

shaped conformationally constrained heterocyclic analogue that

acid derivatives with excess hydrazine hydrate.^3,8Retrosynthetic

analysis of derivatives of 1 (cf. Fig. 1) also reveals that compounds

with up to ﬁve stereogenic centres are available in four steps and

from four building blocks: (a) N-acylglycines, (b) aldehydes (or

ketones) (c) hydrazine hydrate, and (d)

a,b-unsaturated esters.

Thus, combination of just four types of widely available building

blocks enables an easy access to structurally diverse heterocyclic

dipeptides (Fig. 1).

simulates the

b

-turn structure, is of particular interest.¹An im-

portant group of conformationally constrained dipeptide analogues

are azabicycloalkane amino acids, comprising various saturated

4,5-fused, 5,5-fused, 5,6-fused, and related fused heterocycles with

The importance of pyrazolidin-3-one derivatives has grown

increasingly over the last decades due to their synthetic applica-

bility and biological activity.³Recent applications of 3-

pyrazolidinones include their use as templates in enantioselective

DielseAlder,⁴Michael,⁵and ‘click’ reactions,⁶while Eli Lilly’s anti-

biotics are typical examples of bioactive pyrazolo[1,2-a]pyrazolone

derivatives.⁷Thus, the applicability of 3-amino-2-oxo-1,5-

diazabicyclo[3.3.0]octane-7-carboxylic acid (1) for the preparation

of biologically active peptide mimetics has been successfully

demonstrated by Eli Lilly’s researchers almost three decades ago,⁷

however, a literature survey reveals very few other examples of

1-derived compounds since 1990. For example, a substructure

a

bridgehead nitrogen atom.²3-Amino-2-oxo-1,5-diazabicyclo

[3.3.0]octane-8-carboxylic acid (or 6-amino-7-oxotetrahydro-

pyrazolo[1,2-a]pyrazole-1-carboxylic acid) (1) based scaffolds are

a subgroup of 5,5-fused azabicycloalkane amino acids. The most

general and straightforward synthetic approach towards de-

rivatives of 1 includes stereoselective 1,3-dipolar cycloaddition as

the key-step (Fig.1). Generally, derivatives of 1 are prepared from 4-

* Corresponding author. Tel.: þ386 1 2419 254; fax: þ386 1 2419 306; e-mail

address: jurij.svete@fkkt.uni-lj.si (J. Svete).

http://dx.doi.org/10.1016/j.tet.2013.05.122

A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

6649

2. Results and discussion

2.1. Synthesis of racemic pyrazolo[1,2-a]pyrazolone-based

dipeptide building blocks 8e23

The starting model pyrazolidinones 3a (R¼Ph) and 3b (R¼i-Pr)

were prepared by treatment of -dehydro- -amino acid de-

a,

b

a

rivatives 2a,b¹⁴with hydrazine hydrate following the literature

procedure.¹⁵Further acid-catalysed treatment of 3a,b with benz-

aldehyde (4a) and 2,6-dichlorobenzaldehyde (4b) gave the ortho-

unsubstituted azomethine imines 5a,c and their ortho-di-

substituted analogues 5b,d in 73e99% yields.¹⁵First, cycloadditions

of 5-phenyl substituted dipoles 5a,b to tert-butyl acrylate (6a) were

carried out under standard conditions, i.e., in reﬂuxing anisole.⁸

Somewhat unexpectedly,¹⁵reactions of 5-phenyl substituted di-

poles 5a,b furnished, along with cycloadducts 8e11, the hydantoin

derivatives 7a,b in w25% yields. As shown previously,¹⁵formation

of 7a,b is explainable by a ‘ring switching’ transformation consist-

ing of thermal cleavage of the benzylic C(5)eN(1) bond followed by

condensation between the amidic nitrogen N(2) and the benzy-

loxycarbonyl group. Compounds 7a,b were obtained as precipitates

and were removed by ﬁltration. Evaporation of the ﬁltrates fol-

lowed by separation by column chromatography (CC) and medium

pressure liquid chromatography (MPLC) then furnished the corre-

sponding cycloadducts 8ae10a, 9b and 11b in 5e36% yields

(Scheme 1).

Fig. 1. 3-Amino-2-oxo-1,4-diazabicyclo[3.3.0]octane-7-carboxylic acid (1) as scaffold

for preparation of peptide mimetics and retrosynthetic analysis of derivatives of 1.

On the other hand, the C(5)eN(1) bond in 5-isopropyl

substituted dipoles 5c,d were thermally stable and formation of

hydantoins 7 was not observed in cycloadditions of azomethine

imines 5c,d to tert-butyl acrylate (6a), which produced only the

corresponding cycloadducts 8e12. Subsequent chromatographic

separation of isomeric cycloadducts furnished isomerically pure

compounds 8c, 10c and 9de12d in 7e33% yields (Scheme 2).

In contrast, cycloadditions of 5c,d to tert-butyl methacrylate

(6b) were regio- and stereo-selective. Cycloaddition of 6b to dipole

5c followed by chromatographic separation furnished di-

astereomeric cycloadducts 13c and 14c in 35% and 9% yield, re-

spectively, while cycloaddition of 6b to the ortho-disubstituted

dipole 5d gave compound 14d as the only product in 66% yield

(Scheme 3).

The regioselectivity and stereoselectivity of the cycloadditions

and conﬁgurations of the major isomers 8a, 8c, 11b, 13c and 14d

were in agreement with previous results obtained by cycloaddi-

tions of closely analogous dipoles to methyl acrylate¹⁶and methyl

methacrylate.¹⁷The major 1-CO^t₂Bu regioisomers 8a, 8c and 13c

obtained from ortho-unsubstituted dipoles 5a and 5c exhibited

syn-orientation between the substituents at positions 3 and 5 and,

vice versa, the major 2-CO^t₂Bu regioisomers 11b, 11d and 14d ob-

tained from ortho-disubstituted dipoles 5b and 5d exhibited anti-

orientation of the substituents at positions 3 and 5. The trans-

conﬁguration of the substituents at positions 2 and 3 in the 2-

COOBu-t regioisomers 10a, 10c, 10d, 11b and 11d was also in

agreement with closely related literature examples.¹⁶Selectivity of

these cycloadditions was somewhat lower than expected on the

basis of results with analogous reactions.^8,12,16,17Nevertheless,

isolation of multiple isomers may also be advantageous, due to the

increase of the stereochemical diversity of 6-amino-7-

oxotetrahydropyrazolo[1,2-a]pyrazole-1(or 2)-carboxylic acid

scaffold (cf. Schemes 1e3). Having this small, stereochemically

diverse library of dipeptides 8e14 in our hands, we continued

with selective deprotection of the amino and the carboxy function.

Acidolytic deprotection of the carboxy group of dipeptides 8a, 8c,

9b, 11b, 11d, 13c and 14d afforded the corresponding carboxylic

acids 15e19 in 67e100% yields, while hydrogenolytic deprotection

of the amino group of 8a, 8c, 11b, 11d, 13c and 14d afforded the

corresponding free amines 20e23 in 20e94% yields. According to

search on 1 with locked ring fusion gives only one hit, i.e., Eli Lilly’s

patent from 1989.^7eThus, 1 is a practically unexplored scaffold that

should have reasonable applicative potential.

1,3-Dipolar cycloadditions provide an easy access to polyfunc-

tional ﬁve-membered heterocycles with multiple stereogenic cen-

tres, usually with excellent stereocontrol.⁹Asymmetric cycload-

ditions are well elaborated with chiral nitrones, nitrile oxide, and

azomethine ylide series,¹⁰however, far fewer examples of cyclo-

additions to chiral azomethine imines have been reported.¹¹In the

last decade, our studies on [3þ2] cycloadditions of (1Z,4R*,5R*)-1-

arylmethylidene-4-benzoylamino-3-oxo-5-

phenyltetrahydropyrazol-1-ium-2-ides

(1-arylmethylidene-4-

benzoylamino-5-phenylpyrazolidin-3-on-1-azomethine imines)

to various dipolarophiles has revealed general reactivity and se-

lectivity of these cycloadditions,⁸as well as their applicability in

high-throughput synthesis.¹²

Though very useful for the determination of reactivity and se-

lectivity of the above [3þ2] cycloadditions, the obtained cyclo-

adducts were not suitable for incorporation into peptides, since

carboxy and amino functions (COOMe and NHCOPh, respectively)

could not be selectively deprotected without cleaving the pyrazolo

[1,2-a]pyrazolone system as well. Thus, selective deprotection of

a heterocyclic dipeptide was mandatory for a viable method for the

synthesis of peptide mimetics. To do this, we decided to try out

a classical peptide chemistryapproach utilizing a combination of Boc

and Cbz protecting groups. Cycloadditions of 1-arylmethylidene-4-

benzyloxycarbonylamino-3-oxopyrazolidin-1-azomethine imines

to tert-butyl acrylate (or some other tert-butyl 2-alkenoate) would

give selectively deprotectable dipeptides enabling derivatization of

the carboxy and the amino function. Furthermore, coupling of the

racemic dipeptide with an enantiomerically pure reagent (e.g., with

a-amino acid derivative), followed by separation of the so formed

diastereomers would give non-racemic tripeptides.¹³

Herein, we report theresultsof thisstudy, i.e., thesynthesis of tert-

butyl 3-benzyloxycarbonylamino-2-oxo-1,4-diazabicyclo-[3.3.0]oc-

tane-7-carboxylates as heterocyclic peptides with variable amino

acid sequence and conﬁguration and their use as building blocks in

the synthesis of 1-based conformationally constrained peptide

mimetics.

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A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

Scheme 2. Reaction conditions: (i) tert-butyl acrylate (6a), anisole, reﬂux; (ii) chro-

matographic separation (CC, MPLC).

Scheme 1. Reaction conditions: (i) hydrazine hydrate, MeOH, rt (Ref. 15); (ii) ArCHO

(4a,b), EtOH, TFA (cat.), rt (Ref. 15); (iii) tert-butyl acrylate (6a), anisole, reﬂux; (iv)

chromatographic separation (CC, MPLC).

the previously reported results,¹⁵the low yield of compound 20a

could be explained by partial hydrogenolytic cleavage of the C(5)e

N(1) bond, which induces further transformations of the primary

product 20a (Scheme 4).

Scheme 3. Reaction conditions: (i) tert-butyl methacrylate (6b), anisole, reﬂux; (ii)

chromatographic separation (CC, MPLC).

2.3. Synthesis of non-racemic pyrazolo[1,2-a]pyrazolone-

based tripeptides 35e42 and 35⁰e42⁰

2.2. Synthesis of racemic tetrapeptide 28 and hexapeptide 33

To show that compounds 15e23 can serve as useful building

blocks for the synthesis of U-shaped peptides, tetrapeptide 28 and

hexapeptide 33 with 3-amino-2-oxo-1,4-diazabicyclo[3.3.0]oc-

tane-7-carboxylic acid (1) as the central part of the sequence were

prepared. Amidation of dipeptide 18c with glycine methyl ester

(24) gave the tripeptide 25 in 50% yield. Catalytic hydrogenation of

25 followed by acylation of 26 with Boc-glycine (27) in the presence

of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) fur-

nished tetrapeptide 28 in 87% yield. Similarly, acylation of 22c with

N-(N-benzyloxycarbonylglycyl)glycine (29) gave the tetrapeptide

30 in 74% yield. Treatment of 30 with triﬂuoroacetic acid in

dichloromethane afforded the carboxylic acid 31 in 23% yield.

Amidation of the acid 31 with N-(glycyl)glycine methyl ester (32)

furnished the hexapeptide 33 in 62% yield (Scheme 5).

Finally, sixteen non-racemic tripeptides were prepared. The ﬁrst

example was amidation of the racemic N-protected dipeptides

15a,c and 18c with methyl (S)-alaninate (34) giving diastereomeric

tripeptides 35/35⁰and 36/36⁰, which were separated by MPLC to

furnish the non-racemic, diastereomerically pure N-protected tri-

peptide esters 35a,c, 35a,c⁰, 36c and 36c⁰. Similarly, acylation of the

racemic C-protected dipeptide 20c and 22c with (S)-Boc-alanine

(37) in the presence of EEDQ followed by separation of di-

astereomers by MPLC afforded the non-racemic tripeptide esters

38c, 38c⁰, 39c and 39c⁰(Scheme 6).

In the same manner, racemic carboxylic acids 16b and 19d were

transformed with methyl (S)-alaninate (34) into mixtures of di-

astereomeric tripeptides 40b/40b⁰and 41d/41d⁰, while acylation of

the racemic amine 23d with Boc-(S)-alanine afforded a mixture of

A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

6651

Scheme 4. Reaction conditions: (i) CH₂Cl₂eCF₃COOH (2:1), rt; (ii) H₂, PdeC, EtOH, rt.

diastereomeric tripeptides 42d/42d⁰. Separation of the mixtures of

diastereomers by MPLC furnished isomerically pure non-racemic

tripeptide esters 40b, 40b⁰, 41d, 41d⁰, 42d and 42d⁰(Scheme 7).

Scheme 5. Reaction conditions: (i) EEDQ, CH₂Cl₂, rt; (ii) H₂, PdeC, EtOH, rt; (iii)

CH₂Cl₂eCF₃COOH (2:1), rt.

2.4. Structure determination

diffraction, the conﬁgurations of their diastereomers 36c, 39c and

40b were determined unambiguously as well. For the other 3,5-

syn-diastereomers 35a, 35a⁰, 35c, 35c⁰, 38c and 38c⁰, the tentative

conﬁgurations were assigned on the basis of correlation between

speciﬁc rotation and absolute conﬁguration of diastereomeric tri-

peptides 36c, 36c⁰, 39c and 39c⁰. Thus, the tentative

(2S,1⁰R,3⁰R,5⁰R,6⁰R)-conﬁguration was assigned to diastereomers

35a, 35c and 38c exhibiting strong negative speciﬁc rotations,

while tentative (2S,1⁰S,3⁰S,5⁰S,6⁰S)-conﬁguration was assigned to

diastereomers 35a⁰, 35c⁰, and 38c⁰with strong positive speciﬁc

rotations. Such correlation was not clearly applicable for the 3,5-

anti-diastereomers 40/40⁰e42/42⁰, since the diastereomers 40b

and 40b⁰with unambiguously determined absolute conﬁguration

The structures of novel compounds 3b, 5c,d, 8e23, 25, 26, 28,

30, 31, 35/35⁰, 36/36⁰, and 38/38⁰e42/42⁰were determined by

spectroscopic methods (¹H NMR, ¹³C NMR, 2D NMR, NOESY spec-

troscopy, HRMS, IR) and by analyses for C, H, and N. Spectral data for

compounds 3b, 5c,d, 8e23, 25, 26, 28, 30, 31, 35/35⁰, 36/36⁰, and 38/

38⁰e42/42⁰were in agreement with the data for closely related

pyrazolidinone derivatives.^{8,12,13,16e18}The conﬁguration of the

pyrazolo[1,2-a]pyrazole core in compounds 8e23, 25, 26, 28, 30,

33 and 35/35⁰e42/42⁰was determined by NMR. Detailed structure

determination by NMR spectroscopy is given in the Supplementary

data. Finally, the structures of compounds 5c, 5d, 9d, 11b, 11d, 21b,

36c⁰, 39c⁰and 40b⁰were determined by X-ray diffraction (Figs.

2e10). X-ray structures of dipoles 5c and 5d (cf. Figs. 2 and 3) are,

to the best of our knowledge, the ﬁrst examples in the (1Z,4R*,5R*)-

1-arylmethylidene-4-acylamino-3-oxo-5-

exhibited only

a

weak speciﬁc rotation, positive for the

40b and negative for the

(2S,1⁰S,3⁰S,5⁰R,6⁰R)-isomer

(2S,1⁰R,3⁰R,5⁰S,6⁰S)-isomer 40b⁰. This ‘inversion’ of speciﬁc rotation

in 40b/40b⁰with respect to speciﬁc rotation pattern observed in

compounds 35/35⁰, 36/36⁰, 38/38⁰, and 39/39⁰coincides with op-

posite conﬁgurations at positions 1⁰and 3⁰. On the other hand,

other 3,5-anti-diastereomers 41d/41d⁰and 42d/42d⁰are 3⁰-epimers

of 36c/36c⁰and 39/39c⁰and, most probably, the sign of speciﬁc

rotation could not be changed upon reversal of conﬁguration at

phenyltetrahydropyrazol-1-ium-2-ide

ambiguously proved the Z-conﬁguration around the exocyclic C]N

double bond.

series,

which

un-

Since the absolute conﬁgurations of the non-racemic tripeptides

36c⁰, 39c⁰and 40b⁰were unambiguously established by X-ray

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A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

Scheme 7. Reaction conditions: (i) methyl (S)-alaninate (34) or Boc-(S)-alanine (37),

EEDQ, CH₂Cl₂, rt, then chromatographic separation (FC, MPLC).

Scheme 6. Reaction conditions: (i) methyl (S)-alaninate (34) or Boc-(S)-alanine (37),

EEDQ, CH₂Cl₂, rt, then chromatographic separation (FC, MPLC).

only one stereogenic centre (among ﬁve). Therefore, correlation of

speciﬁc rotation of the 3,5-anti-diastereomers 41d/41d⁰and 42d/

42d⁰with epimeric compounds 35/35⁰, 36/36⁰, 38/38⁰and 39/39⁰

Fig. 2. The molecular structure of 5c, showing the atom labelling. The displacement

ellipsoids are drawn with 50% probability and the hydrogen atoms are shown as small

spheres of arbitrary radii.

seems

quite

reasonable.

Accordingly,

the

tentative

(2S,1⁰R,3⁰S,5⁰R,6⁰R)-conﬁguration was proposed for the isomers 41d

and 42d with a strong negative speciﬁc rotation and the tentative

(2S,1⁰S,3⁰R,5⁰S,6⁰S)-conﬁguration for the isomers 41d⁰and 42d⁰with

a strong positive speciﬁc rotation (Table 1, cf. Schemes 6 and 7).

The X-ray structures of dipeptides 11b, 11d and 21b and tri-

peptides 36c⁰, 39c⁰and 40b⁰exhibit the U-shaped structure of the

peptide chain (cf. Figs. 5e10). The U-shape of 36c⁰is additionally

stabilized by intramolecular NeH/O]C hydrogen bond (with

N13/O9 distance of 2.808(3) A) donated by N13 from the alanyl

residue and accepted by O9 of the C]O group. The hydrogen bond

is represented as a dashed line in Fig. 8.

ꢁ

A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

6653

Fig. 6. The molecular structure of 11d, showing the atom labelling. The displacement

ellipsoids are drawn with 50% probability and the hydrogen atoms are shown as small

spheres of arbitrary radii.

Fig. 3. The molecular structure of 5d, showing the atom labelling. The displacement

ellipsoids are drawn with 50% probability and the hydrogen atoms are shown as small

spheres of arbitrary radii.

Fig. 7. The molecular structure of 21b, showing the atom labelling. The displacement

ellipsoids are drawn with 50% probability and the hydrogen atoms are shown as small

spheres of arbitrary radii.

Fig. 4. The molecular structure of 9d, showing the atom labelling. The displacement

ellipsoids are drawn with 50% probability and the hydrogen atoms are shown as small

spheres of arbitrary radii.

Fig. 8. The molecular structure of 36c⁰, showing the atom labelling. The displacement

ellipsoids are drawn with 50% probability and the hydrogen atoms are shown as small

spheres of arbitrary radii. The dashed line represents the intramolecular hydrogen

bond N13eH13/O9 that stabilizes U-shape of the molecule.

Fig. 5. The molecular structure of 11b, showing the atom labelling. The displacement

ellipsoids are drawn with 50% probability and the hydrogen atoms are shown as small

spheres of arbitrary radii.

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A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

In CDCl₃solution, formation of (7⁰)C]O/HeNeC(2) intra-

molecular hydrogen bond in tripeptides 35/35⁰, 36/36⁰, 40/40⁰and

41/41⁰with the C-terminal (S)-alanyl residue was supported by ¹H

NMR spectroscopy. Typically, the signals for the (non-hydrogen

bonded) amidic NH protons in tripeptides 35/35⁰, 36/36⁰and 38/

38⁰e42/42⁰appeared at a chemical shift of

signals for the hydrogen bonded 2-NH protons exhibited higher

d

¼5e7 ppm, while the

chemical shift,

d

¼7.5e9.3 ppm. For example, in the ¹H NMR spec-

trum of tripeptide 36c⁰with the C-terminal (S)-alanine residue,

a doublet for the HeNeC(2) proton at 8.39 ppm indicates non-

covalent interactions of this NH group, explainable by (7⁰)C]

O/HeNeC(2) intramolecular hydrogen bond. In contrast, a broad

singlet for 2-NH proton at 4.94 ppm in the ¹H NMR spectrum of

tripeptide 39c⁰with the N-terminal (S)-alanine residue does not

support hydrogen bonding of this NH group (Fig. 11, Table 2).

Fig. 9. The molecular structure of 39c⁰, showing the atom labelling. The displacement

ellipsoids are drawn with 50% probability and the hydrogen atoms are shown as small

spheres of arbitrary radii.

Fig. 10. The molecular structure of 40b⁰, showing the atom labelling. The displacement

ellipsoids are drawn with 50% probability and the hydrogen atoms are shown as small

spheres of arbitrary radii.

Fig. 11. Chemical shifts of the NH protons in tripeptide 36c⁰(intramolecular hydrogen

bond) and 39c⁰(without intramolecular hydrogen bond).

Table 2

Chemical shifts of the amidic NH protons in tripeptides 35/35⁰, 36/36⁰and 38/

38⁰e42/42⁰

Table 1

Correlation between speciﬁc rotation and absolute conﬁguration of the non-racemic

diastereomeric pairs of tripeptides 35/35⁰, 36/36⁰, 38/38⁰, 39/39⁰and 40/40⁰e42/42⁰

Entry

Compound

d (ppm)

6⁰-NH

2-NH

Entry

Isomer

Conﬁguration

½ ꢀ

a ²_D⁵

1

2

3

4

5

6

7

8

35a/35a⁰

35c/35c⁰

36c/36c⁰

38c/38c⁰

39c/39c⁰

40b/40b⁰

41d/41d⁰

42d/42d⁰

5.48/5.60

5.91/5.99

5.21/5.52

6.51/6.59

6.62/6.55

5.30/5.26

5.53/5.53

6.80/6.97

7.88/8.04

7.84/8.08

8.48/8.39

4.92/5.05

5.11/4.94

7.62/7.53

9.02/9.27

5.09/5.12

1^b

35a

35a⁰

35c

35c⁰

36c

36c⁰

38c

38c⁰

39c

39c⁰

40b

40b⁰

41d

41d⁰

42d

42d⁰

2S,1⁰R,3⁰R,5⁰R,6⁰R^b

2S,1⁰S,3⁰S,5⁰S,6⁰S^b

2S,1⁰R,3⁰R,5⁰R,6⁰R^b

2S,1⁰S,3⁰S,5⁰S,6⁰S^b

2S,1⁰R,3⁰R,5⁰R,6⁰R

2S,1⁰S,3⁰S,5⁰S,6⁰S^a

2S,1⁰R,3⁰R,5⁰R,6⁰R^b

2S,1⁰S,3⁰S,5⁰S,6⁰S^b

2S,1⁰R,3⁰R,5⁰R,6⁰R

2S,1⁰S,3⁰S,5⁰S,6⁰S^a

2S,1⁰S,3⁰S,5⁰R,6⁰R

2S,1⁰R,3⁰R,5⁰S,6⁰S^a

2S,1⁰R,3⁰S,5⁰R,6⁰R^b

2S,1⁰S,3⁰R,5⁰S,6⁰S^b

2S,1⁰R,3⁰S,5⁰R,6⁰R^b

2S,1⁰S,3⁰R,5⁰S,6⁰S^b

ꢁ74.3 (c 0.15, EtOH)

þ62.7 (c 0.15, EtOH)

ꢁ33.0 (c 0.12, CDCl₃)

þ82.9 (c 0.11, CDCl₃)

ꢁ108 (c 0.12, EtOH)

þ81.7 (c 0.16, EtOH)

ꢁ61.6 (c 0.13, CDCl₃)

þ62.6 (c 0.12, CDCl₃)

ꢁ66.2 (c 0.13, EtOH)

þ52.7 (c 0.11, EtOH)

þ6.9 (c 0.12, EtOH)

ꢁ18.2 (c 0.21, EtOH)

ꢁ128 (c 0.10, EtOH)

þ106 (c 0.11, EtOH)

ꢁ93.9 (c 0.41, EtOH)

þ72.7 (c 0.17, EtOH)

2^b

3^b

4^b

5

6^a

7^b

8^b

9

10^a

11

12^a

13^b

14^b

15^b

16^b

2.5. Biological activity

Compounds 8a,c, 9a,b,d, 10a, 11b, 15a, 16b, 17b,d, 20a, 21b,d,

35a, 35a⁰, 40b and 40b⁰were also tested for inhibitory activities on

two bacterial peptidoglycan biosynthesis enzymes: MurD ligase

a

(MurD) and

D-alanine:D

-alanine ligase (DdlB).¹⁸The Malachite

Determined by X-ray diffraction.

Tentative conﬁguration.

green assay,^19,which detects the orthophosphate generated during

b

A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

6655

enzymatic reactions was used. The results are presented as residual

activities (RAs) of the enzyme in the presence of 250 M of each

preparation of structurallyand stereochemically diverse heterocyclic

dipeptides that can be easily incorporated into peptides. Somewhat

low regio- and stereo-selectivity of cycloadditions is not necessarily

disadvantageous, since higher number of separable isomeric cyclo-

adducts increases the stereochemical diversity of heterocyclic di-

peptides and compounds derived thereof.

m

compound. Compounds 9a, 9d, 13c, 18c, 21b, 35a and 35a⁰showed

a weak inhibition of MurD (entries 3, 5, 8, 14, 17, 27 and 28),

whereas compounds 21b and 35a⁰showed a weak inhibition of

DdlB (entries 17 and 28). The other compounds did not inhibit these

two enzymes (Table 3).

4. Experimental

4.1. General

Table 3

Inhibitory activity of compounds 8a,c, 9a,b,d, 10a, 11b, 15a, 16b, 17b,d, 20a, 21b,d,

35a, 35a⁰, 40b and 40b⁰against MurD ligase and

D-Ala:D-Ala ligase

Melting points were determined on a Stanford Research Systems

MPA100OptiMeltautomatedmeltingpointsystem. The NMR spectra

were obtained on a Bruker Avance DPX 300 at 300 MHz for ¹H and

75.5 MHz for ¹³C and on a Bruker Avance III UltraShield 500 plus at

500 MHz for ¹H and 126 MHz for ¹³C, using acetone-d₆, acetonitrile-

d₃, CDCl₃, and DMSO-d₆with Me₄Si as the internal standard, as sol-

vents. Mass spectra were recorded on a Q-Tof Premier spectrometer

and on a Agilent 6224 Accurate Mass TOF LC/MS spectrometer, IR

spectra on a PerkineElmer Spectrum BX FTIR spectrophotometer.

Microanalyses were performed on a PerkineElmer CHN Analyzer

2400 II. Flash column chromatography (FC) was performed on silica

Entry

Compound

% RA

MurD

Ddl

1

2

3

4

5

6

7

8

8a

8c

9a

9b

77^a

93

94

90

98

96

63^a

81^a

47

9d

77

10a

11b

13c

14d

15a

16b

17b

17d

18c

19d

20a

21b

21d

22c

23d

25

81

nd^b

140

86^a

100

88^a

96^a

91

78^a

49^a

101

98^a

97

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

gel (Fluka, Silica gel 60, particle size 35e70

liquid chromatography (MPLC) was performed on a Buchi Flash

m

m). Medium pressure

102^a

109

51^a

69^a

75

46

82

95

65

99

96

107

99

98

101

60^a

63^a

nd^b

77^a

€

100

96

99

87

66

€

Chromatography System (Buchi Fraction Collector C-660, Buchi

€

Pump Module C-605, Buchi Control Unit C-620) on silica gel

(LiChroprep^ÒSi 60, 15e25

backpressure: 10 Bar, detection: UV (254 nm).

mm), column dimensions: 36ꢂ460 mm,

102

98

tert-Butyl acrylate (6a), tert-butyl methacrylate (6b), 10%

PdeC and EEDQ are commercially available (Sigma Aldrich). Methyl

2-(benzyloxycarbonylamino)-3-phenylacrylate (2a), methyl 2-

(benzyloxycarbonylamino)-3-isopropylacrylate (2b),¹⁴pyrazolidi-

none 3a and azomethine imines 5a,b¹⁵were prepared following the

literature procedures.

98

102^a

99

26

28

30

31

93

100

93

33

98

4.2. (4R*,5R*)-4-Benzyloxycarbonylamino-5-

isopropylpyrazolidin-3-one (3b)

35a

35a⁰

40b

40b⁰

92

70^a

96

nd^b

A mixture of 2b (22 g, 80 mmol), ethanol (120 mL) and hydra-

zine hydrate (11.66 mL, 240 mmol) was stirred at rt for 3 h. The

precipitate was collected by ﬁltration and washed with ethanol

(15 mL) to give 3b. Yield: 18.16 g (83%) of pale yellow solid; mp

159e163 ^ꢃC; [Found: C, 60.54; H, 6.84; N, 15.03. C₁₄H₁₉N₃O₃re-

quires: C, 60.63; H, 6.91; N, 15.15%]; n_max(KBr) 3450, 3320, 3213,

2951, 1721 (C]O), 1697 (C]O), 1661, 1635, 1542, 1472, 1457, 1418,

a

Slight precipitation occurred.

Not determined.

b

3. Conclusion

A series of tetrahydropyrazolo[1,2-a]pyrazole-based peptide mi-

metics has been synthesized in ﬁve steps from methyl N-benzylox-

1356, 1292, 1244, 1167, 1061, 978, 775, 756, 730, 696, 645 cm^ꢁ1

; d_H

ycarbonyl-

benzyloxycarbonyl-

with a two step preparation of 5-substituted 1-benzylidene-4-

benzyloxycarbonylamino-3-oxopyrazolidin-1-ium-2-ides 5aed,

a

,

b

-dehydro-

b-phenylalaninate (2a) and methyl N-

(300 MHz, DMSO-d₆) 0.89 and 0.98 (6H, 2d, 1:1, J¼6.7 Hz, Me₂CH),

1.77 (1H, septet, J¼6.4 Hz, CHMe₂), 2.92 (1H, td, J¼8.7, 10.7 Hz, 5-H),

4.02 (1H, dd, J¼9.4, 10.7 Hz, 4-H), 4.89 (1H, d, J¼11.0 Hz, 1-H), 5.04

and 5.07 (2H, 2d, 1:1, J¼12.6 Hz, CH₂Ph), 7.30e7.40 (5H, m, Ph), 7.60

(1H, d, J¼9.4 Hz, NH), 9.22 (1H, s, 2-H); d_C(126 MHz, DMSO-d₆) 18.8,

19.6, 30.10, 56.1, 65.4, 68.0, 127.7, 127.8, 128.4, 137.1, 156.1, 173.7;

HRMS (ESI): MH^þ, found 278.1509. C₁₄H₂₀N₃O₃requires 278.1505.

a,b-dehydroleucinate (2b). The synthesis starts

followed by 1,3-dipolar cycloaddition to tert-butyl acrylate (6a) and

tert-butyl methacrylate (6b) to afford the corresponding racemic

cycloadducts 8e14 as heterocyclic dipeptides that are selectively

deprotectable at the C- and the N-terminal. Using conventional

peptide chemistry protocols, tetrapeptide 28 and hexapeptide 33

with 6-amino-7-oxotetrahydropyrazolo[1,2-a]pyrazole-1-carboxy-

lic acid (1) as the central dipeptide unit were prepared. Besides,

coupling of racemic carboxy-dipeptides 15,16,18 and 19 with methyl

(S)-alaninate (34) and coupling of amino-dipeptides 20, 22 and 23

with N-Boc-(S)-alanine (37) gave the corresponding tripeptides as

1:1 mixtures of diastereomers, which were separated by MPLC to

furnish diastereomerically pure non-racemic title compounds 35/

35⁰, 36/36⁰and 38/38⁰e42/42⁰. The synthesized compounds were

4.3. General procedure for the preparation of azomethine

imines 5c,d

A mixture of 3b (2.77 g, 10 mmol), aromatic aldehyde 4

(12 mmol) and methanol (40 mL) was stirred at room temperature

for 5 min. Then, triﬂuoroacetic acid (20 drops) was added and the

mixture was stirred at rt for 7 h. The precipitate was collected by

ﬁltration and washed with methanol (10 mL) to give 5.

The following compounds were prepared in this manner.

also evaluated for inhibition of MurD ligase and D-alanine:D-alanine

ligase, however, only a few compounds showed a weak inhibition of

these two enzymes. In conclusion, this synthetic method enables

4.3.1. (3R*,4R*,Z)-2-Benzylidene-4-(benzyloxycarbonylami-no)-3-

isopropyl-5-oxopyrazolidin-2-ium-1-ide (5c). Prepared from 3b

6656

A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

(2.77 g, 10 mmol) and benzaldehyde (4a) (1.22 mL, 12 mmol). Yield:

3.50 g (96%) of white solid; mp 209e213 ^ꢃC; [Found: C, 69.13; H,

6.26; N,11.51. C₂₁H₂₃N₃O₃requires C, 69.02; H, 6.34; N,11.50%]; n_max

(KBr) 3420, 3187, 2959, 1713 (C]O), 1656 (C]O), 1589, 1569, 1451,

CDCl₃) 1.55 (9H, s, t-Bu), 2.50 (1H, ddd, J¼0.8, 5.5,13.5 Hz, 2-Ha), 2.74

(1H, ddd, J¼9.3, 11.5, 13.5 Hz, 2-Hb), 3.99 (1H, dd, J¼5.5,11.5 Hz, 3-H),

4.05 (1H, d, J¼12.0 Hz, 5-H), 4.41 (1H, dd, J¼0.8, 9.3 Hz,1-H), 4.97 (2H,

s, CH₂Ph), 5.06 (1H, m, 6-H), 5.30 (1H, br d, J¼8.4 Hz, NH), 7.95e6.68

(15H, m, 3ꢂPh); d_H(500 MHz, MeCN-d₃) 1.53 (9H, s, t-Bu), 2.56 (1H,

ddd, J¼1.2, 5.6,13.2 Hz, 2-Ha), 2.71 (1H, ddd, J¼9.4,11.5,13.2 Hz, 2-Hb),

3.98 (1H, dd, J¼5.6,11.5 Hz, 3-H), 4.09(1H, d, J¼13.0 Hz, 5-H), 4.29(1H,

dd, J¼1.2, 9.4 Hz,1-H), 4.82 (1H, ddd, J¼1.1, 9.8,13.0 Hz, 6-H), 4.96 (2H,

s, CH₂Ph), 6.14 (1H, br d, J¼9.8 Hz, NH), 7.02e7.05 (6H, m, 6H of Ph),

7.18e7.3 (9H, m, 9H of Ph); d_C(126 MHz, CDCl₃) 28.1, 43.0, 55.2, 63.0,

67.1, 68.8, 79.0, 83.3, 127.7, 128.0, 128.0, 128.1, 128.1, 128.1, 128.1, 128.3,

128.5, 135.0,136.2, 136.3, 155.8, 162.9, 168.9; HRMS (ESI): MH^þ, found

528.2493. C₃₁H₃₄N₃O₅requires 528.2498.

1383, 1327, 1276, 1162, 1097, 1042, 747, 676 cm^ꢁ1

; d_H(300 MHz,

DMSO-d₆) 0.76 and 1.04 (6H, 2d, 1:1, J¼6.8 Hz, Me₂CH), 2.52e2.57

(1H, m, CHMe₂), 4.09 (1H, dd, J¼4.4, 8.5 Hz, 4-H), 4.48 (1H, dd,

J¼4.4, 1.1 Hz, 3-H), 5.05 (2H, s, CH₂Ph), 7.30e7.37 (4H, m, 4H of Ph),

7.38 (1H, br s, 1⁰-H), 7.60 (3H, dd, J¼1.9, 5.2 Hz, 3H of Ph), 7.80 (1H, s,

NH), 7.90 (1H, d, J¼8.5 Hz, 1H of Ph), 8.35 (2H, dd, J¼2.8, 6.6 Hz, 2H

of Ph); d_C(75.5 MHz, DMSO-d₆) 14.1,18.0, 30.6, 51.0, 65.6, 78.1,127.5,

127.6, 128.1, 128.3, 129.4, 131.3, 131.4, 133.4, 136.5, 155.6, 180.3;

HRMS (ESI): MH^þ, found 366.1806. C₂₁H₂₄N₃O₃requires 366.1812.

4.3.2. (3R*,4R*,Z)-4-(Benzyloxycarbonylamino)-2-(2,6-dichloro-ben-

zylidene)-3-isopropyl-5-oxopyrazolidin-2-ium-1-ide (5d). Prepared

from 3b (2.77 g, 10 mmol) and 2,6-dichlorobenzaldehyde (4b)

(2.10 g,12 mmol). Yield: 4.30 g (99%) of white solid; mp 180e185 ^ꢃC;

[Found: C, 57.89; H, 4.72; N, 9.61. C₂₁H₂₁Cl₂N₃O₃requires C, 58.07; H,

4.87; N, 9.68%]; n_max(KBr) 3433, 3193, 3027, 2969, 2934, 1720 (C]

O), 1670 (C]O), 1580, 1558, 1463, 1432, 1375, 1330, 1315, 1274, 1258,

4.4.1.3. tert-Butyl (1S*,3S*,5R*,6R*)-6-(benzyloxycarbonylamino)-

7-oxo-3,5-diphenylhexahydropyrazolo[1,2-a]pyrazole-1-carboxylate

(9a). Yield: 53 mg (5%) of colourless oil; n_max(liquid ﬁlm) 3326,

3059, 3033, 2977, 2925, 1731, 1532, 1496, 1456, 1390, 1369, 1287,

1250, 1155, 1063, 1026, 760, 738, 697 cm^ꢁ1

; d_H(500 MHz, CDCl₃)

1.51 (9H, s, t-Bu), 2.31 (1H, br sextet, J¼7.5 Hz, 2-Ha), 2.95 (1H, br td,

J¼9.2, 13.0 Hz, 2-Hb), 3.65 (1H, t, J¼8.1 Hz, 3-H), 4.43 (1H, d,

J¼7.4 Hz, 5-H), 4.47 (1H, dd, J¼6.2, 10.0 Hz,1-H), 5.08 (2H, s, CH₂Ph),

5.04e5.11 (1H, m, 6-H, overlapped by the signal for CH₂Ph), 5.47

(1H, br d, J¼8.5 Hz, NH), 6.74e7.55 (15H, m, 3ꢂPh); d_H(500 MHz,

Me₂CO-d₆) 1.51 (9H, s, t-Bu), 2.21 (1H, ddd, J¼6.1, 8.1, 13.1 Hz, 2-Ha),

3.02 (1H, ddd, J¼8.0, 10.0, 13.1 Hz, 2-Hb), 4.09 (1H, t, J¼8.0 Hz, 3-H),

4.52 (1H, dd, J¼6.1, 10.0 Hz, 1-H), 4.74 (1H, d, J¼11.3 Hz, 5-H), 5.03

and 5.07 (2H, 2d, 1:1, J¼12.5 Hz, CH₂Ph), 5.23 (1H, dd, J¼9.6, 11.3 Hz,

6-H), 6.95e7.07 (8H, m, NH and 7H of Ph), 7.17e7.35 (8H, m, 8H of

Ph); d_C(126 MHz, CDCl₃) 28.1, 42.8, 54.3, 59.2, 62.2, 66.6, 67.4, 83.0,

127.8, 127.8, 128.3, 128.4, 128.5, 128.6, 128.7, 128.8, 128.9, 133.2,

136.1, 137.9, 156.1, 167.3, 168.5; HRMS (ESI): MH^þfound 528.2514.

C₃₁H₃₄N₃O₅requires 528.2498.

1172, 1114, 1092, 1040, 1018, 808, 795, 741, 708, 698, 673 cm^ꢁ1

; d_H

(500 MHz, DMSO-d₆) 0.92 and 1.06 (6H, 2d, 1:1, J¼6.8 Hz, Me₂CH),

2.63e2.70 (1H, m, CHMe₂), 4.17 (1H, dd, J¼4.8, 8.4 Hz, 4-H), 4.85

(1H, dd, J¼4.8, 1.0 Hz, 3-H), 5.06 (2H, s, CH₂Ph), 7.27e7.33 (1H, m, p-

C₆H₃), 7.33e7.37 (4H, m, 4H of Ar), 7.46e7.50 (3H, m, 3H of Ar), 7.88

(1H, br s, 1⁰-H), 7.95 (1H, d, J¼8.4 Hz, NH); d_C(75.5 MHz, DMSO-d₆)

14.1, 18.1, 30.3, 51.4, 65.6, 77.1, 127.4, 127.5, 127.8, 128.0, 128.3, 128.6,

128.9, 132.4, 133.5, 155.71, 180.0; HRMS (ESI): MH^þ, found 434.1031.

C₂₁H₂₂Cl₂N₃O₃requires 434.1033.

4.4. General procedure for the synthesis of tert-butyl 6-

(benzyloxycarbonylamino)-7-oxotetrahydropyrazolo[1,2-a]-

pyrazole-1(or 2) carboxylates (racemic fully protected

dipeptides)

4.4.1.4. tert-Butyl (2R*,3R*,5R*,6R*)-6-(benzyloxycarbonylamino)-

7-oxo-3,5-diphenylhexahydropyrazolo[1,2-a]pyrazole-2-carboxylate

(10a). Yield: 116 mg (11%) of colourless oil; n_max(liquid ﬁlm) 3296,

3064, 2977, 2930,1731,1693,1531,1497,1456,1393,1368,1308,1252,

Under argon, a mixture of dipole 5aed (5 mmol), tert-butyl

acrylate (6a)(0.87 mL,

6 mmol) or tert-butyl methacrylate

(6b)(0.98 mL, 6 mmol) and anisole (25 mL) was heated under reﬂux

for 4e7 h. (Reactions of 5-phenyl substituted dipoles 5a,b produced

the insoluble hydantoin by-products 7a,b, which were isolated by

ﬁltration, while the ﬁltrate was subjected to further workup.)

Volatile components were evaporated in vacuo and the residue was

puriﬁed by FC (EtOAc). Fractions containing the products were

combined and evaporated in vacuo to give a mixture of cyclo-

adducts 8e12, which were separated by MPLC (EtOAc/hexanes).

Fractions containing the products were combined and evaporated

in vacuo to give isomerically pure compounds 8e12.

1217, 1153, 1059, 1029, 1003, 844, 764, 737, 698, 632 cm^ꢁ1

; d_H

(300 MHz, CDCl₃) 1.35 (9H, s, t-Bu), 3.38 (1H, brdt, J¼7.0, 9.2 Hz, 2-H),

3.72 (1H, brt, J¼10.7 Hz,1-Ha), 3.99 (1H, brd, J¼8.2 Hz, 3-H), 4.15 (1H,

br d, J¼11.9 Hz, 5-H), 4.19 (1H, dd, J¼7.0,11.6 Hz,1-Hb), 4.69 (1H, br t,

J¼9.9 Hz, 6-H), 5.05 (2H, s, CH₂Ph), 5.25 (1H, br d, J¼8.0 Hz, NH),

7.07e7.30 (15H, m, 3ꢂPh); d_H(500 MHz, Me₂CO-d₆) 1.32 (9H, s, t-Bu),

3.52 (1H, dt, J¼7.2, 9.5 Hz, 2-H), 3.71 (1H, ddd, J¼1.4, 9.5, 11.2 Hz, 1-

Ha), 4.00 (1H, d, J¼9.5 Hz, 3-H), 4.08 (1H, dd, J¼7.2, 11.2 Hz, 1-Hb),

4.24(1H, d, J¼10.5Hz, 5-H), 4.62(1H, d, J¼10.5 Hz, 6-H), 5.05 and5.06

(2H, 2d,1:1, J¼12.6 Hz, CH₂Ph), 7.06e7.14 (6H, m, 6H of Ph), 7.25e7.36

(9H, m, 9H of Ph), NH exchanged; d_C(126 MHz, CDCl₃) 28.1, 43.3, 54.7,

63.6, 67.3, 73.8, 74.7, 82.4,127.7,128.0,128.2,128.2,128.2,128.2,128.3,

128.3,128.7,136.2,136.3,136.9,155.9,163.1,169.7; HRMS (ESI): MH^þ,

found 528.2508. C₃₁H₃₄N₃O₅requires 528.2498.

The following compounds were prepared in this manner.

4.4.1. Synthesis of hydantoin derivative 7a and cycloadducts 8a, 9a

and 10a. Prepared from 5a (0.799 g, 2 mmol), tert-butyl acrylate

(6a, 0.348 mL, 2.4 mmol) and anisole (12 mL), 4 h, MPLC (EtOAc/

hexanes, 1:3).

4.4.2. Synthesis of hydantoin derivative 7b and cycloadducts 9b and

11b. Prepared from 5b (0.936 g, 2 mmol), tert-butyl acrylate

(6a)(0.348 mL, 2.4 mmol), and anisole (12 mL), 4 h, MPLC (EtOAc/

hexanes, 1:2).

4.4.1.1. (Z)-5-Benzylidene-3-((E)-benzylideneamino)imidazoli-

dine-2,4-dione (7a). Yield: 0.160 g (28%) of white solid. Physical,

spectral and analytical data for compound 7a were in agreement

with the literature data.¹⁵

4.4.2.1. (Z)-5-Benzylidene-3-((E)-(2,6-dichlorobenzylidene)

amino)imidazolidine-2,4-dione (7b). Yield: 0.144 g (20%) of white

solid. Physical, spectral and analytical data for compound 7b were

in agreement with the literature data.¹⁵

4.4.1.2. tert-Butyl (1R*,3R*,5R*,6R*)-6-(benzyloxycarbonylamino)-

7-oxo-3,5-diphenylhexahydropyrazolo[1,2-a]pyrazole-1-carboxylate

(8a). Yield: 0.305 g (29%) of white solid; mp 130e135 ^ꢃC; [Found: C,

70.54;H,6.36;N,7.72.C₃₁H₃₃N₃O₅requiresC,70.57;H,6.30;N,7.96%];

n_max(KBr) 3391, 3064, 3035, 2976, 2930, 1736, 1716, 1542, 1497, 1457,

4.4.2.2. tert-Butyl (1S*,3S*,5R*,6R*)-6-(benzyloxycarbonylamino)-

3-(2,6-dichlorophenyl)-7-oxo-5-phenylhexahydropyrazolo[1,2-a]pyr-

azole-1-carboxylate (9b). Yield: 166 mg (14%) of colourless oil; n_max

1369, 1249, 1229, 1153, 1065, 1057, 754, 742, 697 cm^ꢁ1

; d_H(500 MHz,

A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

6657

(liquid ﬁlm) 3255, 3149, 3064, 2980, 1747, 1726, 1718, 1700, 1686,

J¼8.9 Hz, 6-H), 5.11 and 5.15 (2H, 2d, 1:1, J¼12.3 Hz, CH₂Ph), 5.27

(1H, d, J¼7.8 Hz, NH), 7.30e7.39 (10H, m, 2ꢂ Ph); d_C(126 MHz,

CDCl₃) 17.5, 18.1, 28.0, 29.8, 43.4, 54.8, 56.4, 67.3, 74.1, 75.2, 82.3,

128.1, 128.3, 128.3, 128.6, 128.7, 128.7, 136.2, 138.0, 155.9, 166.3,

170.3; HRMS (ESI): MH^ꢁ, found 492.2526. C₂₈H₃₄N₃O₅requires

492.2504.

1563, 1445, 1408, 1368, 1337, 1214, 1152, 1145, 1131, 1091, 1077, 1048,

1042, 1029, 782, 769, 760, 755, 729, 698 cm^ꢁ1

; d_H(500 MHz, CDCl₃)

1.50 (9H, s, t-Bu), 2.70 (1H, ddd, J¼5.8, 9.8, 13.1 Hz, 2-Ha), 2.75 (1H,

ddd, J¼8.9, 10.1, 13.1 Hz, 2-Hb), 4.39 (1H, t, J¼9.2 Hz, 3-H), 4.72 (1H,

dd, J¼5.8, 10.1 Hz, 1-H), 4.87 (1H, d, J¼10.7 Hz, 5-H), 5.11 (2H, s,

CH₂Ph), 5.15 (1H, d, J¼9.0 Hz, NH), 5.40 (1H, br t, J¼10.7 Hz, 6-H),

6.80e7.72 (13H, m, 2ꢂ Ph and C₆H₃); d_H(500 MHz, Me₂CO-d₆) 1.50

(9H, s, t-Bu), 2.67 (1H, ddd, J¼5.5, 9.5, 12.8 Hz, 2-Ha), 2.93 (1H, ddd,

J¼8.6, 10.7, 12.8 Hz, 2-Hb), 4.64 (1H, t, J¼9.1 Hz, 3-H), 4.69 (1H, dd,

J¼5.5, 10.6 Hz, 1-H), 4.96 (1H, d, J¼12.3 Hz, 5-H), 5.12 (2H, s, CH₂Ph),

5.37 (1H, dd, J¼9.6, 12.3 Hz, 6-H), 6.87 (1H, d, J¼7.8 Hz, 1H of Ph),

6.92e7.03 (5H, m, NH and 4H of Ph), 7.18 (1H, d, J¼7.5 Hz, 1H of Ph),

7.24e7.40 (7H, m, 7H of Ph), 7.64e7.67 (1H, m, 1H of Ph), 7.73e7.77

(1H, m, 1H of Ph); d_C(126 MHz, CDCl₃) 28.1, 36.4, 52.5, 55.4, 59.3,

64.3, 67.6, 82.6, 128.1, 128.30, 128.34, 128.4, 128.6, 128.8, 128.9,

130.8, 131.0, 132.1, 132.6, 136.0, 167.9, 168.7, 175.0; HRMS (ESI):

MH^þ, found 596.1733. C₃₁H₃₂Cl₂N₃O_5:requires 596.1719.

4.4.4. Synthesis of cycloadducts 9de12d. Prepared from 5d

(0.866 g, 2 mmol), tert-butyl acrylate (6a)(0.348 mL, 2.4 mmol), and

anisole (12 mL), 5 h, MPLC (EtOAc/hexanes, 1:3).

4.4.4.1. tert-Butyl (1S*,3S*,5R*,6R*)-6-(benzyloxycarbonylamino)-

3-(2,6-dichlorophenyl)-5-isopropyl-7-oxohexahydropyrazolo[1,2-a]

pyrazole-1-carboxylate (9d). Yield: 101 mg (9%) of pale yellow

crystals; mp 150e155 ^ꢃC; [Found: C, 59.92; H, 6.06; N, 7.43.

C₂₈H₃₃Cl₂N₃O₅requires C, 59.79; H, 5.91; N, 7.47%]; n_max(KBr) 3254,

3057, 2977, 2964, 1879, 1751, 1725, 1682, 1578, 1540, 1455, 1440,

1392, 1369, 1336, 1282, 1235, 1144, 1007, 1084, 1051, 1038, 986, 997,

842, 794, 784, 759, 702 cm^ꢁ1

; d_H(500 MHz, CDCl₃) 0.83 and 0.97

4.4.2.3. tert-Butyl (2S*,3S*,5R*,6R*)-6-(benzyloxycarbonylamino)-

3-(2,6-dichlorophenyl)-7-oxo-5-phenylhexahydropyrazolo[1,2-a]pyr-

azole-2-carboxylate (11b). Yield: 418 mg (36%) of colourless crys-

tals; mp 182e186 ^ꢃC; [Found: C, 62.44; H, 5.20; N, 7.15.

C₃₁H₃₁Cl₂N₃O₅requires C, 62.42; H, 5.24; N, 7.04%]; n_max(liquid

ﬁlm) 3331, 3070, 2977, 2930, 1730, 1705, 1562, 1525, 1476, 1456,

1443, 1370, 1319, 1295, 1253, 1217, 1184, 1157, 1110, 1049, 1023, 977,

(6H, 2d, 1:1, J¼6.8 Hz, Me₂CH), 1.50 (9H, s, t-Bu), 1.80e1.90 (1H, m,

CHMe₂), 2.59 (1H, dd, J¼3.9, 9.0 Hz, 5-H), 2.97e3.06 (2H, m, 2-CH₂),

4.84 (1H, br t, J¼9.0 Hz, 6-H), 4.85 (1H, br dd, J¼3.1, 9.5 Hz, 3-H),

5.05 and 5.10 (2H, 2d, 1:1, J¼12.2 Hz, CH₂Ph), 5.11 (1H, d, J¼9.0 Hz,

NH), 5.55 (1H, dd, J¼5.8, 8.7 Hz, 1-H), 7.19 (1H, t, J¼8.0 Hz, p-C₆H₃),

7.29e7.41 (7H, m, Ph and m-C₆H₃); d_C(126 MHz, CDCl₃) 17.9, 18.1,

28.1, 30.5, 35.1, 53.6, 57.4, 59.5, 67.3, 69.7, 82.9, 128.4, 128.7, 129.8,

130.6, 133.6, 133.9, 136.1, 138.0, 155.9, 162.1, 168.7; HRMS (ESI):

MNa^þ, found 584.1677. C₂₈H₃₃Cl₂N₃NaO₅requires 584.1689.

787, 761, 697, 625 cm^ꢁ1

; d_H(500 MHz, CDCl₃) 1.42 (9H, s, t-Bu), 3.60

(1H, m, 2-H), 3.87 (1H, br t, J¼10.6 Hz, 1-Ha), 4.11 (1H, br dd, J¼4.1,

11.9 Hz, 1-Hb), 4.59 (1H, d, J¼11.1 Hz, 5-H), 4.98 (1H, d, J¼6.8 Hz, 3-

H), 5.07 (2H, s, CH₂Ph), 5.21e5.32 (2H, m, NH and 6-H), 6.86e7.29

(13H, m, 2ꢂ Ph and C₆H₃); d_C(126 MHz, CDCl₃) 27.9, 45.6, 52.3, 54.7,

62.0, 65.8, 67.5, 82.7, 128.17, 128.28, 128.34, 128.4, 128.5, 128.6,

129.31, 129.33, 130.1, 132.1, 132.9, 135.0, 156.2, 170.9, 172.1; HRMS

(ESI): MH^þ, found 596.1742. C₃₁H₃₂Cl₂N₃O_5:requires 596.1719.

4.4.4.2. tert-Butyl (2R*,3R*,5R*,6R*)-6-(benzyloxycarbonylamino)-

3-(2,6-dichlorophenyl)-5-isopropyl-7-oxohexahydropyrazolo[1,2-a]

pyrazole-2-carboxylate (10d). Yield: 101 mg (9%) of colourless oil;

n_max(liquid ﬁlm) 3414, 2962, 1735, 1725, 1682, 1531, 1539, 1250,

1452, 1438, 1384, 1369, 1328, 1261, 1156, 1144, 1087, 1049, 783, 770,

698 cm^ꢁ1

;

d_H(500 MHz, CDCl₃) 0.71 and 0.76 (6H, 2d, 1:1, J¼6.7 Hz,

4.4.3. Synthesis of cycloadducts 8c and 10c. Prepared from 5c

(0.730 g, 2 mmol), tert-butyl acrylate (6a)(0.348 mL, 2.4 mmol), and

anisole (12 mL), 7 h, MPLC (EtOAc/hexanes, 1:3).

Me₂CH), 1.40 (9H, s, t-Bu), 1.54e1.63 (1H, m, CHMe₂), 3.04 (1H, dd,

J¼4.5, 7.6 Hz, 5-H), 3.65 (1H, ddd, J¼1.5, 9.7, 11.6 Hz, 1-Ha), 3.89 (1H,

ddd, J¼3.9, 7.9, 9.7 Hz, 2-H), 4.37 (1H, br dd, J¼3.9, 11.6 Hz, 1-Hb),

4.47 (1H, br t, J¼7.6 Hz, 6-H), 4.95 (1H, br d, J¼7.9 Hz, 3-H), 5.14 (2H,

2d, 1:1, J¼12.4 Hz, CH₂Ph), 5.44 (1H, d, J¼7.6 Hz, NH), 7.27e7.42 (8H,

m, Ph and C₆H₃); d_C(126 MHz, CDCl₃) 17.7, 18.2, 30.9, 43.7, 50.0, 57.2,

67.2, 68.7, 74.6, 77.4, 82.5, 128.2, 128.3, 128.6, 128.7, 130.0, 130.8,

136.2, 136.8, 155.9, 167.4, 170.9; HRMS (ESI): MH^þ, found 562.1868.

C₂₈H₃₄Cl₂N₃O₅requires 562.1870.

4.4.3.1. tert-Butyl (1R*,3S*,5R*,6R*)-6-(benzyloxycarbonylamino)-

5-isopropyl-7-oxo-3-phenylhexahydropyrazolo[1,2-a]pyrazole-1-

carboxylate (8c). Yield: 0.326

g (33%) of white solid; mp

153e156 ^ꢃC; [Found: C, 68.08; H, 7.10; N, 8.51. C₂₈H₃₅N₃O₅requires

C, 68.13; H, 7.15; N, 8.51%]; n_max(KBr) 3370, 2968, 1732, 1715, 1698,

1526,1496,1458,1413,1381,1371,1305,1247,1235,1216,1152,1060,

1022, 752, 701 cm^ꢁ1

;

d_H(500 MHz, CDCl₃) 0.75 and 0.86 (6H, 2d,

4.4.4.3. tert-Butyl (2S*,3S*,5R*,6R*)-6-(benzyloxycarbonylamino)-

3-(2,6-dichlorophenyl)-5-isopropyl-7-oxohexahydropyrazolo[1,2-a]

pyrazole-2-carboxylate (11d). Yield: 292 mg (26%) of colourless

crystals; mp 155e159 ^ꢃC; [Found: C, 59.52; H, 5.87; N, 7.36.

C₂₈H₃₃Cl₂N₃O₅requires C, 59.79; H, 5.91; N, 7.47%]; n_max(KBr) 3329,

2976, 2933, 1728, 1561, 1536, 1454, 1437, 1392, 1368, 1298, 1251,

1:1, J¼6.8 Hz, Me₂CH), 1.37 (1H, septet, J¼6.9 Hz, CHMe₂), 1.52 (9H,

s, t-Bu), 2.49 (1H, ddd, J¼0.7, 5.6, 13.2 Hz, 2-Ha), 2.65 (1H, ddd,

J¼9.3, 11.5, 13.2 Hz, 2-Hb), 3.11 (1H, dd, J¼2.7, 11.8 Hz, 5-H), 3.88

(1H, dd, J¼5.6, 11.5 Hz, 3-H), 4.31 (1H, dd, J¼0.7, 9.3 Hz, 1-H), 4.93

(1H, dd, J¼9.5, 11.8 Hz, 6-H), 5.08 and 5.14 (2H, 2d, 1:1, J¼12.3 Hz,

CH₂Ph), 5.22 (1H, br d, J¼9.5 Hz, NH), 7.04e7.33 (10H, m, 2ꢂ Ph); d_C

(126 MHz, CDCl₃) 16.3, 18.4, 27.4, 28.1, 44.0, 55.3, 56.1, 67.3, 69.0,

80.2, 83.2, 127.5, 128.2, 128.3, 128.6, 128.6, 128.9, 136.3, 138.2, 156.0,

164.3, 169.0; HRMS (ESI): MH^þ, found 494.2641. C₂₈H₃₆N₃O₅re-

quires 494.2649.

1230, 1149, 1121, 1056, 853, 819, 778, 758, 734, 696 cm^ꢁ1

; d_H

(500 MHz, CDCl₃) 0.68 and 0.93 (6H, 2d, 1:1, J¼6.7 Hz, Me₂CH), 1.45

(9H, s, t-Bu), 1.81 (1H, br sextet, J¼6.7 Hz, CHMe₂), 2.78 (1H, dd,

J¼6.4,10.1 Hz, 5-H), 3.69e3.78 (2H, m,1-Ha and 2-H), 4.11e4.19 (1H,

m, 1-Hb), 4.75 (1H, t, J¼9.9 Hz, 6-H), 5.07 (1H, d, J¼8.6 Hz, NH), 5.07

and 5.15 (2H, 2d, 1:1, J¼12.2 Hz, CH₂Ph), 5.49 (1H, d, J¼5.8 Hz, 3-H),

7.21e7.41 (8H, m, Ph and C₆H₃); d_H(500 MHz, Me₂CO-d₆) 0.47 and

0.94 (6H, 2d, 1:1, J¼6.7 Hz, Me₂CH), 1.42 (9H, s, t-Bu), 1.84e1.95 (1H,

m, CHMe₂), 3.31 (1H, ddd, J¼1.7, 10.1, 11.4 Hz, 5-H), 3.53 (1H, ddd,

J¼3.2, 6.6, 9.4 Hz, 2-H), 3.77 (1H, dd, J¼9.4, 11.6, 1-Ha), 4.09 (1H, dd,

J¼3.2, 11.6 Hz, 1-Hb), 4.57 (1H, d, J¼11.4 Hz, 6-H), 5.07 and 5.15 (2H,

2d, 1:1, J¼12.4 Hz, CH₂Ph), 5.14 (1H, d, J¼6.8 Hz, 3-H), 6.82 (1H, d,

J¼9.3 Hz, NH), 7.28e7.44 (6H, m, Ph and p-C₆H₃). 7.47e7.57 (2H, m,

m-C₆H₃); d_C(126 MHz, CDCl₃) 18.5, 19.2, 28.0, 29.9, 43.4, 51.2, 56.2,

62.8, 67.4, 69.7, 82.8, 128.4, 128.7, 129.7, 130.7, 133.3, 134.7, 136.2,

4.4.3.2. tert-Butyl (2R*,3R*,5R*,6R*)-6-(benzyloxycarbonylamino)-

5-isopropyl-7-oxo-3-phenylhexahydropyrazolo[1,2-a]pyrazole-2-

carboxylate (10c). Yield: 69 mg (7%) of colourless oil; n_max(liquid

ﬁlm) 3284, 3062, 3035, 2966, 2875, 1732, 1694, 1682, 1538, 1456,

1392, 1369, 1254, 1153, 1112, 1082, 1056, 737, 700 cm^ꢁ1

; d_H

(500 MHz, CDCl₃) 0.76 and 0.81 (6H, 2d, 1:1, J¼6.6 Hz, Me₂CH), 1.39

(9H, s, t-Bu), 1.55 (1H, m, CHMe₂), 3.04 (1H, dd, J¼3.6, 9.4 Hz, 5-H),

3.27 (1H, br dt, J¼6.2, 8.9, 2-H), 3.56 (1H, br t, J¼10.5 Hz, 1-Ha), 3.90

(1H, d, J¼8.0 Hz, 3-H), 4.14 (1H, dd, J¼6.2, 11.7 Hz, 1-Hb), 4.60 (1H, t,

6658

A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

137.5, 156.0, 167.0, 170.5; HRMS (ESI): MH^þ, found 562.1862.

C₂₈H₃₄Cl₂N₃O₅requires 562.1780.

oxohexahydropyrazolo[1,2-a]pyrazole-1-carboxylate (14d). Under

argon, a mixture of 5d (4.34 g, 10 mmol), tert-butyl methacrylate

(1.96 mL, 12 mmol), and anisole (40 mL) was heated under reﬂux

for 7 h. Volatile components were evaporated in vacuo, the residue

was triturated with diethyl ether (50 mL), and the mixture was

stirred at room temperature for 12 h. The precipitate was collected

by ﬁltration and washed with diethyl ether to give the ﬁrst portion

of 14d. The ﬁltrate was evaporated in vacuo and puriﬁed by FC

(EtOAc). Fractions containing the product were combined and

evaporated in vacuo to give the second portion of 14d. Both por-

tions of the product 14d were combined. Yield: 3.80 g (66%) of

white crystals; mp 161e165 ^ꢃC; [Found C, 60.23; H, 6.22; N, 7.29.

C₂₉H₃₅Cl₂N₃O₅requires C, 60.42; H, 6.12; N, 7.29%]; n_max(KBr) 3427,

3071, 3031, 2975, 2888, 1736, 1682, 1578, 1559, 1512, 1441, 1389,

1367, 1330, 1310, 1276, 1241, 1213, 1147, 1091, 1076, 1044, 966, 938,

4.4.4.4. tert-Butyl (1R*,3S*,5R*,6R*)-6-(benzyloxycarbonylamino)-

3-(2,6-dichlorophenyl)-5-isopropyl-7-oxohexahydropyrazolo[1,2-a]

pyrazole-1-carboxylate (12d). Yield: 135 mg (12%) of colourless oil;

n_max(liquid ﬁlm) 3329, 3066, 2976, 2934, 2873, 1728, 1581, 1561,

1536,1455,1438,1393,1369,1298,1252,1230,1149,1121,1090,1057,

1042, 778, 758, 735, 697 cm^ꢁ1

; d_H(500 MHz, CDCl₃) 0.53 and 0.90

(6H, 2d,1:1, J¼6.7 Hz, Me₂CH),1.68 (9H, s, t-Bu),1.73 (1H, m, CHMe₂),

2.81 (2H, br t, J¼8.5 Hz, 2-CH₂), 3.26 (1H, t, J¼10.1 Hz, 5-H), 4.64 (1H,

br t, J¼9.1 Hz, 3-H), 4.66 (1H, br t, J¼10.1 Hz, 6-H), 4.71 (1H, br t,

J¼7.9 Hz, 1-H), 5.06 (1H, d, J¼9.8 Hz, NH), 5.12 and 5.18 (2H, 2d, 1:1,

J¼12.2 Hz, CH₂Ph), 7.17 (1H, t, J¼8.0 Hz, p-C₆H₃), 7.28e7.43 (7H, m,

Ph and m-C₆H₃); d_H(500 MHz, Me₂CO-d₆) 0.49 and 0.90 (6H, 2d,1:1,

J¼6.7 Hz, Me₂CH), 1.49 (9H, s, t-Bu), 1.68e1.83 (1H, m, CHMe₂), 2.79

(1H, ddd, J¼4.8, 9.2, 12.9 Hz, 2-Ha), 3.02 (1H, ddd, J¼9.2, 10.9,

12.9 Hz, 2-Hb), 3.36 (1H, t, J¼10.9 Hz, 5-H), 4.56 (1H, dd, J¼9.5,

10.9 Hz, 6-H), 4.69 (1H, dd, J¼4.8, 10.9 Hz,1-H), 4.79 (1H, t, J¼9.2 Hz,

3-H), 5.09 and 5.17 (2H, 2d, 1:1, J¼12.6 Hz, CH₂Ph), 6.85 (1H, d,

J¼9.5 Hz, NH), 7.28e7.38 (6H, m, Ph and p-C₆H₃), 7.43e7.49 (2H, m,

m-C₆H₃); d_C(126 MHz, CDCl₃) 19.5, 20.8, 28.1, 28.1, 36.8, 54.0, 55.2,

59.3, 67.5, 68.3, 82.5, 128.2, 128.3, 128.6, 128.9, 129.6, 131.0, 135.6,

136.1, 156.2, 168.8, 176.5; HRMS (ESI): MNa^þ, found 584.1695.

C₂₈H₃₃Cl₂N₃NaO₅requires 584.1689.

914, 901, 847, 791, 757, 717, 726, 696, 667 cm^ꢁ1

; d_H(500 MHz,

CDCl₃) 0.91 and 1.09 (6H, 2d, 1:1, J¼6.8 Hz, Me₂CH), 1.49 (9H, s, t-

Bu), 1.82 (3H, s, 1-CH₃), 1.97e2.05 (1H, m, Me₂CH), 2.48 (1H, dd,

J¼7.7, 12.9 Hz, 2-Ha), 3.02 (1H, br t, J¼2.3 Hz, 5-H), 3.16 (1H, dd,

J¼9.7, 12.9 Hz, 2-Hb), 4.61 (1H, dd, J¼2.3, 9.2 Hz, 6-H), 5.09 in 5.14

(2H, 2d, J¼12.2 Hz, CH₂Ph), 5.20 (1H, dd, J¼7.7, 9.7 Hz, 3-H), 5.47

(1H, d, J¼9.2 Hz, NH), 7.19 (1H, t, J¼8.0 Hz, p-C₆H₃), 7.29e7.38 (7H,

m, Ph and m-C₆H₃); d_C(126 MHz, CDCl₃) 18.0, 20.3, 22.4, 27.3, 38.1,

45.8, 55.1, 59.3, 61.4, 67.0, 67.2, 82.7, 128.3, 128.3, 128.7, 129.1, 129.9,

131.3, 135.8, 136.3, 155.9, 161.9, 170.1; HRMS (ESI): MH^þ, found

576.2054. C₂₉H₃₆Cl₂N₃O₅requires 576.2027.

4.4.5. Synthesis of cycloadducts 13c and 14c. Prepared from 5c

(3.65 g, 10 mmol), tert-butyl methacrylate (6b)(1.96 mL, 12 mmol),

and anisole (40 mL), 7 h, MPLC (EtOAc/hexanes, 1:10).

4.4.6. General procedure for acidolytic deprotection of carboxy

function. Synthesis of carboxylic acids 15a,c, 16b, 17b,d, 18c, 19d and

31. A mixture of tert-butyl ester (0.5 mmol), dichloromethane

(2 mL), and triﬂuoroacetic acid (1 mL) was stirred at room tem-

perature for 24 h. Volatile components were evaporated in vacuo

and the residue was triturated with ether (5 mL). The precipitate

was collected by ﬁltration and washed with ether to give the cor-

responding C-unprotected peptide (carboxylic acid).

4.4.5.1. tert-Butyl (1R*,3R*,5R*,6R*)-6-(benzyloxycarbonylamino)-

5-isopropyl-1-methyl-7-oxo-3-phenylhexahydropyrazolo[1,2-a]pyr-

azole-1-carboxylate (13c). Yield: 1.35 g (35%) of white crystals; mp

133e136 ^ꢃC; [Found: C, 68.68; H, 7.53; N, 8.28. C₂₉H₃₇N₃O₅requires

C, 68.62; H, 7.35; N, 8.28%]; n_max(KBr) 3418, 2974, 2930, 1682, 1651,

1535, 1455, 1300, 1258, 1147, 1089, 1048, 881, 805, 750, 698, 668,

The following compounds were prepared in this manner.

628 cm^ꢁ1

;

d_H(500 MHz, CDCl₃) 0.75 and 0.86 (6H, 2d, 1:1, J¼6.7 Hz,

Me₂CH), 1.32e1.40 (1H, m, Me₂CH), 1.52 (9H, s, t-Bu), 1.68 (3H, s,

CH₃), 2.30 (1H, dd, J¼11.3,13.2 Hz, 2-Ha), 2.66 (1H, dd, J¼5.7,13.2 Hz,

2-Hb), 3.07 (1H, dd, J¼2.9, 11.9 Hz, 5-H), 3.91 (1H, dd, J¼5.7, 11.3 Hz,

3-H), 4.85 (1H, dd, J¼11.9, 9.3 Hz, 6-H), 5.09 and 5.13 (2H, 2d, 1:1,

J¼12.3 Hz, CH₂Ph), 5.11 (1H, d, J¼9.3 Hz, NH), 7.27e7.41 (10H, m, 2ꢂ

Ph); d_C(126 MHz, CDCl₃) 16.4, 18.5, 22.5, 27.4, 28.1, 52.9, 56.0, 62.6,

67.3, 70.1, 79.8, 82.9, 127.5, 128.3, 128.3, 128.6, 128.7, 128.8, 136.4,

138.6, 156.1, 163.3, 170.5; HRMS (ESI): MH^þ, found 508.2808.

C₂₉H₃₈N₃O₅requires 508.2806.

4.4.6.1. (1R*,3R*,5R*,6R*)-6-(Benzyloxycarbonylamino)-7-oxo-

3,5-diphenylhexahydropyrazolo[1,2-a]pyrazole-1-carboxylic

acid

(15a). Prepared from 8a (0.298 g, 0.5 mmol), trituration with ether.

Yield: 0.235 g (100%) of white solid; mp 175e179 ^ꢃC; [Found: C,

68.56; H, 5.54; N, 8.67. C₂₇H₂₅N₃O₅requires C, 68.78; H, 5.34; N,

8.91%]; n_max(KBr) 3331, 3064, 3035, 2893, 2619, 2502, 1751, 1734,

1653, 1537, 1496, 1457, 1449, 1292, 1273, 1261, 1242, 1134, 1066, 751,

696 cm^ꢁ1

;

d_H(500 MHz, CDCl₃) 2.57 (1H, dd, J¼1.0, 5.4, 13.1 Hz, 2-

Ha), 2.73 (1H, ddd, J¼9.4, 11.4, 13.1 Hz, 2-Hb), 3.92 (1H, dd, J¼5.4,

11.4 Hz, 3-H), 4.15 (1H, d, J¼12.7 Hz, 5-H), 4.40 (1H, d, J¼9.4 Hz, 1-

H), 4.70 (1H, dd, J¼ 9.5, 12.7 Hz, 6-H), 4.90 and 4.97 (2H, 2d, 1:1,

J¼12.7 Hz, CH₂Ph), 6.98e7.34 (15H, m, 3ꢂPh), 8.04 (1H, d, J¼9.5 Hz,

NH), 13.25 (1H, br s, COOH); d_C(126 MHz, CDCl₃) 42.4, 53.5, 63.0,

65.5, 68.4, 75.1, 127.6, 127.6, 127.6, 127.7, 127.7, 127.8, 127.8, 127.8,

128.3, 135.9, 136.7, 136.8, 155.9, 161.8, 170.6; HRMS (ESI): MH^ꢁ,

found 470.1717. C₂₇H₂₄N₃O₅requires 470.1716.

4.4.5.2. tert-Butyl (1R*,3S*,5R*,6R*)-6-(benzyloxycarbonylamino)-

5-isopropyl-1-methyl-7-oxo-3-phenylhexahydropyrazolo[1,2-a]pyr-

azole-1-carboxylate (14c). Yield: 0.45 g (9%) of white crystals; mp

95e98 ^ꢃC; [Found: C, 68.24; H, 7.62; N, 8.06. C₂₉H₃₇N₃O₅$¹/₃H₂O re-

quires C, 67.82; H, 7.39; N, 8.18%]; n_max(KBr) 3442, 3240, 3029, 2973,

1731, 1681, 1555, 1537, 1492, 1455, 1392, 1370, 1314, 1269, 1171, 1102,

1037, 985, 927, 853, 819, 743, 702 cm^ꢁ1

; d_H(500 MHz, CDCl₃) 0.80 and

0.98(6H, 2d,1:1, J¼6.8Hz, Me₂CH),1.52(9H, s, t-Bu),1.70 (3H, s,1-Me),

1.80e1.89 (1H, m, Me₂CH), 2.65 (1H, dd, J¼3.5, 8.1 Hz, 5-H), 2.70 (1H,

dd, J¼4.2,13.5 Hz, 2-Ha), 2.78 (1H, dd, J¼8.1, 13.5 Hz, 2-Hb), 4.39 (1H,

dd, J¼4.2, 8.1 Hz, 3-H), 4.63 (1H, t, J¼8.7 Hz, 6-H), 5.07 (2H, s, CH₂Ph),

5.13 (1H, d, J¼9.3 Hz, NH), 7.27e7.39 (8H, m, 8H of Ph), 7.44 (2H, d,

J¼7.2 Hz, 2H of Ph); d_C(126 MHz, CDCl₃) 18.0, 19.0, 22.9, 27.9, 28.0,

50.8, 58.6, 60.8, 61.3, 67.2, 67.4, 83.0, 128.3, 128.3, 128.4, 128.6, 128.7,

128.8, 136.3, 138.2, 155.9, 160.5, 169.6; HRMS (ESI): MH^þ, found

508.2799. C₂₉H₃₈N₃O₅requires 508.2806.

4.4.6.2. (1R*,3R*,5R*,6R*)-6-(Benzyloxycarbonylamino)-5-isopro-

pyl-7-oxo-3-phenylhexahydropyrazolo[1,2-a]pyrazole-1-carboxylic

acid (15c). Prepared from 8c (0.335 g, 0.68 mmol), trituration with

diisopropyl ether. Yield: 0.221

g (74%) of white solid; mp

197e200 ^ꢃC; [Found: C, 65.69; H, 6.15; N, 9.30. C₂₄H₂₇N₃O₅requires

C, 65.89; H, 6.22; N, 9.60%]; n_max(KBr) 3461, 2972, 1748, 1718, 1653,

1545, 1496, 1457, 1292, 1248, 1141, 1056, 765, 746, 693 cm^ꢁ1

; d_H

(500 MHz, CDCl₃) 0.75 and 0.87 (6H, 2d, 1:1, J¼6.8 Hz, Me₂CH),

1.34e1.41 (1H, m, CHMe₂), 2.62e2.72 (2H, m, 2-CH₂), 3.25 (1H, dd,

J¼2.8, 12.4 Hz, 5-H), 4.06 (1H, t, J¼8.4, 3-H), 4.41(1H, br t, J¼5.1 Hz,

1-H), 5.10 and 5.15 (2H, 2d, 1:1, J¼12.7 Hz, CH₂Ph), 5.12 (1H, m, 6-H,

overlapped by the signal for CH₂Ph), 6.65 (1H, d, J¼9.6 Hz, NH),

4.4.5.3. Synthesis of tert-butyl (1R*,3S*,5R*,6R*)-6-(benzyloxy-

carbonylamino)-3-(2,6-dichlorophenyl)-5-isopropyl-1-methyl-7-

A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

6659

6.64e7.38 (10H, m, 2ꢂ Ph), 7.78 (1H, br s, COOH); d_C(126 MHz,

CDCl₃) 16.3, 18.2, 27.1, 43.2, 55.3, 56.4, 66.9, 69.2, 80.8, 127.5, 127.7,

128.0, 128.5, 128.8, 129.0, 136.7, 137.6, 156.5, 165.4, 170.7; HRMS

(ESI): MH^þ, found 438.2022. C₂₄H₂₈N₃O₅requires 438.2023.

2635, 1746, 1731, 1659, 1651, 1538, 1495, 1469, 1455, 1437, 1377, 1322,

1296,1248,1208,1179,1147,1110,1061,1021,1000, 967, 943, 858, 836,

814, 782, 749, 739, 669, 629 cm^ꢁ1

; d_H(500 MHz, DMSO-d₆) 0.62 and

0.71 (6H, 2d, 1:1, J¼7.0 Hz, Me₂CH), 1.15e1.25 (1H, m, Me₂CH), 1.54

(3H, s,1-Me), 2.26 (1H, dd, J¼10.9,13.2 Hz, 2-Ha), 2.78 (1H, dd, J¼5.8,

13.2 Hz, 2-Hb), 3.17 (1H, dd, J¼3.0, 12.1 Hz, 5-H), 3.94 (1H, dd, J¼5.8,

10.9 Hz, 3-H), 4.53 (1H, dd, J¼9.6, 12.1 Hz, 6-H), 5.06 (2H, s, CH₂Ph),

7.29e7.38 (8H, m, 8H of Ph), 7.46 (2H, d, J¼7.3 Hz, 2H of Ph), 7.94 (1H,

d, J¼9.6 Hz, NH), 13.13 (1H, s, COOH); d_C(126 MHz, DMSO-d₆) 16.1,

18.4, 22.3, 26.8, 51.8, 55.1, 60.1, 65.5, 69.5, 75.1, 127.58, 127.63, 127.8,

128.3, 128.4, 128.5, 137.1, 138.5, 155.8, 161.6, 172.1; HRMS (ESI): MH^-,

found 450.2035. C₂₅H₂₈N₃O₅requires 450.2034.

4.4.6.3. (1S*,3S*,5R*,6R*)-6-(Benzyloxycarbonylamino)-3-(2,6-

dichlorophenyl)-7-oxo-5-phenyl-hexahydropyrazolo[1,2-a]pyrazole-

1-carboxylic acid (16b). Prepared from 9b (0.298 g, 0.5 mmol),

trituration with diisopropyl ether. Yield: 0.269 g (100%) of yellow-

ish solid; mp 116e120 ^ꢃC; [Found: C, 59.74; H, 3.96; N, 7.89.

C₂₇H₂₃Cl₂N₃O₅requires C, 60.01; H, 4.29; N, 7.78%]; n_max(KBr) 3481,

3302, 3067, 2953, 1722 (C]O), 1683 (C]O), 1578, 1555, 1456, 1439,

1344, 1288, 1253, 1233, 1210, 1160, 1058, 1004, 786, 779, 762,

697 cm^ꢁ1

;

d_H(500 MHz, DMSO-d₆) 2.96 (1H, ddd, J¼4.1, 9.8,14.2 Hz,

4.4.6.7. (1R*,3S*,5R*,6R*)-6-(Benzyloxycarbonylamino)-5-

isopropyl-1-methyl-7-oxo-3-phenylhexahydropyrazolo[1,2-a]pyr-

azole-1-carboxylic acid (19d). Prepared from 14d (2.88 g, 5 mmol),

trituration with ether. Yield: 3.52 g (67%) of white crystals; mp

159e160 ^ꢃC; n_max(KBr) 3551, 3447, 3262, 3067, 2963, 2933, 2878,

1731,1698,1633,1581,1548,1497,1438,1379,1336,1304,1258,1222,

1190, 1175, 1148, 1133, 1065, 1046, 970, 933, 874, 804, 776, 761, 730,

2-Ha), 3.21 (1H, ddd, J¼4.3, 10.4, 14.2 Hz, 2-Hb), 3.82 (1H, d,

J¼12.1 Hz, 5-H), 4.53 (1H, dd, J¼9.2, 12.1 Hz, 6-H), 4.85 (1H, dd,

J¼4.1, 10.4 Hz, 3-H), 4.91 and 4.96 (2H, 2d, 1:1, J¼12.5 Hz, CH₂Ph),

5.29 (1H, dd, J¼4.3, 9.8 Hz, 1-H), 6.99e7.63 (13H, m, 2ꢂ Ph and

C₆H₃), 8.03 (1H, d, J¼9.2 Hz, NH), 13.27 (1H, br s, COOH); d_C

(126 MHz, DMSO-d₆) 35.1, 52.8, 57.6, 62.9, 65.5, 67.1, 127.4, 127.6,

127.8, 128.2, 128.3, 128.3, 128.9, 130.5, 130.8, 131.6, 134.7, 136.7,

155.9, 160.4, 171.0; HRMS (ESI): MH^þ, found 540.1084.

C₂₇H₂₄Cl₂N₃O₅requires 540.1093.

719, 691, 659 cm^ꢁ1

; d_H(500 MHz, CDCl₃) 0.75 and 1.01 (6H, 2d, 1:1,

J¼6.9 Hz, Me₂CH), 1.74e1.81 (1H, m, Me₂CH), 1.85 (3H, s, 1-Me), 2.79

(1H, dd, J¼3.5,10.1 Hz, 5-H), 2.91 (1H, dd, J¼10.1,14.5 Hz, 2-Ha), 3.37

(1H, dd, J¼4.9, 14.5 Hz, 2-Hb), 5.05 (1H, t, J¼10.1 Hz, 6-H), 5.10 and

5.15 (2H, 2d,1:1, J¼12.1 Hz, CH₂Ph), 5.58 (1H, dd, J¼4.9,10.1 Hz, 3-H),

7.24 (1H, t, J¼8.0 Hz, p-C₆H₃), 7.29e7.42 (7H, m, Ph and p-C₆H₃), NH

and COOH exchanged; d_C(126 MHz, CDCl₃) 16.8,18.5, 25.3, 28.1, 44.8,

55.5, 57.4, 65.9, 67.6, 69.1, 128.2, 128.4, 128.6, 129.7, 130.5, 130.7,

132.0, 134.1, 135.7, 137.4, 155.7, 161.8, 171.5; HRMS (ESI): MH^þ, found

520.1428. C₂₅H₂₈Cl₂N₃O₅requires 520.1401.

4.4.6.4. (2S*,3S*,5R*,6R*)-6-(Benzyloxycarbonylamino)-3-(2,6-

dichlorophenyl)-7-oxo-5-phenylhexahydropyrazolo[1,2-a]pyrazole-

2-carboxylic acid (17b). Prepared from 11b (4.47 g, 7.5 mmol),

trituration with ether. Yield: 3.43 g (84%) of white solid; mp

185e192 ^ꢃC; [Found: C, 59.83; H, 4.25; N, 7.81. C₂₇H₂₃Cl₂N₃O₅re-

quires C, 60.01; H, 4.29; N, 7.78%]; n_max(KBr) 3248, 3039, 1729 (C]

O), 1686 (C]O), 1561, 1537, 1486, 1456, 1437, 1425, 1321, 1278, 1243,

1225, 1187, 1052, 1031, 784, 698, 480 cm^ꢁ1

;

d_H(500 MHz, DMSO-d₆)

4.4.6.8. (1R*,3R*,5R*,6R*)-6-(2-(2-(Benzyloxycarbonylamino)

acetamido)acetamido)-5-isopropyl-1-methyl-7-oxo-3-

3.49 (1H, ddd, J¼3.6, 7.5, 9.3 Hz, 2-H), 3.71 (1H, dd, J¼9.3, 11.7 Hz, 1-

Ha), 4.07 (1H, dd, J¼3.6, 11.7 Hz, 1-Hb), 4.68 (1H, d, J¼7.5 Hz, 3-H),

4.83 (1H, d, J¼12.1 Hz, 5-H), 5.02 (1H, dd, J¼9.0, 12.1 Hz, 6-H), 5.06

(2H, s, CH₂Ph), 7.48e6.72 (13H, m, 2ꢂ Ph and C₆H₃), 7.96 (1H, d,

J¼9.0 Hz, NH), 12.90 (1H, s, COOH); d_C(126 MHz, DMSO-d₆) 45.8,

51.1, 51.3, 61.8, 62.3, 66.0, 127.7, 127.8, 127.9, 128.0, 128.4, 129.7,

130.2, 131.3, 132.5, 134.2, 135.3, 136.6, 156.1, 173.5, 175.2; HRMS

(ESI): MH^þ, found 540.1072. C₂₇H₂₄Cl₂N₃O₅requires 540.1093.

phenylhexahydropyrazolo[1,2-a]pyrazole-1-carboxylic

acid

(31). Prepared from the ester 30 (311 mg, 0.5 mmol). Yield: 65 mg

(23%) of white crystals; mp 128e129 ^ꢃC; [Found: C, 57.58; H, 6.20; N,

11.52. C₂₉H₃₅N₅O₇$2H₂O requires C, 57.89; H, 6.53; N, 11.64%]; n_max

(KBr) 3459, 3069, 2964,1674,1542,1457,1439,1374,1264,1217,1150,

1087, 1051, 1029, 990, 974, 909, 858, 745, 699, 675, 653, 633,

609 cm^ꢁ1

;

d_H(500 MHz, CDCl₃) 0.68 and 0.83 (6H, 2d, 1:1, J¼6.8 Hz,

Me₂CH), 1.25e1.31 (1H, m, Me₂CH), 1.70 (3H, s, CH₃), 2.30 (1H, br dd,

J¼11.5,13.2 Hz, 2-Ha), 2.87 (1H, br dd, J¼5.8,13.2 Hz, 2-Hb), 3.35 (1H,

br d, J¼12.0 Hz, 5-H), 3.84e4.12 (5H, m, 3-H and 2ꢂCH₂NH), 5.05 and

5.13 (2H, 2d, 1:1, J¼12.4 Hz, CH₂Ph), 5.10e5.18 (1H, m, 6-H, over-

lapped by the signals for CH₂Ph), 6.19 (1H, br s, NHCH₂), 7.24e7.38

(10H, m, 2ꢂ Ph), 7.76 (1H, br s, NHCH₂), 8.16 (1H, br d, J¼6.0 Hz,

NHCH), COOH exchanged; d_C(126 MHz, CDCl₃) 16.3, 18.6, 22.5, 27.5,

42.8, 44.6, 52.5, 54.2, 61.7, 67.3, 70.3, 77.4, 127.6, 128.1, 128.3, 128.7,

128.8, 128.9, 136.4,137.8, 157.3,162.9, 170.2, 171.1, 173.0; HRMS (ESI):

MH^þ, found 566.2609. C₂₉H₃₆N₅O₇requires 566.2609.

4.4.6.5. (2S*,3S*,5R*,6R*)-6-(Benzyloxycarbonylamino)-3-(2,6-

dichlorophenyl)-5-isopropyl-7-oxohexahydropyrazolo[1,2-a]pyr-

azole-2-carboxylic acid (17d). Prepared from 11d (510 mg,

0.9 mmol), trituration with diisopropyl ether. Yield: 347 mg (76%)

of white solid; mp 181e185 ^ꢃC; [Found: C, 56.86, H, 4.90; N, 8.17.

C₂₄H₂₅Cl₂N₃O₅requires C, 56.93; H, 4.98; N, 8.30%]; n_max(KBr) 3414,

3302, 3065, 3034, 2966, 2933, 2878, 1741, 1720, 1661, 1654, 1579,

1561,1533,1498,1456,1441,1391,1295,1266,1245,1212,1151,1086,

1055, 1028, 778, 700, 641 cm^ꢁ1

; d_H(500 MHz, CDCl₃) 0.70 and 0.94

(6H, 2d, 1:1, J¼6.8 Hz, Me₂CH), 1.76e1.90 (1H, m, CHMe₂), 2.84 (1H,

dd, J¼6.2, 9.9 Hz, 5-H), 3.81 (1H, dd, J¼6.4, 11.8 Hz, 1-Ha), 3.89 (1H,

td, J¼6.1, 9.5 Hz, 2-H), 4.22 (1H, dd, J¼9.5, 11.8 Hz, 1-Hb), 4.82 (1H, t,

J¼9.9 Hz, 6-H), 5.07 and 5.14 (2H, 2d, 1:1, J¼12.1 Hz, CH₂Ph), 5.29

(1H, d, J¼9.9 Hz, NH), 5.66 (1H, d, J¼5.8 Hz, 3-H), 6.07 (1H, br s,

COOH), 7.13e7.47 (8H, m, Ph and C₆H₃); d_C(126 MHz, CDCl₃) 18.5,

19.2, 29.9, 43.2, 49.7, 56.5, 62.6, 67.6, 69.8, 128.4, 128.5, 128.7, 129.9,

130.6, 130.7, 134.7, 137.9, 156.1, 166.7, 174.5; HRMS (ESI): MNH^þ₄,

found 523.1504. C₂₄H₂₉Cl₂N₄O₅requires 523.151.

4.4.7. General procedure for hydrogenolytic deprotection of amino

function. Synthesis of amines 20a,c, 21b,d, 22c, 23d and 26. A

mixture of a Cbz-protected peptide (1 mmol), ethanol (40 mL), and

10% PdeC (30 mg) was hydrogenated (3.5 bar of H₂) at rt for 3e6 h.

The catalyst was removed by ﬁltration through a fritted funnel,

washed with ethanol (2ꢂ5 mL), and the combined ﬁltrate was

evaporated in vacuo. The residue was puriﬁed by FC (EtOAc).

Fractions containing the product were combined and evaporated in

vacuo to give the N-unprotected peptide (amine).

4.4.6.6. (1R*,3R*,5R*,6R*)-6-(Benzyloxycarbonylamino)-5-

isopropyl-1-methyl-7-oxo-3-phenylhexahydropyrazolo[1,2-a]pyr-

azole-1-carboxylic acid (18c). Prepared from 13c (2.58 g, 5 mmol),

trituration with ether. Yield: 3.39 g (75%) of white crystals; mp

204e205 ^ꢃC; [Found: C, 66.06; H, 6.54; N, 9.34. C₂₅H₂₉N₃O₅$¹/₁₀H₂O

requires C, 66.24; H, 6.49; N, 9.27%]; n_max(KBr) 3297, 3054, 2938,

The following compounds were prepared in this manner.

4.4.7.1. tert-Butyl (1R*,3R*,5R*,6R*)-6-amino-7-oxo-3,5-diphenyl-

hexahydropyrazolo[1,2-a]pyrazole-1-carboxylate

(20a). Prepared

from 8a (0.298 g, 0.5 mmol). Yield: 40 mg (20%) of white solid; mp

114e119 ^ꢃC; [Found: C, 69.61; H, 6.77; N, 10.26. C₂₃H₂₇N₃O₃$¼H₂O

6660

A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

requires C, 69.41; H, 6.96; N, 10.56%]; n_max(KBr) 3440, 3372, 2980,

J¼11.0 Hz, 6-H), 3.92 (1H, dd, J¼5.8, 11.2 Hz, 3-H), 7.28e7.37 (3H, m,

o,p-Ph), 7.37e7.41 (2H, m, m-Ph); d_C(126 MHz, CDCl₃) 16.7, 19.3, 22.5,

27.7, 28.0, 52.9, 56.9, 61.8, 70.4, 80.5, 82.5, 127.5, 128.3, 128.6, 138.5,

166.7, 170.4; HRMS (ESI): MH^þ, found 374.2439. C₂₁H₃₂N₃O₃requires

374.2438.

1728, 1704, 1456, 1426, 1376, 1366, 1358, 1229, 1152, 1140, 1125,

696 cm^ꢁ1

; d_H(500 MHz, CDCl₃) 1.56 (9H, s, t-Bu), 1.61 (2H, s, NH₂),

2.52 (1H, ddd, J¼1.2, 5.5, 13.2 Hz, 2-Ha), 2.78 (1H, ddd, J¼9.2, 11.6,

13.2 Hz, 2-Hb), 3.87 (1H, d, J¼11.8 Hz, 5-H), 3.96 (1H, dd, J¼5.5,

11.6 Hz, 3-H), 4.00 (1H, br d, J¼11.8 Hz, 6-H), 4.41 (1H, br d, J¼9.2 Hz,

1-H), 7.00e7.06, 7.10e7.14, and 7.16e7.19 (10H, 3m, 6:2:2, 2ꢂ Ph); d_C

(126 MHz, CDCl₃) 28.2, 43.1, 55.0, 65.0, 69.0, 81.1, 83.2, 127.8, 127.9,

128.0,128.1,128.1,128.1, 136.4,136.5,166.4,169.0; HRMS (ESI): MH^þ,

found 394.2131. C₂₃H₂₈N₃O₃requires 394.2125.

4.4.7.6. tert-Butyl (1R*,3S*,5R*,6R*)-6-amino-3-(2,6-dichloro-

phenyl)-5-isopropyl-1-methyl-7-oxohexahydropyrazolo[1,2-a]pyr-

azole-1-carboxylate (23d). Prepared from 14d (2.88 g, 5 mmol).

Yield: 0.7 g (93%) of red crystals; mp 109e112 ^ꢃC; [Found: C, 53.73;

H, 6.87; N, 8.95. C₂₁H₂₉Cl₂N₃O₃$1½H₂O requires C, 53.38; H, 6.71; N,

8.93%]; n_max(KBr) 3448, 2969, 2930, 1733, 1699, 1560, 1522, 1454,

1438, 1394, 1369, 1287, 1257, 1204, 1150, 1124, 1085, 930, 845, 788,

757, 701, 648, 618 cm^ꢁ1; NMR (500 MHz, CDCl₃) 0.95 and 1.11 (6H,

2d, 1:1, J¼6.6 Hz, Me₂CH), 1.28 (1H, d, J¼8.6 Hz, Me₂CH), 1.51 (9H, s,

t-Bu), 1.84 (3H, s, 1-Me), 2.08 (2H, br s, NH₂), 2.53 (1H, dd, J¼7.7,

12.9 Hz, 2-Ha), 3.20 (1H, dd, J¼9.9, 12.9 Hz, 2-Hb), 3.42 (1H, br s, 5-

H), 4.20 (1H, br s, 6-H), 5.22 (1H, br t, J¼8.7 Hz, 3-H), 7.21 (1H, t,

J¼8.0 Hz, p-C₆H₃), 7.34 (2H, br t, J¼7.8 Hz, m-C₆H₃); d_C(126 MHz,

CDCl₃) 18.1, 20.3, 22.3, 27.3, 28.1, 45.8, 54.9, 58.6, 61.7, 65.0, 83.3,

129.0, 130.1, 131.5, 136.1, 161.9, 170.4; HRMS (ESI): MH^þ, found

442.1677. C₂₁H₃₀Cl₂N₃O₃requires 442.1659.

4.4.7.2. tert-Butyl (1R*,3R*,5R*,6R*)-6-amino-5-isopropyl-7-oxo-

3-phenylhexahydropyrazolo[1,2-a]pyrazole-1-carboxylate

(20c).

Prepared from 8c (0.300 g, 0.61 mmol). Yield: 0.134 g (62%) of

purplish oil; [Found: C, 66.67; H, 8.08; N,11.55. C₂₀H₂₉N₃O₃requires

C, 66.83; H, 8.13; N, 11.69%]; n_max(liquid ﬁlm) 3462, 2980, 2960,

1733, 1686, 1457, 1420, 1368, 1225, 1155, 1106, 852, 745, 698 cm^ꢁ1

;

d_H(500 MHz, CDCl₃) 0.77 and 0.85 (6H, 2d, 1:1, J¼7.0 Hz, Me₂CH),

1.39 (1H, doublet of quintet, J¼3.1, 6.9 Hz, Me₂CH), 1.53 (9H, s, t-Bu),

1.62 (2H, br s, NH₂), 2.50 (1H, ddd, J¼1.2, 5.5, 13.2 Hz, 2-Ha), 2.66

(1H, ddd, J¼9.3, 11.4, 13.2 Hz, 2-Hb), 2.98 (1H, dd, J¼3.1, 11.3 Hz, 5-

H), 3.83 (1H, br d, J¼11.3 Hz, 6-H), 3.88 (1H, dd, J¼5.5, 11.4 Hz, 3-H),

4.28 (1H, br d, J¼9.3 Hz, 1-H), 7.30e7.41 (5H, m, Ph); d_C(126 MHz,

CDCl₃) 16.8, 19.4, 27.7, 28.1, 44.1, 55.0, 57.4, 69.2, 80.8, 83.0, 127.6,

128.6, 128.8, 138.3, 167.9, 169.0; HRMS (ESI): MH^þ, found 360.2283.

C₂₀H₃₀N₃O₃requires 360.2282.

4.4.7.7. Methyl 2-((1R*,3R*,5R*,6R*)-6-amino-5-isopropyl-1-

methyl-7-oxo-3-phenylhexahydropyrazolo[1,2-a]pyrazole-1-carboxa-

mido)acetate (26). Prepared from N-protected tripeptide 25

(627 mg, 1.19 mmol). Yield: 462 mg (100%) of red oil; n_max(liquid

ﬁlm) 3488, 3416, 3363, 2956, 2873, 1750, 1674, 1525, 1438, 1369,

4.4.7.3. tert-Butyl (2S*,3S*,5R*,6R*)-6-amino-3-(2,6-dichloro-

phenyl)-7-oxo-5-phenylhexahydropyrazolo[1,2-a]pyrazole-2-

carboxylate (21b). Prepared from 11b (4.77 g, 8 mmol). Yield: 2.43 g

(66%) of white solid; mp 138e143 ^ꢃC; n_max(KBr) 3397, 3331, 3067,

3003, 2978, 2956, 2932, 2886, 1734(C]O), 1713, 1582, 1561, 1452,

1440, 1380, 1367, 1353, 1324, 1267, 1239, 1153, 1086, 839, 786, 772,

1307, 1208, 1145, 1021, 976, 902, 873, 748, 701, 671, 656 cm^ꢁ1

; d_H

(500 MHz, CDCl₃) 0.77 and 0.84 (6H, 2d, 1:1, J¼7.0 Hz, Me₂CH), 1.29

(1H, doublet of quintet, J¼2.8, 7.0 Hz, Me₂CH), 1.66 (2H, br s, NH₂),

1.80 (3H, s, CH₃), 2.22 (1H, dd, J¼11.2, 12.7 Hz, 2-Ha), 2.85 (1H, dd,

J¼2.8, 11.3 Hz, 5-H), 3.48 (1H, dd, J¼5.1, 12.7 Hz, 2-Hb), 3.60 (1H, dd,

J¼5.1, 11.2 Hz, 3-H), 3.76 (3H, s, OMe), 3.86 (1H, d, J¼11.3 Hz, 6-H),

3.98 (1H, dd, J¼5.117.9 Hz,1H of CH₂NH), 4.15 (1H, dd, J¼6.2,17.9 Hz,

1H of CH₂NH), 7.28e7.41 (5H, m, Ph), 8.95 (1H, t, J¼5.1 Hz, NHCH₂);

d_C(126 MHz, CDCl₃) 16.7, 19.7, 23.7, 26.8, 41.8, 51.2, 52.5, 58.0, 64.9,

69.0, 79.3, 127.7, 128.6, 128.7, 138.0, 167.3, 170.4, 172.7; HRMS (ESI):

MH^þ, found 389.2187. C₂₀H₂₉N₄O₄requires 389.2183.

757, 703 cm^ꢁ1

; d_H(500 MHz, CDCl₃) 1.40 (9H, s, t-Bu), 1.67 (2H, br s,

NH₂), 3.76 (1H, td, J¼6.6, 7.8 Hz, 2-H), 4.02 (2H, d, J¼7.8 Hz, 1-CH₂),

4.12 (1H, d, J¼11.1 Hz, 6-H), 4.23 (1H, d, J¼11.1 Hz, 5-H), 5.09 (1H, d,

J¼6.6 Hz, 3-H), 6.90e7.37 (8H, m, Ph and C₆H₃); d_C(126 MHz, CDCl₃)

27.9, 44.5, 51.6, 57.5, 61.9, 68.9, 82.4, 127.9, 128.3, 128.3, 128.6, 129.5,

130.2, 132.8, 135.0, 171.0, 172.7; HRMS (ESI): MH^þ, found 462.1355.

C₂₃H₂₆Cl₂N₃O₃requires 462.1351.

4.4.8. General procedure for the synthesis of the racemic tripeptide

25, tetrapeptides 28 and 30, and hexapeptide 33. 4-

Methylmorpholine (0.11 mL, 1 mmol) and EEDQ (247 mg, 1 mmol)

were added to a stirred mixture of carboxylic acid (1 mmol), amine

hydrochloride (1 mmol), and anhydrous dichloromethane (5 mL)

and the mixture was stirred at room temperature for 24 h (Addition

of 4-methylmorpholine was omitted when free amine was used.)

The volatile components were evaporated in vacuo. The residue was

dissolved in EtOAc (5 mL) and then washed, subsequently, with 1 M

aq NaHSO₄(5 mL) and aq NaHCO₃(5 mL), dried over Na₂SO₄, ﬁltered,

and the ﬁltrate vas evaporated in vacuo. The residue was puriﬁed by

FC (EtOAc). Fractions containing the product were combined and

evaporated in vacuo to give the corresponding carboxamide.

The following compounds were prepared in this manner.

4.4.7.4. tert-Butyl (2S*,3S*,5R*,6R*)-6-amino-3-(2,6-dichlorop-

henyl)-5-isopropyl-7-oxohexahydropyrazolo[1,2-a]pyrazole-2-

carboxylate (21d). Prepared from 11d (558 mg, 1 mmol). Yield:

324 mg (76%) of purple oil; n_max(liquid ﬁlm) 3386, 3307, 2974, 2930,

2873,1730,1682,1580,1561,1458,1438,1390,1368,1272,1256,1215,

1154, 1085, 1034, 840, 783, 772, 740, 666 cm^ꢁ1

; d_H(500 MHz, CDCl₃)

0.71 and 0.98 (6H, 2d, 1:1, J¼6.7 Hz, CHMe₂), 1.46 (9H, s, t-Bu), 1.70

(2H, br s, NH₂),1.81 (1H, sextet, J¼6.7 Hz, CHMe₂), 2.75 (1H, dd, J¼7.5,

9.6 Hz, 5-H), 3.67 (1H, d, J¼9.6 Hz, 6-H), 3.75 (1H, td, J¼5.8, 9.5 Hz, 2-

H), 3.84 (1H, dd, J¼5.8, 11.7 Hz, 1-Ha), 4.10 (1H, dd, J¼9.5, 11.7 Hz, 1-

Hb), 5.42 (1H, d, J¼5.8 Hz, 3-H), 7.24 (1H, t, J¼8.0 Hz, p-C₆H₃), 7.40

(2H, dt, J¼1.0, 7.8 Hz, m-C₆H₃); d_C(126 MHz, CDCl₃) 19.2, 20.0, 28.0,

30.2, 43.6, 51.6, 57.0, 62.8, 71.7, 82.5, 129.5, 130.1, 130.7, 133.6, 170.9,

171.8; HRMS (ESI): MH^þ, found 428.1499. C₂₀H₂₈Cl₂N₃O₃requires

428.1502.

4.4.8.1. Methyl 2-((1R*,3R*,5R*,6R*)-6-(benzyloxycarbonylamino)-

5-isopropyl-1-methyl-7-oxo-3-phenylhexahydropyrazolo[1,2-a]pyr-

azole-1-carboxamido)acetate (25). Prepared from carboxylic acid 18c

(451 mg, 1.0 mmol), methyl glycinate hydrochloride (24)(126 mg,

1.0 mmol), 4-methylmorpholine (0.11 mL, 1 mmol), and EEDQ

(247 mg, 1.0 mmol). Yield: 259 mg (50%) of white crystals; mp

171e173 ^ꢃC; n_max(KBr) 3457, 3362, 3245, 3035, 2934, 2850, 1758,

1687,1672,1628,1537,1496,1457,1435,1402,1388,1366,1275,1202,

1179, 1124, 1093, 1056, 1026, 978, 963, 917, 858, 843, 813, 785, 747,

4.4.7.5. tert-Butyl (1R*,3R*,5R*,6R*)-6-amino-5-isopropyl-1-methyl-

7-oxo-3-phenylhexahydropyrazolo[1,2-a]pyrazole-1-carboxylate

(22c). Prepared from 13c (2.54 g, 5 mmol). Yield: 2.58 g (69%) of

reddish oil; n_max(liquid ﬁlm) 3432, 2970, 2925, 2868, 1734, 1684, 1457,

1434, 1390, 1369, 1301, 1253, 1212, 1158, 1070, 974, 847, 751, 701,

607 cm^ꢁ1; NMR (500 MHz, CDCl₃) 0.78 and 0.84 (6H, 2d, 1:1, J¼6.8 Hz,

Me₂CH), 1.31e1.42 (1H, m, Me₂CH), 1.54 (9H, s, t-Bu), 1.62 (2H, s, NH₂),

1.67 (3H, s, 1-Me), 2.30 (1H, dd, J¼11.2, 13.2 Hz, 2-Ha), 2.69 (1H, dd,

J¼5.8, 13.2 Hz, 2-Hb), 2.93 (1H, dd, J¼3.1, 11.0 Hz, 5-H), 3.77 (1H, d,

700, 664, 626 cm^ꢁ1

J¼6.9 Hz, Me₂CH), 1.23e1.38 (1H, m, Me₂CH), 1.79 (3H, s, CH₃), 2.29

; d_H(500 MHz, CDCl₃) 0.73 and 0.82 (6H, 2d, 1:1,

A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

6661

(1H, dd, J¼11.2,12.8Hz, 2-Ha), 3.24 (1H, dd, J¼5.2,12.8 Hz, 2-Hb), 3.31

(1H, dd, J¼3.1,11.4 Hz, 5-H), 3.75 (3H, s, OMe), 3.78 and 4.29 (2H, 2dd,

1:1, J¼5.9,17.7 Hz, CH₂NH), 3.83 (1H, dd, J¼5.2,11.2 Hz, 3-H), 4.56 (1H,

dd, J¼8.7, 11.4 Hz, 6-H), 5.08 and 5.12 (2H, 2d,1:1, J¼12.3 Hz, CH₂Ph),

5.65 (1H, d, J¼8.7 Hz, NHCH), 7.29e7.41 (10H, m, 2ꢂ Ph), 8.56 (1H, t,

J¼5.6 Hz, NHCH₂); d_C(126 MHz, DMSO-d₆) 16.2, 19.1, 22.9, 27.0, 34.1,

41.7, 52.2, 52.4, 57.3, 67.4, 68.9, 75.8, 127.7, 128.1, 128.4, 128.6, 128.7,

128.8,136.2,137.8,156.2,162.6,170.7,172.3; HRMS (ESI): MH^þ, found

523.2545. C₂₈H₃₅N₄O₆requires 523.2551.

1048, 1029, 980, 778, 749, 700, 662, 639 cm^ꢁ1

;

d_H(500 MHz, CDCl₃)

0.72 and 0.84 (6H, 2d, 1:1, J¼6.8 Hz, Me₂CH), 1.20e1.26 (1H, m,

Me₂CH), 1.75 (3H, s, CH₃), 2.37 (1H, dd, J¼11.0, 13.4 Hz, 2-Ha), 2.89

(1H, dd, J¼6.0, 13.4 Hz, 2-Hb), 3.61 (3H, s, OMe), 3.61e3.77 (3H, m,

CH₂NH and 5-H), 3.81 (1H, br dd, J¼6.2, 17.2 Hz, 1H of CH₂NH), 3.89

(1H, br dd, J¼5.6, 17.2 Hz, 1H of CH₂NH), 3.96 (1H, br dd, J¼5.8,

16.7 Hz, 1H of CH₂NH), 4.00e4.07 (1H, m, 1H of CH₂NH overlapped

by the signal for 3-H), 4.04 (1H, br dd, J¼6.0, 11.0 Hz, 3-H), 4.22 (1H,

br dd, J¼7.3, 9.3 Hz, 6-H), 4.32 (1H, dd, J¼7.1, 17.8 Hz, 1H of CH₂NH),

4.42 (1H, m, J¼8.3, 17.1 Hz, 1H of CH₂NH), 5.06e5.14 (2H, m, CH₂Ph),

6.03 (1H, t, J¼5.0 Hz, NHCH₂), 7.28e7.40 (10H, m, 2ꢂ Ph), 7.51 (1H, t,

J¼6.0 Hz, NHCH₂), 7.82 (1H, d, J¼7.3 Hz, NHCH), 7.99 (1H, t, J¼5.2 Hz,

NHCH₂), 8.68 (1H, dd, J¼3.9, 7.4 Hz, NHCH₂); d_C(126 MHz, CDCl₃)

15.8, 18.9, 23.1, 28.2, 41.0, 42.8, 43.5, 44.9, 52.3, 52.9, 57.2, 62.2, 67.4,

69.5, 71.7, 127.8, 128.2, 128.5, 128.7, 128.9, 128.9, 136.0, 136.9, 157.4,

160.9, 170.3, 170.8, 170.9, 171.1, 171.6; HRMS (ESI): MH^þ, found

694.3191. C₃₄H₄₄N₇O₉requires 694.3195.

4.4.8.2. Methyl 2-((1R*,3R*,5R*,6R*)-6-(2-(tert-butoxycarbonyl-

amino)acetamido)-5-isopropyl-1-methyl-7-oxo-3-phenylhexahydro-

pyrazolo[1,2-a]pyrazole-1-carboxamido)acetate (28). Prepared from

amine 26 (370 mg, 0.95 mmol), Boc-glycine (27)(175 mg, 1 mmol),

4-methylmorpholine (0.11 mL, 1 mmol), and EEDQ (247 mg,

1.0 mmol). Yield: 517 mg (87%) of red crystals; 104e108 ^ꢃC; n_max

(KBr) 3491, 3426, 3069, 2964, 2875, 1755, 1682, 1538, 1456, 1438,

1392, 1367, 1249, 1206, 1172, 1050, 1027, 980, 946, 862, 748, 702,

667, 630 cm^ꢁ1

;

d_H(500 MHz, CDCl₃) 0.70 and 0.81 (6H, 2d, 1:1,

4.4.9. General procedure for the synthesis of non-racemic tripeptides

35, 36, 38e42, 35⁰, 36⁰and 38⁰e42⁰. 4-Methylmorpholine (0.11 mL,

1 mmol) and EEDQ (247 mg,1 mmol) were added to a stirred mixture

of carboxylic acid (1 mmol), amine hydrochloride (1 mmol) and an-

hydrous dichloromethane (5 mL) and the mixture was stirred at

room temperature for 24 h. (Addition of 4-methylmorpholine was

omitted when free amine was used.) The volatile components were

evaporated in vacuo. The residue was puriﬁed by FC. Fractions con-

taining the product were combined and evaporated in vacuo to give

a mixture of diastereomeric carboxamides, which were separated by

MPLC. Fractions containing the products were evaporated invacuo to

give two diastereomeric non-racemic tripeptides.

J¼6.8 Hz, Me₂CH), 1.20e1.28 (1H, m, Me₂CH), 1.43 (9H, s, Boc), 1.79

(3H, s, CH₃), 2.32 (1H, dd, J¼11.2, 13.0 Hz, 2-Ha), 3.17 (1H, dd, J¼5.1,

13.0 Hz, 2-Hb), 3.42 (1H, dd, J¼3.3, 11.2 Hz, 5-H), 3.74 (1H, dd, J¼5.7,

16.8 Hz, 1H of CH₂NH), 3.78 (3H, s, OMe), 3.83 (1H, dd, J¼6.2,

16.8 Hz, 1H of CH₂NH), 3.85 (1H, dd, J¼5.2, 17.4 Hz, 1H of CH₂NH),

3.95 (1H, dd, J¼5.1, 11.2 Hz, 3-H), 4.32 (1H, dd, J¼6.6, 17.4 Hz, 1H of

CH₂NH), 4.61 (1H, br t, J¼9.4 Hz, 6-H), 5.42 (1H, t, J¼5.9 Hz, NHCH₂),

7.29e7.35 (3H, m, o,p-Ph), 7.37e7.41 (2H, m, m-Ph), 7.48 (1H, br d,

J¼6.6 Hz, NHCH), 8.58 (1H, t, J¼5.7 Hz, NHCH₂); d_C(126 MHz, CDCl₃)

16.0, 19.2, 22.7, 27.2, 28.5, 41.8, 44.5, 52.5, 52.9, 56.3, 63.8, 68.8, 74.2,

80.4, 127.7, 128.7, 128.8, 137.6, 156.3, 161.7, 170.6, 170.8, 172.1; HRMS

(ESI): MH^þ, found 546.2917. C₂₇H₄₀N₅O₇requires 546.2922.

The following compounds were prepared in this manner.

4.4.8.3. tert-Butyl (1R*,3R*,5R*,6R*)-6-(2-(2-(benzyloxycarbonyl-

amino)acetamido)acetamido)-5-isopropyl-1-methyl-7-oxo-3-pheny-

4.4.9.1. Synthesis of tripeptides 35a in 35a⁰. Prepared from the

carboxylic acid 15a (0.838 g, 1.55 mmol), methyl (S)-alaninate hy-

drochloride (34)(0.217 g, 1.55 mmol), 4-methylmorpholine

(0.216 mL, 1.55 mmol) and EEDQ (0.915 g, 1.63 mmol); FC

(dichloromethane/methanol, 30:1), MPLC (EtOAc/hexanes, 1:1).

4.4.9.1.1. Methyl (S)-2-((1R,3R,5R,6R)-6-(benzyloxycarbonylam-

ino)-7-oxo-3,5-diphenylhexahydropyrazolo[1,2-a]pyrazole-1-

carboxamido)propanoate (35a). Yield: 368 mg (43%) of white solid;

mp 160e166 ^ꢃC; [Found: C, 66.43; H, 6.07; N, 9.71. C₃₁H₃₂N₄O₆re-

lhexahydropyrazolo[1,2-a]pyrazole-1-carboxylate

(30). Prepared

from amine 22c (374 mg, 1 mmol), N-(Z-glycyl)glycine (29)(266 mg,

1 mmol), 4-methylmorpholine (0.11 mL, 1 mmol), and EEDQ

(246 mg, 1 mmol). Yield: 466 mg (74%) of pink crystals; mp

90e94 ^ꢃC; [Found: C, 63.05; H, 7.23; N, 10.73. C₃₃H₄₃N₅O₇$½H₂O

requires C, 62.84; H, 7.03; N, 11.10%]; n_max(KBr) 3432, 3067, 2973,

2925, 2873,1731, 1693, 1681, 1651, 1537, 1455, 1393, 1369, 1257, 1148,

1085, 1048, 1028, 988, 915, 885, 844, 745, 698, 651, 611 cm^ꢁ1

;

d_H

quires C, 66.89; H, 5.79; N, 10.07%]; [

(KBr) 3316, 3064, 3036, 2950, 1737, 1715, 1687, 1681, 1548, 1541,

1454, 1437, 1243, 1217, 1156, 1058, 753, 698 cm^ꢁ1

d_H(500 MHz,

a]

²⁴e74.3 (c 0.146, EtOH); n_max

D

(500 MHz, CDCl₃) 0.70 and 0.81 (6H, 2d, 1:1, J¼6.6 Hz, Me₂CH),

1.24e1.30 (1H, m, Me₂CH), 1.48 (9H, s, t-Bu), 1.64 (3H, s, CH₃), 2.28

(1H, dd, J¼11.2, 13.3 Hz, 2-Ha), 2.67 (1H, dd, J¼5.4, 13.3 Hz, 2-Hb),

3.25 (1H, dd, J¼3.2, 11.8 Hz, 5-H), 3.77 (1H, dd, J¼5.3, 16.5 Hz, 1H of

CH₂NH), 3.83 (1H, dd, J¼5.4, 17.1 Hz, 1H of CH₂NH), 3.91 (1H, dd,

J¼5.4, 11.2 Hz, 3-H), 3.93 (1H, dd, J¼5.8, 17.7 Hz, 1H of CH₂NH), 4.09

(1H, dd, J¼5.9, 17.7 Hz, 1H of CH₂NH), 5.11 (1H, dd, J¼7.2, 11.8 Hz, 6-

H), 5.11 and 5.15 (2H, 2d,1:1, J¼12.4 Hz, CH₂Ph), 6.16 (1H, t, J¼5.8 Hz,

NHCH₂), 7.27e7.39 (10H, m, 2ꢂ Ph), 7.62e7.70 (2H, m, NHCH and

NHCH₂); d_C(126 MHz, CDCl₃) 15.3, 16.2, 18.4, 22.3, 27.3, 42.7, 44.4,

52.3, 54.0, 61.5, 65.9, 67.1, 70.1, 127.4, 128.0, 128.1, 128.5, 128.7, 136.2,

137.6, 157.2, 162.7, 169.9, 170.1, 170.8, 171.0, 172.8; HRMS (ESI): MH^þ,

found 622.3241. C₃₃H₄₄N₅O₇requires 622.3235.

;

CDCl₃) 1.51 (3H, d, J¼7.2 Hz, 2⁰-Me), 2.70 (1H, br q, J¼11.5 Hz, 2-Ha),

2.85e2.92 (1H, m, 2-Hb), 3.75 (3H, s, OMe), 3.92 (1H, dd, J¼11.5,

5.3 Hz, 3-H), 4.28 (1H, d, J¼11.7 Hz, 5-H), 4.53e4.61 (2H, m, 1-H and

3⁰-H), 4.68 (1H, br t, J¼9.9 Hz, 6-H), 5.04 (2H, s, CH₂Ph), 5.48 (1H, d,

J¼8.3 Hz, NH), 6.97e7.13 (10H, m, 2ꢂ Ph), 7.26e7.34 (5H, m, Ph), 7.88

(1H, d, J¼6.4 Hz, NH); d_C(126 MHz, CDCl₃) 17.6, 42.5, 49.0, 52.6, 55.8,

64.7, 67.3, 68.6, 75.3, 127.8, 128.0, 128.0, 128.0, 128.0, 128.1, 128.1,

128.3, 128.6, 135.3, 135.8, 136.1, 156.0, 162.1, 168.4, 173.0; HRMS

(ESI): MH^þ, found 557.2400. C₃₁H₃₃N₄O₆requires 557.2395.

4.4.9.1.2. Methyl (S)-2-((1S,3S,5S,6S)-6-(benzyloxycarbonylam-

ino)-7-oxo-3,5-diphenylhexahydropyrazolo[1,2-a]pyrazole-1-

carboxamido)propanoate (35a⁰). Yield: 420 mg (48%) of white solid;

mp 70e74 ^ꢃC; [Found: C, 66.68; H, 5.98; N, 9.84. C₃₁H₃₂N₄O₆re-

4.4.8.4. Methyl 2-(2-((1R*,3R*,5R*,6R*)-6-(2-(2-(benzyloxycarbonyl-

amino)acetamido)acetamido)-5-isopropyl-1-methyl-7-oxo-3-

phenylhexahydropyrazolo[1,2-a]pyrazole-1-carboxamido)acetamido)ac-

etate (33). Prepared from carboxylic acid 31 (175 mg, 0.3 mmol),

methyl N-(glycyl)glycinate hydrochloride (32)(56 mg, 0.3 mmol), 4-

methylmorpholine (0.033 mL, 0.3 mmol), and EEDQ (76 mg,

0.3 mmol); General procedure A. Yield: 135 mg (62%) of red crystals;

mp 114e117 ^ꢃC; [Found: C, 56.60; H, 6.42; N, 13.14. C₃₄H₄₃N₇O₉$1³/₄

H₂O requires C, 56.30; H, 6.46; N, 13.52%]; n_max(KBr) 3390, 3068,

2958, 2355, 2317, 1681, 1650, 1537, 1454, 1410,1371, 1337, 1228, 1150,

quires C, 66.89; H, 5.79; N, 10.07%]; [

(KBr) 3384, 3065, 3037, 1743, 1728, 1705, 1695, 1684, 1676, 1558,

1539,1498,1454,1438,1382,1340,1254,1216,1150,1066,1058, 760,

a

]

²⁴þ62.7 (c 0.15, EtOH); n_max

D

754, 697 cm^ꢁ1

;

d_H(500 MHz, CDCl₃) 1.51 (3H, d, J¼7.4 Hz, 2⁰-Me),

2.70e2.92 (2H, m, 2-CH₂), 3.78 (3H, s, OMe), 3.99 (1H, dd, J¼5.6,

11.0 Hz, 3-H), 4.42 (1H, d, J¼11.3 Hz, 5-H), 4.50 (1H, dd, J¼7.6,

11.3 Hz, 6-H), 4.58 (1H, br d, J¼8.8 Hz, 1-H), 4.66 (1H, quintet,

J¼5.6 Hz, 3⁰-H), 5.07 (2H, s, CH₂Ph), 5.60 (1H, d, J¼7.6 Hz, NH),

6.95e7.13 (10H, m, 2ꢂ Ph), 7.32 (5H, m, Ph), 8.04 (1H, d, J¼7.1 Hz,

6662

A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

NH); d_C(126 MHz, CDCl₃) 17.2, 43.4, 48.8, 52.5, 55.5, 65.6, 67.4, 68.3,

74.1, 127.9, 128.0, 128.0, 128.1, 128.1, 128.1, 128.3, 128.4, 128.7, 135.6,

135.7, 135.9, 156.2, 161.1, 168.6, 173.5; HRMS (ESI): MH^þ, found

557.2401. C₃₁H₃₃N₄O₆requires 557.2400.

CH₂Ph), 5.21 (1H, d, J¼8.8 Hz, NH), 7.29e7.41 (10H, m, 2ꢂ Ph), 8.48

(1H, d, J¼6.5 Hz, NH); d_C(126 MHz, CDCl₃) 16.0, 17.3, 18.9, 23.4, 26.7,

49.0, 51.5, 52.3, 56.9, 64.3, 67.2, 68.9, 75.5, 127.5, 128.0, 128.2, 128.5,

128.5, 128.6, 136.0, 137.7, 155.8, 162.4, 171.2, 173.0; HRMS (ESI): MH^þ,

found 537.2707. C₂₉H₃₇N₄O₆requires 537.2708.

4.4.9.2. Synthesis of tripeptides 35c in 35c⁰. Prepared from the

carboxylic acid 15c (143 mg, 0.33 mmol), methyl (S)-alaninate hy-

drochloride (34)(46 mg, 0.33 mmol), 4-methylmorpholine

(0.036 mL, 0.33 mmol) and EEDQ (86 mg, 0.35 mmol); FC

(EtOAc), MPLC (EtOAc/hexanes, 1:1).

4.4.9.3.2. Methyl (S)-2-((1S,3S,5S,6S)-6-(benzyloxycarbonylam-

ino)-5-isopropyl-1-methyl-7-oxo-3-phenylhexahydropyrazolo[1,2-a]

pyrazole-1-carboxamido)propanoate (36c⁰). Yield: 181 mg (17%) of

white solid; mp 159e162 ^ꢃC; [Found: C, 64.75; H, 6.76; N, 10.44.

24

C₂₉H₃₆N₄O₆requires C, 64.91; H, 6.76; N, 10.44%]; [

a

]

þ81.7 (c

D

24

4.4.9.2.1. Methyl (S)-2-((1R,3R,5R,6R)-6-(benzyloxycarbonylam-

ino)-5-isopropyl-7-oxo-3-phenylhexahydropyrazolo[1,2-a]pyrazole-

1-carboxamido)propanoate (35c). Yield: 55 mg (32%) of colourless

0.158, EtOH); [

a

]

D

þ85.4 (c 0.125, CH₂Cl₂); n_max(KBr) 3624, 3583,

3301, 3065, 2958, 1748, 1711, 1693, 1650, 1547, 1430, 1375, 1334,

1277, 1205, 1155, 1109, 1084, 1064, 1026, 992, 961, 916, 900, 867,

oil; [

a

]

²⁴ꢁ33.04 (c 0.115, CDCl₃); n_max(liquid ﬁlm) 3300, 3064, 3034,

848, 833, 783, 753, 720, 697, 664 cm^ꢁ1

; d_H(500 MHz, CDCl₃) 0.73

D

2956, 2873,1718,1681,1540,1495,1455,1387,1369,1294,1248,1212,

and 0.82 (6H, 2d, 1:1, J¼6.9 Hz, Me₂CH), 1.23e1.33 (1H, m, Me₂CH),

1.48 (3H, d, J¼7.4 Hz, 2⁰-Me), 1.79 (3H, s, 1-Me), 2.27 (1H, dd, J¼11.1,

13.0 Hz, 2-Ha), 3.21 (1H, dd, J¼5.2, 13.0 Hz, 2-Hb), 3.31 (1H, dd,

J¼2.7, 11.4, Hz, 5-H), 3.75 (3H, s, OMe), 3.81 (1H, dd, J¼5.2, 11.1 Hz,

3-H), 4.52e4.62 (2H, m, 6-H and 2⁰-H), 5.10 (2H, s, CH₂Ph), 5.52 (1H,

d, J¼8.6 Hz, NH), 7.27e7.42 (10H, m, 2ꢂ Ph), 8.39 (1H, d, J¼7.2 Hz,

NH); d_C(126 MHz, CDCl₃) 16.0, 17.0, 19.0, 22.6, 26.9, 48.8, 52.2, 52.4,

57.2, 63.8, 67.1, 68.7, 75.2, 127.5, 127.9, 128.2, 128.4, 128.6, 128.6,

136.0, 137.7, 155.9, 162.1, 171.4, 173.7; HRMS (ESI): MH^þ, found

537.2699. C₂₉H₃₇N₄O₆requires 537.2708.

1155,1145,1109,1055, 977, 847, 735, 700 cm^ꢁ1

; d_H(500 MHz, CDCl₃)

0.74 and 0.83 (6H, 2d, 1:1, J¼6.9 Hz, Me₂CH), 1.23e1.37 (1H, m,

CHMe₂), 1.47 (3H, d, J¼7.3 Hz, 2⁰-Me), 2.63 (1H, ddd, J¼9.3, 11.2,

13.0 Hz, 2-Ha), 2.76 (1H, ddd, J¼0.9, 5.5,13.0 Hz, 2-Hb), 3.31 (1H, dd,

J¼2.7,11.7 Hz, 5-H), 3.73 (3H, s, OMe), 3.92 (1H, dd, J¼5.5,11.2 Hz, 3-

H), 4.44 (1H, br d, J¼9.3 Hz,1-H), 4.59 (1H, br d, J¼10.1 Hz, 6-H), 4.53

(1H, quintet, J¼7.4 Hz, 3⁰-H), 5.08 and 5.13 (2H, 2d, 1:1, J¼12.4 Hz,

CH₂Ph), 5.91 (1H, d, J¼9.1 Hz, NH), 7.29e7.40 (10H, m, 2ꢂ Ph), 7.84

(1H, d, J¼6.9 Hz, NH); d_C(126 MHz, CDCl₃),

d 16.3,17.8,18.9, 27.2, 43.6,

49.0, 52.7, 56.0, 56.9, 67.4, 68.8, 77.4, 127.7, 128.2, 128.4, 128.7, 128.8,

128.9,136.3,138.0,156.0,163.6,168.4,173.1; HRMS (ESI): MH^þ, found

523.2553. C₂₈H₃₅N₄O₆requires 523.2551.

4.4.9.4. Synthesis of tripeptides 38c in 38c⁰. Prepared from the

amine 20c (88 mg, 0.25 mmol), (S)-Boc-alanine (37)(47 mg,

0.25 mmol) and EEDQ (65 mg, 0.26 mmol); FC (EtOAc), MPLC (EtOAc/

hexanes, 1:4).

4.4.9.4.1. tert-Butyl (1R,3R,5R,6R)-6-((S)-2-(tert-butoxycarbonyla-

mino)propanamido)-5-isopropyl-7-oxo-3-phenylhexahydropyrazolo

[1,2-a]pyrazole-1-carboxylate (38c). Yield: 58 mg (44%) of purple oil;

4.4.9.2.2. Methyl

(S)-2-((1S,3S,5S,6S)-6-(benzylox-

ycarbonylamino)-5-isopropyl-7-oxo-3-phenylhexahydropyrazolo

[1,2-a]pyrazole-1-carboxamido)propanoate (35c⁰). Yield: 35 mg

24

(20%) of colourless oil; [

a

]

D

þ82.86 (c 0.105, CDCl₃); n_max(liquid

ﬁlm) 3334, 3063, 3034, 2958, 2873, 1739, 1716, 1696, 1681, 1538,

1496, 1455, 1388, 1369, 1292, 1256, 1212, 1173, 1144, 1057, 990, 848,

[

a

]

²⁴ꢁ61.6 (c 0.125, CDCl₃); n_max(liquid ﬁlm) 3310, 2976, 2935, 2878,

D

736, 701, 667, 606 cm^ꢁ1

;

d_H(500 MHz, CDCl₃): d_H0.74 and 0.80 (6H,

1738,1704,1698,1684,1558,1538,1520,1505,1496,1456,1446,1392,

1368,1244,1226,1159,1112,1070,1041,1023, 993, 853, 843, 734, 701,

2d, 1:1, J¼6.9 Hz, Me₂CH), 1.26e1.29 (1H, m, CHMe₂), 1.45 (3H, d,

J¼7.4 Hz, 2⁰-Me), 2.66 (1H, ddd, J¼9.5, 11.2, 12.9 Hz, 2-Ha), 2.80 (1H,

ddd, J¼0.9, 5.3,12.9 Hz, 2-Hb), 3.45 (1H, dd, J¼3.1,11.2 Hz, 5-H), 3.90

(3H, s, COOMe), 3.92 (1H, dd, J¼5.3, 11.2 Hz, 3-H), 4.47 (1H, br d,

J¼9.5 Hz, 1-H), 4.55 (1H, br d, J¼8.6, 11.2 Hz, 6-H), 4.60 (1H, quintet,

J¼7.4 Hz, 2⁰-H), 5.07 and 5.10 (2H, 2d, 1:1, J¼12.5 Hz, CH₂Ph), 5.99

(1H, d, J¼8.4 Hz, NH), 7.28e7.40 (10H, m, 2ꢂ Ph), 8.08 (1H, d,

J¼7.3 Hz, NH); d_C(126 MHz, CDCl₃) 16.2, 17.2, 19.1, 27.2, 44.1, 48.8,

52.6, 55.6, 57.3, 67.2, 68.3, 75.4, 127.7, 127.9, 128.3, 128.7, 128.7,

128.8, 136.2, 137.7, 156.3, 162.8, 168.8, 173.5; HRMS (ESI): MH^þ,

found 523.2548. C₂₈H₃₅N₄O₆requires 523.2551.

666 cm^ꢁ1

;

d_H(500 MHz, CDCl₃) 0.68 and 0.85 (6H, 2d, 1:1, J¼6.9 Hz,

Me₂CH), 1.28e1.35 (1H, m, Me₂CH),1.36 (3H, d, J¼7.0 Hz, 2⁰-Me), 1.42

and 1.53 (18H, 2s, 1:1, 2ꢂ t-Bu), 2.49 (1H, ddd, J¼0.7, 5.5, 13.2 Hz, 2-

Ha), 2.66 (1H, ddd, J¼9.3, 11.4, 13.2 Hz, 2-Hb), 3.13 (1H, dd, J¼2.7,

11.7Hz, 5-H), 3.89(1H, dd, J¼5.5,11.4Hz, 3-H), 4.11e4.18 (1H, m, 2⁰-H),

4.30 (1H, d, J¼9.3 Hz, 1-H), 4.92 (1H, br s, NH), 5.17 (1H, dd, J¼9.2,

11.7 Hz, 6-H), 6.51 (1H, br s, NH), 7.30e7.40 (5H, m, Ph); d_C(126 MHz,

CDCl₃) 16.3,18.4, 27.5, 28.1, 28.2, 28.5, 44.1, 50.2, 53.9, 55.3, 69.1, 79.7,

80.3, 83.3, 127.6, 128.7, 128.9, 138.3, 155.5, 164.0, 169.1, 172.3; HRMS

(ESI): MH^þ, found 531.3171. C₂₈H₄₃N₄O₆requires 531.3177.

4.4.9.4.2. tert-Butyl (1S,3S,5S,6S)-6-((S)-2-((tert-butoxycarbonyl)

amino)propanamido)-5-isopropyl-7-oxo-3-phenylhexahydropyrazolo

[1,2-a]pyrazole-1-carboxylate (38c⁰). Yield: 38 mg (29%) of purple

4.4.9.3. Synthesis of tripeptides 36c in 36c⁰. Prepared from the

carboxylic acid 18c (904 mg, 2 mmol), methyl (S)-alaninate hydro-

chloride (34)(287 mg, 2 mmol), 4-methylmorpholine (0.220 mL,

2 mmol) and EEDQ (0.518 g, 2.1 mmol); FC (EtOAc), MPLC (EtOAc/

hexanes, 1:4).

oil; [

a

]

²⁴þ62.6 (c 0.115, CDCl₃); n_max(liquid ﬁlm) 3321, 2978, 2930,

D

2873, 1739, 1713, 1695, 1683, 1495, 1455, 1392, 1368, 1246, 1227,

1155, 1110, 1068, 1050, 1027, 843, 741, 701 cm^ꢁ1

; d_H(500 MHz,

4.4.9.3.1. Methyl (S)-2-((1R,3R,5R,6R)-6-(benzyloxycarbonylam-

ino)-5-isopropyl-1-methyl-7-oxo-3-phenylhexahydropyrazolo[1,2-a]

pyrazole-1-carboxamido)propanoate (36c). Yield: 320 mg (30%) of

CDCl₃) 0.70 and 0.85 (6H, 2d, 1:1, J¼6.9 Hz, Me₂CH), 1.36 (3H, d,

J¼7.1 Hz, 2⁰-Me), 1.30e1.36 (1H, m, Me₂CH), 1.45 and 1.53 (18H, 2s,

1:1, 2ꢂ t-Bu), 2.49 (1H, ddd, J¼1.2, 5.6, 13.2 Hz, 2-Ha), 2.67 (1H, ddd,

J¼9.3,11.4,13.2 Hz, 2-Hb), 3.12 (1H, dd, J¼2.8,11.8 Hz, 5-H), 3.88 (1H,

dd, J¼5.6, 11.4 Hz, 3-H), 4.19e4.24 (1H, m, 2⁰-H), 4.31 (1H, d,

J¼9.3 Hz,1-H), 5.05 (1H, d, J¼5.1 Hz, NH), 5.18 (1H, dd, J¼9.2,11.8 Hz,

6-H), 6.59 (1H, d, J¼9.2 Hz, NH), 7.30e7.40 (5H, m, Ph); d_C(126 MHz,

CDCl₃) 16.3, 18.5, 27.5, 28.1, 28.2, 28.5, 44.1, 50.4, 53.9, 55.2, 69.0,

79.7, 80.3, 83.3, 127.6, 128.7, 128.9, 138.2, 155.6, 164.2, 169.2, 172.5;

HRMS (ESI): MH^þ, found 531.3167. C₂₈H₄₃N₄O₆requires 531.3177.

white solid; mp 186e188 ^ꢃC; [Found: C, 64.70; H, 6.81; N, 10.36.

24

C₂₉H₃₆N₄O₆requires C, 64.91; H, 6.76; N, 10.44%]; [

a

]

ꢁ107.5 (c

D

24

0.120, EtOH); [

a

]

ꢁ107.4 (c 0.125, CH₂Cl₂); n_max(KBr) 3354, 3067,

D

2971, 2956, 2872, 1756, 1716, 1681, 1654, 1573, 1514, 1459, 1430, 1387,

1347, 1337, 1306, 1292, 1258, 1215, 1188, 1153, 1130, 1080, 1053, 1025,

976, 965, 936, 917, 885, 849, 817, 779, 751, 701, 659 cm^ꢁ1

; d_H

(500 MHz, CDCl₃) 0.72 and 0.82 (6H, 2d, 1:1, J¼7.0 Hz, Me₂CH), 1.26

(1H, td, J¼7.0, 3.5 Hz, Me₂CH), 1.48 (3H, d, J¼7.3 Hz, 2⁰-Me), 1.80 (3H,

s, 1-Me), 2.25 (1H, dd, J¼11.2, 13.0 Hz, 2-Ha), 3.23 (1H, dd, J¼2.9,

11.6 Hz, 5-H), 3.31 (1H, dd, J¼5.3, 13.0 Hz, 2-Hb), 3.70 (1H, dd, J¼5.3,

11.2 Hz, 3-H), 3.74 (3H, s, OMe), 4.47 (1H, quintet, J¼7.2 Hz, 2⁰-H),

4.59 (1H, dd, J¼8.8, 11.6 Hz, 6-H), 5.09 and 5.12 (2H, 2d, J¼12.4 Hz,

4.4.9.5. Synthesis of tripeptides 39c in 39c⁰. Prepared from the

amine 22c (373 mg, 1 mmol), (S)-Boc-alanine (37)(190 mg,

1 mmol) and EEDQ (247 mg, 1 mmol); FC (EtOAc), MPLC (EtOAc/

hexanes, 1:4).

A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

6663

4.4.9.5.1. tert-Butyl (1R,3R,5R,6R)-6-((S)-2-(tert-butoxycarbonyl-

amino)propanamido)-5-isopropyl-1-methyl-7-oxo-3-phenylhexahy-

dropyrazolo[1,2-a]pyrazole-1-carboxylate (39c). Yield: 117 mg (21%)

(3H, d, J¼7.2 Hz, 2⁰-Me), 3.05 (1H, ddd, J¼4.2, 10.2, 13.9 Hz, 2-Ha),

3.43 (1H, ddd, J¼4.0, 10.2, 13.9 Hz, 2-Hb), 3.76 (1H, d, J¼12.0 Hz, 5-

H), 3.79 (3H, s, OMe), 4.61 (1H, quintet, J¼7.3 Hz, 2⁰-H), 4.93 (1H, br

t, J¼10.4 Hz, 6-H), 4.98 (1H, dd, J¼4.0, 10.2 Hz, 3-H), 4.99 (2H, s,

CH₂Ph), 5.26 (1H, d, J¼9.2 Hz, NH), 5.50 (1H, dd, J¼4.2,10.2 Hz,1-H),

7.41e6.98 (13H, m, 2ꢂ Ph and C₆H₃), 7.53 (1H, d, J¼7.4 Hz, 2⁰-NH);

d_C(126 MHz, CDCl₃) 18.2, 33.4, 48.8, 52.7, 55.1, 58.2, 63.9, 67.4, 70.6,

127.7, 128.3, 128.6, 128.6, 128.8, 129.4, 130.2, 130.4, 132.3, 134.7,

135.7, 138.9, 155.6, 161.2, 168.6, 172.8; HRMS (ESI): MH^þ, found

625.1599. C₃₁H₃₁Cl₂N₄O₆requires 625.1621.

24

of pink crystals; mp 76e80 ^ꢃC; [

a

]

ꢁ66.2 (c 0.130, EtOH); [

a]

D

ꢁ58.8 (c 0.110, CH₂Cl₂); n_max(KBr) 3388, 2978, 2925, 2873, 1693,

1681, 1504, 1455, 1390, 1368, 1293, 1251, 1219, 1165, 1088, 1070,

1028, 989, 941, 846, 753, 701, 651, 613 cm^ꢁ1

; d_H(500 MHz, CDCl₃)

0.69 and 0.86 (6H, 2d, 1:1, J¼6.9 Hz, Me₂CH), 1.29e1.35 (1H, m,

Me₂CH), 1.36 (3H, d, J¼7.1 Hz, 2⁰-Me), 1.45 and 1.54 (18H, 2s, 1:1, 2ꢂ

t-Bu), 2.05 (3H, s, 1-Me), 2.31 (1H, dd, J¼11.4, 13.2 Hz, 2-Ha), 2.67

(1H, dd, J¼5.7, 13.2 Hz, 2-Hb), 3.09 (1H, dd, J¼2.8, 11.7 Hz, 5-H), 3.92

(1H, dd, J¼5.7, 11.4 Hz, 3-H), 4.17e4.26 (1H, m, 2⁰-H), 5.11 (1H, dd,

J¼9.2, 11.7 Hz, 6-H), 5.11 (1H, br s, NH), 6.62 (1H, br d, J¼9.2 Hz, NH),

7.26e7.36 (3H, m, o,p-Ph), 7.36e7.41 (2H, m, m-Ph); d_C(126 MHz,

CDCl₃) 14.4, 16.3, 18.5, 21.3, 22.5, 27.5, 28.2, 28.5, 52.9, 53.8, 60.6,

62.7, 70.0, 79.3, 83.0, 127.5, 128.6, 128.8, 138.5, 163.3, 170.6, 171.4,

172.5; HRMS (ESI): MH^þ, found 545.3335. C₂₉H₄₅N₄O₆requires

545.3334.

4.4.9.7. Synthesis of tripeptides 41d in 41d⁰. Prepared from the

carboxylic acid 19d (260 mg, 0.5 mmol), methyl (S)-alaninate hy-

drochloride (34) (63 mg, 0.5 mmol), 4-methylmorpholine

(0.055 mL, 0.5 mmol) and EEDQ (0.113 g, 0.5 mmol); FC (EtOAc),

MPLC (EtOAc/hexanes, 1:6).

4.4.9.7.1. Methyl (S)-2-((1R,3S,5R,6R)-6-(benzyloxycarbonylam-

ino)-3-(2,6-dichlorophenyl)-7-oxo-5-phenylhexahydropyrazolo[1,2-

a]pyrazole-1-carboxamido)propanoate (41d). Yield: 46 mg (15%) of

4.4.9.5.2. tert-Butyl (1S,3S,5S,6S)-6-((S)-2-(tert-butoxycarbonyla-

mino)propanamido)-5-isopropyl-1-methyl-7-oxo-3-phenylhexahyd-

ropyrazolo[1,2-a]pyrazole-1-carboxylate (39c⁰). Yield: 150 mg (28%)

white solid; mp 48e49 ^ꢃC; [

a]

²⁴ꢁ128.2 (c 0.104, EtOH); n_max(KBr)

D

3308, 3065, 3033, 2957, 2935, 2875, 1716, 1673, 1580, 1537, 1532,

1454, 1436, 1393, 1375, 1252, 1204, 1187, 1150, 1117, 1083, 1041, 984,

24

of pink crystals; mp 167e169 ^ꢃC; [

a

]

D

²⁴þ52.7 (c 0.112, EtOH); [

a]

D

þ35.0 (c 0.100, CH₂Cl₂); n_max(KBr) 3418, 2974, 1681, 1651, 1514,

950, 939, 848, 779, 750, 735, 697, 652, 635, 607 cm^ꢁ1

; d_H(500 MHz,

1454, 1369, 1288, 1251, 1162, 1084, 1048, 880, 840, 794, 746, 700,

CDCl₃) 0.82 and 1.08 (6H, 2d, 1:1, J¼6.8 Hz, Me₂CH), 1.44 (3H, d,

J¼7.2 Hz, 2⁰-Me), 1.83 (3H, s, 1-Me), 1.85e1.92 (1H, m, Me₂CH), 2.63

(1H, dd, J¼8.6, 13.7 Hz, 2-Ha), 3.01 (1H, dd, J¼3.4, 6.5 Hz, 5-H), 3.31

(1H, dd, J¼7.7, 13.7 Hz, 2-Hb), 3.71 (3H, s, OMe), 4.54 (1H, quintet,

J¼7.4 Hz, 2⁰-H), 4.76 (1H, br t, J¼7.9 Hz, 6-H), 5.12 (2H, s, CH₂Ph),

5.36 (1H, t, J¼8.2 Hz, 3-H), 5.53 (1H, br d, J¼9.2 Hz, NH), 7.20 (1H, t,

J¼8.0 Hz, p-C₆H₃), 7.29e7.40 (7H, m, Ph and m-C₆H₃), 9.02 (1H, d,

J¼7.0 Hz, NH); d_C(75.5 MHz, CDCl₃) 17.2, 17.9, 19.6, 23.8, 27.5, 45.4,

48.6, 52.6, 55.3, 58.1, 64.3, 67.4, 67.6,128.3, 128.4,128.6,129.3,130.0,

134.9, 136.1, 136.8, 155.9, 162.3, 171.5, 173.5; HRMS (ESI): MH^ꢁ,

found 603.1783. C₂₉H₃₃Cl₂N₄O₆requires 603.1788.

667 cm^ꢁ1

;

d_H(500 MHz, CDCl₃) 0.68 and 0.86 (6H, 2d, 1:1, J¼6.9 Hz,

Me₂CH), 1.28e1.33 (1H, m, Me₂CH), 1.36 (3H, d, J¼7.0 Hz, 2⁰-Me),

1.42 and 1.55 (18H, 2s, 2ꢂ t-Bu), 2.05 (3H, s, 1-Me), 2.30 (1H, dd,

J¼11.3, 13.2 Hz, 2-Ha), 2.67 (1H, dd, J¼5.7, 13.2 Hz, 2-Hb), 3.09 (1H,

dd, J¼2.6, 11.8 Hz, 5-H), 3.92 (1H, dd, J¼5.7, 11.3 Hz, 3-H), 4.12e4.20

(1H, m, 2⁰-H), 4.94 (1H, br s, NH), 5.09 (1H, dd, J¼9.5, 11.8 Hz, 6-H),

6.55 (1H, d, J¼9.5 Hz, NH), 7.27e7.36 (3H, m, o,p-Ph), 7.37e7.41 (2H,

m, m-Ph); d_C(126 MHz, CDCl₃) 11.0, 16.1, 17.7, 18.3, 22.3, 23.0, 28.0,

28.3, 30.4, 52.8, 53.6, 60.4, 62.4, 70.0, 79.1, 82.8, 127.4, 128.4, 128.6,

138.4, 162.9, 170.3, 172.1; HRMS (ESI): MH^þ, found 545.3330.

C₂₉H₄₅N₄O₆requires 545.3334.

4.4.9.7.2. Methyl (S)-2-((1S,3R,5S,6S)-6-(benzyloxycarbonylam-

ino)-3-(2,6-dichlorophenyl)-7-oxo-5-phenylhexahydropyrazolo[1,2-

a]pyrazole-1-carboxamido)propanoate (41d⁰). Yield: 49 mg (16%) of

4.4.9.6. Synthesis of tripeptides 40b in 40b⁰. Prepared from the

carboxylic acid 16b (1.08 g, 2 mmol), methyl (S)-alaninate hydro-

chloride (34) (287 mg, 2 mmol), 4-methylmorpholine (0.220 mL,

2 mmol) and EEDQ (0.518 g, 2.1 mmol); FC (EtOAc), MPLC (EtOAc/

hexanes, 2:1).

white solid; mp 49e50 ^ꢃC; [

a

]

²⁴þ105.7 (c 0.106, EtOH); n_max(KBr)

D

3305, 3066, 3034, 2957, 2934, 2877, 1715, 1673, 1580, 1531, 1454,

1436, 1393, 1374, 1250, 1205, 1152, 1116, 1082, 1041, 983, 951, 912,

862, 846, 779, 751, 736, 699, 668, 632 cm^ꢁ1

; d_H(500 MHz, CDCl₃)

4.4.9.6.1. Methyl (S)-2-((1S,3S,5R,6R)-6-(benzyloxycarbonylam-

ino)-3-(2,6-dichlorophenyl)-7-oxo-5-phenylhexahydropyrazolo[1,2-

a]pyrazole-1-carboxamido)propanoate (40b). Yield: 442 mg (35%) of

0.80 and 1.08 (6H, 2d, 1:1, J¼6.8 Hz, Me₂CH), 1.47 (3H, d, J¼7.3 Hz,

2⁰-Me), 1.83 (3H, s, 1-Me), 1.85e1.93 (1H, m, Me₂CH), 2.62 (1H, dd,

J¼8.9, 13.8 Hz, 2-Ha), 3.00 (1H, dd, J¼3.0, 6.6 Hz, 5-H), 3.50 (1H, dd,

J¼7.5, 13.8 Hz, 2-Hb), 3.70 (3H, s, OMe), 4.55 (1H, quintet, J¼7.3 Hz,

2⁰-H), 4.73 (1H, br t, J¼7.8 Hz, 6-H), 5.11 (2H, s, CH₂Ph), 5.38 (1H, t,

J¼8.2 Hz, 3-H), 5.53 (1H, br d, J¼8.4 Hz, NH), 7.18 (1H, t, J¼8.0 Hz, p-

C₆H₃), 7.28e7.38 (7H, m, Ph and m-C₆H₃), 9.27 (1H, d, J¼7.1 Hz, NH);

d_C(75.5 MHz, CDCl₃) 17.0, 17.6, 19.7, 24.5, 27.4, 44.8, 48.6, 52.5, 54.9,

58.3, 64.8, 67.3, 67.6, 128.3, 128.6, 129.1, 129.9, 131.0, 135.5, 136.2,

136.6, 155.9, 162.2, 171.8, 173.4; HRMS (ESI): MH^þ, found 605.1930.

C₂₉H₃₅Cl₂N₄O₆requires 605.1928.

pale yellowish solid; mp 86e90 ^ꢃC; [Found: C, 59.32; H, 4.99; N, 8.78.

24

C₃₁H₃₀Cl₂N₄O₆requires C, 59.53; H, 4.83; N, 8.96%]; [

a

]

D

þ6.9 (c

0.124, EtOH); n_max(KBr) 3314, 3065, 3036, 2953, 1725, 1710, 1686,

1578,1560,1536,1456,1440,1380,1344,1290,1246,1213,1152,1057,

783, 753, 698 cm^ꢁ1

;

d_H(500 MHz, CDCl₃) 1.49 (3H, d, J¼7.2 Hz, 2⁰-

Me), 2.98 (1H, ddd, J¼4.1, 10.4, 14.1 Hz, 2-Ha), 3.48 (1H, ddd, J¼4.3,

10.2, 14.1 Hz, 2-Hb), 3.76 (1H, d, J¼11.7 Hz, 5-H), 3.77 (3H, s, OMe),

4.61 (1H, quintet, J¼7.0 Hz, 2⁰-H), 4.92 (1H, br t, J¼10.1 Hz, 6-H), 4.99

(2H, s, CH₂Ph), 5.01 (1H, dd, J¼4.3,10.4 Hz, 3-H), 5.30 (1H, d, J¼9.2 Hz,

NH), 5.49 (1H, dd, J¼4.1,10.2 Hz,1-H), 7.00e7.40 (13H, m, 2ꢂ Ph and

C₆H₃), 7.62 (1H, d, J¼5.4 Hz, NH); d_C(75.5 MHz, CDCl₃) 18.2, 33.6,

49.0, 52.7, 55.3, 58.1, 63.8, 67.4, 71.1, 127.8, 128.4, 128.7, 128.7, 128.7,

128.9,129.5,130.2,130.5,132.4,134.8,138.9,155.7,161.1,168.5,173.0;

HRMS(ESI): MH^þ, found 625.1620. C₃₁H₃₁Cl₂N₄O₆requires625.1621.

4.4.9.6.2. Methyl (S)-2-((1R,3R,5S,6S)-6-(benzyloxycarbonylam-

ino)-3-(2,6-dichlorophenyl)-7-oxo-5-phenylhexahydropyrazolo[1,2-

a]pyrazole-1-carboxamido)propanoate (40b⁰). Yield: 336 mg (27%)

4.4.9.8. Synthesis of tripeptides 42d in 42d⁰. Prepared from the

amine 23d (856 mg, 1.9 mmol), Boc-(S)-alanine (37) (360 mg,

2 mmol), 4-methylmorpholine (0.22 mL, 2 mmol) and EEDQ

(469 mg, 2 mmol); FC (EtOAc), MPLC (EtOAc/hexanes, 1:4).

4.4.9.8.1. tert-Butyl (1R,3S,5R,6R)-6-((S)-2-(tert-butoxycarbonyl-

amino)-propanamido)-3-(2,6-dichlorophenyl)-5-isopropyl-1-methyl-

7-oxohexahydropyrazolo[1,2-a]pyrazole-1-carboxylate (42d). Yield:

24

115 mg (10%) of white solid; mp 55e57 ^ꢃC; [

a]

ꢁ107.5 (c 0.120,

D

of pale yellowish solid; mp 72e78 ^ꢃC; [Found: C, 59.41; H, 4.90; N,

EtOH); n_max(KBr) 3455, 2975, 2930, 2878, 1699, 1581, 1561, 1520,

24

8.78. C₃₁H₃₀Cl₂N₄O₆requires C, 59.53; H, 4.83; N, 8.96%]; [

a]

1452, 1438, 1392, 1368, 1290, 1251, 1162, 1085, 1070, 1046, 884,

D

ꢁ18.2 (c 0.208, EtOH); n_max(KBr) 3328, 3065, 3036, 2953, 1724,

783, 763, 668, 656, 622, 605 cm^ꢁ1

; d_H(500 MHz, CDCl₃) 0.88 and

1690, 1578, 1543, 1533, 1499, 1455, 1441, 1381, 1335, 1247, 1213,

1.08 (6H, 2d, 1:1, J¼6.8 Hz, Me₂CH), 1.39 (3H, d, J¼7.3 Hz, 2⁰-Me),

1.42 and 1.52 (18H, 2s, 2ꢂ t-Bu), 1.83 (3H, s, 1-Me), 1.95e2.04 (1H,

1154, 1064, 1051, 912, 778, 766, 698 cm^ꢁ1

; d_H(500 MHz, CDCl₃) 1.49

6664

A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

m, Me₂CH), 2.49 (1H, dd, J¼8.0, 13.0 Hz, 2-Ha), 2.99 (1H, br t,

J¼3.0 Hz, 5-H), 3.14 (1H, dd, J¼9.5, 13.0 Hz, 2-Hb), 4.15e4.25 (1H,

m, 2⁰-H), 4.83 (1H, dd, J¼3.0, 8.8 Hz, 6-H), 5.09 (1H, br d, J¼6.7 Hz,

NH), 5.18 (1H, dd, J¼8.0, 9.5 Hz, 3-H), 6.80 (1H, br d, J¼7.8 Hz, NH),

7.18 (1H, t, J¼8.0 Hz, p-C₆H₃), 7.30 and 7.33 (2H, 2d, 1:1, J¼8.0 Hz,

m-C₆H₃); d_C(75.5 MHz, CDCl₃) 18.0, 18.9, 20.3, 22.4, 27.4, 28.1, 28.4,

45.8, 50.2, 55.1, 57.4, 66.8, 77.2, 79.9, 82.8, 129.0, 129.8, 131.3, 136.2,

155.4, 162.9, 170.3, 172.3; HRMS (ESI): [MeH]^ꢁ, found 613.2610.

C₂₉H₄₁Cl₂N₄O₆requires 613.2554.

MurD from Escherichia coli²⁵(diluted in 50 mM Hepes, and 1 mM

dithiothreitol), and 250 M of the compound dissolved in DMSO.

DdlB: 50 mM Hepes, pH 8.0, 5 mM MgCl₂, 6.5 mM (NH₄)₂SO₄,

10 mM KCl, 0.005% Triton X-114, 700 -Ala, 500 M ATP, puri-

ﬁed DdlB from E. coli (diluted in 50 mM Hepes, and 1 mM dithio-

threitol), and 250 M of compound dissolved in DMSO.

m

mM

D

m

The ﬁnal concentration of DMSO was 5% (v/v). The reaction

mixture was incubated at 37 ^ꢃC for 15 (MurD) and 20 min (DdlB),

respectively, then quenched with 100 mL of Biomol reagent. Ab-

4.4.9.8.2. tert-Butyl (1S,3R,5S,6S)-6-((S)-2-(tert-butoxycarbonyl-

amino)-propanamido)-3-(2,6-dichlorophenyl)-5-isopropyl-1-methyl-

sorbance at 650 nm was read after 5 min. Residual activities were

calculated with respect to control assay without the compound and

with DMSO.

7-oxohexahydropyrazolo[1,2-a]pyrazole-1-carboxylate (42d⁰). Yield:

24

195 mg (17%) of white solid; mp 76e80 ^ꢃC; [

a

]

þ72.7 (c 0.172,

D

EtOH); n_max(KBr) 3430, 2978, 2932, 1707, 1579, 1561, 1507, 1452,

Acknowledgements

1438, 1393, 1367, 1289, 1249, 1163, 1129, 1087, 1065, 1023, 929, 845,

784, 763, 721, 688, 661, 639 cm^ꢁ1

; d_H(500 MHz, CDCl₃) 0.89 and

The ﬁnancial support from the Slovenian Research Agency

through grants P1-0179, J1-6689-0103-04, X-2000, and L1-4039 is

gratefully acknowledged. The authors thank Dr. Didier Blanot for

providing MurD and Professor Ian Chopra for providing DdlB.

1.09 (6H, 2d, 1:1, J¼6.8 Hz, Me₂CH), 1.39 (3H, d, J¼7.3 Hz, 2⁰-Me),

1.45 and 1.52 (18H, 2s, 2ꢂ t-Bu), 1.83 (3H, s, 1-Me), 1.95e2.05 (1H,

m, Me₂CH), 2.50 (1H, dd, J¼8.0, 12.9 Hz, 2-Ha), 2.98 (1H, br t,

J¼2.5 Hz, 5-H), 3.14 (1H, dd, J¼9.5,12.9 Hz, 2-Hb), 4.24e4.34 (1H, m,

2⁰-H), 4.81 (1H, dd, J¼2.5, 8.7 Hz, 6-H), 5.12 (1H, br d, J¼6.2 Hz, NH),

5.18 (1H, dd, J¼8.0, 9.5 Hz, 3-H), 6.97 (1H, d, J¼8.7 Hz, NH), 7.18 (1H,

t, J¼8.0 Hz, p-C₆H₃), 7.29 and 7.34 (2H, 2d, 1:1, J¼8.0 Hz, m-C₆H₃); d_C

(75.5 MHz, CDCl₃) 18.1, 19.1, 20.3, 22.3, 28.1, 28.5, 45.9, 50.4, 55.0,

57.4, 66.7, 77.2, 80.0, 82.7, 129.0, 129.9, 131.3, 134.2, 136.2, 155.4,

163.1, 170.2, 172.3; HRMS (ESI): MH^ꢁ, found 611.2426.

C₂₉H₄₁Cl₂N₄O₆requires 611.2403.

Supplementary data

Supplementary data associated with this article can be found in

the online version, at http://dx.doi.org/10.1016/j.tet.2013.05.122.

These data include MOL ﬁles and InChiKeys of the most important

compounds described in this article.

References and notes

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positions with bond lengths CeH of 0.96 A for methyl, 0.97 A for

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M UDP-MurNAc- -Ala, 100 -Glu, 400 M ATP, puriﬁed

mL was used, containing:

80

m

L

mM

D

m

A. Novak et al. / Tetrahedron 69 (2013) 6648e6665

6665

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Article Doi

Synthesis of pyrazolo[1,2-a]pyrazole-based peptide mimetics

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