New tripeptide-based macrocyclic calpain inhibitors formed by n-alkylation of histidine

Article abstract of DOI:10.1002/cbdv.201200320

Two new series of 15-membered macrocyclic peptidomimetics, in which the P1 and P3 residues of the peptide backbone are linked by a bridge containing a 1,4-disubstituted 1H-imidazole, are reported. The structure with an aldehyde at the C-terminus and the i

Full text of DOI:10.1002/cbdv.201200320

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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New Tripeptide-Based Macrocyclic Calpain Inhibitors Formed by
N-Alkylation of Histidine
by Hongyuan Chena)1), Wanting Jiaoa), Matthew A. Jonesa)b), James M. Coxon*a), James D.
Mortonc), Roy Bickerstaffec), Ashok D. Pehered), Ondrej Zvarecd)2), and Andrew D. Abell*a)3)
a) Chemistry Department, University of Canterbury, Christchurch, New Zealand
(e-mail: jim.coxon@canterbury.ac.nz; andrew.abell@adelaide.edu.au)
b) University of Roehampton, London, UK
c) Lincoln University, Lincoln, New Zealand
d) School of Chemistry and Physics, University of Adelaide, South Australia, Australia
Dedicated to Professor Dieter Seebach in celebration of his 75th birthday
Two new series of 15-membered macrocyclic peptidomimetics, in which the P1 and P3 residues of the
peptide backbone are linked by a bridge containing a 1,4-disubstituted 1H-imidazole, are reported. The
structure with an aldehyde at the C-terminus and the imidazole at P3, i.e., 4c, shows significant inhibitory
activity against calpain 2, with an IC50 value of 238 nm. The macrocyclic aldehyde with the imidazole at
the alternative P1 position, i.e., 5c, is significantly less active. The relative activities are linked to the
ability of the component macrocycles to mimic a b-strand geometry that is known to favor active-site
binding. This ability is defined by conformational searches and docking studies with calpain.
1. Introduction. – Much work has been done on constraining the backbone of
peptide-based protease inhibitors into a biologically active b-strand conformation by
chemically linking the P1 and P3 residue [1–3]; for example, see the C-terminal
aldehyde macrocycles CAT811 (1) [1], 2, and 3 [2] in Fig. 1. This geometry is almost
universally recognized by proteases for active-site binding, with a preorganisation of
conformation facilitating active-site binding [1][3]. Such macrocyclic inhibitors also
offer enhanced resistance to proteolytic degradation and thus an improved pharma-
cokinetic profile [4]. The introduction of a conformational constraint of this type into
peptidomimetics is also of wider interest as a basis of important biological probes and
scaffolds [2].
A number of approaches have been reported for introducing such a macrocycle into
peptidomimetics, including ring-closing metathesis [1][5], Huisgen cycloaddition
[2][6], and intramolecular nucleophilic cyclization [3][7]. Here, we extend these
studies with methodology for the preparation of histidine-containing macrocycles 4 and
1
)
)
)
Present address: Department of Biological Chemistry, School of Medicine, University of California,
Irvine, USA.
Present address: School of Materials, Science and Engineering, Nanyang Technological University,
Singapore.
Present address: School of Chemistry and Physics, University of Adelaide, South Australia,
Australia.
2
3
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 1. Key macrocycles
5 (see Fig. 1). These structures provide a new ring system to complement and extend
those found in CAT811 [1], and also the triazole-containing macrocycles 2 and 3 [2].
These known macrocyclic peptidomimetics have been shown to inhibit the cysteine
protease calpain 2, and as such the new macrocycles reported here (4b and 4c, and 5b
and 5c) have also been tested against this enzyme. Calpain is of particular therapeutic
significance due to its involvement in critical human diseases, including the develop-
ment of cortical cataract [1][8], the leading cause of blindness in humans [9]. CAT811
is a potent inhibitor of ovine calpain 2 (o-CAPN2), and it slows the development of
cataract in sheep with a genetic disposition to cataract [10]. Modelling studies [11] have
shown that the peptide backbone of CAT811 is constrained into the required b-strand
conformation, with the benzylic group at P3 playing an important role [1]. In this
article, we also report molecular-modelling and docking studies with calpain to better
understand and characterize their potential as protease inhibitors and more generally
as b-strand mimics.
2. Results and Discussion. – 2.1. Computation. Molecular modelling was carried out
on the macrocycle 4a–4c and 5a–5c to establish their propensity to adopt a b-strand
conformation within the peptide backbone, with the results compiled in Table 1.
Compounds 4a–4c gave rise to a conformational ensemble with a high percentage of b-
strand conformers (> 70%). However, while 5b exhibited a modest percentage of b-
strand conformers (41%), 5a and the aldehyde 5c gave low percentages of b-strand
conformations. This suggested that the macrocyclic aldehyde 5c would be a less potent
inhibitor of calpain compared to the aldehyde 4c. The target macrocycles were also
docked into the calpain active site in a calpain construct model defined previously
[11][12], and the resulting poses were evaluated with respect to two main criteria; 1)
the ability to form three key H-bonds (designated as A, B, and C) with the active site

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Table 1. Molecular-Modelling Data for 4a–4c and 5a–5c
Compound
% b-strand
Essential H-bondsa)
WHDb) [ꢂ]
4a
4b
4c
5a
5b
5c
96.1
73.1
74.6
12.1
41.6
4.2
A,Cþ2
3.51
3.69
3.33
5.51
3.79
10.7
A,B,Cþ1
A,B,Cþ1
A,Bþ1
Aþ1
NONEþ3
a) The H-bonds from the CO group of Gly208, the NH group of Gly208, and the CO group of Gly271 of
the o-CAPN1 homology model are labeled A, B, and C, resp. b) War head distance (WHD) is the
distance between the C-terminal C-atom and the active site cysteine S-atom in ꢂ.
that are critical for defining a bound b-strand conformation, and 2) the relative position
of an active site cysteine S-atom and the p* orbital of the CO, recognizing the Dunitz
trajectory for nucleophilic attack.
Results of the docking studies suggest that the structures 4b and 4c exhibit poses in
which the three essential H-bonds are formed with the active site Gly271 and Gly208
(see Table 1) [4][11][13]. The ester 4a docks with only two of the essential H-bonds,
while macrocycles 5a–5c do not adopt poses containing a b-strand conformation as
evidenced by a lack of all three H-bonds in the docked structures. Again this suggests
that these compounds are likely to be less potent inhibitors of calpain, supporting the
earlier conformational analysis results. All the synthesized macrocycles were assayed
against calpain 2 as discussed later. Derivatives 5b and 5c were synthesized to extend
the scope of the new intramolecular lactamization methodology and to provide
macrocycles that have a reduced b-strand character for comparative assay against
calpain.
2.2. Synthesis. The macrocycle of the first series (derivatives 4) was introduced by
lactamization of a key amino acid intermediate 11 as shown in Scheme 1. Coupling of
commercially available N-Cbz-His-OH (6) with Leu-OtBu (7), in the presence of 1-
ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDCl) and 1-hydroxy-7-azabenzo-
triazole (HOAt), gave the dipeptide 8. The 1H-imidazole 8 was deprotonated on
treatment with NaH and the resulting anion alkylated with the iodide 9 [14] using
general methodology reported by Kaur et al. [15] to give the key intermediate 10.
Mono-alkylation of the 1H-imidazole ring was confirmed by the presence of the
appropriate parent-ion peak in the mass spectrum (m/z 702.4080 [MþH]þ ).
Alkylation at N(1) was confirmed by 2D-HMBC analysis of 10 that showed that
C(6) H-atoms at C(6) (3.90 ppm) were coupled with C(7) (a two-bond coupling), and
with C(8) (22.6 ppm) and two imadazole C-atoms, namely C(2) (137.1 ppm) and C(5)
(117.8 ppm) (all involve three-bond coupling). A correlation between HꢀC(6) and the
imidazole C-atom C(4) was not apparent, which would be expected in the alternative
N(3)-alkylated product. Selective alkylation at N(1) suggests that this site is sterically
more accessible than N(3).
t
The Boc and Bu ester protecting groups of 10 were simultaneously removed on
treatment with TFA in CH2Cl2 to give 11, which was cyclized on treatment with EDCI
and HOAt to give the key macrocycle 4a. The mass spectrum of 4a showed the expected

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Scheme 1
a) 7, 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDCI), 1-hydroxy-7-azabenzotriazale
(HOAt), DMF, EtNiPr2 (DIPEA). b) NaH, DMF, then 9. c) CF3COOH (TFA), CH2Cl2. d) 2-(1H-7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), DIPEA, DMF. e)
LiBH4, THF. f) DessꢀMartin periodinane, CH2Cl2.
parent-ion peak at m/z 528.2830, [MþH]þ and 1H-NMR analysis showed the signals of
the three a-H-atoms at 4.64, 4.54, and 4.35 ppm due to the leucine, histidine, and
glycine fragments respectively. Reduction of the ester of 4a with LiBH4 gave alcohol 4b,
and this was oxidized under DessꢀMartin conditions to give the target macrocyclic
aldehyde 4c.
The second series of macrocycles, 5a–5c, was prepared using a similar strategy as
outlined in Scheme 2. Commercially available His-OMe (12) was coupled with Boc-
Leu-OH (13), in presence of EDCI and HOAt, to give the dipeptide 14. The 1H-
imidazole moiety of 14 was N(1)-monoalkylated with the iodide 15, under the
conditions used to prepare 10, to give 16. The formation of 16 was confirmed by mass
spectrometry (m/z 702.4044 ([MþH]þ )) and 2D-HMBC that showed that HꢀC(6)
(3.94 ppm) was coupled with C(7) (29.8 ppm), C(8) (22.6 ppm), and the 1H-imidazole
C(2) (136.7 ppm) and C(5) (117.2 ppm). Again HꢀC(6) was not coupled with the
imidazole C(4) (137.0 ppm) as would be expected if alkylation had occurred at the
alternative and more sterically hindered N(3) position.
Removal of the N-Boc and tBu ester protecting groups of 16 on treatment with TFA
in CH2Cl2 gave 17, which was cyclized on treatment with EDCI and HOAt to give the
key macrocycle 5a. The ester of 5a was reduced with LiBH4, and the resulting primary

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2477
Scheme 2
a) 13, EDCI, HOAt, DMF, DIPEA. b) NaH, DMF, then 15. c) TFA, CH2Cl2. d) HATU, DIPEA, DMF.
e) LiBH4, THF. f) DessꢀMartin periodinane, CH2Cl2. Cbz¼(Benzyloxy)carbonyl.
alcohol 5b was oxidized under DessꢀMartin conditions to give the desired macrocyclic
aldehyde 5c. 1H-NMR Analysis of the macrocyclic esters, 4a and 5a, and the
3
corresponding alcohols, 4b and 5b, in (D6)DMSO revealed J(NH,HꢀC(a)) coupling
constant >8 Hz, which suggests that their peptide backbones can adopt a b-strand
conformation [16].
The alkyl iodide 15 [17] used for alkylation of the imidazole moiety of 14 was
prepared from amine 18 by reaction with sodium nitroprusside to give 19, followed by
t
esterification with BuBr bromide according to Chevalletꢃs method [18] to give 20.
Reaction with I2 in the presence of PPh3 and 1H-imidazole then gave 15 as shown in
Scheme 3.
2.3. Assay against Calpain 2. The macrocycles 4b, 4c, 5b, and 5c were assayed against
calpain 2 using the method described in [19], and the results are compiled in Table 2.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Scheme 3
a) Sodium nitroprusside. b) tBuBr, N,N,N’-triethylbenzenemethanaminium chloride (BTEAC), K2CO3,
4-(dimethylamino)cinnamaldehyde (DMAC). c) PPh3, 1H-imidazole, I2, THF.
Table 2. Calpain 2 Assay Results for Compounds 4b and 4c, and 5b and 5c
Compound
IC50 [nm]
CAT811 (1)
30
29300
238
>50000
1000
4b
4c
5b
5c
Aldehyde 4c showed significant inhibitory activity against calpain 2 with an IC50
value of 238 nm. As shown in the docking studies discussed earlier, 4c forms the key H-
bonds that define active site binding in a b-strand conformation (see Fig. 2). It is
notable that the imidazole of 4c (and also its alcohol analog 4b) is positioned
perpendicular to and pointing away from the cleft, and as such it does not participate in
additional H-bonds with the enzyme.
The macrocyclic alcohol 4b was considerably less active than the aldehyde 4c, with
an IC50 value of 2.93 mm. Both 4b and 4c have a high propensity to adopt a b-strand
geometry based on the conformation search data given in Table 1. While the docking
studies predict that the alcohol 4b can adopt similar poses to 4c in the active site, it lacks
the aldehyde warhead that is critical for inactivation of the enzyme through the
formation of a hemithioacetal with the active-site cysteine [20]. A parallel, but less
marked difference in reactivity is observed for CAT811 (1; IC50 30 nm) and its alcohol
analog (IC50 700 nm) [1]. Docking studies show that the aryl group of CAT811 (1) is
positioned perpendicular to and pointing away from the active-site cleft, as was
observed for the imidazole of 4b and 4c.
The second macrocyclic aldehyde 5c was significantly less active with an IC50 value
of 1000 nm against calpain 2, while its corresponding alcohol 5b was devoid of activity
(IC50 >50 mm). Docking studies again show the imidazole moiety of 5b and 5c is
positioned perpendicular to and pointing away from the cleft. The poses suggest that
these two compounds do not adopt a b-strand conformation in the calpain active site,
which perhaps reflects their diminished propensity (relative to 4a and 4c, resp.) to
adopt this geometry based on the conformation search data (see Table 1). The pose for
5c shows the structure to be positioned more loosely and less deeply in the active cleft
when compared to the other compounds. It thus appears that having an imidazole

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2479
Fig. 2. Poses from molecular modelling for compounds 4b (magenta C-atoms) and 4c (yellow C-atoms).
The active site of calpain construct is displayed by surface representation.
moiety at the P1 position as in 5b and 5c is not favored for b-strand formation and
active-site binding via this normally preferred geometry. One thing is clear and that is
that a C-terminal aldehyde is required for potent inhibition.
3. Conclusions. – We presented the synthesis and biological properties of two new
series of macrocyclic peptidomimetics, 4a–4c and 5a–5c, in which the P1 and P3
residues of the peptide backbone are linked by a bridge containing a 1,4-disubstituted
imidazole moiety. The aldehyde 4c, with the imidazole moiety at P3, shows significant
inhibitory activity against calpain 2 with an IC50 value of 238 nm. The macrocyclic
aldehyde 5c with the imidazole moiety at the alternative P1 site is significantly less
active. The relative activities are linked to the ability of the component macrocycles to
mimic a b-strand geometry that is known to favor active site binding. This ability to
define a particular geometry is defined by conformational searches and docking studies
with calpain. The corresponding macrocyclis alcohols, 4b and 5b, lack the key C-
terminal aldehyde and are as such significantly less active.
We gratefully acknowledge a grant from the Foundation for Research Science and Technology of New
Zealand (LINX0205 and LINX03007), the Auckland Centre for Molecular Biodiscovery, the Australian
Research Council (ARC), and Douglas Pharmaceuticals Ltd. for student support.

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Experimental Part
General. Amino acid starting materials were purchased from GL Biochem (Shanghai) Limited.
Reagents and other chemicals were purchased from Aldrich Chemicals. TLC was performed on
1
aluminium-backed Merck Kieselgel KG60F254 silica plates. H-NMR Spectra: Inova 500 spectrometer
operating at 500 MHz or Varian Gemini 2000 (300 MHz). 13C-NMR Spectra: Varian Unity XL 300 MHz
Fourier transform spectrometer operating at 75 MHz; chemical shifts are reported in ppm referenced
relative to the residual solvent peak; coupling constants J are reported in Hz. ESI-MS: Micromass liquid
chromatography time of flight (TOF) mass spectrometer, with a probe voltage of 3200 V, temp. of 1508,
and a source temp. of 808. Direct ionization used with 10 ml of a 10 mg mlꢀ1 soln., using a carrier solvent of
50% MeCN/H2O at a flow rate of 20 ml minꢀ1. Ionization was assisted by the addition of 0.5% HCOOH.
Molecular Modelling. Conformational Search Method. Conformational searches were carried out
with MacroModel 9.1 [21] to generate an ensemble of low-energy conformers. The searches were
conducted with the MCMM method using a GB/SA water model and the OPLS2001 force field, with
3000 steps for the conformational search and up to 5000 iterations for the minimization of each generated
structure. The minimization was stopped with the default gradient convergence threshold of d 0.05 kJ/
(mol·ꢂ). The default PolakꢀRibiere conjugate gradient method was used for all minimizations.
Refinement of the Glide and Induced-Fit Model from 1KXR. The crystal structure of mini CAPN1
(pdb code 1KXR) [22] was prepared by deleting H2O molecules and ions, and mutation of Ser115 to
Cys115. This structure was minimized using the OPLS2001 force field with a GB/SA water model over
500 iterations. All residues within a 5-ꢂ distance to the calcium ions of the crystal structure were kept
frozen during this minimization. The RMSD of the minimized structure to the crystal structure was
0.96 ꢂ for the heavy atoms (C, N, O, S). The structure was refined by the Prime preparation and
refinement tool. The cysteine S of Cys115 was deprotonated.
Ligand Docking Using GLIDE. The receptor grid used in ligand docking calculation was generated
using GLIDE 4.0 [23]. The centre of the docking grid was defined as the centroid of the residues Cys115,
Gly208, Gly271, and Lys347. The midpoint of each docked ligand was set to a 12ꢁ12ꢁ12 ꢂ box. Van der
Waals radii of the receptor atoms were scaled to 1.0. Ligands were docked flexibly in extra precision
mode (XP) and with a Van der Waals scaling of 0.8. The structure output was set to write out 10 or 20
poses per ligand.
Inhibition Assay. The biological activity of the calpain inhibitors was determined by measuring the
inhibition constants (IC50) in an in vitro assay using an established assay protocol. This assay utilized a
quenched fluorogenic substrate, casein labeled with the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-
3a,4a-diaza-s-indacene-3-propanoic acid (BODIPY) [18].
Syntheses. Methyl (6S,9S,12S)-12-{[(Benzyloxy)carbonyl]amino}-9-(2-methylpropyl)-8,11-dioxo-
1,7,10,15-tetraazabicyclo[12.2.1]heptadeca-14(17),15-diene-6-carboxylate (4a). To a soln. of 11 (1.22 g,
1 equiv.) in DMF (30 ml), under N2, was added HATU (1.05 g, 1.2 equiv.) and DIPEA (0.96 ml,
2.4 equiv.), and the soln. stirred at r.t. overnight. The mixture was diluted with AcOEt (300 ml), and
washed with H2O and brine. The org. layer was dried (MgSO4) and concentrated in vacuo, and the
residue was absorbed onto Si2O, and elution with 5% MeOH in CH2Cl2 gave 4a (620 mg, 52%). White
solid. 1H-NMR (CD3OD, 500 MHz): 8.56 (s, HꢀC(16)); 7.35–7.37 (m, 5 arom. H); 7.17 (s, HꢀC(17)); 5.16
(d, J ¼ 12.2, 1 H of OCH2Ph); 5.08 (d, J ¼ 12.5, 1 H of OCH2Ph); 4.63–4.65 (m, HꢀC(9)); 4.53–4.55 (m,
HꢀC(12)); 4.34–4.37 (m, HꢀC(6)); 4.23–4.26 (m, 1 H of CH2(2)); 4.10–4.13 (m, 1 H of CH2(2)); 3.71 (s,
MeO); 3.12–3.14 (m, CH2(13)); 2.12–2.15 (m, 1 H of CH2(3)); 1.95–1.99 (m, 1 H of CH2(3)); 1.63–1.73
(m, CH2(Leu), Me2CH(Leu)); 1.51–1.54 (m, CH2(5)); 1.21–1.26 (m, 1 H of CH2(4)); 1.08–1.12 (m, 1 H
of CH2(4)); 0.94 (d, J ¼ 6.4, Me(Leu)); 0.89 (d, J ¼ 6.4, Me(Leu)). 13C-NMR (CDCl3, 75 MHz): 173.0;
172.4; 170.1; 156.1; 137.8; 137.7; 137.0; 129.0; 128.5; 128.4; 116.7; 66.1; 54.0; 52.6; 51.5; 49.4; 46.3; 41.7;
41.0; 31.0; 30.6; 28.1; 24.7; 23.2; 23.0; 21.3. HR-ESI-MS: 528.2830 ([MþH]þ , C27H38N5O6þ ; calc.
528.2822).
Benzyl [(6S,9S,12S)-6-(Hydroxymethyl)-9-(2-methylpropyl)-8,11-dioxo-1,7,10,15-tetraazabicy-
clo[12.2.1]heptadeca-14(17),15-dien-12-yl]carbamate (4b). To a soln. of 4a (200 mg) in anh. THF
(10 ml) under N2 was added LiBH4 (17.4 mg), and the mixture was stirred at r.t. overnight. The reaction
was quenched by the addition of MeOH (2 ml), and the solvent was removed in vacuo. The residue was

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2481
dissolved in AcOEt (2ꢁ10 ml), washed with dist. H2O (5 ml), and the aq. layer was further extracted
with CHCl3 (2ꢁ5 ml), and the combined org. phase was washed with brine, dried (MgSO4), and
concentrated in vacuo to give a crude product that was recrystallized from EtOH to give 4b (185 mg,
98%). White solid. 1H-NMR (DMSO, 500 MHz): 8.15 (s, HꢀC(16)); 7.95 (d, J¼8.6, NH); 7.78 (d, J ¼ 9.0,
NH); 7.50 (d, J ¼ 7.1, NH); 7.29–7.35 (m, 5 arom. H); 7.13 (s, HꢀC(17)); 4.97–5.02 (m, OCH2Ph); 4.59–
4.61 (m, CH2OH); 4.55–4.58 (m, HꢀC(12)); 4.27–4.29 (m, HꢀC(9)); 3.94–3.97 (m, CH2(2)); 3.77–3.81
(m, HꢀC(6)); 3.21–3.25 (m, 1 H of CH2OH); 3.14–3.18 (m, 1 H of CH2OH); 3.07–3.10 (m, 1 H of
CH2(13)); 2.65–2.67 (m, 1 H of CH2(13)); 1.85–1.97 (m, CH2(3)); 1.53–1.55 (m, 1 H of CH2(5)); 1.30–
1.45 (m, CH2(Leu), CH(Leu)); 1.15–1.18 (m, 1 H of CH2(5)); 1.05–1.09 (m, 1 H of CH2(4)); 0.94–0.96
(m, 1 H of CH2(4)); 0.80–0.86 (m, 2 Me(Leu)). 13C-NMR (CDCl3, 75 MHz): 171.7; 160.6; 156.1; 137.9;
137.8; 134.0; 129.0; 128.4; 128.3; 118.9; 66.0; 64.8; 51.7; 53.1; 51.7; 48.0; 42.3; 41.0; 30.5; 28.7; 27.1; 24.8;
23.2. HR-ESI-MS: 500.2870 ([MþH]þ , C26H38N5O5þ ; calc. 500.2873).
Benzyl [(6S,9S,12S)-6-Formyl-9-(2-methylpropyl)-8,11-dioxo-1,7,10,15-tetraazabicyclo[12.2.1]hepta-
deca-14(17),15-dien-12-yl]carbamate (4c). Compound 4b (30 mg) was suspended in anh. CH2Cl2 (20 ml)
under N2. DessꢀMartin periodinane (76 mg) was added to the soln. with vigorous stirring under N2. The
mixture was stirred for 1 h at r.t., and the reaction was then quenched with sat. aq. NaHCO3 soln.
containing Na2S2O5 (10%). The aq. layer was extracted with CH2Cl2 (3ꢁ ), and the combined org. phase
was washed with H2O and brine, and dried (Na2SO4). The solvent was evaporated in vacuo, and the crude
product was purified by CC (Si2O; MeOH/CH2Cl2 1:99) to give 4c (14 mg, 48%). White solid. 1H-NMR
(300 MHz, CDCl3): 9.54 (s, 1 H); 7.47–7.29 (m, 6 H); 6.64 (s, 1 H); 6.38 (d, J¼8.7, 1 H); 6.23 (d, J¼8.0,
1 H); 6.17 (d, J¼7.3, 1 H); 5.12 (q, J¼12.2, 2 H); 4.63 (dd, J¼21.2, 11.1, 2 H); 4.30 (dd, J¼12.6, 5.3, 1 H);
4.09–3.92 (m, 1 H); 3.92–3.77 (m, 1 H); 3.08–2.91 (m, 2 H); 2.16–1.86 (m, 3 H); 1.85–1.50 (m, 4 H);
1.18–1.00 (m, 2 H); 0.93 (d, J¼4.2, 3 H); 0.88 (d, J¼3.7, 3 H). 13C-NMR (75 MHz, (CD3)2SO): 201.0;
172.2; 171.6; 157.7; 136.8; 135.1; 128.3; 127.8; 127.7; 118.6; 65.6; 55.7; 54.3; 52.7; 51.0; 41.0; 35.1; 29.0; 24.1;
22.7; 22.2; 13.9. HR-ESI-MS: 498.27140 ([MþH]þ , C27H38N5O6þ ; calc. 498.2716).
Methyl (6S,9S,12S)-6-{[(Benzyloxy)carbonyl]amino}-9-(2-methylpropyl)-7,10-dioxo-1,8,11,15-tet-
raazabicyclo[12.2.1]heptadeca-14(17),15-diene-12-carboxylate (5a). To a soln. of 16 (430 mg) in CH2Cl2
(20 ml), under N2, was added TFA (8 ml), and the soln. was stirred at r.t. for 3 h. The solvent was
removed in vacuo and the residue dissolved in toluene, and the soln. was re-evaporated to give 17 that
was used without further purification. DMF (15 ml), HATU (285 mg, 0.75 mmol, 1.2 equiv.), and
DIPEA (260 ml, 1.49 mmol, 2.4 equiv.) were added, and the soln. was stirred at r.t. overnight. AcOEt was
added, and the org. layer was washed successively with aq. HCl (1m), sat. aq. NaHCO3, and brine. The
org. phase was dried (MgSO4), and the solvent was removed in vacuo and the residue absorbed onto
1
Si2O, and elution with MeOH/CH2Cl2 1:20 gave 5a (167 mg, 51%). White solid. H-NMR (CD3OD,
500 MHz): 7.57 (s, HꢀC(16)); 7.29–7.33 (m, 5 arom. H); 6.81 (s, HꢀC(17)); 5.06 (s, OCH2Ph); 4.84–4.86
(m, HꢀC(12)); 4.52–4.54 (m, HꢀC(6)); 4.06 (dd, J ¼ 4.2, 9.7, HꢀC(9)); 3.97–3.99 (m, CH2(2)); 3.76 (s,
MeO); 3.19 (dd, J ¼ 2.6, 15.0, 1 H of CH2(13)); 2.78–2.82 (m, 1 H of CH2(13)); 1.78–1.81 (m, 1 H of
CH2(3)); 1.55–1.70 (m, 1 H of CH2(3), 1 H of CH2(4), HꢀC(Leu), CH2(Leu)); 1.50–1.55 (m, CH2(5));
1.09–1.11 (m, 1 H of CH2(4)); 0.96 (d, J ¼ 6.4, Me(Leu)); 0.89 (d, J ¼ 6.4, Me(Leu)). 13C-NMR (CDCl3,
75 MHz): 172.7; 172.3; 171.8; 156.7; 137.0; 136.8; 136.2; 128.3; 127.8; 127.7; 117.5; 66.4; 54.1; 51.8; 51.4;
51.2; 45.8; 41.1; 31.3; 30.3; 28.3; 24.5; 21.8; 21.6; 21.3. HR-ESI-MS: 528.2799 ([MþH]þ , C27H38N5O6þ ;
calc. 528.2822).
Benzyl [(6S,9S,12S)-12-(Hydroxymethyl)-9-(2-methylpropyl)-7,10-dioxo-1,8,11,15-tetraazabicy-
clo[12.2.1]heptadeca-14(17),15-dien-6-yl]carbamate (5b). To a suspension of 5a (160 mg, 1 equiv.) in
dry THF (5 ml) was added MeOH (2 equiv., 24 ml), followed by a soln. of LiBH4 in THF (2.0m; 0.3 ml,
2 mol-equiv.). The resulting mixture was stirred overnight at r.t. and the THF was evaporated in vacuo.
The residue was diluted with AcOEt, and the soln. was washed with aq. HCl (1m), H2O, dried (MgSO4),
and the solvent was removed in vacuo. The crude product was purified by CC (Si2O; MeOH/CH2Cl2
1:10) to give 5b (116 mg, 72%). White solid. 1H-NMR (CD3OD, 500 MHz): 8.44 (s, HꢀC(16)); 7.29–7.34
(m, 5 arom. H); 7.08 (s, HꢀC(17)); 5.06 (s, OCH2Ph); 4.40–4.42 (m, HꢀC(6)); 4.21–4.23 (m, HꢀC(12));
4.11–4.15 (m, CH2(2)); 4.06 (dd, J ¼ 4.7, 10.2, HꢀC(9)); 3.58–3.60 (m, CH2OH); 2.98 (d, J ¼ 14.1, 1 H of
CH2(13)); 2.65–2.68 (m, 1 H of CH2(13)); 1.85–1.90 (m, 1 H of CH2(3)); 1.71–1.73 (m, 1 H of CH2(3),
1 H of CH2(4)); 1.60–1.70 (m, 1 H of CH2(4), HꢀC(Leu), 1 H of CH2(Leu)); 1.54–1.59 (m, 1 H of

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
CH2(5)); 1.47–1.51 (m, 1 H of CH2(5)); 1.18–1.21 (m, 1 H of CH2(Leu)); 0.89–0.93 (m, 2 Me(Leu)).
13C-NMR (CDCl3, 75 MHz): 171.9; 171.2; 160.6; 156.0; 138.8; 137.8; 129.0; 128.5; 128.4; 123.1; 116.6;
65.9; 64.7; 51.3; 49.7; 47.7; 42.6; 40.8; 30.7; 24.8; 23.3; 23.1; 22.2. HR-ESI-MS: 500.2850 ([MþH]þ
,
C27H38N5Oþ6 ; calc. 528.2873).
Benzyl [(6S,9S,12S)-12-Formyl-9-(2-methylpropyl)-7,10-dioxo-1,8,11,15-tetraazabicyclo[12.2.1]hep-
tadeca-14(17),15-dien-6-yl]carbamate (5c). Compound 5b (25 mg) was suspended in anh. CH2Cl2
(20 ml), under N2, and DessꢀMartin periodinane (63 mg) was added with vigorous stirring. The mixture
was stirred for 1 h, and then the reaction was quenched with sat. aq. NaHCO3 soln. containing Na2S2O5
(10%). The aq. layer was extracted with CH2Cl2, and the org. phase was washed with H2O and brine, and
dried (Na2SO4). The solvent was removed in vacuo, and the residue absorbed onto Si2O, and elution with
MeOH/CH2Cl2 1:99 gave 5c (9.9 mg, 40%). White solid. 1H-NMR (300 MHz, CDCl3): 9.71 (s, 1 H); 7.34
(m, J¼23.4, 4.8, 6 H); 6.83 (d, J¼6.0, 1 H); 6.67 (s, 1 H); 6.30 (d, J¼7.2, 1 H); 5.53 (d, J¼7.2, 1 H); 5.10
(d, J¼23.5, 2 H); 4.80 (dd, J¼11.9, 8.0, 1 H); 4.46 (dd, J¼14.8, 8.3, 1 H); 4.09 (dd, J¼11.3, 7.7, 1 H); 3.89
(m, 2 H); 3.39 (dd, J¼15.2, 4.1, 1 H); 2.81 (dd, J¼14.9, 8.9, 1 H); 2.17–1.93 (m, 2 H); 1.90–1.42 (m,
4 H); 1.40–1.03 (m, 3 H); 0.96–0.77 (m, 6 H).13C-NMR (75 MHz, CDCl3): 198.3; 171.9; 170.4; 155.6;
137.0; 136.6; 135.9; 128.5; 128.2; 128.0; 116.9; 67.0; 58.5; 54.3; 51.6; 46.3; 41.6; 32.0; 29.7; 24.7; 22.7; 22.1;
14.1. HR-ESI-MS: 498.27110 ([MþH]þ , C27H38N5O6þ ; calc. 498.2716).
tert-Butyl N-[(Benzyloxy)carbonyl]-l-histidyl-l-leucinate (8). To a soln. of N-Cbz histidine (¼ N-
[(benzyloxy)carbonyl]-l-histidine; 6; 10 g, 1 equiv.) and Leu-OtBu (¼tert-butyl l-leucinate; 7; 7.75 g,
1 equiv.) in anh. DMF (100 ml), under N2, were added EDCI (8 g, 41.5 mmol, 1.2 equiv.), HOAt (5.65 g,
41.5 mmol, 1.2 equiv.), and DIPEA (14.5 ml, 2.4 equiv.), and the mixture was stirred at r.t. overnight. The
soln. was diluted with AcOEt (500 ml), washed with dist. H2O (2ꢁ50 ml), and brine (2ꢁ50 ml), and
dried (MgSO4). The org. layer was evaporated in vacuo, and the product absorbed onto Si2O, and elution
with MeOH in CH2Cl2 1:10 gave 8 (14.1 g, 89%). White solid. 1H-NMR (CDCl3, 500 MHz): 7.50 (app. d,
J ¼ 5.8, NH); 7.43 (s, CH(2His)); 7.25 (m, 5 arom. H); 6.74 (s, CH(5His)); 5.08 (s, OCH2Ph); 4.49 (m,
CH(aHis)); 4.34–4.37 (m, CH(2Leu)); 3.10 (dd, J ¼ 4.2, 14.5, 1 H of CH2(bHis)); 3.01 (dd, J ¼ 6.1, 14.8, 1 H
t
of CH2(bHis)); 1.45–1.54 (3 H, m, CH(4Leu), CH2(3Leu)); 1.43 (s, Bu); 0.82–0.85 (m, 2 Me(5Leu)).
13C-NMR (CDCl3, 75 MHz): 172.6; 171.5; 156.7; 136.4; 135.3; 128.7; 128.4; 128.2; 104.9; 82.3; 67.2; 54.8;
52.0; 41.2; 28.2; 24.9; 23.0; 22.0. HR-ESI-MS: 459.2600 ([MþH]þ , C24H35N4O5þ ; calc. 459.2607).
tert-Butyl N-[(Benzyloxy)carbonyl]-1-((5S)-5-{[(tert-butoxy)carbonyl]amino}-6-methoxy-6-oxo-
hexyl)-l-histidyl-l-leucinate (10). NaH (0.92 g, 60% suspension, 3 equiv.) was washed with dry hexane
(10 mlꢁ3) and dried under vacuum. Dipeptide 8 (3.5 g, 1 equiv.) in DMF (30 ml) was added under N2 at
ꢀ158, and the mixture was stirred for 30 min at ꢀ158, followed by the addition of methyl N-[(tert-
butoxy)carbonyl]-6-iodo-l-norleucinate (9; 5.67 g, 2 equiv.) [14]. The temp. was raised to ꢀ58 (ice-salt),
and the mixture was stirred for 4 h. The reaction was quenched by the addition of MeOH (5 ml), and the
solvent was removed under reduced pressure. The residue was extracted with CHCl3 (4ꢁ50 ml) and
dried (MgSO4), and the solvent was removed under reduced pressure. The residue was absorbed onto
Si2O, and elution with AcOEt/petroleum ether 6 :1 gave 10 (2.66 g, 50%). Yellow oil. 1H-NMR (CD3OD,
500 MHz): 7.58 (s, CH(2His)); 7.29–7.32 (m, 5 arom. H); 6.91 (s, CH(5His)); 5.04 (s, OCH2Ph); 4.38–4.41
(m, CH(aHis)); 4.31–4.32 (m, CH(2Leu)); 4.08–4.12 (m, CH(10His)); 3.90–3.93 (m, CH2(6His)); 3.68 (s,
MeO); 3.03 (dd, J ¼ 4.8, 14.9, 1 H of CH2(bHis)); 2.84 (dd, J ¼ 9.2, 14.9, 1 H of CH2(bHis)); 1.72–1.80 (m,
t
t
CH2(7His), HꢀC(4Leu)); 1.62–1.65 (m, CH2(3Leu)); 1.57–1.62 (m, CH2(9His)); 1.45 (s, Bu); 1.43 (s, Bu);
0.94 (d, J ¼ 6.4, Me(5Leu)); 0.89 (d, J ¼ 6.4, Me(5Leu)). 13C-NMR (CDCl3, 75 MHz): 173.6; 172.8; 172.0;
156.9; 137.0; 136.8; 136.7; 128.3; 127.8; 127.7; 117.4; 110.4; 81.4; 79.4; 66.4; 55.0; 53.6; 51.7; 51.4; 46.7; 40.4;
30.9; 30.5; 30.2; 27.5; 27.1; 24.7; 22.6; 22.1; 20.7. HR-ESI-MS: 702.4080 ([MþH]þ , C36H56N5O9þ ; calc.
702.4078).
(2S)-2-({1-[(5S)-5-Ammonio-6-methoxy-6-oxohexyl]-N-[(benzyloxy)carbonyl]-l-histidyl}amino)-
4-methylpentanoate (11). A soln. of 10 (1.6 g, 2.3 mmol) in CH2Cl2 (50 ml) and TFA (30 ml) was stirred,
under N2, at r.t. for 3 h. The solvent was removed in vacuo, the residue was dissolved in toluene, and the
soln. was re-evaporated to give 11 (1.22 g, 99%) as a colorless oil which was used without further
purification. 1H-NMR for major product from the mixture (CD3OD, 500 MHz): 8.78 (s, CH(2His)); 7.40 (s,
CH(5His)); 7.34–7.36 (m, 5 arom. H); 5.08 (s, OCH2Ph); 4.49–4.52 (m, CH(aHis)); 4.40–4.43 (m,
CH(2Leu)); 4.15–4.18 (m, CH2(6His)); 4.03–4.06 (m, CH(10His)); 3.83 (s, MeO); 3.29 (dd, J ¼ 6.8, 15.4, 1 H

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2483
of CH2(bHis)); 3.04 (dd, J ¼ 7.2, 15.6, 1 H of CH2(bHis)); 1.85–2.00 (CH2(9His), CH2(8His)); 1.62–1.75 (m,
CH(4Leu), CH2(3Leu)); 1.41–1.49 (m, CH2(7His)); 0.95 (d, J ¼ 6.3, Me(5Leu)); 0.91 (d, J ¼ 6.1, Me(5Leu)).
13C-NMR (CD3OD, 75 MHz): 174.8; 171.1; 169.6; 158.5; 136.9; 134.5; 130.4; 127.8; 125.1; 120.2; 114.1;
66.7; 53.8; 52.5; 51.0; 45.8; 40.1; 29.7; 29.2; 27.6; 24.8; 22.2; 21.7; 20.5; 20.3. HR-ESI-MS: 546.2914 ([Mþ
H]þ , C27H40N5Oþ7 ; calc. 546.2928).
Methyl N-[(tert-Butoxy)carbonyl]-l-leucyl-l-histidinate (14). To a stirred soln. of the N-[(tert-
butoxy)carbonyl]-l-leucine (13; 11.1 g, 44.5 mmol, 1.0 equiv.) in dry DMF (100 ml), His-OMe (¼ methyl
l-histidinate; 12; 10.7 g, 44.5 mmol, 1 equiv.), DIPEA (18.6 ml, 107 mmol, 2.4 equiv.), EDCI (10.2 g,
53.4 mmol, 1.2 equiv.), and HOAt (7.27 g, 53.4 mmol, 1.2 equiv.) were added. The soln. was stirred at r.t.
overnight, diluted with AcOEt, and the separated org. layer was washed twice with aq. HCl (1m), sat. aq.
NaHCO3, and brine. The org. layer was dried (MgSO4), and the solvent was removed in vacuo. The
residue was absorbed onto Si2O, and elution with MeOH/CH2Cl2 1:10 gave 14 (14.4 g, 85%). White solid.
1H-NMR (CDCl3, 500 Hz): 7.48 (s, CH(2His)); 7.36 (s, NH); 6.71 (s, CH(5His)); 5.26 (d, J ¼ 7.2, NH); 4.72–
4.76 (m, CH(aHis)); 4.04–4.05 (m, CH(2Leu)); 3.65 (s, MeO); 3.07–3.10 (m, CH2(bHis)); 1.61–1.67 (m,
CH(4Leu)); 1.53–1.60 (m, 1 H of CH2(3Leu)); 1.41–1.48 (m, 1 H of CH2(3Leu)); 1.38 (s, tBu); 0.86–0.89 (m,
2 Me(5Leu)). 13C-NMR (CDCl3, 75 MHz): 173.2; 171.6; 156.3; 135.6; 130.7; 120.0; 80.4; 53.7; 52.9; 52.6;
41.4; 28.5; 24.8; 23.2; 23.0; 22.2. HR-ESI-MS: 383.2279 ([MþH]þ , C18H31N4O5þ ; calc. 383.2294).
Methyl N-[(tert-Butoxy)carbonyl]-l-leucyl-1-[(5S)-5-{[(benzyloxy)carbonyl]amino}-6-(tert-bu-
toxy)-6-oxohexyl]-l-histidinate (16). NaH (358 mg, 60% suspension, 8.9 mmol, 3 equiv.) was washed
with dry hexane (3ꢁ10 ml) and dried under vacuum. The mixture cooled was to ꢀ158, and a soln. of 14
(1.14 g, 3.0 mmol, 1 equiv.) in DMF (30 ml) was added. The mixture was stirred for 30 min at this temp.
and then tert-butyl N-[(benzyloxy)carbonyl]-6-iodo-l-norleucinate (15; 2.66 g, 5.96 mmol, 2 equiv.) [17]
was added. The temp. was raised to ꢀ58 (ice-salt), and the mixture was stirred for 4 h. The reaction was
quenched with MeOH (5 ml), and the solvent was removed under reduced pressure. The residue was
extracted with CHCl3 (4ꢁ50 ml), and the combined org. phase was dried (MgSO4). The solvent was
removed under reduced pressure, and the residue was absorbed onto Si2O, and elution with MeOH/
CH2Cl2 1:20 gave 16 (860 mg, 41%). Yellow oil. 1H-NMR (CD3OD, 500 MHz): 7.52 (s, CH(2His)); 7.32–
7.37 (m, 5 arom. H); 6.93 (s, CH(5His)); 5.09–5.11 (m, OCH2Ph); 4.85–4.87 (m, CH(5His)); 4.07–4.10 (m,
CH(2Leu)); 4.00–4.03 (m, CH(10His)); 3.92–3.95 (m, CH2(6His)); 3.67 (s, MeO); 3.05 (dd, J ¼ 4.9, 14.8, 1 H
of CH2(bHis)); 2.82–2.85 (m, 1 H of CH2(bHis)); 1.74–1.80 (m, CH2(7His), 1 H of CH2(9His)); 1.63–1.69
t
t
(m, 1 H of CH2(9His), CH(4Leu)); 1.45–1.50 (m, CH2(3Leu)); 1.43 (s, Bu); 1.42 (s, Bu); 1.32–1.39 (m,
CH2(8His)); 0.91–0.95 (m, 2 Me(5Leu)). 13C-NMR (CD3OD, 126 MHz): 174.4; 172.1; 171.9; 157.4; 156.5;
137.0; 136.7; 128.3; 127.8; 127.7; 117.2; 81.5; 79.3; 66.4; 54.8; 53.2; 52.6; 51.5; 46.6; 40.9; 31.0; 30.3; 29.8;
27.6; 27.0; 24.6; 22.6; 22.3; 20.7. HR-ESI-MS: 702.4044 ([MþH]þ , C36H56N5O9þ ; calc. 702.4078).
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Received September 12, 2012