A combinatorial approach towards pharmaceutically relevant cyclic peptides
Springer, J.
Publication date
2008
Link to publication
Citation for published version (APA):
Springer, J. (2008). A combinatorial approach towards pharmaceutically relevant cyclic
peptides.
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Introduction
Small cyclic peptides constitute an important class of compounds.
1-4Naturally
occurring cyclic peptides often possess potent biological activities.
4Because of their restricted
conformational flexibility they often show enhanced receptor selectivity.
5-7Compared to their
linear counterparts, cyclic peptides are more resistant against enzymatic degradation and show
a better membrane permeability improving their bioavailability.
8,9Besides pharmaceutical
research, cyclic peptides find application in fields such as materials science
10,11and
catalysis.
12Despite their interesting properties, especially 7-15-membered cyclic peptides find limited
application mainly because of their difficult synthesis. Ring-closure is often hampered by the
backbone amide bonds possessing a strong
π-character with preferentially a transoid
conformation leading to an extended structure with the mutually reactive terminal groups far
apart.
13As has been outlined in the previous chapter, traditional lactamization strategies often
lead to low yields.
14,15Other strategies have been developed based on auxiliaries,
16-19cyclizations on solid phase,
20intramolecular Staudinger ligation reactions,
21chemoenzymatic
cyclizations
22-24and on the site-isolation effect exerted within dendrimeric nanoreactors,
25but
each application is still limited to specific sequences.
The smallest cyclic peptides that are difficult to address via classical direct lactamization are
the seven-membered bis(lactams) (homodiketopiperazines), made up by a combination of an
α and β amino acid.
26The difficulties in the synthesis of such cyclic peptides have been
illustrated by linear head-to-tail cyclization of several precursors leading to the
homodiketopiperazine cyclo-[Phe
−βAla] 2 (Scheme 2.1).
19Both linear precursors 1a and 4a
did not result in the formation of any cyclic product using the classical cyclization conditions.
N-Benzylation of the internal amide bond of both starting materials leading to precursors 1b
and 4b did affect the yield for the cyclization, leading to 11% and 30% yield of the cyclized
products 3a and 3b respectively, depending on the closure site. N-Benzylation of the
intermediate amide bond shows the principle of shifting of the transoid to cisoid ratio of this
amide bond, but the yields are still low and sequence dependent.
Scheme 2.1 Synthesis of 1,4-diazepine-2,5-diones by direct cyclization.
H2N N O R1 CO2H Ph N N O R1 Ph O R2 H2N Ph O N R2 CO2H 1a, R1 = H 1b, R1 = Bn 4a, R2 = H 4b, R2 = Bn 2, R1 = H, R2 = H 3a, R1 = Bn, R2 = H 3b, R1 = H, R2 = Bn EDCI, HOBt DMF, CH2Cl2 10-3 M EDCI, HOBt DMF, CH2Cl2 10-3 M
The natural homodiketopiperazines TAN-1057C 7a and TAN-1057D 7b were isolated from
Flexibacter sp. PK-74 and PK-176 and show potent antibacterial activity against
methicillin-resistent Staphylococcus aureus (MRSA).
27The synthesis has been described by means of
TBTU-mediated cyclization of the appropriate intermediate 5 to obtain the
homodiketopiperazine 6 in moderate 43% yield (Scheme 2.2) making use of an N-alkylated
internal amide bond.
Scheme 2.2 Synthesis of TAN-1057C and D by head-to-tail cyclization.
CbzHN N NH Cbz BocHN N O Me CO2tBu H PhtHN CbzHN N NH Cbz HN N O H NHPht Me O H2N N H NH HN N O H N H Me O H N HN NH2 O 1) TFA 2) TBTU 43% + H2N N H NH HN N O H N H Me O H N HN NH2 O TAN-1057C 7a 5 6 TAN-1057D 7b
The synthesis of several 1,4-diazepine-2,5-diones 9a-c in solution has been described by
Riche et al. by means of a classical head-to-tail cyclization of the linear precursors 8a-c
(Scheme 2.3).
28,29Bop mediated cyclization led to the homodiketopiperazines in moderate
yields of 41-68%. In this case, alkylation of the intermediate amide bond also proved to be
necessary.
Scheme 2.3 Synthesis of substituted diazepinediones by Riche et al..
N CO2H R2 R1 O H2N MeO2C MeO2C TFA BOP, DIPEA CHCl3, -30 οC 41-68% N H N MeO2C MeO2C O R1 R2 O 8a, R1 = Bn, R2 = H 8b, R1 = Bn, R2 = CH 3 8c, R1 = CH 2Ph-2,4-(OMe)2, R2 = Bn 9a-c
For a combinatorial access towards these molecules several strategies have been developed
using immobilization on resins. These ‘on resin’ cyclizations also benefit from the so-called
pseudo-dilution effect exerted within the resin during the cyclization, favouring the formation
of monomeric cyclization products. The solid phase synthesis of several substituted
1,4-diazepine-2,5-diones has been described by means of a backbone amide linkage (Scheme 2.4).
The reductive amination of the first amino acid on the resin 10, coupling and subsequent
deprotection of the second amino acid led to the desired linear peptides 11, which could be
cyclized on the solid support by treatment with sodium methoxide in a mixture of NMP and
methanol to obtain the homodiketopiperazines 12. Cyclization was further enhanced due to
the internal tertiary amide bond. Although side reactions were observed both during
cyclization and cleavage and some of the products were not isolated, several
homodiketopiperazines could be made. In a similar strategy by Martinez et al. cyclization on
the resin was conducted by basic treatment of the amine/benzyl ester linear peptide on the
resin. However, only one successful example was shown.
Scheme 2.4 Synthesis of diverse 1,4-diazepane-2,5-dione by backbone amide linkage (BAL)
strategy.
H N O O CHO MeO OMe H N O O MeO OMe N CO2Et R1 O H2N R2 H N O O MeO OMe N R1 O NH R2 O 0.3M NaOMe in NMP/MeOH (4:1) 10 11 12In a strategy by Houghten et al. (Scheme 2.5) the linear peptide 14 was build up on the solid
phase via attachment of the aspartic acid side chain. The HATU mediated cyclization of the
amine of the second amino acid to the side chain of aspartic acid resulted in formation of the
1,4-diazepane-2,5-dione 15. With this method 40 different diazepines were made with purities
ranging from 15-78%.
Scheme 2.5 Cyclization via the side chain of aspartic acid.
H N O NHFmoc CO2tBu H N O N tBuO2C R1 O H N R2 R3 HN O N R1 O N R2 R3 O 13 14 15
However, both amide bonds had to be alkylated for a successful cyclization, by-products were
observed from unsuccessful couplings, and the method was limited to aspartic acid. Similar to
this, Weichsel et al. reported the synthesis of a library of 2,700 compounds by cyclization
with DPPA, although individual products were not isolated and purities were estimated from
HPLC traces.
The synthesis of 1,4-diazepine-2,5-diones has also been described by cyclization under
Mitsunobu conditions (Scheme 2.6). In this case, a linear serine-containing dipeptide 17 was
assembled on the solid support via a hydroxylamine resin 16. Cyclization of the hydroxamate
under Mitsunobu conditions led to the desired homodiketopiperazines 18, although forcing
Scheme 2.6 Synthesis of 1,4-diazepine-2,5-dione by Mitsunobu conditions.
O NH2 O N H O H N O Ph NHFmoc OH O N O NH O Ph FmocHN DIAD, PPh3 DMF, MW, 60 W 16 17 18The synthesis of 1,4-diazepine-2,5-diones has also been described by means of an
intramolecular Staudinger ligation (Scheme 2.7).
21Coupling of the borane-protected auxiliary
to azido acid dipeptides resulted in the formation of the linear cyclization precursors 19 and
20, after which the intramolecular Staudinger ligation could be effected after liberation of the
phosphane. The 1,4-diazepine-2,5-dione 21 was obtained from both linear precursors in
reasonable yield.
Scheme 2.7 Synthesis of 1,4-diazepine-2,5-dione by intramolecular Staudinger ligation.
N3 Ph N H O S O PPh2 BH3 N Ph NH O S O Ph2P HN Ph NH O O dabco H2O, 35% N3 H N S O PPh2 BH3 O Ph N NH S O Ph2P O Ph dabco H2O, 29% 19 20 2
Besides the direct lactamization strategies described above, these compounds could also be
accessed by an auxiliary mediated approach based on a concept developed by Meutermans et
al. (Route B, Scheme 2.8).
16The auxiliary is incorporated at the N-terminus of the linear
precursor 27, facilitating the cyclization forming first the macrolactone 25, which collapses in
a ring-contractive O
→N acyl shift providing the desired lactams 26. However, from several
sequences removal of the auxiliary resulted in a N
→O acyl shift back to the lactones, driven
by strain relief. In these cases N-alkylation of the intermediate amide bonds was necessary.
Based on the method developed by Meutermans, an auxiliary-based cyclization method was
developed in our group.
19In contrast to Meutermans who adds the auxiliary 21 to the
N-terminus of the linear precursor, in this strategy a salicylaldehyde-derived auxiliary 23 is
incorporated within the backbone of a linear peptide 24 (Route C, Scheme 2.8). A
macrolactamization reaction, facilitated by a combination of the templating effect of the
auxiliary and an extension of the backbone by four atoms, resulted in the formation of lactam
25. Liberation of the N-Boc protected amine induced a ring-contractive O
→N acyl shift
providing the target strained lactam 26. Final removal of the auxiliary residue gave the lactam
28.
In this study it was found that introduction of a sterically demanding substituent flanking the
aryl ester (Scheme 2.8, 23 R
1= H, R
2= OtBu) was necessary to prevent premature
intermolecular aminolysis during the macrolactamization step.
19A drawback of this auxiliary,
however, is the harsh conditions required for removal of its remainings after the final ring
contraction step liberating the lactam 28. More importantly, the sterically demanding
OtBu-substituent prevented incorporation of two
α-substituted amino acids flanking the auxiliary,
thereby severely limiting the synthetic scope of the target compounds.
Scheme 2.8 General strategy for the synthesis of bis(lactams).
CO2PG NHPG HO2C H N O H2N CO 2PG NH N O O H R2 R1 O NH HN O O NH N O O HO R2 R1 O R2 R1 BocN NHPG H2N CO2PG O + C 1 H N O H N CO2H O2N OH A 8 i-iii iv, iii R3 R3 macrolactonization macro-lactamization v, vi iv CHO OH R2 R1 CHO OH NO2 B vii i 3 5 6 7 2 ring contraction
Reaction conditions: (i) reductive amination; (ii) (Boc)2O; (iii) couple; (iv) PG-removal; (v) TFA; (vi) base; (vii)
auxiliary removal
In this chapter, we present 2-hydroxy-3-isopropoxy-4-methoxybenzaldehyde 31 as an
improved auxiliary overcoming these problems as will be shown by the synthesis of several
substituted seven- and eight-membered bis(lactams).
2.2
Development and synthesis of the different auxiliaries
To avoid racemization in peptide synthesis, removal of protective groups is preferably
done by treatment with strong acid. It was anticipated that incorporation of a methoxy
substituent on the para position with respect to the aldehyde on the auxiliary (i.e. R
1= OMe,
Scheme 2.8) should render the benzylic amide bond susceptible for acidolytic cleavage.
30To
reduce the steric hindrance of substituent R
2such that it prevents premature aminolysis of the
phenolic ester during the macrolactamization step but still allowing the introduction of two
α-substituted amino acids we chose for replacement of the tBu group by a TBS group (auxiliary
32) and an isopropyl group (auxiliary 31) (Scheme 2.9).
22 23 24 25 26 27 28 21
Scheme 2.9 Synthesis of the auxiliaries 31 and 32.
H O OMe OMe MeO 30 BCl3, CH2Cl2 80% NaH, iPrBr KI, DMSO 38% H O OH OH MeO H O OH OiPr MeO 31 29 H O OH OTBS MeO H O OMOM OTBS MeO H O OMOM OH MeO H O OMOM OiPr MeO 32 35 33 34 HCl, MeOH 95% K2CO3, iPrBr KI, DMF 86% TBDMSCl, imidazole, DMF 97% NaH, MOMCl DMF 99% TBAF, THF 79%The synthesis of auxiliaries 31 and 32 started from commercially available
2,3,4-trimethoxybenzaldehyde 29. Selective BCl
3mediated demethylation of 29 resulted in the
formation of diol 30.
31From this diol both auxiliaries could be obtained by two distinct
routes. In the first route, direct alkylation with isopropyl bromide resulted in a mixture
containing auxiliary 31
32,33and the expected by-products resulting from alkylation on the
ortho position and alkylation on both hydroxyl groups. From this mixture 31 could be isolated
easily although in a moderate 38% yield.
In a second route, the alcohol flanking the methoxy group was selectively functionalized with
a tert-butyldimethylsilyl group
34to provide auxiliary 32. Auxiliary 32 could also serve as
starting material for auxiliary 31. Protection of the second hydroxyl group with a
MOM-group
35and TBS-group removal
36furnished compound 34. Alkylation with isopropyl
bromide
33and removal of the MOM-group by treatment with conc. HCl in methanol
37resulted in the formation of the auxiliary 31 in a good 62% overall yield from 30.
2.3
Evaluation of the TBS-substituted auxiliary
As a model target compound to test the methodology
(S)-3-benzyl-[1,4]-diazepine-2,5-dione 2 was chosen (Scheme 2.10). To date, this seven-membered bis(lactam) could not be
ring-closed via direct head-to-tail cyclization conditions at both possible ring-closure sites
(Scheme 2.1).
The synthesis of the linear cyclization precursor peptides was accomplished by reductive
amination of auxiliary 32 with H–
βAla–OBn (Scheme 2.10), followed by N-Boc protection of
the resulting secondary amine to give phenol 37 in an overall yield of 76%. Consecutive
EDCI/DMAP mediated esterification of the phenol with N-Cbz–Phe–OH provided the
protected linear precursor peptide 38 in a moderate yield of 45%.
Scheme 2.10 Synthesis of 1,4-diazepine-2,5-dione with auxiliary 32.
OH OTBS MeO H O OH OTBS MeO N Boc CO2Bn O OTBS MeO N Boc CO2Bn O NHCbz Ph N OMe OTBS OH NH O Ph O HN H N O O Ph 1) H−βAla−OBn, Na2SO4, THF then NaBH(OAc)3 2) (Boc)2O, CH2Cl2 N-Cbz−Phe−OH EDCI, DMAP 45% 1) H2, Pd/C 2) EDCI, HOBt 3) TFA, CH2Cl2 4) NaHCO3 29% over 3 steps TFA, anisole 60 οC N OMe OH OH NH O Ph O TFA, anisole 60 οC 76% 32 37 38 39 40 2
Hydrogenolytic removal of the Bn and N-Cbz groups from 38 provided the precursor for the
tethered macrolactamization step. Activation of the carboxylic acid by EDCI/HOBt under
dilute conditions (10
–3M) gave the medium sized lactam, without detectable formation of
dimers or oligomers. TFA mediated removal of the N-Boc group from the crude lactam was
followed by NaHCO
3mediated neutralization inducing the final ring contraction providing
the N-benzyl substituted 1,4-diazepine-2,5-dione 39 in an overall yield of 29% over four
steps. However, TFA treatment of the bis(lactam) in order to remove the auxiliary remainings
led to premature loss of the TBS group from the auxiliary providing diol 40. All further
cleavage attempts failed.
2.4
The isopropyl-substituted auxiliary proved to be optimal
As the silyl group on the auxiliary 32 proved to be too labile the isopropyl substituted
auxiliary 31 was evaluated. The synthesis of the linear cyclization precursor peptides was
accomplished by reductive amination of auxiliary 31 with H–Phe–OBn or H–
βAla–OBn
(Scheme 2.11, route I and II), followed by N-Boc protection of the resulting secondary amine
to give the phenols 41 and 42 in overall yields of 91% and 78%, respectively.
Consecutive EDCI/DMAP mediated esterification of the phenols 41 and 42 with N-Cbz–
βAla–OH or acyl fluoride coupling
38,39with N-Cbz–Phe–F in the presence of DIPEA,
provided the protected linear precursor peptides 43 and 44 in yields of 99% and 84%,
respectively. These two coupling methods are complementary, although acyl fluoride
couplings are preferred in the case of sterically hindered couplings (vide infra).
Scheme 2.11 Synthesis of the linear precursors.
H O OH OiPr MeO N OH OiPr MeO N OH OiPr MeO Boc Boc CO2Bn CO2Bn Ph N O OiPr MeO Boc CO2Bn N O OiPr MeO Boc CO2Bn Ph O NHCbz O NHCbz Ph 1) H−Phe−OBn Na2SO4, THF then NaBH(OAc)3 2) (Boc)2O, CH2Cl2 91% N-Cbz−βAla−OH EDCI, DMAP 99% 1) H−βAla−OBn Na2SO4, THF then NaBH(OAc)3 2) (Boc)2O, CH2Cl2 78% N-Cbz−Phe−OH EDCI, DMAP 84% Route I Route II 31 41 42 43 44Hydrogenolytic removal of the Bn and N-Cbz groups from 43 and 44 provided the precursors
for the tethered macrolactamization step (Scheme 2.12). Activation of the carboxylic acid by
EDCI/HOBt under dilute conditions (10
–3M) smoothly gave the medium sized lactams 45
and 46, respectively, without detectable formation of dimers or oligomers. Also, no premature
intermolecular aminolysis was observed, indicating that the isopropyl group exerts sufficient
steric bulk to shield the ester.
Scheme 2.12 Cyclization of the linear precursors and removal of the auxiliary to obtain the
desired bis(lactams).
N HN O O O iPrO MeO N NH O O iPrO MeO Boc O Boc N NH O O N NH O O 46 48 47 HO iPrO OMe HO iPrO OMe Ph Ph Ph Ph 1) H2, Pd/C 2) EDCI, HOBt, CH2Cl2, DMF 10–3 M 1) TFA, CH2Cl2 2) NaHCO3, EtOAc68% over four steps
2 NH HN O O Ph TFA, anisole 60 οC N O OiPr MeO Boc CO2Bn N O OiPr MeO Boc CO2Bn Ph O NHCbz O NHCbz Ph 1) H2, Pd/C 2) EDCI, HOBt, CH2Cl2, DMF 10–3 M 1) TFA, CH2Cl2 2) NaHCO3, EtOAc
68% over four steps
TFA, anisole 60 οC Route I Route II 43 44 45
The obtained lactams 45 and 46 were not purified, as this led to inevitable loss of the
products, so the crude mixture was carried on in further reactions. TFA mediated removal of
neutralization inducing the final ring contraction providing the N-benzyl substituted
1,4-diazepine-2,5-diones 47 and 48 in overall yields of 72% and 85%, respectively, over four
steps.
In the case of the seven-membered bis(lactams) the auxiliary remainings were removed by
treatment of both 47 and 48 with TFA in the presence of anisole at 60 °C liberating the target
(S)-3-benzyl-[1,4]-diazepine-2,5-dione 2 in nearly quantitative yields after precipitation with
ether/pentane (Scheme 2.12). The optical rotation of the target molecule
(S)-3-benzyl-[1,4]-diazepine-2,5-dione 2 ([
α]
26D =
–10.2 (c 0.5, MeOH)) is comparible with the rotation found in
literature of diazepinedione 2 (([
α]
26D =
–13.3 (c 0.65, MeOH)
21
or ([
α]
26D =
–10.8 (c 1.8,
MeOH)
19). Although the
1H spectrum of (S)-3-benzyl-[1,4]-diazepine-2,5-dione 2 in CDCl
3gives broad lines due probably due to multiple conformations, the
1H in MeOD provides a
clear spectrum comparable with literature values (Table 2.1).
40Table 2.1 Comparison of the
1H spectrum of (S)-3-benzyl-[1,4]-diazepine-2,5-dione 2 in
MeOD with literature.
Spectrum Chemical shift (ppm)
Lita 7.34-7.20 (m, 5 H) 4.68 (t, 1 H) 3.75-3.74 (m, 1 H) 3.30-3.22 (m, 2 H) 2.91 (dd, 1 H) 2.73-2.69 (m, 1 H) 2.57-2.55 (m, 1 H) Expa 7.35-7.20 (m, 5 H) 4.68 (t, 1 H) 3.75 (qd, 1 H) 3.27-3.21 (m, 2 H) 2.91 (dd, 1 H) 2.72 (dt, 1 H) 2.56 (qd, 1 H) a in MeOD, 400 MHz
With this auxiliary mediated route the target compound
(S)-3-benzyl-[1,4]-diazepine-2,5-dione 2 was obtained over 8 steps in 43% and 55% overall yield for route I and II,
respectively. As was shown earlier (Scheme 2.1), the classical head-to-tail cyclization did not
provide any of the target compound. Synthesis of the target compound via alkylation of the
intermediate amide bond did provide product, but taken into account the alkylation of the
amide bonds and the removal afterwards, yields would be around 10-20% for this five step
sequence. With this in mind, the newly developed auxiliary-mediated synthesis of this
bis(lactams) efficiently provided the target molecules in good overall yield, although the
reaction sequence is relatively long.
Evaluation of the enantiomeric purity of the product 2 by chiral HPLC showed that no
extensive racemization had occurred especially during the coupling of the
α-substituted
amino acid (Scheme 2.11, ee = 97 % for route I and ee = 90 % for route II). Compared to
experiments using more hindered templates (i.e. R
2= OtBu, OTIPS, data not shown) giving
extensive epimerization due to slow esterification reactions (ee’s only up to ~ 60%), the
enantiomeric purity had greatly improved, especially during the hindered coupling via route
II.
2.5
Synthesis of eight-membered bis(lactams)
We then turned our attention to the eight-membered bis(lactam) series. Only a few
examples of substituted 1,4-diazocane-3,8-diones are known (Scheme 2.13, 50 and 52)
41-44which show interesting biological activities and are made by a classical head-to-tail
cyclization. Moreover, the cyclizations of the linear precursors 49 and 51 gave low yields
only.
Scheme 2.13 Literature syntheses of eight-membered bis(lactams).
HN N H O H2O3PO O NH O HN O CO2H Me Me HO2C O O H N O N H O H N O NH2 Me Me CO2tBu CO2H OPO3H2 1) HBTU, NMM 2) TFA, anisole H2O
15% over two steps
BocHN O O HN O Ph Ph N O CO2H N N H O O NH2 O 1) DCC, NHS 2) 4 M HCl/dioxane 3) DIPEA 4) NH4OH no yield mentioned 49 50 51 52
To show the applicability of the method, two substituted 1,4-diazocane-3,8-diones 56 and 57
were addressed. The latter example also bears substituents next to the amine and carboxyl
auxiliary connecting groups, a sequence that was inaccessible using the previously described
auxiliary 23 (Scheme 2.18, R
1= H, R
2= OtBu).
The reductive amination of
γ−Abu−OBn and Glu(OMe)−OBn with the auxiliary 31 and
subsequent N-Boc protection smoothly gave the phenols in good yields of 69% and 54%,
respectively (Scheme 2.14). For these two examples esterification using the coupling reagents
EDCI and DMAP only gave low yields. To our delight the more powerful acyl fluoride
coupling
45-47using N-Cbz–Phe–F and DIPEA gave the esters 53 and 55 in good yields of 61%
and 99% for these very hindered couplings, respectively. Starting from phenol 41 substituted
with phenylalanine (see Scheme 2.11) N-Cbz
−γAbu−OH was coupled using EDCI and
DMAP to provide ester 54 in 68% overall yield.
Scheme 2.14 Synthesis of substituted eight-membered ring bis(lactams).
MeO OiPr OH H O MeO OiPr O N Boc CO2Bn O NHCbz Ph MeO OiPr O N Boc CO2Bn O NHCbz Ph CO2Me MeO OiPr O N Boc CO2Bn O Ph NHCbz N NH MeO OiPr OH O O Ph MeO2C N NH MeO OiPr OH O O Ph 1) reductive amination 2) Boc-protection 3) esterification 68% over 3 steps 1) Bn/Cbz-deprotection 2) macrolactamization 3) Boc-removal 4) ring-contraction 30% over 4 steps 1) reductive amination 2) Boc-protection 3) esterification 42% over 3 steps 1) reductive amination 2) Boc-protection 3) esterification 53% over 3 steps 1) Bn/Cbz-deprotection 2) macrolactamization 3) Boc-removal 4) ring-contraction 30% over 4 steps 31 53 54 55 56 57
The rest of the sequence was continued using the optimized conditions resulting in the
formation of the products 56 and 57 in yields of 30% over four steps. Macrolactamization
reactions were run at a concentration of 10
–3M to avoid oligomerization. These two examples
have shown the efficiency of our method for substituted eight-membered bis(lactams) giving
the products in acceptable overall yields.
Removal of the protective groups from linear peptide 54 did result in the formation of the
unprotected peptide (Scheme 2.15). However, under the cyclization conditions employed to
obtain the desired macrolactam, formation of the five-membered pyrrolidinone 59 could not
be prevented by shielding of the iPr group. The disability of the use of
γ-amino acids as
phenol esters has to be taken into account for further synthesis planning of bis(lactams).
Scheme 2.15 Aminolysis of the linear peptide 54.
MeO OiPr O N Boc CO2Bn O Ph NHCbz MeO OiPr O N Boc CO2H O Ph NH2 MeO OiPr OH N Boc CO2H Ph NH O 1) H2, Pd/C 99% 2) base 54 58 59 60
Evaluation of the enantiomeric purity of the products 56 and 57 was performed by hydrolysis
of the products in strong acidic media and subsequent GC-MS analysis of the enantiomeric
purity of phenylalanine. This revealed an enantiomeric excess of 80% for 56 and 97% for 57,
indicating that some racemization had occurred but not as much as previously was found (R
2= OTIPS, enantiomeric excess of the final bis(lactams) proved to be ca. 60%). The low
enantiomeric excess indicates the thin line between hindrance of the i-Pr group preventing
unwanted aminolysis and also preventing fast esterifications leading to possible racemization.
Unexpectedly, removal of the auxiliaries from both the eight-membered bis(lactams) 56 and
57 was unsuccessful. Treatment of 56 and 57 with TFA in the presence of anisole at 60 °C
resulted, as became clear by careful LC-MS and MS-MS analysis, in re-attack of the phenol
on the tertiary amide forming back the ester 62 with the amine as its TFA-salt (Scheme 2.16,
56 shown) and the hydrolysis product 63. Most probably this is due to the unfavoured eight-
membered ring. To prevent these side reactions it was tried to methylate the phenolic
hydroxyl groups of both 56 and 57. Several conditions were tried, from K
2CO
3, MeI in
acetone, to TMSCHN
2in MeOH and also (OMe
3)BF
4, molecular sieves (4Å) in CH
2Cl
2, but
all attempts were unsuccessful.
Scheme 2.16 Possible auxiliary mediated side reactions from 56.
N MeO OiPr OH NH O O Ph N MeO OiPr O N H HO O Ph H H2 N MeO OiPrO NH O O Ph N H2 MeO OiPr OH H N O O Ph HO + H - H 56 61 62 63
Furthermore, by LC-MS imidazolone 66 was characterized. Formation of 66 can be
envisioned by an equilibrium of 64 with the bicyclic hemi aminal 65 via transannular attack
of the
γ-amino acid amide nitrogen on the opposite amide carbonyl moiety followed by loss of
water (Scheme 2.17), also observed by Núñez et al..
44,48This may also explain the difficulties
in the removal of the template from both substituted products 56 and 57 by treatment with
TFA.
Scheme 2.17 Possible transannulation process providing 66.
N H NH O O Ph N NH O OH Ph N N O Ph 64 65 66
2.6
Towards a solid phase immobilized auxiliary
To allow for a combinatorial approach of the auxiliary mediated synthesis of the
bis(lactams), the auxiliary had to be modified for the attachment to a solid phase resin. Initial
experiments were conducted on the liberation and modification of the methoxy group para
with respect to the aldehyde on the original auxiliary. However, all attempts were
unsuccessful and because of the availability of the auxiliaries like 30 and 34, modification of
the isopropyl group to allow for resin attachment seemed logical. The linkage of the auxiliary
to the resin had to be stable during the complete synthesis protocol towards the bis(lactams).
Thus, the linkage had to be stable towards TFA, base and the removal conditions of both the
protective groups. Similar to the work of Lober et al. (Scheme 2.18), a linkage was chosen
based on the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction.
49,50Scheme 2.18 Use of the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction for
the attachment of a linker by Lober et al..
H O O OMe H O O OMe N N N N3 N O OMe N N N R1 R2 O + 67 68 69 70
In that case, 4-hydroxy-3-methoxybenzaldehyde was alkylated with propargyl bromide.
Merrifield polystyrene can easily be converted to Merrifield azide 67 by treatment with
sodium azide at elevated temperature. The alkyne containing auxiliary 68 was coupled by
treatment with CuI in THF to provide triazole 69 and was used for the synthesis of a library of
amides.
Scheme 2.19 Synthesis of solution phase ‘modified’ auxiliary.
MeO OH OMOM H O MeO O OMOM H O Me MeO O OH H O Me MeO O OMOM H O Me N N N MeO O OH H O Me N N N Ph Me HO Br Me PBr3 45% K2CO3, 72 KI, DMF 98% HCl, MeOH 99% CuI, DIPEA 74, 99% CuI,DIPEA 74, 99% HCl,MeOH Cl N3 NaN3 DMSO 34 71 72 73 74 75 76 77 78
To have enough steric bulk compared to the isopropyl group in the original design, the
auxiliary 34 was alkylated with 3-bromo-1-butyne (72) in the presence of potassium
carbonate in acetone to provide auxiliary 75 in 68% yield. In contrast to isopropyl bromide,
direct alkylation of the diol 30 with the bromide 72 did not result in a separable mixture of
products. As a solution phase substitute for Merrifield azide the MOM-protected auxiliary 34
was first coupled successfully to benzyl azide 74 to provide auxiliary 75 in quantitative yield.
However, deprotection of the MOM group in 75 did not give the desired auxiliary 78.
Thus, prior to the cycloaddition reaction the MOM protective group was removed in 68%
yield by treatment of 75 with conc. HCl in MeOH, now delivering the auxiliary 77. The
subsequent cycloaddition reaction of the alkyne of 77 with benzyl azide 74 was accomplished
in quantitative yield by treatment with CuI and DIPEA in THF to provide the
triazole-containing auxiliary 78. A crystal of 78 was subjected to X-ray structure determination which
revealed the proper structural arrangement (Figure 2.1).
Figure 2.1 Graphical representation of the crystal structure of the ‘modified’ auxiliary 78.
The reaction sequence towards the bis(lactams) was first conducted in solution with auxiliary
78. This allowed us to carefully investigate the required modifications for a solid phase
approach. As hydrogenolysis on resins is complicated and not widely applied because of
precipitation of palladium black on the resin, the original benzyl ester and N-Cbz protective
groups were substituted for the complementary allyl ester and N-Alloc protective groups.
51These can be simultaneously removed by homogeneous Pd(0) catalysis and an a nucleophilic
additive. Thus, starting from the auxiliary 78 H
−Phe−OAll was attached by reductive
amination using trimethyl orthoformate instead of Na
2SO
4as the water scavenger (Scheme
2.20). N-Boc protection resulted in the formation of phenol 79 in 55% yield over two steps.
EDCI mediated esterification with N-Alloc
−βAla−OH furnished the linear precursor 80 in
84% yield.
Scheme 2.20 Synthesis of linear precursor with the modified auxiliary.
MeO O OH H O Me N N N Ph MeO O OH N Me N N N Ph Boc CO2All Ph MeO O O N Me N N N Ph Boc CO2All Ph O NHAlloc 1) H−Phe−OAll TMOF then NaBH(OAc)3 2) (Boc)2O, CH2Cl2 55% over 2 steps N-Alloc−βAla−OH EDCI, DMAP 84% 78 79 80Several conditions for the simultaneous deprotection of the allyl ester and the N-Alloc group
from 80 were tried (Scheme 2.21). Direct addition of Pd(PPh
3)
4with phenylsilane as the
scavenger of the
π-allyl complex resulted in the clean formation of the deprotected linear
precursor 81 in quantitative yield.
Scheme 2.21 Synthesis of seven-membered bis(lactam) with the modified auxiliary.
MeO O O N Me N N N Ph Boc CO2All Ph O NHAlloc MeO O O N Me N N N Ph Boc CO2H Ph O NH2 MeO O OH N Me N N N Ph H N Ph O O N H H N Ph O O Pd(PPh3)4 PhSiH3, CH2Cl2 99% 1) DIC, HOBt 2) TFA, CH2Cl2 3) DIPEA, DMF 25% TFA anisole 60 οC 80 81 82 2
The deprotection could also be accomplished by in situ formation of Pd(PPh
3)
4by reduction
of PdCl
2with tributyltin hydride
52with the addition of triphenylphosphine. In this case,
tributyl tinhydride also acted as the
π-allyl scavenger.
The deprotected linear precursor 81 was cyclized by treatment with DIC and HOBt to provide
the macrolactam. Removal of the N-Boc protective group by treatment with TFA and
subsequent neutralization to induce the O
→N acyl transfer reaction resulted in the formation
of the desired lactam 82, to which the remainings of the auxiliary were still attached
analogous to the earlier sequence. Treatment of the lactam 82 with TFA and anisole liberated
the target bis(lactam) 2 in quantitative yield after precipitation. This proved the effectiveness
of both the replacement of the isopropyl group by the triazole linkage and the modifications in
Our attention now turned towards the solid phase. Lober’s conditions for the copper-catalyzed
azide-alkyne 1,3-dipolar cycloaddition reaction for attachment of the auxiliary 77 to
Merrifield azide 67 resulted in the disappearance of the azide signal at 2090 cm
-1, as
monitored by solid state IR spectroscopy (Scheme 2.22). However, the reaction was generally
slow (five days). To optimize the reaction and to carefully determine the yield of the
azide-alkyne cycloaddition reaction, a simple model substrate was used.
Scheme 2.22 Copper-catalyzed azide-alkyne cycloaddition reaction on the solid phase.
OH O MeO O Me H CuI, DIPEA THF OH O MeO O Me N N N H N3 IR = 2090 cm-1 Cl NaN3, DMSO 77 82 67 83 +
Thus, N-Fmoc protected propargyl amine 84
53was coupled to Merrifield azide 67 allowing
monitoring of the reaction by measuring the intensive UV absorption of the fluorenyl group
after N-Fmoc removal (Scheme 2.23). Because of the poor solubility of CuI in THF and its
sensitivity in solution to air oxidation, a complex of copper and a pybox-type bisoxazoline
ligand was made in MeCN. CuI and an excess of pybox were heated in MeCN and the
resulting complex was added to the azide resin 67, swollen in a mixture of toluene and THF
and premixed with the alkyne 84. To monitor the reaction, the N-Fmoc group was cleaved
from 85 by treatment with piperidine in DMF and the absorption of the resulting
N-(9-fluorenylmethyl)piperidine was measured at 301 nm. The reaction proved to be much faster
(overnight) and a good yield of 84% was obtained.
Scheme 2.23 Optimization of the ‘on resin’ copper catalyzed azide alkyne cycloaddition
reaction.
FmocHN
CuI, PyBox, DIPEA THF, toluene, MeCN 4.5 : 4.5 : 1 FmocHN N N N N3 + H2N N N N N-(9-fluorenylmethyl)piperidine DMF, piperidine + 84 67 85 86
With the optimized reaction conditions in hand, auxiliary 77 was coupled to the resin, until IR
spectroscopy indicated complete disappearance of the azide signal (Scheme 2.24). To ensure
complete reaction, the number of equivalents of the reagents were doubled and each single
step was conducted twice. However, by applying the cleavage conditions after performing the
complete reaction sequence to the desired 91, no identifiable products were obtained.
Scheme 2.24 Attempts towards the solid phase synthesis of bis(lactams).
OH O MeO O Me N N N H N H NH O Ph O TFA, anisole 60 οC OH O MeO O Me H N3 +CuI, PyBox, DIPEA THF, toluene, MeCN 4.5 : 4.5 : 1 OH O MeO Me N N N N HN O Ph O OH O MeO Me N N N BocN CO2All Ph OH O MeO Me BocN CO2All Ph N3 + O O MeO Me N N N BocN CO2All Ph O NHAlloc 0.79 mmol/g, 98 % yield O O MeO Me BocN CO2All Ph O NHAlloc 7 steps
CuI, PyBox, DIPEA THF, toluene, MeCN 4.5 : 4.5 : 1
5 steps
N3 +
CuI, PyBox, DIPEA THF, toluene, MeCN 4.5 : 4.5 : 1 77 67 67 2 67 83 87 88 89 90 91
To get more insight in to the problems of the reaction sequence on the resin, the phenol 87
was made in solution and successfully coupled to the resin with the optimized cycloaddition
conditions to provide resin bound 89. Continuation of the final steps of the sequence to this
resin and applying the cleavage conditions, also did not result in the formation of any product.
This indicated that the problems on the resin arise somewhere later in the sequence.
Figure 2.2 Comparison of the
1H spectrum of 80 (top) and Magic Angle Spinning NMR
(Nanoprobe) spectrum of 90 (bottom).
The full linear precursor 88 was made in solution and was coupled to the resin 67 to provide
resin bound linear precursor 90 in an excellent yield of 98%, as determined by weighing.
NMR spectroscopy was performed on this resin. By means of magic angle spinning NMR
spectroscopy, the spectrum of 90 was compared with the spectrum of 80 in solution (Figure
2.2). In this case, large similarities could be seen between the two spectra, indicating a
successful attachment of the precursor on the resin.
Starting from the linear precursor on the resin 90, the rest of the sequence was completed.
However, following the reactions by NMR did not clearly show considerable changes. By
applying the cleavage conditions, the linear dipeptide 93 was obtained as the only identifiable
product.
Scheme 2.25 Cleavage of linear dipeptide from the resin-bound auxiliary.
O O MeO Me N N N BocN CO2All Ph O
NHAlloc 1) DIC, HOBt
2) TFA, CH2Cl2 3) DIPEA N H CO2All Ph O AllocHN O O MeO Me N N N BocN CO2All Ph O NHAlloc Pd(PPh3)4 Bu3SnH TFA, anisole 60 οC OH O MeO Me N N N N AllO2C Ph O NHAlloc 90 90 92 93
This indicated incomplete removal of the allyl ester and N-Alloc protective groups from 90.
The macrolactamization conditions performed on 90 then obviously could not lead to the
desired lactam. Removal of the N-Boc protective group from 90 and base-induced O
→N acyl
shift consequently led to the unwanted substituted dipeptide 92, which after treatment with
TFA and anisole liberated the linear dipeptide 93. Further evaluation of the protective group
removal conditions should eventually lead to the desired bis(lactams).
2.7 Conclusions
In conclusion, we have optimized our auxiliary-based approach towards medium sized
bis(lactams) that are difficult to access using traditional methodologies. Auxiliary 31 allows
straightforward synthesis of the cyclization precursors and its remainings can be efficiently
removed from the seven-membered bis(lactams). However, removal of the auxiliary from the
eight-membered bis(lactams) is hampered by unwanted side reactions and poor product
stability. The auxiliary was developed further to allow solid phase combinatorial access
towards a library of medium-sized bis(lactams). Although initial solution phase experiments
were successful, attempts of on resin auxiliary mediated formation of bis(lactams) so far have
been unsuccessful, mainly due to problems with allyl and N-Alloc protective group removal.
2.8 Acknowledgments
M. Boesten (DSM research, Geleen) is kindly acknowledged for the measurement of
enantiomeric purity by LC-MS. E. van der Klift and Dr. T. van Beek (Wageningen
University) are kindly acknowledged for the measurement of the enantiomeric purity by
GC-MS. J.A.J. Geenevasen is kindly acknowledged for the help with the measurement of the
Magic Angle Spinning Nanoprobe NMR experiments. Dr. R. de Gelder (Radboud University
Nijmegen) is kindly acknowledged for the crystal structure determination.
2.9 Experimental
section
General remarks. All reactions were conducted under nitrogen unless stated otherwise and monitored
by TLC on silica gel coated aluminium sheets. Flash column chromatography was performed on silica gel 300– 400 mesh using the indicated solvent mixtures. All solvents were distilled from sodium/benzophenone ketyl (THF, Et2O) or CaH2 (DMF, CH2Cl2). The NMR spectra were determined in CDCl3 solutions, using a Bruker
ARX 400 and a Varian Inova 500 spectrometer unless indicated otherwise. Spectra are reported in δ units (ppm) and J values (Hz) with Me4Si as the internal standard. HRMS data (FAB+) were recorded with a JEOL JMS
SX/SX 102A four-sector mass spectrometer. The LC-MS experiments were performed with the use of a Finnigan LXQ Ion Trap apparatus. LC was carried out with an ODS-3 column using gradients between H2O/0.1% HCOOH (solvent A) and CH3CN/0.1% HCOOH (solvent B). The electronspray ionization mass
spectra (positive ions) were recorded in full scan mode (m/z = 100–2000). The MS-MS experiments were performed by collision-induced dissociation with an indicated collision energy of 30 eV. Infrared (IR) spectra were obtained with a Bruker IFS 28 FTIR spectrometer and are reported in wave numbers [cm–1]. Melting points were recorded with a Büchi melting point apparatus B-545 and are uncorrected. HPLC analyses were measured with an Agilent HPLC system equipped with a C-18 column (Varian-Chrompack, inertsil-ODS-3, 3μ, 50 × 4.6 mm), a maximum flowspeed of 2.0 mL/min, the UV/Vis detector at λ = 220 or 254 nm as indicated, and a gradient of 100% A to 100% B [A: H2O/CH3CN/HCOOH (95:5:0.04); B: H2O/ CH3CN/HCOOH (5:95:0.04)] as
the eluent in 5 min. The enantiomeric excess values of compounds 2a were determined at DSM Pharma Chemicals and obtained from chiral HPLC analyses. Measurements were taken on I.D. Chiralpack AD column (25 × 0.46 cm), a flowspeed of 1 mL/min, the UV/Vis detector at λ = 210 nm, and n-heptane/i-PrOH (55:45 %v/v) as the eluent.
2,3-Dihydroxy-4-methoxybenzaldehyde (30). According to the literature procedure,
providing the product 30 (7.51 g, 89%) as purple crystals. 1H NMR (400 MHz, CDCl3) δ 11.12
(s, 1 H), 9.80 (s, 2 H), 7.13 (d, J = 8.4 Hz, 1 H), 6.64 (d, J = 8.4 Hz, 1 H), 3.98 (s, 3 H) ppm. RP-HPLC: Rt 3.10 min (λ = 254). H O OH OH MeO
2-Hydroxy-3-isopropoxy-4-methoxybenzaldehyde (31). Compound 30 (0.5 g, 3 mmol) was
added in small portions to a solution of sodium hydride (0.240 g, 60% w/w in mineral oil, 6 mmol) in dry DMSO (8 mL). The solution was stirred for 20 min, after which potassium iodide (0.498 g, 3 mmol) and 2-bromopropane (0.282 mL, 3 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours after which it was diluted with EtOAc (30 mL) and washed with water (2 × 40 mL). The organic phase was then washed with brine, dried with Na2SO4 and concentrated in vacuo. The crude product was purified by flash column chromatography [silica gel, ethyl acetate/petroleum ether
(boiling range 40–65 °C), 1:9] to give aldehyde 31 as yellowish needles (0.193 g, 0.92 mmol, 31%). M.p. 81–83 °C. IR (neat) ν = 1639, 1502, 1439 cm-1 . 1H NMR (400 MHz, CDCl3) δ 11.14 (s, 1 H), 9.74 (s, 1 H), 7.27 (d, J = 8.4 Hz, 1 H), 6.59 (d, J = 8.8 Hz, 1 H), 4.46 (sept, J = 6.4 Hz, 1 H), 3.92 (s, 3 H), 1.32 (d, J = 6.4 Hz, 6 H) ppm. 13 C NMR (100 MHz, CDCl3) δ 194.9, 160.0, 156.3, 134.1, 129.9, 116.5, 103.9, 75.3, 56.2, 22.5 ppm. 3-(tert-Butyldimethylsilanyloxy)-2-hydroxy-4-methoxybenzaldehyde (32). Diol 30 (1.25 g,
7.4 mmol, 1 equiv) was dissolved in DMF (6 mL). Imidazole (0.558 g, 8.2 mmol, 1.1 equiv) was added and the mixture was stirred for 10 minutes at room temperature. TBDMSCl (1.23 g, 8.2 mmol, 1.1 equiv) was added in portions and the mixture was stirred at room temperature overnight. The mixture was extracted with Et2O (3 × 100 mL). The combined organic layer was washed with
water (3 × 250 mL) and brine (250 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo.
The residue was purified by flash column chromatography [silica gel, ethyl acetate/petroleum ether, boiling range 40-65 οC, 1:9] to obtain the product 32 (2.029 g, 97%) as a yellow oil. IR (neat) ν = 2930, 2858, 1660, 1507, 1442, 1293, 1118, 1045, 862 cm-1. 1H NMR (400 MHz, CDCl3) δ 11.04 (s, 1 H), 9.70 (s, 1 H), 7.13 (d, J =
8.4 Hz, 1 H), 6.55 (d, J = 8.8 Hz, 1 H), 3.87 (s, 3 H), 1.02 (s, 9 H), 0.16 (s, 6 H) ppm. 13C NMR (100 MHz, CDCl3) δ 194.8, 157.3, 153.8, 132.2, 127.5, 116.2, 103.5, 55.5, 25.5, 18.6 ppm. RP-HPLC: Rt 6.35 min (λ =
254).
3-(tert-Butyldimethylsilanyloxy)-4-methoxy-2-methoxymethoxybenzaldehyde (33).
Sodium hydride, 60% in mineral oil (1.86 g, 46 mmol, 1 equiv) was washed with pentane (3 × 20 mL). DMF (150 mL) was added and phenol 32 (13.11 g, 46 mmol, 1 equiv) was added slowly in portions. After stirring for 30 minutes at room temperature MOMCl (3.43 mL, 46 mmol, 1 equiv) was added and the mixture was stirred at room temperature for 2 days. The mixture was extracted with Et2O (3 × 400 mL) and the combined organic layer was washed with water (3 × 500 mL) and
brine (500 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo to obtain the product 33
(16.1 g, 99%) as yellow oil. IR (neat) ν = x cm-1
. 1H NMR (400 MHz, CDCl3) δ 10.32 (s, 1 H), 7.53 (d, J = 8.8
Hz, 1 H), 6.77 (d, J = 8.8 Hz), 5.22 (s, 2 H), 3.89 (s, 3 H), 3.55 (s, 3 H), 1.04 (s, 9 H), 0.19 (s, 6 H) ppm. RP-HPLC: Rt 6.27 min (λ = 254).
3-Hydroxy-4-methoxy-2-methoxymethoxybenzaldehyde (34). Silyl ether 33 (1.934 g, 6
mmol, 1 equiv) was dissolved in THF (20 mL). TBAF (1 M in THF) (12 mL, 12 mmol, 2 equiv) was added and the mixture was stirred for two hours at room temperature. Solvents were evaporated in vacuo. The residue was dissolved in ethyl acetate (100 mL). The organic layer was washed with water (2 × 100 mL) and brine (100 mL). The organic layer was dried over Na2SO4 and
concentrated in vacuo. The residue was purified by flash column chromatography [silica gel, ethyl acetate/petroleum ether, boiling range 40-65 οC, 1:2] to obtain the product 34 (1.002 g, 79%) as a pink solid. 1H
H O OH OTBS MeO H O OMOM OTBS MeO H O OMOM OH MeO H O OH OiPr MeO
NMR (400 MHz, CDCl3) δ 10.25 (s, 1 H), 7.43 (d, J = 8.8 Hz, 1 H), 6.76 (d, J = 8.8 Hz, 1 H), 6.53 (bs, 1 H),
5.27 (s, 2 H), 3.95 (s, 3 H), 3.61 (s, 3 H) ppm. RP-HPLC: Rt 3.38 min (λ = 254).
3-Isopropoxy-4-methoxy-2-methoxymethoxybenzaldehyde (35). To a solution the phenol 34 (0.540 g, 2.7 mmol, 1 equiv) in DMF (15 mL) was added K2CO3 (1.466 g, 10.8 mmol, 4
equiv) in portions under stirring at room temperature. The mixture was stirred for 20 minutes, after which potassium iodide (1.494 g, 10.3 mmol, 3.8 equiv) and isopropyl bromide (0.996 ml, 10.8 mmol, 4 equiv) were added. The resulting suspension was stirred at 60 οC overnight. After conversion of the starting material, the mixture was allowed to cool to room temperature. The mixture was diluted with water (100 mL) and EtOAc (100 mL) and the aqueous layer was extracted with EtOAc (3 × 75 mL). The combined organic layer was washed with water (3× 200 mL) and brine (200 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The product was purified by flash column
chromatography [silica gel, ethyl acetate/petroleum ether, boiling range 40-65 οC, 1:2] to obtain the product 35 (0.588 g, 86%). 1H NMR (400 MHz, CDCl3) δ 10.10 (s, 1 H), 7.61 (d, J = 8.8 Hz, 1 H), 6.78 (d, J = 8.6 Hz, 1
H), 5.27 (s, 2 H), 4.41 (sept, J = 6.2 Hz, 1 H), 3.91 (s, 3 H), 3.54 (s, 3 H), 1,30 (d, J = 6.2 Hz, 6 H) ppm. 13C NMR (125 MHz, CDCl3) δ 189.4, 159.9, 154.9, 139.9, 124.3, 124.2, 107.9, 100.2, 76.1, 58.2, 56.4, 22.7 ppm.
2-Hydroxy-3-isopropoxy-4-methoxybenzaldehyde (31). Compound 35 (0.588 g, 2.3 mmol,
1 equiv) was dissolved in MeOH (15 mL). A solution of 1.5 M hydrochloric acid (1.54 mL, 2.3 mmol, 1 equiv) was added to the solution and it was stirred at 60 οC overnight. The purple reaction mixture was concentrated in vacuo. The mixture was diluted with EtOAc (50 mL) and washed with water (2 × 50 mL) and brine (50 mL). The organic layer was dried over Na2SO4, filtered and
concentrated in vacuo. Product 31 (0.070 g, 85%) was obtained as purplish crystals. Spectroscopic data, see above.
General procedure A for reductive amination and N-Boc protection. The appropriate amine (1.1 equiv.) and
Na2SO4 (excess) was added to a solution of the aldehyde in THF. The reaction mixture was then stirred at room
temperature for 3–5 hours after which Na(OAc)3BH (4 equiv.) was added and stirring was continued for 16
hours. The reaction mixture was then quenched by pouring it into saturated ammonium chloride solution and stirring for 30 min. The remaining mixture was quenched with saturated sodium hydrogen carbonate solution and extracted with EtOAc. The organic phase was then washed with brine, dried with Na2SO4 and concentrated in vacuo. The product was used without purification in the next step. If necessary the resulting oil can be purified
by flash column chromatography [silica gel, ethyl acetate/petroleum ether (40:65), 1:3]. To a solution of the amine in dry CH2Cl2 was added (Boc)2O (1.1 – 1.5 equiv.), and the reaction mixture was stirred at room
temperature until completed. The reaction mixture was then washed with a saturated solution of sodium bicarbonate and brine, dried with Na2SO4 and concentrated in vacuo. The crude product was purified by flash
column chromatography [silica gel, ethyl acetate/petroleum ether (boiling range 40– 65 °C), 1:9, then 1:4].
Benzyl 3-[
tert-butoxycarbonyl(3-{tert-butyldimethylsilanyloxy}-2-hydroxy-4-methoxybenzyl)amino]propionate (37). According to the general procedure A,
using aldehyde 32 (1.00 g, 3.5 mmol) and H2N–βAla–OBn (0.627 g, 3.5 mmol), the
product 37 (1.459 g, 2.67 mmol, 76%) was obtained as a yellow oil. IR (neat) ν 3172, H O OMOM iPrO MeO H O OH OiPr MeO MeO OTBS OH N Boc CO2Bn
7.33 (m, 5 H), 6.70 (d, J = 8.4 Hz, 1 H), 6.36 (d, J = 8.4 Hz, 1 H), 5.11 (s, 2 H), 4.34 (s, 2 H), 3.77 (s, 3 H), 3.51 (s, 2 H), 2.57 (s, 2 H), 1.47 (s, 9 H), 1.00 (s, 9 H), 0.18 (s, 6 H) ppm. 13C NMR (100 MHz, CDCl3) δ 171.4,
155.6, 135.5, 128.3, 128.2, 128.0, 116.8, 102.8, 79.6, 65.7, 55.2, 42.6, 33.4, 28.2, 25.8, 18.5, -4.6 ppm.
General procedure B for esterification. To a solution of the phenol in dry MeCN was added the appropriate
acid followed by EDCI and DMAP. The reaction mixture was then stirred at room temperature until completion, after which the solvent was evaporated and the residue taken up in EtOAc and washed with a solution of potassium hydrogensulphate (0.5 M, 2 ×) and brine. The organic phase was dried with Na2SO4 and concentrated in vacuo. The crude product was purified by flash column chromatography [silica gel, ethyl acetate/petroleum
ether (boiling range 40–65 °C), 1:3–4] to yield the product.
6-({[2-(Benzyloxycarbonyl)ethyl]( tert-butoxycarbonyl)amino}-methyl)-2-{tert-butyldimethylsilanyloxy}-3-methoxyphenyl 2-(benzyloxycarbonylamino)-3-phenylpropionate (38). According to the general procedure B, using phenol 37
(0.982 g, 1.8 mmol, 1 equiv) and N-Cbz–Phe–OH (2.154 g, 7.2 mmol, 4 equiv), the product 38 (0.663 g, 0.80 mmol, 45%) was obtained as a colourless oil. IR (neat) ν 1768, 1731, 1695, 1504, 1455 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.37–7.26 (m, 15 H), 6.84 (m, 1 H), 6.72 (d, J
= 8.0 Hz, 1 H), 5.12–4.99 (m, 5 H), 4.28–4.12 (m, 2 H), 3.79 (s, 3 H), 3.46–3.12 (m, 4 H), 2.59–2.49 (m, 2 H), 1.48 (s, 9 H), 0.95 (s, 9 H), 0.16 (s, 6 H) ppm. 13C NMR (100 MHz, CDCl3) δ 171.8, 169.1, 155.5, 155.3, 150.7,
141.8, 136.4, 135.5, 129.2, 127.9, 127.4, 126.8, 122.8, 116.8, 109.2, 80.0, 67.7, 66.2, 55.6, 54.6, 44.7, 42.7, 38.0, 33.3, 28.8, 26.1, 18.4, -4.6 ppm.
General procedure C for tethered lactamization and consecutive ring contraction. The starting material was
dissolved in EtOAc/iPrOH (4:1). Pd/C (20%) was then added, and the mixture was brought under hydrogen and stirred for 16 hours. The reaction mixture was then filtered through Celite and concentrated in vacuo. The remaining residue was dissolved in CH2Cl2/DMF (4:1; c = 10-3 M) followed by addition of EDCI (4 equiv.) and
HOBt (4 equiv.). The mixture was then stirred at room temperature for 4–6 hors, after which the reaction mixture was diluted with diethyl ether, extracted (3 ×) and washed with saturated sodium hydrogencarbonate solution, a potassium hydrogensulphate solution (0.5 M) and brine. The organic phase was then dried with Na2SO4 and
concentrated in vacuo to give the crude medium-sized lactam, which was used in the consecutive ring contraction without purification. The medium-sized lactam was dissolved in a mixture of TFA/CH2Cl2 (1:1) and
stirred at room temperature for 16 hours. The solvent was evaporated, after which the remaining TFA salt was dissolved in EtOAc (c = 10-2 M) and an excess of solid sodium hydrogencarbonate was added. After 4–5 hours, the reaction mixture was washed with a solution of potassium hydrogensulphate (0.5 M, 2 ×) and brine. The organic phase was dried with Na2SO4 and concentrated in vacuo. The remaining residue can be purified by flash
column chromatography [silica gel, ethyl acetate/petroleum ether (boiling range 40–65 °C), 1:1 → ethyl acetate/iPrOH, 9:1] to yield the product.
3-Benzyl-1-(2-hydroxy-3-{ tert-butyldimethylsilanyloxy}-4-methoxybenzyl)-1,4-diazepane-2,5-dione (39). According to the general procedure C, using 38 (0.663 g,
0.8 mmol), the product 39 (0.107 g, 0.22 mmol, 29%) was obtained as a white amorphous solid. 1H NMR (400 MHz, CDCl3) δ 7.34–7.22 (m, 5 H), 6.83 (s, 1 H), MeO OTBS O N Boc CO2Bn O NHCbz Ph MeO OTBS OH N NH O O Ph
6.79 (d, J = 8.5 Hz, 1 H), 6.39 (d, J = 8.5 Hz, 1 H), 5.71 (s, 1 H), 4.57 (m, 3 H), 3.85 (m, 2 H), 3.78 (s, 3 H), 3.43 (m, 2 H), 2.89 (m, 1 H), 2.50 (m, 2 H), 1.01 (s, 9 H), 0.19 (s, 6 H) ppm.
Benzyl 2-[tert-Butoxycarbonyl(2-hydroxy-3-isopropoxy-4-methoxybenzyl)amino]-3-phenylpropionate (41). According to the general procedure A, using aldehyde 31
(0.070 g, 0.33 mmol) and H2N–Phe– OBn (0.084 g, 0.33 mmol), the product 41 (0.164
g, 0.30 mmol, 91%) was obtained as a colourless oil. IR (neat) ν 1710, 1697, 1616, 1506, 1455 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.32–7.14 (m, 10 H), 6.76 (d, J = 8.5
Hz, 1 H), 6.27 (d, J = 8.3 Hz, 1 H), 5.85 (br. s, 1 H), 5.07–4.95 (m, 2 H), 4.60–4.24 (m, 3 H), 3.79 (s, 3 H), 3.70 (m, 1 H), 3.38 (dd, J = 13.8, J = 5.1 Hz, 1 H), 3.25–3.13 (m, 1 H), 1.42 (s, 9 H), 1.27 (d, J = 6.3 Hz, 6 H) ppm.
13C NMR (100 MHz, CDCl3) δ 170.6, 155.7, 152.7, 149.3, 137.9, 135.2, 129.2, 128.3, 128.2, 127.9, 127.8,
126.4, 125.3, 116.2, 103.0, 81.4, 74.9, 66.5, 61.7, 55.6, 47.4, 36.2, 28.1, 22.4 ppm. HRMS (FAB) calcd. for C32H40O7N [MH+] 550.2810, found 550.2801. RP-HPLC: Rt 6.69 min (λ = 254).
Benzyl 3-[tert-butoxycarbonyl(2-hydroxy-3-isopropoxy-4-methoxybenzyl)amino]propionate (42). According to the general procedure A,
using aldehyde 31 (0.169 g, 0.8 mmol) and H2N–βAla–OBn (0.179 g, 1 mmol), the
product 42 (0.416 g, 0.73 mmol, 91%) was obtained as a colourless oil. IR (neat) ν 1734, 1688, 1652, 1506, 1456 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.64 (br. s, 1 H), 7.40–7.35 (m, 5 H), 6.85 (d, J = 8.4 Hz, 1 H), 6.42 (d, J = 8.4 Hz, 1 H), 5.12 (s, 2 H), 4.52 (br. s, 1 H), 4.38 (s, 2 H), 3.78 (s, 3 H), 3.53 (br. s,
2 H), 2.60 (br. s, 2 H), 1.48 (s, 9 H), 1.31 (d, J = 6.0 Hz, 6 H) ppm. 13C NMR (100 MHz, CDCl3) δ 171.6, 135.7,
128.5, 128.2, 128.2, 127.6, 127.0, 124.6, 117.0, 103.2, 75.0, 66.3, 60.4, 55.8, 46.3, 42.8, 33.6, 28.4, 22.5 ppm. HRMS (FAB) calcd. for C26H36O7N [MH+] 474.2490, found 474.2429.
Benzyl
2-[{2-[3-(Benzyloxycarbonylamino)propionyloxy]-3-isopropoxy-4-methoxybenzyl}(tert-butoxycarbonyl)amino]-3-phenylpropionate (43).
According to the general procedure B, using phenol 41 (0.100 g, 0.18 mmol) and
N-Cbz–βAla–OH (0.080 g, 0.36 mmol), the product 43 (0.096 g, 0.13 mmol, 71%)
was obtained as a colourless oil. IR (neat) ν 1723, 1698, 1498, 1454 cm-1
. 1H NMR (500 MHz, CDCl3) δ 7.36–7.04 (m, 15 H), 6.80 (d, J = 8.5 Hz, 1 H), 6.53 (d, J = 8.1 Hz, 1 H), 5.65 (br. s, 1 H),
5.13–5.05 (m, 5 H), 4.36 (m, 2 H), 3.88 (br. s, 1 H), 3.79 (s, 3 H), 3.55 (br. s, 3 H), 3.34 (m, 1 H), 3.06 (m, 1 H), 2.71 (br. m, 2 H), 1.44 (s, 9 H), 1.16 (br. d, J = 6.1 Hz, 6 H) ppm. 13C NMR (100 MHz, CDCl3) δ 170.5, 169.8,
156.3, 154.6, 153.0, 143.6, 138.2, 136.4, 135.3, 129.2, 129.1, 128.3, 128.1, 127.9, 126.4, 124.5, 122.2, 109.4, 80.9, 75.2, 66.7, 66.4, 60.9, 55.7, 46.9, 36.5, 36.3, 34.2, 28.2, 22.4 ppm. HRMS (FAB) calcd. for C43H51O10N2
[MH+] 755.3545, found 755.3527. RP-HPLC: Rt 6.83 min (λ = 254).
6-({[2-(Benzyloxycarbonyl)ethyl]( tert-butoxycarbonyl)amino}-methyl)-2-isopropoxy-3-methoxyphenyl 2-(benzyloxycarbonylamino)-3-phenylpropionate (44). N-Cbz–Phe–F (0.127 g, 0.42 mmol) and DIPEA (0.073 mL, 0.42 mmol) were
added to a solution of the phenol 42 (0.100 g, 0.21 mmol) in dry CH2Cl2 (4 mL). The
MeO OiPr OH N CO2Bn Ph Boc MeO OiPr OH N Boc CO2Bn MeO OiPr O N CO2Bn Ph Boc O NHCbz MeO OiPr O N Boc CO2Bn O NHCbz Ph
reaction mixture was stirred at room temperature until completion. The mixture was diluted with EtOAc (15 mL) and washed with water (2 × 15 mL) and brine. The organic phase was dried with Na2SO4 and concentrated in vacuo. The crude product was purified by flash column chromatography [silica gel, ethyl acetate/petroleum ether
(boiling range 40–65 °C), 1:4] to yield the product 44 (0.133 g, 0.18 mmol, 84%) as a colourless oil. IR (neat) ν 1769, 1727, 1693, 1498, 1454 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.33–7.15 (m, 15 H), 7.03 (bd, 1 H), 6.80 (d, J = 8.0 Hz, 1 H), 5.17–4.99 (m, 5 H), 4.51 (br. s, 1 H), 4.28–4.17 (br. m, 2 H), 3.86 (s, 3 H), 3.46–3.14 (br. m, 4
H), 2.59–2.50 (br. m, 2 H), 1.48 (s, 9 H), 1.23 (br. d, J = 14.8 Hz, 6 H) ppm. 13C NMR (100 MHz, CDCl3) δ
171.8, 169.5, 155.8, 155.4, 152.1, 141.8, 137.0, 135.7, 129.5, 129.3, 128.6, 128.5, 128.4, 128.4, 128.1, 128.0, 127.0, 126.9, 124.0, 120.1, 110.2, 80.1, 75.2, 66.9, 66.3, 60.4, 55.8, 54.8, 45.2, 43.1, 37.8, 33.5, 28.4, 22.4 ppm. HRMS (FAB) calcd. for C43H50O10N2 [MH+] 754.3465, found 754.3450.
3-Benzyl-4-(2-hydroxy-3-isopropoxy-4-methoxybenzyl)-1,4-diazepan-2-one (47).
According to the general procedure C, using 43 (0.089 g, 0.12 mmol), the product 47 (0.033 g, 0.080 mmol, 68%) was obtained as a white amorphous solid. IR (neat) ν 2923, 2852, 1654, 1503, 1458 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.58 (br. s, 1 H), 7.38–7.20 (m, 5 H), 6.66 (d, J =
8.6 Hz, 1 H), 6.36 (d, J = 8.6 Hz, 1 H), 4.85 (AB, J = 14.5 Hz, 1 H), 4.59 (X part of ABX, JAX + JBX = 14.3 Hz, 1
H), 4.53 (sept, J = 6.2 Hz, 1 H), 3.82 (m, 1 H), 3.80 (s, 3 H), 3.49–3.33 (m, 2 H), 3.37 and 3.24 (AB part of ABX, JAB = 13.6, JBX = 9.8, JAX = 4.5 Hz, 2 H), 3.20 (AB, J = 14.4 Hz, 1 H), 2.79 (m, 1 H), 1.23 (d, J = 6.2 Hz,
6 H) ppm. 13C NMR (100 MHz, CDCl3) δ 172.7, 172.3, 152.7,149.0, 136.6, 133.3, 129.5, 129.3, 129.2, 129.0,
128.8, 128.6, 127.4, 125.3, 115.2, 103.4, 75.1, 55.8, 55.7, 48.7, 40.4, 40.0, 36.1, 22.5 ppm. HRMS (FAB) calcd. for C23H29O5N2 [MH+] 413.2078, found 413.2095
.
RP-HPLC: Rt 4.32 min (λ = 254).3-Benzyl-1-(2-hydroxy-3-isopropoxy-4-methoxybenzyl)-1,4-diazepane-2,5-dione (48). According to the general procedure C, using 44 (0.075 g, 0.1 mmol), the product 48 (0.028 g, 0.068 mmol, 68%) was obtained as a white amorphous solid. IR (neat) ν
2925, 2853, 1649, 1504, 1454 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.35–7.24 (m, 5
H), 6.95 (d, J = 8.6 Hz, 1 H), 6.75 (s, 1 H), 6.43 (d, J = 8.6 Hz, 1 H), 5.59 (s, 1 H), 4.68 (AB, J = 14.3 Hz, 1 H), 4.56 (m, 2 H), 4.54 (AB, J = 12.0 Hz, 1 H) 3.87 (m, 1 H), 3.82 (s, 3 H), 3.49 (m, 1 H), 3.41 and 2.89 (AB part of ABX, JAB = 14.7, JBX = 9.6, JAX = 4.7 Hz, 2 H), 2.50 (m, 2 H), 1.27 (d, J = 12 Hz, 6 H) ppm. 13C NMR (100
MHz, CDCl3) δ 171.3, 170.2, 152.8, 149.0, 136.1, 133.6, 129.2, 129.1, 127.3, 125.3, 115.4, 103.8, 75.2, 55.8,
53.9, 45.8, 42.2, 36.9, 35.1, 22.5 ppm. HRMS (FAB) calcd. for C23H29O5N2 [MH+] 413.2078, found 413.2069.
RP-HPLC: Rt 4.57 min (λ = 254).
3-Benzyl-1,4-diazepane-2,5-dione (2). Bis(lactams) 46, 48 and 82 were dissolved in TFA (ca. 2–4
mL), and anisole (10 equiv.) was added. The reaction mixture was stirred at 60 °C for 16 hours, after which it was concentrated in vacuo. The pure product was isolated by precipitation with diethyl ether and pentane, to provide 2 as a grey solid (analytical data according to literature). [α]26D –10.2 (c 0.5, MeOH). 1H NMR (400 MHz, CD3OD) δ 7.35-7.20 (m, 5 H), 4.68 (t, J = 7.0 Hz, 1 H), 3.75 (qd, J = 11.6 Hz, J = 3.7 Hz, 1 H), 3.27-3.21 (m, 2 H), 2.91 (dd, J = 14.2 Hz, J = 7.8 Hz, 1 H), 2.72 (dt, J = 17.8 Hz, J = 3.8 Hz, 1 H), 2.56 (qd, J = 11.7 Hz, J = 5.8 Hz, 1 H). 13C NMR (100 MHz, CD3OD) δ 175.4, 174.7, 138.7, 130.6, 129.8, 128.0, 55.2, 37.5, 37.0, 37.0. RP-HPLC: Rt 3.05 min (λ = 220). MeO iPrO OH N NH O O Ph MeO iPrO OH N NH O O Ph HN NH O O Ph
Benzyl 4-[tert-Butoxycarbonyl(2-hydroxy-3-isopropoxy-4-methoxybenzyl) amino]butyrate (53a). According to the general procedure A, using aldehyde 31
(0.300 g, 1.4 mmol) and H2N–γAbu–OBn (0.348 g, 1.8 mmol), the product 53a
(0.468 g, 0.96 mmol, 69%) was obtained as a colourless oil. IR (neat) ν 1737, 1690, 1654, 1504, 1455 cm-1. 1H NMR (400 MHz, CDCl3) δ 8.02 (br. s, 1 H), 7.39–7.33 (m, 5 H), 6.81 (d, J =
8.8 Hz, 1 H), 6.40 (d, J = 8.4 Hz, 1 H), 5.13 (s, 2 H), 4.50 (br. s, 1 H), 4.35 (s, 2 H), 3.83 (s, 3 H), 3.25 (br. s, 2 H), 2.36 (t, J = 7.2 Hz, 2 H), 1.89 (quint, J = 7.2 Hz, 2 H), 1.47 (s, 9 H), 1.32 (d, J = 6.0 Hz, 6 H) ppm. 13C NMR (100 MHz, CDCl3) δ 172.9, 156.0, 140.9, 135.9, 133.0, 128.6, 128.6, 128.2, 127.6, 127.0, 124.7, 117.1, 103.1, 75.0, 66.4, 66.3, 65.4, 55.8, 45.7, 31.5, 28.4, 23.3, 22.6 ppm. HRMS (FAB) calcd. for C27H37O7N [MH+]
487.2570, found 487.2568.
Benzyl
4-[{2-[2-(benzyloxycarbonylamino)-3-phenylpropionyloxy]-3-isopropoxy-4-methoxybenzyl}(tert-butoxycarbonyl)amino]butyrate (53).
N-Cbz–Phe–F (0.579 g, 1.92 mmol) and DIPEA (0.334 mL, 1.92 mmol) were added to a solution of the phenol 53a (0.468 g, 0.96 mmol) in dry CH2Cl2 (15 mL). The
reaction mixture was stirred at room temperature until completion. The mixture was diluted with EtOAc (45 mL) and washed with water (2 × 45 mL) and brine. The organic phase was dried with Na2SO4 and concentrated in vacuo. The crude product was purified by flash column chromatography [silica gel, ethyl acetate/petroleum ether
(boiling range 40–65 °C), 1:4] to yield the product 53 (0.447 g, 0.58 mmol, 61%) as a colourless oil. IR (neat) ν 1769, 1729, 1694, 1498, 1455 cm-1. 1H NMR (400 MHz, CDCl3) δ 7.34– 7.18 (m, 15 H), 6.97 (br. d, 1 H), 6.81 (d, J = 8.4 Hz, 1 H), 5.13–5.09 (m, 4 H), 4.99 (q, J = 7.6 Hz, 1 H), 4.52 (br. s, 1 H), 4.25–4.13 (br. m, 2 H), 3.86 (s, 3 H), 3.45–3.14 (br. m, 4 H), 2.30 (m, 2 H), 1.88–1.63 (m, 2 H), 1.49 (br. s, 9 H), 1.36–1.22 (br. m, 6 H) ppm. 13 C NMR (100 MHz, CDCl3) δ 172.9, 169.5, 155.8, 135.9, 129.5, 128.7, 128.5, 128.4, 128.4, 128.1, 128.0, 127.1, 126.9, 124.0, 120.1, 110.2, 79.8, 75.2, 66.9, 66.2, 60.3, 56.0, 54.9, 45.2, 44.2, 37.9, 31.4, 28.4, 23.2, 22.4 ppm. HRMS (FAB) calcd. for C44H52O10N2 [MH+] 768.3622, found 768.3624.
6-({[1-(Benzyloxycarbonyl)-2-phenyl-ethyl]( tert-butoxycarbonyl)amino}-methyl)-2-isopropoxy-3-methoxyphenyl 4-(benzyloxycarbonylamino)butyrate (54). According to the general procedure B, using phenol 41 (0.638 g, 1.2 mmol, 1
equiv) and N-Cbz–γAbu–OH (0.570 g, 2.4 mmol, 2 equiv), the product 54 (0.691 g, 0.90 mmol, 75%) was obtained as a colourless oil. IR (neat) ν 2975, 1725, 1698, 1498, 1455 cm-1. 1H NMR (500 MHz, CDCl3) δ 7.28-7.01 (m, 15 H), 6.92 (d, J = 6.4 Hz, 1 H), 6.67 (d, J = 8.4
Hz, 1 H), 6.42 (d, J = 8.4 Hz, 1 H), 5.71 (s, 1 H), 5.13-4.89 (m, 5 H), 4.32-4.19 (m, 2 H), 3.77 (m, 1 H), 3.65 (s, 3 H), 3.32-3.18 (m, 4 H), 2.91 (m, 1 H), 2.51-2.35 (m, 2 H), 1.78 (m, 2 H), 1.27 (s, 9 H), 1.06 (dd, J = 6.8 Hz, J = 6.8 Hz, 6 H) ppm. 13C NMR (100 MHz, CDCl3) δ 170.8, 170.6, 156.7, 154.7, 153.5, 243.9, 138.4, 136.8,
135.5, 129.4, 128.3, 128.1, 126.6, 124.8, 123.0, 122.5, 109.4, 81.1, 75.4, 66.8, 66.3, 60.9, 55.9, 47.3, 39.8, 36.7, 30.4, 28.3, 24.6, 22.6 ppm. HRMS (FAB) calcd. for C44H53O10N2 [MH+] 769.3702, found 769.3698.
5-Benzyl 1-Methyl-2-[tert-Butoxycarbonyl(2-hydroxy-3-isopropoxy-4-methoxybenzyl)amino]pentanedioate (55a). According to the general procedure
A, using aldehyde 31 (0.200 g, 0.95 mmol) and H2N–Glu(OBn)–OMe (0.475 g, 1.3
mmol), the product 55a (0.277 g, 0.51 mmol, 54%) was obtained as a colourless MeO OiPr OH N Boc CO2Bn MeO OH N Boc CO2Bn CO2Me MeO OiPr O N Boc CO2Bn O NHCbz Ph MeO OiPr O N Boc CO2Bn O Ph NHCbz