Synthesis of external β-turn templates by reaction of protected dehydroamino acids with cyclic enaminoesters

Article abstract of DOI:10.1016/j.tet.2004.09.036

Two external β-turn templates have been synthesised and one of them has been derivatised as a GLDV-tetrapeptide. In the course of the synthesis an interesting dichotomy was observed in the condensation of exocyclic enamines such as 4 and 19 with protected

Full text of DOI:10.1016/j.tet.2004.09.036

Tetrahedron 61 (2005) 287–299
Synthesis of external b-turn templates by reaction of protected
dehydroamino acids with cyclic enaminoesters*
*
Jane M. Berry, Paul M. Doyle and Douglas W. Young
Department of Chemistry, Sussex Centre for Biomolecular Design and Drug Development, University of Sussex, Falmer,
Brighton BN1 9QJ, UK
Received 15 July 2004; revised 7 September 2004; accepted 9 September 2004
Available online 6 October 2004
Abstract—Two external b-turn templates have been synthesised and one of them has been derivatised as a GLDV-tetrapeptide. In the course
of the synthesis an interesting dichotomy was observed in the condensation of exocyclic enamines such as 4 and 19 with protected
dehydroamino acids using phosphorus trichloride. When dehydroamido acids were condensed with the enamines 4 and 19 then 6/6 and 6/5
fused bicyclic compounds such as 5 and 20, respectively, were obtained, whereas, when dehydroamino acid urethanes were used, the 5/6 and
5/5 fused products 7 and 23 were obtained. The bicyclic template 20 was converted to the GLDV-tetrapeptide derivative 31 but the sensitivity
to base of the acyl–enamine system of the template reduced the yield in the synthesis of the external turn 35.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
successfully to replace the D-Phe-Pro dipeptide sequence
in gramicidin S;5 to prepare biologically active mimetics of
enkephalin and luteinising hormone releasing factor
(LRF);6 and to replace residues 19 and 20 or 33 and 34 of
ha CGRP8–37, thus modelling the bioactive structure of the
natural vasodilator peptide.7
Molecular recognition processes involving interaction
between (i) proteins and nucleic acids; (ii) proteins and
proteins; and (iii) proteins and small molecules are
important in many biological regulatory mechanisms.
Reverse turns connect elements of protein secondary
structure such as a-helices and b-sheets and, since they
usually occur on the surface of the protein, these turns are
often important in molecular recognition processes,2,3 such
as those involved in immunological recognition, and in
protein phosphorylation and glycosylation.
Native proteins are large, have poor transport properties and
are prone to proteolysis and so their therapeutic use is
limited. Small synthetic molecules which mimic the turns
responsible for molecular recognition in these proteins may
have the therapeutic effect of the protein they mimic but
have none of the transport or other associated problems.
Thus these compounds have become interesting as potential
drugs and indeed incorporation of some dipeptide mimetics
into peptide sequences has given compounds with highly
constrained geometries and useful biological activity. The
external turn mimetic BTD, 1, prepared by Nagai4–6 was an
early example of such a compound and it was used
During our work on the synthesis of the bridged b-lactam 2,8
we developed9 a facile one-step synthesis of a series of
bicyclic compounds 5 by the method summarised in Scheme
1. The products of this synthesis were all esters at C-4 and
some, 5a, 5b, 5c and 5d, were substituted at C-7 with
protected amino groups. It was evident, therefore, that
suitably protected derivatives of these compounds might act
as external b-turn mimetics, since selective deprotection
might allow us to build an appropriate cyclic peptide on this
framework. The fact that the compounds 5a–d were not
homochiral was evidently a drawback but we hoped that this
problem might be solved by separation of the diastereo-
isomers produced as the turn was built up by reaction of the
mimetic with suitably protected L-amino acids.
* See Ref. 1.
Keywords: GLDV-tetrapeptide; b-Turn templates; Annelation.
* Corresponding author. Tel.: C44 1273 678327; fax: C44 1273 677196;
e-mail: d.w.young@sussex.ac.uk
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.09.036

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J. M. Berry et al. / Tetrahedron 61 (2005) 287–299
Scheme 1. Reagents and conditions—see Ref. 9.
Scheme 2. Reagents and conditions: (i) PCl3/dioxan/benzene/reflux (7a 57%, 7b 53%).
2. Results and discussion
Our well-proven synthesis of pyridothiazines9 had thus
proceeded in an anomalous manner when the dehydroamino
acid used was protected as a urethane. The structure of the
product suggested that the reaction had proceeded by
nucleophilic attack of the enamine 4 on the imine tautomer
6b rather than by nucleophilic attack at C-3 of the tautomer
6a, as shown in Scheme 3.
Our first targets were the benzyloxycarbonyl derivatives, 5g
and 5h, and so we reacted 2-benzyloxycarbonylamino-
acrylic acid 610 with the esters 4g and 4h, respectively, in
the presence of PCl3 as shown in Scheme 2. The products,
obtained in ca. 70% yields, were not the reduced
pyridothiazines, 5g or 5h, expected by analogy with our
previous work,9 since the characteristic signals for H-7 and
H-8, seen in the analogous compounds 5a, 5b, 5c and 5d,
1
The H NMR spectrum of the urethane 6 suggested that
some of the imine tautomer 6b might be present in
(C2H3)2SO with time, as a singlet appeared at 1.89 ppm in
1
were absent in the H NMR spectra. An additional methyl
1
the H NMR spectrum on standing in this solvent. It is
singlet was observed in the 1H NMR spectra of both
compounds, suggesting that the products were in fact the
pyrrolinothiazines 7a and 7b, an assignment which was
confirmed by single crystal X-ray structure analysis of the
methyl ester 7b.1
interesting to note that intramolecular attack by a nitrogen
nucleophile has been observed to occur at the a-carbon atom
of the dehydrodipeptide, 10, giving the product 11, as shown
in Scheme 4.11
Scheme 3.

J. M. Berry et al. / Tetrahedron 61 (2005) 287–299
289
of the dehydroamino acid. The esters 4g and 4h were,
therefore, reacted with 2-chloroacetylaminoacrylic acid
1514 in the presence of PCl3 as shown in Scheme 6. The
products obtained were those expected from ‘normal’
cyclisation, having all the spectral and analytical charac-
teristics of the pyridothiazines 5i and 5j. An attempt to
resolve these racemic products by selective hydrolysis of the
L-chloroacetamide using immobilised acylase I from
Aspergillus proved unsuccessful. The chloroacetylamine
5j was converted to the free amine 16 by reaction with
ortho-phenylenediamine.
Scheme 4. Reagents and conditions—see Ref. 11.
Further, it has been shown that reaction of acetylamino-
acrylic ester with HBr first gives the 2-bromo compound as
the kinetic product and that this then rearranges to the
3-bromo compound, the product of thermodynamic con-
trol.12 The nitrogen lone pair is certainly more available in
the urethane derivative 6a, allowing tautomerism to 6b, than
it is in the compounds 3a and 3b which form six membered
ring products by Michael attack on 6a. However, when
Millet et al.13 reacted the urethane 6 with the vinylogous
urethane 12 using 1-ethyl-3-(30-N,N-dimethylaminopro-
pyl)-carbodiimide and HOBt, Michael addition occurred
to give the 6/5 system 13 as shown in Scheme 5.
As we wished to make a series of b-turn templates, the
6/5-system 21 seemed to be a useful second series. It was
also of interest to see whether the cyclisation method used in
our synthesis might exhibit the same dichotomy between
urethane protected amines and amides that we had observed
in synthesis of the 6/6-ring system. Our starting point for the
6/5-system 21 was to prepare the imino ether 17 from ethyl
(2S)-pyroglutamate15 in 92% yield using Meerwein’s
reagent (Scheme 7). This was then reacted with tert-butyl
cyanoacetate and triethylamine to yield the vinylogous
urethane 18 as a single geometrical isomer in 58% yield.
This was assumed to be the Z-isomer which was expected to
be stabilised by hydrogen bonding. Treatment with
trifluoroacetic acid gave a 1:1 mixture of the E and Z
isomers 19a and 19b. The olefinic and NH protons in the 1H
NMR spectrum of the mixture diminished in intensity in the
presence of 2H2O, indicating a dynamic equilibrium
between the two isomers via the intermediate imine. The
spectrum was assigned with the aid of a 2D-COSY
spectrum, and the spectral peaks associated with the
individual isomers were assigned with the aid of the NOE
studies shown in Fig. 1 and reported in Section 3.1.9.
Scheme 5. Reagents and conditions—see Ref. 13.
Since the pyrrolinothiazines 7 might themselves be useful
b-turn mimetics, attempts were made to remove the
benzyloxycarbonyl group from 7a and 7b under a variety
of conditions, but to no avail. When the methyl ester was
treated with aqueous KOH in ethanol a crude product was
obtained which had two sharp singlets at 6.1 and 6.2 ppm in
the 1H NMR spectrum characteristic of the protons H-2 and
H-4 in double bond rearranged compounds such as 14.8
The mixed geometric isomers 19 were now reacted with
chloroacetylaminoacrylic acid 15 and PCl3 to afford the
protected hexahydroindolizine 20 in 41% yield. Treatment
of this with ortho-phenylenediamine gave the template
amino acid 21.
We had now prepared two b-turn templates 16 and 21 and
were interested to see if an ‘anomalous’ cyclisation reaction
might be observed if benzyloxycarbonylaminoacrylic acid 6
were reacted with the enamine 19. When these compounds
were reacted in the presence of PCl3, a product was obtained
in 21% yield which appeared to be a mixture of pairs of
diastereoisomers of 22 and 23 (Scheme 8). The only pure
product to be obtained by repeated column chroma-
tography was one of the diastereoisomers of the ‘anomalous
product’ 23.
Because of the anomalous reaction observed when urethane
protection was used, we decided to investigate the reaction
using the chloroacetyl group to protect the amine function
Scheme 6. Reagents and conditions: (i) PCl3/dioxan/benzene/reflux (5g 72%, 5h 71%); (ii) ortho-phenylenediamine/EtOH/aq LiOH/100 8C/48 h (51%).

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J. M. Berry et al. / Tetrahedron 61 (2005) 287–299
Scheme 7. Reagents and conditions: NCCH2COt2Bu/NEt3/60 8C/24 h (92%); (ii) CF3CO2H/room temperature/8 min (68%); (iii) 15CPCl3/dioxan/benzene/
reflux/90 min (41%); (iv) ortho-phenylenediamine/EtOH/aq LiOH/100 8C/48 h (43%).
peptide was built up. We chose the diastereoisomers 21 as
our template and opted to build the cyclic peptide GLDV
between the amino and carboxylic groups, since a turn
incorporating this motif has been shown to inhibit the
interaction of the integrin a4b1 with vascular cell adhesion
molecule-1.16 The amine 21 was therefore reacted with
(2S)-N-tert-butoxycarbonylvaline and 2-(1H-benzotriazol-
1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU)
Figure 1. NOE experiments on the mixture of geometric isomers 19a and
in DMF to give the adduct 24 in 76% yield as shown in
19b.
Scheme 9. Deprotection with trifluoroacetic acid gave the
We were unable to resolve the enantiomers 16 or separate
the diastereoisomers 21, and so we decided to build an
external turn motif on one of these templates and to attempt
separation of the various diastereoisomers prepared as the
amine 25 in 99% yield and condensation with 4-tert-butyl
(2S)-N-Fmoc-aspartic acid gave the dipeptide adduct 26 in
70% yield. This was deprotected using piperidine in DMF to
give a 70% yield of the amine 27 which was condensed with
Scheme 8. Reagents and conditions: (i) PCl3/dioxan/benzene/reflux 24 h (21%).
Scheme 9. Reagents and conditions: (i) TBTU/DIPEA/DMF/Boc-Val-OH/room temperature/12 h (76%); (ii) CF3CO2H/0 8C/15 min (74%);
(iii) TBTU/DIPEA/Fmoc-Asp(OtBu)-OH/room temperature/12 h (70%); (iv) piperidine/DMF/room temperature/90 min (O70%); (vi) TBTU/DIPEA/DMF/
Fmoc-Leu-OH/room temperature/12 h (97%); (vii) TBTU/DIPEA/Fmoc-Gly-OH/room temperature/12 h (97%).

J. M. Berry et al. / Tetrahedron 61 (2005) 287–299
291
Scheme 10. Reagents and conditions: (i) NaOH/H2O/EtOH/pH 11/room temperature/24 h (32, 3%; 33, 14%).
(2S)-N-Fmoc-leucine to give the tripeptide adduct 28 in
97% yield. Deprotection to the amine 29 in 78% yield and
condensation with N-Fmoc-glycine gave the tetrapeptide
adduct 30 in 97% yield. None of the diastereoisomeric
compounds in this synthesis could be separated.
Finally the GLDV-tetrapeptide amine 31 was prepared in
95% yield by treating the compound 30 with piperidine in
dimethylformamide. The next stage was to hydrolyse the
ethyl ester which we accomplished, obtaining the acid 32 in
3% yield (Scheme 10). The major hydrolysis product was
the diacid 33, the result of hydrolysis of the bicyclic lactam.
The sensitivity of this functionality to base was presumably
due to the fact that this was an enaminamide, a functionality
which is thought to make the lactam system in cephalo-
sporin more reactive.17 Attempts to improve the yield of the
acid 32 were unsuccessful. The small amount of acid 32
available was reacted with TBTU and DMAP in DMF at
high dilution. The crude product showed an ion at m/z 667 in
the mass spectrum corresponding [MCNa]C for the desired
cyclic peptide 34 and further purification by HPLC gave a
sample with m/z 645 ([MCH]C for 34). There was however
insufficient material for further characterisation. Reaction
with trifluoroacetic acid and purification by HPLC
gave material with m/z 610 ([MCNa]C for 35) and 626
([MCK]C for 35) but again there was insufficient material
for further characterisation.
In conclusion we have discovered an interesting dichotomy
in the annelation of exocyclic enamines, depending on
whether dehydroamino acid urethanes or dehydroamido
acids are used. We have synthesised a series of potential
external b-turn templates but when one of these was
converted to a GLDV-tetrapeptide derivative, the sensitivity
of the conjugated lactam system to base prevented the
accumulation of sufficient bicyclic acid to fully characterise
the product. In no case was separation of any of the
diastereoisomeric products achieved.
3. Experimental
3.1. General
Melting points were determined on a Kofler hot-stage
apparatus and are uncorrected. Optical rotation measure-
ments (in units of 10K1 deg cm2 gK1) were obtained on a
Perkin Elmer PE241 polarimeter, using a 1 dm path length
micro cell. IR spectra were recorded on Perkin Elmer 1720
and Bruker IFS 66 Fourier transform instruments. 1H NMR
spectra were recorded on Bruker WM360 (360 MHz),
AMX500 (500 MHz), AC200 (200 MHz), AMX360
(360 MHz) and AMX600 (600 MHz) Fourier transform
instruments. J values are given in Hz. NOE experiments
were carried out on a Bruker AMX500 (500 MHz) Fourier
transform instrument, and COSY experiments on a Bruker

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J. M. Berry et al. / Tetrahedron 61 (2005) 287–299
AMX600 (600 MHz) Fourier transform instrument. 13C
NMR spectra were recorded on Bruker AMX500
(125.8 MHz) and Bruker AC200 (50 MHz) Fourier trans-
form instruments. INEPT experiments were used to help
assign 13C NMR resonances where necessary. Residual
solvent peaks were used as internal reference for all NMR
spectra. Mass spectra were recorded on Kratos MS80F,
MS50 and MS25 double focusing spectrometers and a VG
Bio-Q spectrometer. Accurate mass measurements were
recorded on a Concept Kratos spectrometer by Dr S. Chotai
(Wellcome Research Laboratories). Microanalyses were
performed by Miss M. Patel (Sussex) and Mrs. C. Longhurst
and Miss W. C. Mann (Wellcome). Column chromatog-
raphy was carried out on Merck Kieselgel 60 (239–240
mesh—ART 9385). Petroleum ether refers to the fraction of
alkanes of bp 60–80 8C.
(C-4), 154.13 (C-8) and 162.13, 162.20, 162.26 and 173.22
(4!C]O).
3.1.3. 8-Benzyl 4-methyl (7RS)-7-benzyloxycarbonyl-
amino-6,7-dihydro-3,7-dimethyl-6-oxo-2H-pyrrolino-
[2,1b][1,3]-thiazine-4,8-dicarboxylate (7b). Methyl 2-ben-
zyloxycarbonylmethylene-3,6-dihydro-5-methyl-2H-[1,3]-
thiazine-4-carboxylate 4h9 (1.0 g, 3.13 mmol) and 2-benzyl-
oxycarbonylaminoacrylic acid 6 (1.4 g, 6.3 mmol) were
dissolved in dioxane (15 ml) and benzene (20 ml). Phos-
phorus trichloride (900 mg, 6.3 mmol) was added and the
solution was heated at reflux under nitrogen for 12 h. The
solvent was removed in vacuo to afford a red oil, which was
dissolved in ethyl acetate. The solution was washed with
saturated aqueous sodium hydrogen carbonate, 1 M hydro-
chloric acid and saturated aqueous sodium chloride and
dried (MgSO4). The solvent was removed in vacuo and the
residue was purified by column chromatography on silica
gel, eluting with petroleum ether/ethyl acetate (3:2). The
resulting orange foam was recrystallised from chloroform to
yield 8-benzyl 4-methyl (7RS)-7-benzyloxycarbonylamino-
6,7-dihydro-3,7-dimethyl-6-oxo-2H-pyrrolino-[2,1b][1,3]-
thiazine-4,8-dicarboxylate 7b as white crystals (870 mg,
53%), mp 147-149 8C; (Found: C, 61.6; H, 4.9; N, 5.2.
C27H26N2O7S requires C, 62.05; H, 5.0; N, 5.4%); m/z [Cve
FAB (thioglycerol)] 545 ([MCNa]C) and 523 ([MCH]C);
nmax (KBr)/cmK1 3311 (br, NH), 1764 and 1739 (ester) and
1691 (lactam); lmax (MeOH)/nm 324 (3 21,800); dH
(360 MHz, C2HCl3) 1.55 (3H, s, CH3), 2.12 (3H, s, CH3),
3.1.1. 2-Benzyloxycarbonylaminoacrylic acid (6). This
was prepared by the method of Frankel and Reichmann10 as
a white solid in 53% yield, mp 110–112 8C (lit.10 105 8C);
m/z [Cve FAB (thioglycerol)] 244 ([MCNa]C); nmax
(KBr)/cmK1 3424 (br, NH), 3300–2500 (NH, OH), 1749
(ester) and 1699 (acid); dH (360 MHz, (C2H3)2SO) 6a
5.09 (2H, s, OCH2Ph), 5.60 (1H, s, olefinic), 5.83 (1H, s,
olefinic), 7.30–7.40 (5H, m, ArH) and 8.53 (1H, br s, NH);
signals for 6b appeared with time 1.89 (3H, s, CH3), 4.95
(2H, s, OCH2Ph) and 7.4 (5H, m, ArH).
3.13 (1H, d, J2A,2BZ15.6 Hz, H-2A), 3.59 (1H, d, J2B,2A
15.6 Hz, H-2B), 3.80 (3H, s, OCH3), 4.96 (2H, 2!d, JAB
Z
Z
3.1.2. 8-Benzyl4-ethyl(7RS)-7-benzyloxycarbonylamino-
6,7-dihydro-3,7-dimethyl-6-oxo-2H-pyrrolo[2,1b]-[1,3]-
thiazine-4,8-dicarboxylate (7a). Ethyl 2-benzyloxycarbo-
nylmethylene-3,6-dihydro-5-methyl-2H-[1,3]-thiazine-4-
carboxylate 4g9 (500 mg, 1.5 mmol) and 2-benzyloxycar-
bonylaminoacrylic acid 6 (500 mg, 2.25 mmol) were
dissolved in dioxane (7.5 ml) and benzene (10 ml).
Phosphorus trichloride (310 mg, 2.25 mmol) was added
and the solution was heated at reflux under nitrogen for 5 h.
The solvent was removed in vacuo to afford a dark red oil,
which was dissolved in ethyl acetate. The solution was
washed with saturated aqueous sodium hydrogen carbonate,
1 M hydrochloric acid and saturated aqueous sodium
chloride and dried (MgSO4). The solvent was removed in
vacuo to afford an orange solid, which was recrystallised
from dichloromethane/diethyl ether to yield 8-benzyl
4-ethyl (7RS)-7-benzyloxycarbonylamino-6,7-dihydro-3,7-
dimethyl-6-oxo-2 -pyrrolo[2,1b][1,3]thiazine-4,8-dicarb-
oxylate 7a as a beige solid (460 mg, 57%), mp 158–160 8C;
(Found: C, 63.05; H, 4.9; N, 5.1. C28H28N2O7S requires C,
62.7; H, 5.0; N, 5.2%); m/z [Cve FAB (3-NBA)] 536
([M]C); nmax (KBr)/cmK1 3414 (br, NH), 1764 and 1732
(ester) and 1687 (lactam); lmax (MeOH)/nm 328 (3 6500);
dH (360 MHz, C2HCl3) 1.27 (3H, t, JZ7.1 Hz, CH3), 1.55
(3H, s, CH3), 2.14 (3H, s, CH3), 3.10 (1H, d, J2A,2BZ15 Hz,
H-2A), 3.60 (1H, d, J2B,2AZ15 Hz, H-2B), 4.20–4.35
(2H, m, JZ7.1 Hz, OCH2), 4.99 (1H, d, JABZ14 Hz,
OCH[A]Ph), 5.01 (1H, d, JBAZ14 Hz, OCH[B]Ph), 5.20
(1H, d, JCDZ14 Hz, OCH[C]Ph), 5.31 (1H, d, JDCZ14 Hz,
OCH[D]Ph), 5.46 (1H, br s, NH, exchanges in 2H2O)
and 7.29–7.35 (10H, m, ArH); dC (125.8 MHz, C2HCl3)
13.96 (CH3), 18.74 (CH3), 22.43 (CH3), 30.59 (C-2), 59.48
(C-7), 61.69 (OCH2), 65.99 and 67.07 (OCH2Ph), 124.95
(C-9), 127.19 (C-3), 127.99–136.02 (aromatics), 153.00
12 Hz, OCH2Ph), 5.28 (2H, 2!d, JABZ12 Hz, OCH2Ph),
5.36 (1H, br s, NH) and 7.27–7.34 (10H, m, ArH); dC
(125.8 MHz, C2HCl3) 18.77 (CH3), 22.43 (CH3), 30.44
(C-2), 52.30 (OCH3), 59.48 (C-7), 65.97 and 66.89
(OCH2Ph), 123.85 (C-9), 127.06 (C-3), 127.95–136.18
(aromatics), 152.55 (C-4), 154.19 (C-8) and 156.62,
162.25, 162.72 and 173.33 (4!C]O).
3.1.4. 9-Benzyl 4-ethyl (7RS)-7-(2-chloroacetylamino)-
7,8-dihydro-3-methyl-6-oxo-2H,6H-pyrido[2,1b][1,3]-
thiazine-4,9-dicarboxylate (5i). Ethyl 2-benzyloxycarbo-
nylmethylene-3,6-dihydro-5-methyl-2H-[1,3]-thiazine-4-
carboxylate 4g9 (2.0 g, 6 mmol) and 2-chloroacetylamino-
acrylic acid 1514 (1.1 g, 6.6 mmol) were dissolved in
dioxane (30 ml) and benzene (40 ml). Phosphorus trichlo-
ride (900 mg, 6.6 mmol) was added and the solution was
heated at reflux under nitrogen for 156 h. The solvent was
removed in vacuo to afford an oil which was dissolved in
ethyl acetate. The solution was washed successively with
saturated aqueous sodium hydrogen carbonate, 1 M hydro-
chloric acid and saturated aqueous sodium chloride and
dried (MgSO4). The solvent was removed in vacuo and the
residue was purified by column chromatography on silica
gel, eluting with ethyl acetate/chloroform (1:1) to yield
9-benzyl 4-ethyl (7RS)-7-(2-chloroacetylamino)-7,8-dihy-
dro-3-methyl-6-oxo-2H,6H-pyrido[2,1b][1,3]thiazine-4,9-
dicarboxylate 5i as a pale yellow foam (2.1 g, 72%), mp 64–
65 8C; m/z [EI] Found: 478.09564. C22H23N2O6S35Cl
requires 478.09654; m/z [Cve FAB (EtOAc/3-NBA)] 479
and 481 ([MCH]C); nmax (KBr)/cmK1 3414 (br, NH), 1725
(ester) and 1681 (lactam); lmax (MeOH)/nm 316 (3 17,800);
dH (500 MHz, C2HCl3) 1.28 (3H, t, JZ7.1 Hz, CH3), 2.28

J. M. Berry et al. / Tetrahedron 61 (2005) 287–299
293
(3H, s, CH3), 2.48 (1H, t, J8A,8BZJ8A,7Z15 Hz, H-8A),
the extracts were washed with saturated aqueous sodium
hydrogen carbonate and saturated aqueous sodium chloride
and dried (MgSO4). The solvent was removed in vacuo and
the residue was purified by column chromatography on
silica gel, eluting with methanol/dichloromethane (4:96), to
yield 9-benzyl 4-methyl (7RS)-7-amino-7,8-dihydro-3-
methyl-6-oxo-2H,6H-pyrido[2,1b][1,3]thiazine-4,9-dicar-
boxylate 16 as a pale yellow foam (250 mg, 51%), mp 58–
60 8C; m/z [EI] Found 388.10836. C19H20N2O5S requires
388.10929; nmax (KBr)/cmK1 3413 (br, NH), 1728 (ester)
and 1681 (lactam); lmax (MeOH)/nm 232 and 315.0 (3
11,900 and 3400); dH (360 MHz, C2HCl3) 2.28 (3H, s, CH3),
2.51 (1H, t, J8A,7ZJ8A,8BZ15 Hz, H-8A), 2.87 (1H, d,
J2A,2BZ13.7 Hz, H-2A), 3.39 (1H, d, J2B,2AZ13.7 Hz,
H-2B), 3.48 (1H, dd, J8B,7Z5.6 Hz, J8B,8AZ15 Hz, H-8B),
3.58 (1H, dd, J7,8AZ15 Hz, J7,8BZ5.6 Hz, H-7), 3.77 (3H,
s, OCH3), 5.25 (2H, s, OCH2Ph) and 7.35–7.48 (5H, m,
ArH); dC (125.8 MHz, C2HCl3) 18.67 (CH3), 28.94 (C-2),
31.79 (C-8), 50.87 (OCH3), 52.2 (C-7), 66.72 (OCH2Ph),
106.22 (C-10), 126.89 (C-4), 128.22–135.87 (aromatics),
140.91 (C-3), 151.63 (C-9), 162.90, 165.64 and 172.35
(3!–C]O).
2.87 (1H, d, J2A,2BZ13.7 Hz, H-2A), 3.50 (1H, d, J2B,2A
13.7 Hz, H-2B), 3.52 (1H, AB, J8B,7Z5.9 Hz, J8B,8A
Z
Z
15 Hz, H-8B), 4.09 (2H, s, CH2Cl), 4.14–4.36 (2H, m,
OCH2), 4.59–4.64 (1H, m, J7,8AZ15 Hz, J7,8BZ5.9 Hz,
H-7), 5.16 (1H, d, JABZ12.4 Hz, OCH[A]Ph), 5.32 (1H, d,
JBAZ12.4 Hz, OCH[B]Ph) and 7.29–7.40 (5H, m, ArH);
dC (125.8 MHz, C2HCl3) 14.13 (CH3), 18.74 (CH3), 26.40
(C-2), 32.04 (C-8), 42.43 (CH2Cl), 49.63 (C-7), 61.50
(OCH2), 66.90 (OCH2Ph), 106.44 (C-10), 126.76 (C-4),
128.39–135.84 (aromatics), 142.31 (C-3), 152.27 (C-9)
and 162.13, 165.50, 166.38 and 167.97 (4!C]O).
3.1.5. 9-Benzyl 4-methyl (7RS)-7-(2-chloroacetylamino)-
7,8-dihydro-3-methyl-6-oxo-2H,6H-pyrido[2,1b][1,3]-
thiazine-4,9-dicarboxylate (5j). Methyl 2-benzyloxycar-
bonylmethylene-3,6-dihydro-5-methyl-2H-[1,3]-thiazine-4-
carboxylate 4h9 (4.0 g, 13 mmol) and 2-chloroacetylami-
noacrylic acid 1514 (2.3 g, 14 mmol) were dissolved in
dioxane (60 ml) and benzene (80 ml). Phosphorus trichlo-
ride (1.9 g, 14 mmol) was added and the solution was heated
at reflux under nitrogen for 90 min. The solvent was
removed in vacuo to afford an oil, which was dissolved in
ethyl acetate. The solution was washed with saturated
aqueous sodium hydrogen carbonate, 1 M hydrochloric acid
and saturated aqueous sodium chloride and dried (MgSO4).
The solvent was removed in vacuo and the residue was
purified by column chromatography on silica gel, eluting
with petroleum ether/ethyl acetate (3:2) to afford an off-
white solid which was recrystallised from chloroform to
yield 9-benzyl 4-methyl (7RS)-7-(2-chloroacetylamino)-
7,8-dihydro-3-methyl-6-oxo-2H,6H-pyrido[2,1b][1,3]thia-
zine-4,9-dicarboxylate 5j as a white solid (4.1 g, 68%), mp
202–204 8C; (Found: C, 53.9; H, 4.5; N, 5.9. C21H21N2O6-
SCl requires C, 54.25; H, 4.55; N, 6.0%); m/z [Cve FAB
(thioglycerol)] 487 and 489 ([MCNa]C) and 465 and 467
([MCH]C); nmax (KBr)/cmK1 3354 (br, NH), 1730 (ester)
and 1676 (lactam); lmax (MeOH)/nm 318 (3 9000); dH
3.1.7. Ethyl (2S)-5-ethoxy-3,4-dihydro-2H-pyrrole-2-
carboxylate (17). Ethyl (2S)-pyroglutamate15 (44.4 g,
0.28 mol) was dissolved in dichloromethane (600 ml) and
a solution of triethyloxonium tetrafluoroborate (76.7 g,
0.4 mol) in dichloromethane (300 ml) was added under
nitrogen. The solution was stirred at room temperature for
40 h and saturated aqueous potassium hydrogen carbonate
(225 ml) was added. When effervescence ceased, the
organic layer was separated and the aqueous layer was
extracted with dichloromethane. The combined organic
layers were dried (MgSO4) and the solvent was removed in
vacuo to yield ethyl (2S)-5-ethoxy-3, 4-dihydro-2H-pyrrole-
2-carboxylate 17 as a dark yellow oil (48.3 g, 92%); m/z [EI]
Found: 185.10494. C9H15NO3 requires 185.10519; m/z
[Cve FAB (3-NBA)] 186 ([MCH]C); nmax (film)/cmKl
1740 (ester) and 1684 (C]N); dH (360 MHz, C2HCl3) 1.17
and 1.25 (6H, 2!t, JZ7.1 Hz, 2!CH3), 2.00–2.52 (4H, m,
H-3CH-4), 4.07–4.22 (4H, 2!q, JZ7.1 Hz, 2!OCH2)
and 4.42 (1H, dd, J2,3AZ1.9 Hz, J2,3BZ1.2 Hz, H-2); dC
(125.8 MHz, C2HCl3) 12.17 and 13.95 (2!CH3), 29.52
(C-3), 36.48 (C-4), 57.93 and 60.65 (2!OCH2), 59.25
(C-2), 171.89 (C-5) and 175.0 (C]O).
(360 MHz, C2HCl3) 2.25 (3H, s, CH3), 2.51 (1H, t, J8A,7
Z
J8A,8BZ17.5 Hz, H-8A), 2.88 (1H, d, J2A,2BZ15 Hz,
H-2A), 3.46 (1H, d, J2B,2AZ15 Hz, H-2B), 3.51 (1H, dd,
J8B,7Z7 Hz, J8B,8AZ17.5 Hz, H-8B), 3.80 (3H, s, OCH3),
4.10 (2H, s, CH2Cl), 4.64 (1H, m, J7,8AZ17.5 Hz, J7,8B
Z
7 Hz, H-7), 5.15 (1H, d, JABZ12 Hz, OCH[A]Ph), 5.38
(1H, d, JBAZ12 Hz, OCH[B]Ph) and 7.35–7.45 (5H, m,
ArH); dC (125.8 MHz, C2HCl3) 18.73 (CH3), 26.28 (C-2),
31.85 (C-8), 42.32 (CH2Cl), 49.38 and 49.48 (C-7), 52.33
(OCH3), 66.77 (OCH2Ph), 106.40 (C-10), 126.32 (C-4),
128.22–135.68 (aromatics), 142.15 (C-3), 151.97 (C-9) and
162.66, 165.27, 166.24 and 167.72 (4!C]O).
3.1.8. Ethyl (2S)-5-(tert-butoxycarbonylcyanomethyl-
ene)-pyrrolidine-2-carboxylate (18). Ethyl (2S)-5-
ethoxy-3, 4-dihydro-2H-pyrrole-2-carboxylate 17 (45 g,
0.24 mol), tert-butyl cyanoacetate (68.6 g, 0.49 mol) and
triethylamine (2.5 g, 24 mmol) were heated at 60 8C under
nitrogen, for 24 h. The solvent was removed by distillation
under reduced pressure. The resulting orange oil was
purified by column chromatography on silica gel, eluting
with diethyl ether/petroleum ether (1:1). The resulting
yellow oil crystallised on standing and was recrystallised
from dichloromethane/petroleum ether to yield ethyl (2S)-5-
(tert-butoxycarbonylcyanomethylene)-pyrrolidine-2-car-
boxylate 18 as white crystals (39.1 g, 58%), mp 77–79 8C;
[a]2D4ZK6.25 (c 1, CHCl3); (Found: C, 60.2; H, 6.8; N, 9.8.
C14H20N2O4 requires C, 60.0; H, 7.2; N, 10.0%); m/z [Cve
FAB (thioglycerol)] 281 ([MCH]C); nmax (KBr)/cmK1
3344 (br, NH), 2199 (CN) and 1736 (ester); lmax (MeOH)/
3.1.6. 9-Benzyl 4-methyl (7RS)-7-amino-7,8-dihydro-3-
methyl-6-oxo-2H,6H-pyrido[2,1b][1,3]thiazine-4,9-di-
carboxylate (16). 9-Benzyl 4-methyl (7RS)-7-chloroaceta-
mido-3-methyl-6-oxo-2H,6H-pyrido[2,1b][l,3] thiazine-
4,9-dicarboxylate 5j (600 mg, 1.25 mmol) was dissolved
in ethanol (60 ml). A solution of ortho-phenylenediamine
dihydrochloride (530 mg, 2.9 mmol) and lithium hydroxide
(180 mg, 7.3 mmol) in water (50 ml) was added and the
solution was stirred and heated at 100 8C for 48 h. The
solution was concentrated in vacuo and the resulting
aqueous solution was washed with diethyl ether. The
aqueous phase was extracted with dichloromethane and

294
J. M. Berry et al. / Tetrahedron 61 (2005) 287–299
nm 273 (3 16,300); dH (360 MHz, C2HCl3) 1.23 (3H, t, JZ
7.1 Hz, CH3), 1.43 (9H, s, C(CH3)3), 2.15 (1H, m, H-3A),
2.37 (1H, m, H-3B), 2.90 (2H, m, H-4), 4.17 (2H, q, JZ
7.1 Hz, OCH2), 4.48 (1H, dd, J2,3AZ5.5 Hz, J2,3BZ8.7 Hz,
H-2) and 9.08 (1H, br s, NH, exchanges in 2H2O); dC
(25.8 MHz, C2HCl3) 14.0 (CH3), 25.2 (C-3), 28.2
(C(CH3)3), 32.3 (C-4), 61.96 (OCH2), 62.02 (C-2), 70.4
(C-6), 81.2, (OC(CH3)3), 118.4 (CN), 166.9 (C-5) and 170.3
and 172.7 (2!C]O).
and benzene (225 ml). Phosphorus trichloride (4.6 g,
34 mmol) was added and the solution was heated at reflux
under nitrogen for 90 min. The solvent was removed in
vacuo to afford a dark red oil which was dissolved in ethyl
acetate. The solution was washed with saturated aqueous
sodium hydrogen carbonate, 1 M hydrochloric acid and
saturated aqueous sodium chloride and dried (MgSO4). The
solvent was removed in vacuo and the residue was purified
by column chromatography on silica gel, eluting with
dichloromethane/diethyl ether (1:1), to afford ethyl
(3S,6RS)-6-chloroacetylamino-8-cyano-5-oxo-1,2,3,5,6,7-
hexahydroindolizine-3-carboxylate 20 as a pale yellow
foam (4.51 g, 41%), mp 40–42 8C; (Found: C, 51.8; H,
4.9; N, 12.9. C14H16N3O4C1 requires C, 51.6; H, 5.0; N,
12.9%); m/z [Cve FAB (CHCl3/3-NBA)] 326 and 328
([MCH]C); nmax (KBr)/cmK1 3302 (br, NH), 2214 (CN),
1746 (ester) and 1670 (lactam); lmax (MeOH)/nm 277 (3
19,000); dH (500 MHz, C2HCl3, two diastereoisomers) 1.30
(3H, 2!t, JZ7 Hz, CH3), 2.24 (1H, m, J2A,3Z2.3, 3.1 Hz,
J2A,2BZ13.5 Hz, H-2A), 2.41 (1H, m, J2B,3Z9.2, 9.4 Hz,
J2B,2AZ13.5 Hz, H-2B), 2.55 (1H, m, J7A,6Z14.2 Hz,
J7A,7BZ16 Hz, H-7A), 2.90–3.06 (2H, m, H-1), 3.15 (1H,
2!AB, J7B,6Z7.6 Hz, J7B,7AZ16 Hz, H-7B), 4.09 and
4.10 (2H, 2!s, CH2Cl), 4.23 (2H, 2!q, JZ7 Hz, OCH2),
4.59–4.70 (1H, m, J6,7AZ14.2, 14.6 Hz, J6,7BZ7.4, 7.7 Hz,
J6,NHZ6 Hz, 6 (m, simplifies to 2!q in 2H2O), H-7B), 4.80
(1H, 2!q, J3,2AZ2, 3 Hz, J3,2BZ9.1, 9.5 Hz, H-3) and
7.30–7.35 (1H, 2!d.br, JNH,6Z5.2, 5.8 Hz, NH exchanges
3.1.9. Ethyl (2S)-5-cyanomethylenepyrrolidine-2-car-
boxylate (19). Ethyl (2S)-5-(tert-butoxycarbonylcyano-
methylene)-pyrrolidine-2-carboxylate 18 (16 g, 57 mmol)
was stirred vigorously at room temperature in trifluoroacetic
acid (80 ml) under argon for 8 min. Ice cold dichloro-
methane (200 ml) and saturated aqueous sodium hydrogen
carbonate (200 ml) were carefully added. The reaction was
cooled in an ice bath and solid sodium hydrogen carbonate
was added until pH 6.0 was reached. Solid sodium chloride
was added until the solution was saturated and it was filtered
through Celitew. The aqueous layer was extracted with
dichloromethane and the combined organic layers were
washed with aqueous sodium hydrogen carbonate (2 g/
100 ml H2O) and dried (MgSO4). The solvent was removed
in vacuo to yield a yellow oil which was purified by column
chromatography on silica gel, eluting with ethyl acetate/
toluene (1:1), to afford ethyl (2S)-5-cyanomethylenepyrro-
lidine-2-carboxylate 19 as a pale oil (7.0 g, 68%); [a]2D9ZC
6.75 (c 1, MeOH); (Found: C, 60.25; H, 7.0; N, 15.8.
C9H12N2O2 requires C, 60.0; H, 6.7; N, 15.5%); m/z [Cve
FAB (thioglycerol)] 181 ([MCH]C); nmax film)/cmK1 3344
(br, NH), 2192 (CN) and 1738 (ester); lmax (MeOH)/nm 263
(3 21,000); dH (500 MHz, C2HCl3, mixture of E and Z
isomers) 1.23 and 1.27 (3H, 2!t, JZ7.1 Hz, CH3, E/Z),
2.10 (1H, m, J3A,2Z5.4 Hz, J3A,3BZ13.0 Hz, H-3A E/Z),
2.34 (1H, m, J3B,2Z8.3 Hz, J3B,3AZ13.0 Hz, H-3B E/Z),
2.60 (1H, m, H-4Z), 2.78 (1H, m, H-4E), 3.76 (0.5H, s,
2
in H2O); dC (125.8 MHz, C2HCl3) 14.05 (CH3) 26.00 and
26.34 (C-2), 27.41 and 28.38 (C-1), 28.88 and 29.06 (C-7),
42.30 (CH2Cl), 48.99 and 49.73 (C-6), 59.77 and 60.00
(C-3), 62.27 (OCH2), 80.38 and 80.48 (C-8), 117.11 and
117.17 (CN), 154.59 and 154.81 (C-9) and 165.59–169.76
(3!C]O). The yellow foam was recrystallised from ethyl
acetate/chloroform, affording one diastereoisomer as a
white solid, mp 147–150 8C; lmax (MeOH)/nm 277; dH
(360 MHz, C2HCl3, single diastereoisomer) 1.28 (3H, t, JZ
2
7 Hz, CH3), 2.26–2.41 (2H, m, H-2), 2.46 (1H, m, J7A,6
Z
H-6Z, exchanges slowly in H2O), 4.09 (0.5H, s, H-6E,
2
14.3 Hz, J7A,7BZ16 Hz, H-7A), 2.98 (2H, m, H-1), 3.18
(1H, AB, J7B,6Z7.4 Hz, J7B,7AZ16 Hz, H-7B), 4.09 (2H, s,
exchanges slowly in H2O), 4.13 and 4.22 (2H, m, JZ
7.1 Hz, 2!OCH2 E/Z), 4.25 (0.5H, dd, J2,3AZ5.4 Hz,
J2,3BZ8.3 Hz, H-2Z), 4.30 (0.5H, dd, J2,3AZ5.4 Hz,
J32,3BZ8.3 Hz, H-2E), 5.94 (0.5H, br, NH(Z), exchanges
slowly in 2H2O) and 5.96 (0.5H, br, NH(E), exchanges
slowly in 2H2O); dC (125.8 MHz, C2HCl3 mixture of E and
Z isomers) 14.1 (CH3), 26.7 and 27.1 (C-3), 29.98 and 30.5
(C-4), 55.7 and 58.1 (C-6), 60.0 and 60.4 (C-2), 61.6
(OCH2), 119.9 and 121.3 (CN), 166.0 and 166.5 (C-5) and
CH2Cl), 4.22 (2H, q, JZ7 Hz, OCH2), 4.62 (1H, m, J6,7A
Z
14.3 Hz, J6,7BZ7.4 Hz, H-6, simplifies in 2H2O), 4.84 (1H,
dd, J3,2AZ1.5 Hz, J3,2BZ8 Hz, H-3) and 7.35 (1H, br d,
2
JNH.6Z4.3 Hz, NH, exchanges in H2O); dC (125.8 MHz,
C2HCl3) 14.05 (CH3), 26.35 (C-2), 27.49 (C-1), 29.07 (C-7),
42.30 (CH2Cl), 49.77 (C-6), 59.82 (C-3), 62.26 (OCH2),
80.50 (C-8), 117.15 (CN), 154.78 (C-9) and 166.26 and
169.26 (3!C]O).
1
171.6 and 171.96 (ester). Assignment of H NMR spectral
values to the individual isomers were made by a 2D COSY
experiment and the following NOE experiment; (i)
irradiation at d 3.76 ppm for H-6Z gave a 4% NOE in
both of H-4Z at d 2.60 ppm; (ii) irradiation at d 5.96 ppm for
NH(E) gave an NOE at 4.30 for H-2E and at d 4.09 for
H-6E; (iii) irradiation at d 4.09 ppm for H-6E resulted in a
2.6% NOE of NH(E) at d 5.96 ppm; and (iv) irradiation at
NH(Z) at d 5.94 ppm resulted in a NOE at H-2Z (d
4.25 ppm).
3.1.11. Ethyl (3S,6RS)-6-amino-8-cyano-5-oxo-1,2,3,
5,6,7-hexahydroindolizine-3-carboxylate (21). Ethyl
(3S,6RS)-8-cyano-6-chloroacetylamino-5-oxo-1,2,3,5,6,7-
hexahydroindolizine-3-carboxylate 20 (4.54 g, 13 mmol)
was dissolved in ethanol (30 ml). ortho-Phenylenediamine
dihydrochloride (4.7 g, 26 mmol) and lithium hydroxide
(1.54 g, 64 mmol) were added in water (80 ml) and the
solution was stirred and heated at 100 8C for 48 h. The
solution was concentrated in vacuo and the resulting
aqueous solution was washed with ethyl acetate. The
solvent was removed from the aqueous phase in vacuo to
afford a brown foam. This was purified by preparative
reverse phase HPLC on a Zorbax C8 column (25 cm!
22.2 mm), eluting with 0.2% TFA in acetonitrile and 0.2%
3.1.10. Ethyl (3S,6RS)-6-chloroacetylamino-8-cyano-5-
oxo-1,2,3,5,6,7-hexahydroindolizine-3-carboxylate (20).
Ethyl (2S)-5-cyanomethylenepyrrolidine-2-carboxylate 19
(5.5 g, 31 mmol) and 2-chloroacetylaminoacrylic acid
1514 (5.5 g, 34 mmol) were dissolved in dioxane (170 ml)

J. M. Berry et al. / Tetrahedron 61 (2005) 287–299
295
TFA/water, fractionating at 30 s intervals. Samples, judged
to be homogeneous by analytical reverse phase HPLC on a
Zorbax C8 column (25 cm!4.6 mm), were pooled and
lyophilised. This resulted in ethyl (3S,6RS)-6-amino-8-
cyano-5-oxo-1,2,3,5,6,7-hexahydroindolizine-3-carboxyl-
ate 21 as a pale yellow foam (1.5 g, 43%), mp 32–34 8C; m/z
[EI] Found: 249.11155. C12H15N3O3 requires 249.11134;
m/z [Cve FAB (thioglycerol)] 250 ([MCH]C); nmax (KBr)/
cmK1 3370 (br, NH), 2207 (CN), 1741 (ester) and 1664
(lactam); lmax (MeOH)/nm 275 (3 14,000); dH (360 MHz,
C2HCl3) 1.27–1.32 (3H, 2!t, JZ7.1 Hz, CH3), 2.21 (1H,
m, J2A,3Z2, 3 Hz, J2A,2BZ13 Hz, H-2A), 2.31–2.49 (1H,
m, J2B,3Z9.1, 9.3 Hz, J2B,2AZ13 Hz, H-2B), 2.51–2.62
(1H, m, J7A,6Z12.1 Hz, J7A,7BZ13.8 Hz, H-7A), 2.75–2.83
(1H, m, J7B.6Z7.3 Hz, J7B,7AZ13.8 Hz, H-7B), 2.88–3.00
(2H, m, H-1), 3.59–3.74 (1H, 2!q, J6,7AZ12.1, 13.6 Hz,
J6,7BZ7.3, 7.6 Hz, H-6), 4.18–4.26 (2H, m, JZ7.1 Hz,
OCH2) and 4.72–4.82 (1H, 2!q, J3,2AZ2, 3 Hz, J3,2BZ9.1,
9.3 Hz, H-3); dC (50 MHz, C2HCl3) 14.08 (CH3), 26.13 and
26.34 (C-2), 29.16 (C-1), 30.50 and 30.94 (C-7), 50.51 and
50.84 (C-3), 59.63 and 59.74 (C-6), 62.06 (OCH2), 102.0
(C-8), 117.0 (CN), 121.0 (C-9), 155.0 and 171.0 (2!
C]O).
1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU)
(1.2 g, 3.6 mmol) and di-isopropylethylamine (470 mg,
3.6 mmol) were added and the solution was stirred under
nitrogen at room temperature for 12 h. The solvent was
removed in vacuo (!50 8C) and the resulting oil was
dissolved in ethyl acetate. The solution was successively
washed with 5% aqueous citric acid, 5% aqueous sodium
hydrogen carbonate and saturated aqueous sodium chloride
and dried (MgSO4). The solvent was removed in vacuo and
the resulting foam was purified by column chromatography
on silica gel, eluting with methanol/dichloromethane (4:96),
to yield ethyl (3S,6RS)-6-[(2S)-2-tert-butoxycarbonyl-
amino-3-methylbutanamido]-8-cyano-5-oxo-1,2,3,5,6,7-
hexahydroindolizine-3-carboxylate 24 as a pale yellow
powder (1.2 g, 76%), mp 83–84 8C; [a]2D8ZK32.5 (c 1,
CHCl3); m/z [EI] Found: 448.23069, C22H32N4O6 requires
448.23219; m/z [EI] 487 ([MCK]C), 471 ([MCNa]C) and
449 ([MCH]C); nmax (KBr)/cmK1 3412 (br, NH), 2210
(CN), 1748 (ester) and 1665 (lactam); lmax (MeOH)/nm 277
(3 14,300); dH (360 MHz, C2HC13) 0.91–0.99 (6H, m, JZ
6.7 Hz, C(CH3)2), 1.25–1.33 (3H, 2!t, JZ7.1 Hz, CH3),
1.45–1.46 (9H, 2!s, OC(CH3)3), 2.15–2.30 (2H, m, J7A,6
7.1 Hz, Jb,aZ5.4 Hz, H-7ACH-b), 2.39 (1H, m, J2A,3
9.1 Hz, J2A,2BZ13.3 Hz, H-2A), 2.42–2.61 (1H, m, J7B,6
Z
Z
Z
14.1 Hz, J7B,7AZ16.7 Hz, H-7B), 2.94–3.14 (2H, m, H-1),
3.17–3.20 (1H, m, J2B,3Z1.7 Hz, J2B,2AZ13.3 Hz, H-2B),
3.90–4.10 (1H, m, Ja,bZ5.4 Hz, H-a), 4.21–4.28 (2H, m,
JZ7.1 Hz, OCH2), 4.56–4.71 (1H, 2!q, J6,7AZ7.1 Hz,
3.1.12. Reaction of 2-benzyloxycarbonylaminoacrylic
acid with ethyl (2S)-5-cyanomethylenepyrrolidine-2-
carboxylate. Ethyl (2S)-5-cyanomethylenepyrrolidine-2-
carboxylate 19 (500 mg, 2.8 mmol) and 2-benzyloxycarbo-
nylaminoacrylic acid 610 (920 mg, 4.2 mmol) were dis-
solved in dioxane (15 ml) and benzene (20 ml). Phosphorus
trichloride (570 mg, 4.2 mmol) was added and the solution
was heated at reflux under nitrogen for 24 h. The solvent
was removed in vacuo affording a dark orange oil, which
was dissolved in ethyl acetate. The solution was succes-
sively washed with saturated aqueous sodium hydrogen
carbonate, 1 M hydrochloric acid and saturated aqueous
sodium chloride and dried (MgSO4). The solvent was
removed in vacuo and was purified by column chromatog-
raphy on silica gel, eluting with dichloromethane/diethyl
ether (2:1), yielding a mixture of four compounds as a foam
(230 mg, 21%); m/z [Cve FAB (EtOAc/3-NBA)] 384
([MCH]C); nmax (nujol)/cmK1 3350 (br, NH), 2210 (CN),
1733 (ester), 1716 (g-lactam) and 1665 (d-lactam); lmax
(MeOH)/nm 282 and 283. Repeated column chromatog-
raphy separated one diastereoisomer of the g-lactam 23 as
an off-white foam (44 mg, 5%); lmax (MeOH)/nm 282; dH
(360 MHz, C2HCl3) 1.28 (3H, t, JZ7.1 Hz, CH3), 1.50 (3H,
s, CH3), 2.51–3.00 (4H, m, H-1CH-2), 4.17–4.29 (2H, m,
JZ7.1 Hz, OCH2), 4.53 (1H, m, H-3), 5.08 (2H, d, JZ
7.4 Hz, OCH2Ph), 5.47 (1H, br s, NH) and 7.29–7.36 (5H,
m, ArH); dC (125.8 MHz, C2HCl3) 13.9 (CH3), 22.56 (CH3),
23.93 (C-2), 30.88 (C-1), 55.65 (C-3), 62.23 (OCH2), 65.84
(C-6), 67.24 (OCH2Ph), 113.83 (CN), 127.99–135.8 (aro-
matics), 136.15 (C-7), 153.77 (C-8) and 161.7, 168.42 and
172.2 (3!C]O).
2
J6,7BZ14.1 Hz, H-6, simplifies to q in H2O), 4.73–4.76
(1H, 2!q, J3,2AZ9.1 Hz, J3,2BZ1.7 Hz, H-3), 5.02 (1H, br
s, NH, exchanges in 2H2O) and 6.60–6.80 (1H, 2!br s, NH,
exchanges in 2H2O); dC (125.8 MHz, C2HC13) 14.05 (CH3),
17.41, 17.54, 19.18 and 19.22 (C(CH3)2), 26.06 and 26.41
(C-2), 27.70 and 28.74 (C-1), 28.30 (OC(CH3)3), 28.91 and
29.10 (C-7), 30.73 and 30.90 (C-6), 31.23 (OC(CH3)3),
48.72 (C-3), 49.47 (C-b), 59.81 and 60.03 (C-a), 62.20
(OCH2), 80.15 and 80.52 (C-8), 117.20 and 117.29 (CN),
154.49 and 154.72 (urethane), 155.80 (C-9), and 166.03,
166.62, 169.34, 169.83, 171.89 and 172.05 (3!C]O).
3.1.14. Ethyl (3S,6RS)-6-[(2S)-2-amino-3-methylbutana-
mido]-8-cyano-5-oxo-1,2,3,5,6,7-hexahydroindolizine-3-
carboxylate (25). Ethyl (3S,6RS)-6-[(2S)-2-tert-butoxycar-
bonylamino-3-methylbutanamido]-8-cyano-5-oxo-1,2,3,
5,6,7-hexahydroindolizine-3-carboxylate 24 (300 mg,
0.67 mmol) was cooled to 0 8C. Trifluoroacetic acid (8 ml)
was added and the solution was stirred under nitrogen at
0 8C, for 15 min. Stirring was continued at room tempera-
ture for a further 2 h. The solvent was removed in vacuo
(!50 8C), affording an orange oil. This was triturated with
diethyl ether and the solvent was decanted off, yielding the
TFA salt of ethyl (3S,6RS)-6-[(2S)-2-amino-3-methylbuta-
namido]-8-cyano-5-oxo-1,2,3,5,6,7-hexahydroindolizine-3-
carboxylate 25 as an off-white solid (230 mg, 74%), mp
194–196 8C; [a]2D6ZK125.53 (c 0.7, MeOH); m/z [EI]
Found: 348.17809, C17H24N4O4 requires 348.17976; m/z
[Cve FAB (thioglycerol)] 697 ([2MCH]C), 371 ([MC
Na]C) and 349 ([MCH]C); nmax (KBr)/cmK1 3414 (br,
NH), 2213 (CN), 1750 (ester) and 1672 (lactam); lmax
(MeOH)/nm 276 (3 5700); dH (360 MHz, C2HC13) 0.89–
0.99 (6H, m, C(CH3)2), 1.22–1.38 (3H, 2!t, JZ7.1 Hz,
CH3), 2.12–2.30 (2H, m, J7A,6Z5.8 Hz, J7A,7BZ14.2 Hz,
Jb,aZ3.2 Hz, H-7ACH-b), 2.37 (1H, m, J2A,3Z8.8 Hz,
3.1.13. Ethyl (3S,6RS)-6-[(2S)-2-tert-butoxycarbonyl-
amino-3-methylbutanamido]-8-cyano-5-oxo-1,2,3,5,6,7-
hexahydroindolizine-3-carboxylate (24). Ethyl (3S,6RS)-
6-amino-8-cyano-5-oxo-1,2,3,5,6,7-hexahydroindolizine-3-
carboxylate 21 (900 mg, 3.6 mmol) and (2S)-N-tert-butoxy-
carbonylvaline (780 mg, 3.6 mmol) were dissolved in
dimethylformamide (35 ml). 2-(1H-Benzotriazol-1-yl)-

296
J. M. Berry et al. / Tetrahedron 61 (2005) 287–299
J2A,2BZ15.8 Hz, H-2A), 2.43–2.62 (1H, m, J7B,6Z10.6 Hz,
J7B,7AZ14.2 Hz, H-7B), 2.88–3.03 (2H, m, H-1), 3.04–3.22
(1H, m, J2B,3Z2.4 Hz, J2B,2AZ15.8 Hz, H-2B), 4.15–4.32
(2H, m, JZ7.1 Hz, OCH2), 4.53–4.84 (3H, m, Ja,bZ3.6 Hz,
3.1.16. Ethyl (3S,6RS)-6-[(2S)-2-((2S)2-amino-4-tert-
butoxycarbonylpropanamido)-3-methylbutanamido]-8-
cyano-5-oxo-1,2,3,5,6,7-hexahydroindolizine-3-carboxyl-
ate (27). Ethyl (3S,6RS)-6-[(2S)-2-((2S)-2-(9H)-fluoren-9-
ylmethoxycarbonylamino-4-tert-butoxycarbonylpropana-
mido)-3-methylbutanamido]-8-cyano-5-oxo-1,2,3,5,6,7-
J6,7AZ5.8 Hz, J6,7BZ10.6 Hz, J3,2AZ8.8 Hz, J3,2B
Z
2.4 Hz, H-aCH-6CH-3) and 6.30–6.40 (1H, br s, NH,
exchanges in 2H2O); dC (125.8 MHz, C2H3O2H) 14.85
(CH3), 18.37 and 19.37 (C(CH3)2), 27.49 and 27.61 (C-2),
29.15 and 29.72 (C-1), 30.61 and 31.26 (C-7), 32.05 and
32.20 (C-6), 50.94, 60.48 and 60.55 (C-3), 61.97 and 62.16
(C-a), 63.50 and 63.57 (OCH2), 79.98 and 80.39 (C-8),
119.08 and 119.92 (CN), 157.64 and 157.88 (C-9), and
163.40, 163.67, 167.97, 168.40, 172.00 and 172.49 (3!
C]O).
hexahydroindolizine-3-carboxylate
26
(750 mg,
1.01 mmol) was dissolved in dimethylformamide (12 ml)
and piperidine (3 ml; 20% by volume). The solution was
stirred at room temperature under a nitrogen atmosphere for
1.5 h. The solvent was removed in vacuo to yield an off-
white solid, which was purified by column chromatography
on silica gel, eluting with a gradient of methanol/
dichloromethane (1:99) to methanol in dichloromethane
(1:9). This yielded ethyl (3S,6RS)-6-[(2S)-2-((2S)-2-amino-
4-tert-butoxycarbonylpropanamido)-3-methylbutanamido]-
8-cyano-5-oxo-1,2,3,5,6,7-hexahydroindolizine-3-carbox-
ylate 27 as a white powder (370 mg, 70%), mp 102–104 8C;
[a]2D8ZK72.46 (c 1, CHC13); m/z [EI] Found: 519.26745.
C25H37N5O7 requires 519.26930; m/z [Cve FAB (thio-
glycerol)] 520 ([MCH]C); nmax (KBr)/cmK1 3411 (br,
NH), 2210 (CN), 1710 (ester) and 1664 (lactam); lmax
(MeOH)/nm 275 (3 5300); dH (360 MHz, C2HC13) 0.94–
0.99 (6H, m, JZ6.7 Hz, C(CH3)2), 1.23–1.33 (3H, m, JZ
7.1 Hz, CH3), 1.45 (9H, 2!s, OC(CH3)3), 2.19–2.29 (2H,
m, J7A,6Z7.0 Hz, J7A,7BZ13.3 Hz, Jb,aZ3.7 Hz, H-7AC
H-b), 2.36–2.49 (1H, m, J2A,3Z9.2 Hz, J2A,2BZ13.6 Hz,
3.1.15. Ethyl (3S,6RS)-6-[(2S)-2-((2S)-2-(9H)-fluoren-9-
ylmethoxycarbonylamino-4-tert-butoxycarbonylpropa-
namido)-3-methylbutanamido]-8-cyano-5-oxo-1,2,3,5,
6,7-hexahydroindolizine-3-carboxylate (26). Ethyl
(3S,6RS)-6-[(2S)-2-amino-3-methylbutanamido]-8-cyano-
5-oxo-1,2,3,5,6,7-hexahydroindolizine-3-carboxylate 25
(560 mg, 1.6 mmol) and 4-tert-butyl (2S)-N-(9H)-fluoren-
9-ylmethoxycarbonylaspartate (660 mg, 1.6 mmol) were
dissolved in dimethylformamide (22 ml). TBTU (520 mg,
1.6 mmol) and di-isopropylethylamine (210 mg, 1.6 mmol)
were added and the solution was stirred under nitrogen at
room temperature for 12 h. The solvent was removed in
vacuo (!50 8C) and the resulting oil was dissolved in
dichloromethane. The solution was successively washed
with 5% aqueous citric acid, 5% aqueous sodium hydrogen
carbonate and saturated aqueous sodium chloride and dried
(MgSO4). The solvent was removed in vacuo and the
resulting foam was triturated with diethyl ether, affording
ethyl (3S,6RS)-6-[(2S)-2-((2S)-2-(9H)-fluoren-9-ylmethoxy-
carbonylamino-4-tert-butoxycarbonylpropanamido)-3-
methylbutanamido]-8-cyano-5-oxo-1,2,3,5,6,7-hexahydro-
indolizine-3-carboxylate 26 as a pale yellow solid (840 mg,
70%), mp 100–102 8C; [a]2D9ZK51.3 (c 1, CHCl3); m/z [EI]
Found: 741.34168. C40H47N5O9 requires 741.33738; m/z
[Cve FAB (thioglycerol)] 764 ([MCNa]C); nmax (KBr)/
cmK1 3414 (br, NH), 2210 (CN), 1710 (ester) and 1652
(lactam); lmax (MeOH)/nm 265 (3 24,000); dH (360 MHz,
C2HC13) 0.92–0.98 (6H, m, JZ6.7 Hz, C(CH3)2), 1.21–
1.33 (3H, m, JZ7.1 Hz, CH3), 1.43–1.45 (9H, 2!s,
0
0
H-2A), 2.53–2.84 (3H, m, J2B,3Z1.9 Hz, Jb ,a Z3.8 Hz,
H-2BCH-b0), 2.91–2.96 (2H, m, H-1), 3.00–3.12 (1H, m,
0
0
0
H-7B), 3.68–3.74 (1H, m, Ja ,b Z3.8 Hz, H-a ), 4.18–4.28
(3H, m, JZ7.1 Hz, OCH2CH-a), 4.60–4.69 (1H, m,
J6,7AZ7.0 Hz, J6,7BZ2.2 Hz, J6,NHZ5.7 Hz, H-6), 4.72–
4.83 (1H, 2!d, J3,2AZ9.2 Hz, J3,2BZ1.9 Hz, H-3), 6.67–
2
6.68 (1H, br d, JZ6.2 Hz, NH, exchanges in H2O), 6.90–
6.91 (1H, br d, JNH,6Z5.7 Hz, NH, exchanges in 2H2O) and
7.91–7.99 (1H, 2!br d, JZ9 Hz, NH, exchanges in 2H2O);
dC (125.8 MHz, C2HC13) 14.05 (CH3), 18.01 and 19.21
(C(CH3)2), 26.11 and 26.36 (C-2), 27.63 and 28.49 (C-l),
28.06 (OC(CH3)3), 28.92 and 29.14 (C-7), 29.67 (C-b0),
30.25 (C-a), 30.61 and 31.21 (C-b), 48.61 and 49.22 (C-6),
57.5 and 58.8 (C-3), 59.77 and 60.01 (C-a0), 62.18 (OCH2),
80.31 (C-8), 117.20 and 117.29 (CN), 154.58 and 154.79
(C-9), and 166.0, 167.55, 169.91, 171.0, 171.1, 171.2 and
171.45 (5!C]O).
OC(CH3)3), 2.16–2.21 (2H, m, J7A,6Z6.6 Hz, Jb,a
Z
4.4 Hz, H-7ACH-b), 2.27–2.45 (1H, m, J2A,3Z9.2 Hz,
3.1.17. Ethyl (3S,6RS)-6-[(2S)-2-(((2S)-2-((2S)-2-(9H)-
fluor-9-enylmethoxycarbonylamino-4-methylpentan-
amido)-4-tert-butoxycarbonylpropanamido)-3-methyl-
butanamido]-8-cyano-5-oxo-1,2,3,5,6,7-hexahydroindo-
lizine-3-carboxylate (28). Ethyl (3S,6RS)-6-[(2S)-2-((2S)-
2-amino-4-tert-butoxycarbonylpropanamido)-3-methyl-
butanamido]-8-cyano-5-oxo-1,2,3,5,6,7-hexahydroindoli-
zine-3-carboxylate 27 (300 mg, 0.58 mmol) and N-(9H)-
J2A,2BZ13.4 Hz, H-2A), 2.54–3.00 (6H, m, J2B,3Z3.6 Hz,
0
0
0
J2B,2AZ13.4 Hz, Jb ,a Z6 Hz, H-2BCH-7BCH-1CH-b ),
4.15–4.24 (3H, m, JZ7.1 Hz, OCH2,CJa,bZ4.4 Hz, H-a),
0
0
4.28–4.75 (6H, m, J6,7AZ6.6, J6,7BZ2.3 Hz, Ja ,b Z6 Hz,
J3,2AZ9.2 Hz, J3,2BZ3.6 Hz, H-6CH-a0CH-3CAr2CHC
Ar2CHCH2), 6.12–6.14 (1H, br d, JNH,6Z8.4 Hz, NH,
exchanges in 2H2O), 6.87–7.17 (2H, br m, 2!NH
exchanges in 2H2O) and 7.29–7.78 (8H, ArH); dC
(125.8 MHz, C2H3O2H) 14.86 (CH3), 18.87 and 18.93
(C(CH3)2), 27.47 and 27.55 (C-2), 28.85 (OC(CH3)3), 29.64
and 29.74 (C-1), 30.57 (C-7), 32.41 and 32.76 (C-b), 38.66
and 38.88 (C-b0), 39.03 and 39.37 (C-6), 53.53 (CAr2),
58.81 (OC(CH3)3), 60.40 (C-3), 61.86 and 62.09 (C-a0),
63.43 and 63.51 (OCH2), 68.71 (CCHAr2), 79.96 (C-a),
82.92 and 83.01 (C-8), 119.11(CN), 121.44, 126.72, 128.69,
130.37 and 143.05 (Ar), 145.63 and 145.74 (C-9), and
157.5-173.9 (6!C]O).
fluor-9-enylmethoxycarbonyl-(2S)-leucine
(200 mg,
0.58 mmol) were dissolved in dimethylformamide (40 ml).
TBTU (190 mg, 0.58 mmol) and di-isopropylethylamine
(75 mg, 0.58 mmol) were added and the solution was stirred
under nitrogen at room temperature for 12 h. The solvent
was removed in vacuo (!50 8C) and the resulting oil was
dissolved in dichloromethane. The solution was succes-
sively washed with 5% aqueous citric acid, 5% aqueous
sodium hydrogen carbonate and saturated aqueous sodium
chloride and dried (MgSO4). The solvent was removed in

J. M. Berry et al. / Tetrahedron 61 (2005) 287–299
297
vacuo affording ethyl (3S,6RS)-6-[(2S)-2-(((2S)-2-((2S)-2-
(9H)-fluor-9-enylmethoxycarbonylamino-4-methylpentana-
mido)-4-tert-butoxycarbonylpropanamido)-3-methylbuta-
namido]-8-cyano-5-oxo-1,2,3,5,6,7-hexahydroindolizine-3-
carboxylate 28 as a pale yellow solid (480 mg, 97%), mp
162–163 8C; [a]2D8ZK48.6 (c 1, CHCl3); m/z [Cve FAB
(3-NBA)] 855 ([MCH]C); nmax (KBr)/cmK1 3289 (br,
NH), 2210 (CN), 1710 (ester) and 1641 (lactam); lmax
(MeOH)/nm 264 (3 29,950); dH (360 MHz, C2HC13) 0.92–
0.96 (12H, m, 2!C(CH3)2), 1.25–1.30 (3H, m, JZ7.1 Hz,
CH3), 1.34 and 1.39 (9H, 2!s, OC(CH3)3), 1.55–1.73 (3H,
17.17, 17.61, 19.21, 21.28 and 21.32 (2!C(CH3)2), 23.36
and 24.85 (C-6), 26.12 and 26.45 (C-2), 27.67 and 28.62
(C-1), 28.02 (OC(CH3)3), 28.90 and 29.13 (C-b0), 29.69 and
30.36 (C-3), 36.53 and 36.76 (C-7), 43.87 and 43.97 (C-b00),
48.70 and 49.10 (C-g00) 49.32 and 49.76 (C-b), 53.39 and
53.47 (C-a), 58.48 and 58.85 (C-a0), 59.77 and 59.99
(C-a00), 62.15 (OCH2), 64.0 (OC(CH3)3), 80.22 and 80.38
(C-8), 117.24 and 117.41 (CN), 154.55 and 154.75 (C-9)
and 165.98-176.58 (6!C]O).
3.1.19. Ethyl (3S,6RS)-6-[(2S)-2-((((2S)-2-(((2S)-2-(2-
(9H)-fluor-9-enylmethoxycarbonylaminoacetamido)-4-
methylpentanamido)-4-tert-butoxycarbonylpropana-
mido)-3-methylbutanamido]-8-cyano-5-oxo-1,2,3,5,6,7-
hexahydroindolizine-3-carboxylate (30). Ethyl (3S,6RS)-
6-[(((2S)-2-((2S)-2-((2S)-2-amino-4-methylpentanamido)-
4-tert-butoxycarbonylpropanamido)-3-methylbutanamido]-
8-cyano-5-oxo-1,2,3,5,6,7-hexahydroindolizine-3-carboxy-
late 29 (200 mg, 0.32 mmol) and N-(9H)-fluor-9-enyl-
methoxycarbonylglycine (94 mg, 0.32 mmol) were
dissolved in dimethylformamide (25 ml). TBTU (100 mg,
0.32 mmol) and di-isopropylethylamine (41 mg,
0.32 mmol) were added and the solution was stirred under
nitrogen at room temperature for 12 h. The solvent was
removed in vacuo (!50 8C) and the resulting oil was
dissolved in dichloromethane. The solution was succes-
sively washed with 5% aqueous citric acid, 5% aqueous
sodium hydrogen carbonate and saturated aqueous sodium
chloride and dried (MgSO4). The solvent was removed in
vacuo affording a pale yellow foam. This was triturated with
diethyl ether and dried in vacuo to yield ethyl (3S,6RS)-6-
[((((2S)-2-(((2S)-2-((2S)-2-(2-(9H)-fluor-9-enylmethoxy-
carbonylacetamido)-4-methylpentanamido)-4-tert-butoxy-
carbonylpropanamido)-3-methylbutanamido]-8-cyano-5-
oxo-1,2,3,5,6,7-hexahydroindolizine-3-carboxylate 30 as a
cream solid (280 mg, 97%), mp 171–172 8C; [a]2D6Z
K36.13 (c 0.3, CHC13); m/z [Cve FAB (3-NBA)] 912
([MCH]C); nmax (KBr)/cmK1 3282 (br, NH), 2211 (CN),
1710 (ester) and 1638 (lactam); lmax (MeOH)/nm 265 (3
29,400); dH (360 MHz, C2H3O2H) 0.94–0.99 (12H, m, 2!
C(CH3)2),1.25–1.40 (3H, m, JZ7.1 Hz, CH3), 1.44 and 1.45
(9H, 2!s, OC(CH3)3), 1.50–1.62 (3H, m, H-bCH-b0),
m, H-b0CH-b), 2.19–2.36 (3H, m, J2A,3Z8.8 Hz, J2B,3
Z
2.8 Hz, J2A,2BZ13.3 Hz, H-2CH-g00), 2.61–2.72 (2H, m,
J7A,6Z7.2 Hz, J7B,6Z4.4 Hz, J7A,7BZ12.6 Hz, H-7), 2.81–
3.00 (3H, m, H-1CH-a00), 4.18–4.79 (11H, m, J3,2A
Z
8.8 Hz, J3,2BZ2.8 Hz, J6,7AZ7.2 Hz, OCH2CH-aCH-6C
H-a0CH-3CH-b00CAr2CHCAr2CHCH2), 5.27–5.29 (1H,
br d, NH, exchanges in 2H2O), 6.86–7.24 (3H, 6!br d, 3!
2
NH, exchange in H2O) and 7.29–7.78 (8H, m, ArH); dC
(125.8 MHz, C2H3O2H) 14.86 (CH3), 18.87, 20.26, 22.47
and 23.92 (2!C(CH3)2), 26.37 (C-6), 27.5 (C-2), 28.83
(OC(CH3)3), 29.67 (C-1), 30.59 (C-7), 32.30 (C(Ar)2),
32.61 (C-3), 37.91 (C-b00), 42.46 (C-b0), 51.90 (C-g00), 55.52
(C-b), 58.82 (OC(CH3)3), 60.56 (C-a00), 61.70 (C-a0), 62.11
(C-a), 63.45 (OCH2), 68.55 (CCHAr2), 80.37 and 80.74
(C-8), 83.04 (quat C), 119.15 (CN), 121.43, 126.75, 128.69
and 129.30 (FmocCH), 143.10 (quat C), 145.62 and 145.89
(C-9), and 157.83–176.00 (6!C]O).
3.1.18. Ethyl (3S,6RS)-6-[(2S)-2-(((2S)-2-((2S)-2-amino-
4-methylpentanamido)-4-tert-butoxycarbonylpropana-
mido)-3-methylbutanamido]-8-cyano-5-oxo-1,2,3,5,6,7-
hexahydroindolizine-3-carboxylate (29). Ethyl (3S,6RS)-
6-[(2S)-2-(((2S)-2-((2S)-2-(9H)-fluor-9-enylmethoxycarbo-
nylamino-4-methylpentanamido)-4-tert-butoxycarbonyl-
propanamido)-3-methylbutanamido]-8-cyano-5-oxo-1,2,3,
5,6,7-hexahydroindolizine-3-carboxylate 28 (400 mg,
0.47 mmol) was dissolved in dimethylformamide (12 ml)
and piperidine (3 ml, 20% by volume). The solution was
stirred at room temperature under a nitrogen atmosphere for
1.5 h. The solvent was removed in vacuo to yield a pale
yellow solid, which was purified by column chromatog-
raphy on silica gel, eluting with a gradient of methanol/
dichloromethane (1:99) to methanol in dichloromethane
(4:96). This yielded ethyl (3S,6RS)-6-[(2S)-2-(((2S)-2-((2S)-
2-amino-4-methylpentanamido)-4-tert-butoxycarbonylpro-
panamido)-3-methylbutanamido]-8-cyano-5-oxo-1,2,3,5,
6,7-hexahydroindolizine-3-carboxylate 29 as a cream
powder (260 mg, 87%), mp 92–94 8C; [a]2D9ZK86.94 (c
1, CHC13); m/z [EI] Found: 632.35412. C31H48N6O8
requires 632.35336; m/z [Cve FAB (3-NBA)] 633 ([MC
H]C); nmax (KBr)/cmK1 3316 (br, NH), 2210 (CN), 1710
(ester) and 1662 (lactam); lmax (MeOH)/nm 277 (3 18,600);
dH (360 MHz, C2HC13) 0.94–0.97 (12H, m, 2!C(CH3)2),
1.26–1.33 (3H, m, JZ7.1 Hz, CH3), 1.44 and 1.45 (9H, 2!
s, OC(CH3)3), 1.65–1.70 (3H, m, H-bCH-b0, masked by
water peak), 2.22–2.26 (1H, m, JZ7 Hz, H-g00), 2.35–2.38
(1H, m, J2A,3Z8.3 Hz, H-2A), 2.50–2.90 (3H, m, H-7C
H-2B), 2.94–3.03 (3H, m, H-a00CH-1), 3.43–3.45 (2H, d,
2.10–2.25 (2H, m, H-g00CH-2A), 2.30–2.48 (1H, m, J2B,3
Z
3.7 Hz, H-2B), 2.55–2.75 (2H, m, J7A,6Z7.5 Hz, H-7),
2.84–3.02 (3H, m, H-1CH-a00), 3.75 (2H, s, H-a000), 4.15–
4.85 (11H, m, J6,7AZ7.5 Hz, J6,7BZ5.2 Hz, J3,2AZ8.6 Hz,
J3,2BZ3.7 Hz, OCH2CH-aCH-6CH-3CH-a0CH-b00C
Ar2CHCAr2CHCH2) and 7.25–7.80 (8H, m, ArH); dC
(125.8 MHz, C2H3O2H) 14.85 (CH3), 15.92–22.41 (2!
C(CH3)2), 23.90 (C-6), 26.32 (CAr2), 27.44 and 27.55
(C-a000), 28.83 (C(CH3)3), 29.22 and 29.70 (C-2), 30.52 and
30.60 (C-1), 32.12 and 32.52 (C-3), 37.91 and 38.12 (C-7),
42.11 (C-b0), 45.46 (C-b00), 50.58 (C-g00), 52.06 and 52.49
(C-b), 53.86 and 54.03 (C-a00), 60.52 and 60.85 (C-a0), 61.82
and62.02(C-a), 63.41and63.49(OCH2), 67.36(OC(CH3)3),
68.75 (CCHAr2), 80.36 and 80.71 (C-8), 82.94 and 83.05
(quat C), 119.09 (CN), 121.40–129.27 (FmocCH), 143.63
(quat C), 145.63 (C-9) and 157.50–175.19 (8!–C]O).
JZ7.3 Hz, H-b00), 4.20–4.84 (6H, m, J6,7AZ9.9 Hz, J6,7B
Z
3.1.20. Ethyl (3S,6RS)-6-[((((2S)-2-(((2S)-2-(((2S)-2-(2-
aminoacetamido)-4-methylpentanamido)-4-tert-butoxy-
carbonylpropanamido)-3-methylbutanamido]-8-cyano-
5-oxo-1,2,3,5,6,7-hexahydroindolizine-3-carboxylate
6.3 Hz, J3,2AZ8.3 Hz, OCH2CH-aCH-a0CH-6CH-3),
6.60, 7.0–7.1, 7.2 and 8.25–8,4 (4H, 4!br d, 4!NH,
exchange in 2H2O); dC (125.8 MHz, C2HC13) 14.06 (CH3),

298
J. M. Berry et al. / Tetrahedron 61 (2005) 287–299
(31). Ethyl (3S,6RS)-6-[((((2S)-2-(((2S)-2-(((2S)-2-(2-(9H)-
fluor-9-enylmethoxycarbonylacetamido)-4-methylpentana-
mido)-4-tert-butoxycarbonylpropanamido)-3-methylbutan-
amido]-8-cyano-5-oxo-1,2,3,5,6,7-hexahydroindolizine-3-
carboxylate 30 (250 mg, 0.27 mmol) was dissolved in
dimethylformamide (8 ml) and piperidine (2 ml, 20% by
volume). The solution was stirred at room temperature
under a nitrogen atmosphere for 2 h. The solvent was
removed in vacuo to yield a cream solid, which was purified
by column chromatography on silica gel, eluting with a
gradient of methanol/dichloromethane (1:99) to methanol/
dichloromethane (1:9). This yielded ethyl (3S,6RS)-6-
[((((2S)-(((2S)-2-(((2S)-2-(2-aminoacetamido)-4-methyl-
pentanamido)-4-tert-butoxycarbonylpropanamido)-3-methyl-
butanamido]-8-cyano-5-oxo-1,2,3,5,6,7-hexahydroindoli-
zine-3-carboxylate 31 as a white powder (180 mg, 95%), mp
103–105 8C; [a]2D6ZK65.24 (c 1, CHC13); m/z [EI] Found:
689.37462. C33H51N7O9 requires 689.37483; m/z [Cve
FAB (3-NBA)] 712 ([MCNa]C) and 690 ([MCH]C); nmax
(KBr)/cmK1 3413 (br, NH), 2210 (CN), 1710 (ester) and
1639 (lactam); lmax (MeOH)/nm 275 (3 23,600); dH
(600 MHz, C2H3O2H) 0.90–1.05 (12H, m, 2!C(CH3)2),
((2S)-2-(2-aminoacetamido)-4-methylpentanamido)-4-tert-
butoxycarbonylpropanamido)-3-methylbutanamido]-8-cyano-
5-oxo-1,2,3,5,6,7-hexahydroindolizine-3-carboxylic acid
32 as a white solid (2 mg, 3%), mp 180–183 8C; m/z [C
ve FAB (thioglycerol)l 662 ([MCH]C); nmax (KBr)/cmK1
3290 (br, NH), 3000–2600 (COOH), 2200 (CN), 1664
(CONH) and 1645 (C]O); lmax (MeOH)/nm 275 (3
12,000); dH (600 MHz, C2H3O2H) 0.91–0.99 (12H, m,
2!C(CH3)2), 1.44 and 1.45 (9H, 2!s, OC(CH3)3), 1.59–
1.65 (2H, m, H-b0), 1.67–1.75 (1H, m, H-b), 2.10–2.19 (1H,
m, H-g00), 2.20–2.29 (1H, m, H-1A), 2.39–2.48 (1H, m,
H-1B), 2.61–2.69 (1H, m, H-2A), 2.70–2.79 (1H, m, H-7A),
2.81–2.89 (1H, m, H-2B), 2.90–2.98 (1H, m, H-7B), 2.99–
3.08 (1H, m, H-a00), 3.71 (2H, s, H-a000), 4.21–4.29 (2H,
m, H-b00), 4.40–4.48 (2H, m, H-aCH-a0), 4.62–4.68 (0.5H,
m, H-6), 4.70–4.74 (1H, m, H-3) and 4.75–4.78 (0.5H, m,
H-6).
3.1.22. (3S,6RS)-6-[((((2S)-2-(((2S)-2-((2S)-2-(2-amino-
acetamido)-4-methylpentanamido)-4-tert-butoxycarbo-
nylpropanamido)-3-methylbutanamido]-6-cyano-5-pyr-
rolidinylidine-2,8-dicarboxylate (33). Ethyl (3S,6RS)-6-
[((((2S)-2-(((2S)-((2S)-2-(2-aminoacetamido)-4-methylpen-
tanamido)-4-tert-butoxycarbonylpropanamido)-3-methyl-
butanamido]-8-cyano-5-oxo-1,2,3,5,6,7-hexahydroindoli-
zine-3-carboxylate 31 (80 mg, 0.12 mmol) was dissolved in
ethanol/water (5:1) (24 ml) and stirred at room temperature.
Aqueous sodium hydroxide (0.05 mol dmK3) (8.2 ml) was
added dropwise by means of an autoburette (Radiometer)
with a fixed endpoint of pH 11.0 for 24 h. The solution was
acidified to pH 5.0 by addition of 5% aqueous citric acid and
concentrated in vacuo. The resulting aqueous solution was
washed with ethyl acetate and freeze-dried to a white solid.
This was purified by preparative reverse phase HPLC on a
Zorbax C8 column (25 cm!22.2 mm), eluting with 0.2%
trifluoroacetic acid in acetonitrile and 0.2% trifluoroacetic
acid in water, fractionating at 30 s intervals. Samples judged
to be homogeneous by analytical reverse phase HPLC on a
Zorbax C8 column (25 cm!4.6 mm) were pooled and
lyophilised. This resulted in (3S,6RS)-6-[((((2S)-2-(((2S)-2-
((2S)-2-(2-aminoacetamido)-4-methylpentanamido)-4-tert-
butoxycarbonylpropanamido)-3-methylbutanamido]-6-cyano-
5-pyrrolidinylidine-2,8-dicarboxylate 33 as a white solid
(11 mg, 14%), mp 203–205 8C; m/z [Cve FAB (thio-
glycerol)] 680 ([MCH]C); nmax (KBr)/cmK1 3292 (br,
NH), 3000–2600 (COOH), 2200 (CN), 1718 (COOH), 1670
(CONH) and 1659 (C]O); lmax (MeOH)/nm 266 (3
21,500); dH (600 MHz, C2H3O2H) 0.91–1.01 (12H, m,
2!OC(CH3)2), 1.44 and 1.45 (9H, 2!s, OC(CH3)3), 1.60–
1.62 (2H, m, H-b0), 1.65–1.72 (1H, m, H-b), 2.06–2.20
(2H, m, H-g00CH-4A), 2.36–2.42 (1H, m, H-4B), 2.52–2.59
(1H, m, H-7A), 2.62–2.71 (2H, m, H-3A,CH-7B), 2.74–
2.80 (1H, m, H-a00), 2.85–2.91 (1H, m, H-3B), 3.72 (2H, s,
H-a000), 4.23–4.28 (0.5H, m, H-8), 4.29–4.38 (2H, m, H-b00),
4.40–4.45 (1H, m, H-a0), 4.48–4.51 (0.5H, m, H-8), 4.52–
4.59 (1H, m, H-a) and 4.68–4.72 (1H, m, H-2); dC
(128.5 MHz, C2H3O2H) 18.73–23.84 (C(CH3)2), 26.39
(C-8), 28.59 (C-4), 28.88 (OC(CH3)3), 31.43 (C-3), 32.44
(C-b00), 32.59 (C-g00). 38.32 (C-7), 41.94 (C-b0), 42.59
(C-a000), 51.97 (C-2), 53.48 (C-b), 53.89 (C-a00), 60.21
(C-a0), 62.49 (C-a), 67.07 (OC(CH3)3), 83.22 (C-6), 119.75
(CN), 166.81 (C-5) and 167.81-176.86 (7!C]O).
1.25–1.30 (3H, m, JZ7.1 Hz, CH3), 1.44 and 1.45 (9H, 2!
0
0
0
s, OC(CH3)3), 1.58–1.65 (2H, m, Jb ,a Z8.8 Hz, H-b ),
1.65–1.72 (1H, m, H-b), 2.12–2.26 (1.5H, m, H-g00CH-1A),
2.40–2.50 (1H, m, H-1B), 2.61–2.77 (3H, m, J2A,3Z8 Hz,
J7A,6Z8.4 Hz, H-2ACH-7), 2.80–3.03 (2.5H, m, H-2BC
H-1ACH-a00), 3.38 (2H, s, H-a000), 4.18–4.29 (4H, m,
OCH2CH-b00), 4.39–4.45 (2H, m, Ja ,b Z8.8 Hz, H-aC
H-a0) and 4.63–4.75 (2H, m, J6,7AZ8.4 Hz, J3,2AZ8 Hz,
H-6CH-3); dC (125.8 MHz, C2HC13) 13.93 (CH3), 17.74–
22.66 (2!C(CH3)2), 24.79 (C-6), 26.03 and 26.21 (C-a000),
27.60 and 28.52 (C-2), 27.86 (OC(CH3)3), 28.85 and 29.04
(C-b0), 30.41 and 30.83 (C-g00), 37.07 and 37.56 (C-1),
41.36 and 41.74 (C-b00), 44.63 (C-7), 48.17 and 49.02 (C-3),
49.43 and 49.70 (C-b), 51.57 and 51.74 (C-a00), 58.48
(C-a0), 59.67 and 59.87 (C-a), 61.87 (OCH2), 79.69 and
79.75 (OC(CH3)3), 81.41 and 81.55 (C-8), 117.41 and
117.53 (CN), 154.79 (C-9) and 166.16-173.27 (7!C]O).
0
0
3.1.21. (3S,6RS)-6-[((((2S)-2-(((2S)-2-((2S)-2-(2-amino-
acetamido)-4-methylpentanamido)-4-tert-butoxycarbo-
nylpropanamido)-3-methylbutanamido]-8-cyano-5-oxo-
1,2,3,5,6,7-hexahydroindolizine-3-carboxylic acid (32).
Ethyl (3S,6RS)-6-[((((2S)-2-(((2S)-((2S)-2-(2-aminoaceta-
mido)-4-methylpentanamido)-4-tert-butoxycarbonylpropa-
namido)-3-methylbutanamido]-8-cyano-5-oxo-1,2,3,5,6,7-
hexahydroindolizine-3-carboxylate 31 (80 mg, 0.12 mmol)
was dissolved in methanol/water (5:1, 24 ml) and the
solution was stirred at room temperature. Aqueous sodium
hydroxide (0.05 mol dmK3) (7 ml) was added dropwise by
means of an autoburette (Radiometer) with a fixed endpoint
of pH 11.0 for 24 h. The solution was then acidified to pH
5.0 by addition of 5% aqueous citric acid and concentrated
in vacuo. The resulting aqueous solution was washed with
ethyl acetate and freeze-dried to yield a white solid. This
was purified by preparative reverse phase HPLC on a
Zorbax C8 column (25 cm!22.2 mm), eluting with 0.2%
trifluoroacetic acid in acetonitrile and 0.2% trifluoroacetic
acid in water, fractionating at 30 s intervals. Samples judged
to be homogeneous by analytical reverse phase HPLC on a
Zorbax C8 column (25 cm!4.6 mm) were pooled and
lyophilised. This resulted in (3S,6RS)-6-[((((2S)-2-(((2S)-2-

J. M. Berry et al. / Tetrahedron 61 (2005) 287–299
299
7. Wisskirchen, F. M.; Doyle, P. M.; Gough, S. L.; Harris, C. J.;
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Acknowledgements
We thank the EPSRC and GlaxoSmithKline for a CASE
award (to J. M. B.), Drs. A. G. Avent and P. N. Sanderson
for NMR spectra and Dr. S. Chotai for mass spectra.
10. Frankel, M.; Reichmann, M. E. J. Chem. Soc. 1952, 289–291.
¨
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