Synthesis of an external β-turn based on the GLDV motif of cell adhesion proteins

Article abstract of DOI:10.1016/j.tetlet.2003.09.170

The (3S,6S,10S)-7/5 bicyclic lactam 4, designed as an external turn constraint, was synthesised by a new stereoselective route involving Eschenmoser condensation. Calculated preferred conformations compare well with the preferred solid state conformation,

Full text of DOI:10.1016/j.tetlet.2003.09.170

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 8887–8891
Synthesis of an external b-turn based on the GLDV motif of cell
adhesion proteins
David E. Davies,a Paul M. Doyle,b R. Duncan Farrant,a Richard D. Hill,c Peter B. Hitchcock,c
Paul N. Sandersona and Douglas W. Youngc,*
aGlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK
bBioFocus Discovery PLC, Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AZ, UK
cSussex Centre for Biomolecular Design and Drug Development, Department of Chemistry, University of Sussex,
Falmer, Brighton BN1 9QJ, UK
Received 18 August 2003; revised 8 September 2003; accepted 19 September 2003
Abstract—The (3S,6S,10S)-7/5 bicyclic lactam 4, designed as an external turn constraint, was synthesised by a new stereoselective
route involving Eschenmoser condensation. Calculated preferred conformations compare well with the preferred solid state
conformation, obtained by X-ray crystallography. The lactam 4 was not a turn mimic in its own right but could be used as an
external constraint to prepare the cyclic peptide 29 containing the integrin recognition motif GLDV. High-resolution NMR
measurements were consistent with this compound having a single backbone conformation.
© 2003 Elsevier Ltd. All rights reserved.
Reverse turns connect elements of protein secondary
structure and usually occur on the surface of the
protein. They can, therefore, be important in molecular
recognition processes.1,2 Synthetic molecules which
mimic these turns, such as the classic 5/6 bicyclic lactam
BTD, 1, prepared by Nagai,3–5 have been used to
replace b-turns in various proteins without affecting
their overall biological properties.4–6 BTD, 1, has been
shown to be a type II% b-turn mimic from the angles „
Amino acid 7/5-bicyclic lactams of type 2 have found
use as dipeptide surrogates in angiotensin-converting
enzyme (ACE) inhibitors9 and have more recently been
used as external constraints for tripeptide RGD turns
which were inhibitors of the aVb3 integrin receptor.10
The 7/5 bicyclic lactam 3 was synthesised using an
electrochemical approach and conformational analysis
showed that the (4S,7S,10S)-stereoisomer had families
of minimum conformations with torsion angles close to
those of classical b-turns.11 In continuation of our work
on externally constrained GLDV turns,8 we have now
developed a new and stereoselective synthesis of the 7/5
bicyclic lactam, 4, via a route involving Eschenmoser
coupling. The crystal structure of our new constraint
will indicate its promise as a turn mimic, and attach-
(−161°) and ƒ (−69.4°) in the X-ray crystal structure.27
3
One of us has used BTD8 as a strong structural con-
straint on which to build a cyclic GLDV motif. This
was shown to be a type I b-turn and inhibited the
interaction of the integrin a4b1 with vascular cell adhe-
sion molecule-1.8
Keywords: b-turn; protein mimetic; amino acid; bicyclic lactam.
* Corresponding author.
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.09.170

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D. E. Da6ies et al. / Tetrahedron Letters 44 (2003) 8887–8891
Scheme 1. Reagents and conditions: (i) HBr/CHCl3, rt, 89%; (ii) 7/MeCN, reflux, then Ph3P/Et3N, 25%.
Scheme 2. Reagents and conditions:(i) NaHMDS/PhCH2Br/THF/−78°C, 64%; (ii) P4S10/THF/rt, 95%; (iii) 6/Ph3P/Et3N/CH3CN,
rt, 91%.
ment of the GLDV recognition motif to it will allow it
to be assessed as an external turn constraint. GLDV is
present in the integrin family of cell adhesion
proteins.12
tuted,16 we prepared the N-benzyl thiolactam 11 as
shown in Scheme 2. This compound had previously
been prepared by Rapoport17 by a synthesis involving
N-alkylation of glutamic acid prior to cyclisation to
avoid the danger of racemisation. We were able to
alkylate tert-butyl pyroglutamate 918 directly to the
N-benzyl derivative 10 in 64% yield and reaction with
P4S10 gave the thiolactam 11 in 95% yield with spectro-
scopic and optical properties similar to those reported
by Rapoport.17 The sulfur contraction reaction with the
bromide 6, using Ph3P/Et3N in CH3CN then gave the
vinylogous amide 12,† as an oil, [h]2D8 +122.6 (c 1.025,
CHCl3), in 91% yield.
As a first step in our synthesis of the bicyclic lactam 4,
we attempted to prepare the bromide 6 by the method
used by Weygand13 to prepare the corresponding ethyl
ester bromide but without success. Modification of the
method by reacting the diazoketone 514 with HBr in
CHCl3, however, gave the bromide 6,† mp 117–8°C,
[h]3D0 +3 (c 1.01, CH2Cl2), in 89% yield, as shown in
Scheme 1. The bromide was heated at reflux with the
thiolactam 715 in CH3CN, followed by addition of Ph3P
and triethylamine to give the desired product 8† as an
oil, [h]3D0 +12.3 (c 1, CHCl3). This was shown to be the
Z-isomer, since irradiation of the olefinic proton, H-6,
caused enhancements to all four protons at C-4 and C-8
When the olefinic proton, H-6, in the N-benzyl deriva-
tive 12 was irradiated, both of the protons H-8 and one
of the benzylic CH2 protons showed enhancement,
implying that, unlike the N-H analogue 8, the N-benzyl
compound 12 had E-geometry. In order to reduce the
vinylogous amide and hydrogenolyse the N-benzyl
group in 12, it was necessary to hydrogenate the com-
pound at a pressure of 60–200 psi for 50 hours using
10% palladium on activated carbon (75% by weight) in
methanol containing trifluoroacetic acid as shown in
Scheme 3. The resultant single diastereoisomeric
product 13† was obtained as an oil, [h]2D8 +8.5 (c 1,
1
in the H NMR spectrum. The hydrogen bond between
the N–H of the vinylogous amide and its carbonyl
group, as shown, presumably accounts for this specific-
ity. The yield of the product 8 could not be optimised
above 25%.
Since sulfur contraction reactions have been reported to
be more effective when the thiolactam is trisubsti-
t
Scheme 3. Reagents and conditions: (i) H2/Pd-C/60–200 psi, MeOH, TFA, 65%; (ii) BuMgCl/Et2O/0°C, 29%; (iii) K2CO3/MeOH/
D, 86%.
† These compounds had the appropriate analytical and spectroscopic properties.

D. E. Da6ies et al. / Tetrahedron Letters 44 (2003) 8887–8891
8889
compound 4, BTD, 1, and compound, 3, are shown
in Figure 1. Except for minor conformers, those tor-
sional minima found for compound 4 are not com-
patible with b-turns, and this agrees with the X-ray
crystal structure analysis (Fig. 2)20 which showed tor-
CHCl3), in 65% yield but NOE experiments were
inconclusive as to its stereochemistry. Interestingly, in
some hydrogenation reactions, a small yield of a by-
product was obtained. This proved to be the bicyclic
lactam alcohol 14,† mp 141–3°C, [h]2D8 +14.6 (c 1,
CHCl3). The (3S,6S,8S,10S)-stereochemistry of this
compound was implied from NOE studies, since irra-
diation at H-3 caused enhancement of H-10 and irra-
diation at H-10 caused enhancements at both H-8
and H-6. It was not possible to optimise the yield of
the bicyclic compound 14 but the mechanism sug-
gested in Scheme 3 would account for its formation.
Here, incomplete reduction to the alcohol 15 would
be followed by lactonisation, giving the product 16
from which final cyclisation would give the product
14.
sion angles ƒ −86.5° and „ +169° which are high-
2
lighted in F3igure 1. The conformational minima
found for BTD, 1, are almost entirely b-turns, whilst
the dispersion of minima indicate a more flexible scaf-
fold than our compound 4. The torsion angles found
for the X-ray structure of 17 are somewhat shifted
relative to those calculated but this is likely to be due
to the combination of a flexible scaffold and strong
lattice interactions, since the crystal structure was that
of the free acid. The oxa compound 3 is seen to have
more rigidity than compound 1 whilst maintaining
three distinct local minima that enable the scaffold to
mimic both the torsional regions displayed by com-
pound 4 and the region associated with b-turns.
The amino ester 13 was cyclised using tBuMgCl in
ether at 0°C, giving the bicyclic lactam 4,† mp 127–
9°C, [h]2D8 −20.6 (c 1, CHCl3), in 29% yield. Calcula-
tions of the conformational preferences19 of the
We now wished to examine the usefulness of our turn
mimic as an external constraint by adding GLDV
across the amino and carboxyl groups. Treatment of
the lactam 4 with K2CO3 in methanol at reflux, as
shown in Scheme 3, gave the free amine 17† in 86%
yield and reaction of this with Fmoc-valine, TBTU
and DIPEA gave the product 18,† mp 129–131°C,
[h]2D8 −24.4 (c 1.1, CHCl3), in 80% yield, as shown in
Scheme 4. Given the need to protect the b-carboxyl
of the aspartate residue in GLDV as the tert-butyl
ester to prevent intramolecular cyclisation reactions,
we now needed to change the orthogonality of the
protecting groups. The Fmoc-tert-butyl ester 18 was
therefore deprotected using TFA to give the acid 19,†
mp 131–2°C, [h]2D8 −69.6 (c 0.47, CHCl3), in quantita-
tive yield. Reprotection using diphenyldiazomethane
yielded the ester 20† as an oil, [h]3D0 −32.6 (c 1,
CHCl3) in 64% yield. This was now sequentially
deprotected with piperidine and reacted in turn with
Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH and Cbz-Gly-
OH to give the protected tetrapeptide 26,† as sum-
marised in Scheme 4. Hydrogenolysis, cyclisation and
final deprotection were then carried out, as in Scheme
4, and the final cyclic peptide 29 was purified by
repeated reverse phase HPLC.
Figure 1. Calculated conformations based on energy min-
imisation for compounds 1, 3, and 4. Large circles denote
conformations within 5 kCal of minimum energy, smaller
circles within 12 kCal. X-ray derived angles for 1 and 4 are
also shown.
High-resolution NMR spectroscopic data were
obtained to establish whether the constrained peptide
29 adopted a dominant conformation in solution.
Inter-proton distance constraints derived from 2D-
ROESY data were incorporated into molecular
dynamics calculations, but were not sufficient to
define a unique solution conformation of the back-
bone. However, additional NMR spectral parameters
including the wide range of amide proton temperature
coefficients, the non-‘conformationally averaged’
amide to a-H coupling constants and the approxi-
mately 0.4 ppm difference in the glycine methylene
proton chemical shifts together with their ‘non-equal’
vicinal coupling constants to the glycine amide proton
(Table 1) were consistent with a single backbone con-
formation.
Figure 2. X-ray crystal structure of the bicyclic lactam 4.

8890
D. E. Da6ies et al. / Tetrahedron Letters 44 (2003) 8887–8891
Scheme 4. Reagents and conditions; (i) TBTU/DIPEA/DMF/Fmoc-Val-OH, 80%; (ii) CF3CO2H/CH2Cl2, quant; (iii) Ph2CHN2/
CH2Cl2, 64%; (iv) piperidine/DMF, >82%; (v) TBTU/DIPEA/Fmoc-Asp(OtBu)-OH, 88%; (vi) TBTU/DIPEA/DMF/Fmoc-Leu-
OH, 82%; (vii) TBTU/DIPEA/Cbz-Gly-OH, 81%; (viii) H2/Pd-C, 92%; (ix) TBTU/DMAP/DMF; (x) CF3CO2H/ TES.
Table 1. 1H NMR data for 29 in 90% H2O:10% D2O (ref l 4.70) at 298 K
AA unit
NH
aH
bH
gH
dH
oH
3JNH,aH (Hz)
Dl/DT (ppb/K)
Gly (G)
Leu (L)
Asp (D)
Val (V)
cLys (cK)
Pro (P)
8.68
8.40
8.58
7.24
7.53
–
3.62 and 4.01
4.78
4.53
4.35
4.45
–
–
1.50
–
2.40
1.88
1.61 and 1.67
–
–
–
–
–
4.48
–
5.4 and 7.0
−7.8
−2.8
−4.3
−2.6
−1.7
–
1.42 and 1.75
2.81 and 2.91
2.36
1.61
1.94 and 2.10
0.83 and 0.89
–
0.77 and 0.84
1.39
9.0
5.5
9.5
5.6
–
4.47
–
The concentration of monomeric peptide was approximately 0.6 mM; a substantial amount of aggregated peptide material was also present.
Acknowledgements
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D. E. Da6ies et al. / Tetrahedron Letters 44 (2003) 8887–8891
8891
of 20 kCal. Energy minimisations were performed with
the Tripos forcefield with Gasteiger-Hu¨ckel charges and
1000 iterations.
were R1=0.058, wR2=0.152; Data collection was car-
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analysis using program package WinGX, and refinement
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20. Crystal data: compound 4, C16H23F3N2O4, M=364.4,
monoclinic, space group P21 (No 4), a=5.485(2), b=
3
,
,
11.970(4), c=14.437(5) A, V=947.9(6) A , Z=2, Dcalc
=
1.28 mg/m3, v (Mo-Ka) 0.11 mm−1, T=293(2) K, 1757
independent reflections, 1295 with I>2|(I), final residuals