Synthesis and biological properties of macrolactam analogs of the natural product macrolide (-)-A26771B

Article abstract of DOI:10.1016/j.bmcl.2011.06.073

Promising synthetic derivatives of macrolactone natural product (-)-A26771B have been designed and synthesized both from semisynthesis and total synthesis. Further optimization led to the first synthesis of macrolactam analogs of (-)-A26771B with improved antibacterial activity and metabolic stability.

Full text of DOI:10.1016/j.bmcl.2011.06.073

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4768–4772
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological properties of macrolactam analogs of the natural
product macrolide (À)-A26771B
Sophie Canova b, Renaud Lépine a, , Amber Thys c, Anne Baron b, Didier Roche b
⇑
a Galapagos, 102 Avenue Gaston Roussel, 93230 Romainville, France
b Edelris, 115 Avenue Lacassagne, 69003 Lyon, France
c Galapagos, Industriepark Mechelen Noord, General De Wittelaan L11 A3, B-2800 Mechelen, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Promising synthetic derivatives of macrolactone natural product (À)-A26771B have been designed and
synthesized both from semisynthesis and total synthesis. Further optimization led to the first synthesis
of macrolactam analogs of (À)-A26771B with improved antibacterial activity and metabolic stability.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 28 April 2011
Revised 14 June 2011
Accepted 15 June 2011
Available online 23 June 2011
Keywords:
Natural products
(À)-A26771B
Antibacterial
Macrolactams
Metathesis
Natural product (À)-A26771B produced by Penicillium turbatum
has been identified as an attractive target in the development of
new antibiotics.1 (À)-A26771B exhibits moderate in vitro activity
against Gram-positive bacteria, mycoplasma and fungi. Despite
its attractive biological properties, (À)-A26771B suffers from poor
pharmacokinetic properties which translates into a lack of in vivo
activity after subcutaneous administration against Staphylococcus
aureus, Streptococcus pyogenes and Streptococcus pneumoniae infec-
tions in mice. Promising results obtained from a screening cam-
paign with synthetic derivatives of (À)-A26771B encouraged us
to explore further the structure–activity relationship (SAR) in order
to identify candidates with improved biological profile and phar-
macokinetic properties. We wish to report herein our results on
the synthesis and evaluation of the biological activity of new ana-
logs which led to the first total synthesis of macrolactam analogs of
(À)-A26771B.
metabolic stability and potentially toxicological perspective (gen-
eral Michael acceptor). Our first goal aimed to measure the impact
of replacing this reactive functionality. We explored at first a semi-
synthetic strategy to access the first analogs. For this purpose, mul-
tigram quantities of pure 1 could be obtained from P. turbatum
fermentation.
The first analogs were prepared from the t-butyl succinate ana-
log 2 (Scheme 1), easier to handle on a chemistry perspective and
which turned out to be at least as active as (À)-A26771B against a
panel of bacteria (Table 1). Hydrogenation of 2 provided the satu-
rated analog 3 with good yield. 1,4-Addition of various amines to
the
a,b-unsaturated carbonyl system was regioselective and pro-
ceed smoothly to provide compounds 4 and 5a. Compound 5b
was obtained by acylation in the presence of acetic anhydride.
No control was observed on the newly created stereogenic center
during 1,4-addition. Reaction of 2 with TMS-diazomethane fol-
lowed by treatment with tetrabutyl ammonium fluoride (TBAF)
provided the heterocyclic pyrazole derivatives 6 over two steps al-
(À)-A26771B is a structurally unique 16 member ring macro-
lactone ( Fig. 1) possessing a highly oxidized
c-oxo-d-hydroxy-
a,b-unsaturated carboxyl system found in many other natural
macrocycles such as cladospolides, cytochalasins, colletodiols and
macrosphelides.2 Although the total synthesis of (À)-A26771B
has been well covered,3 limited SAR was available from literature
data. We anticipated that the sensitivity of the 4-oxygenated 2-
enoic carboxyl functionality could be a risk from a chemical and
O
O
O
O
O
OH
O
⇑
Corresponding author. Tel.: +33 149424716; fax: +33 149424658.
E-mail address: renaud.lepine@glpg.com (R. Lépine).
Figure 1. (À)-A26771B (1).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.073

S. Canova et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4768–4772
4769
O
O
O
O
O
O
a)
c)
b)
O
O
O
O
O
O
O
O
O
O
O
OH
O
O
O
O
1
2
3
f), g)
d)
O
H
N
O
O
H
N
R
O
O
O
N
O
N
Ph
H
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
4
5a, R =H
6
e)
5b, R =Ac
Scheme 1. Analog synthesis via semisynthesis. Reagents and conditions: (a) t-BuOCNHCCl3, CH2Cl2, 40 °C; 88%; (b) H2, Pd/C, EtOAc, rt; 94%; (c) N-methyl piperazine, CH2Cl2,
rt; 62%; (d) C6H5OC2H4NH2, CH2Cl2, rt; used as crude; (e) Ac2O, Et3N, CH2Cl2, 0 °C to rt; 46% over two steps; (f) TMS-diazomethane, Pd(OAc)2, CH2Cl2/Et2O, 0 °C; used as crude;
(g) TBAF, THF, rt; 24% over two steps.
Table 1
MIC values (l
g/mL)a
Bacterial strains
1
2
4
5b
6
7
8
9
10
13
14
15
16a 16b 17
18
19
28a
28b
Escherichia coli ATCC 25922
Enterococcus faecalis ATCC29212
Enterococcus faecium 1
Haemophilus influenzae ATCC 31517
Haemophilus influenzae LS2 efflux knock- 16
>32 >32 >32 >32 >64 >32 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64
>64
2
0.5
2
16
4
2
nd
4
4
>32 >64
>32 >64
2
8
2
2
32
4
4
2
32
4
32
16
>64 >64 >64 64
>64 >64 >64 32
>64 >64
>64 >64
4
4
4
4
32
2
2
0.5
2
>32 >32 >32 >32 >64 16
>64 >64 >64 >64 >64 >64 >64 32
16 64 >64 >64 >64 >64
8
nd
>32
8
1
2
2
0.25 0.25
out
Pseudomonas aeruginosa ATCC 27853
Staphylococcus aureus ATCC13709
Staphylococcus aureus Sa2 MRSAc
Staphylococcus aureus ATCC25923
Staphylococcus aureus ATCC25923
+10%SHb
>32 >32 >32 >32 >64 >32 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64
>64
0.5
0.5
1
2
2
4
2
0.5
8
2
2
4
>32 >64
>32 >64
>32 >64
1
2
2
1
0.5
1
0.5
0.5
1
32
32
32
>64 >64 >64 32
>64 >64 >64 >64 >64
>64 >64 >64 64 >64
>64
1
2
1
1
2
1
1
1
1
1
1
1
8
>32 >32 >32 >32 >64 32
16
16
>64 >64 >64 >64 >64 >64 16
32
32
8
Staphylococcus aureus Oxford
Streptococcus pneumoniae ATCC49619
Streptococcus pneumoniae Pen9 IV381-
221
2
16
8
2
2
1
2
4
2
>32 >64
>32 >64
>32 32
1
2
2
1
2
2
1
2
4
32
16
8
>64 >64 >64 64
>64 >64 >64 64
>64 >64 >64 64
>64
>64
>64
1
2
1
1
4
2
1
4
4
1
2
8
0.5
2
8
a
b
c
Minimum inhibitory concentration. Determined according to the CLSI method by broth microdilution.
10% of human serum were added to the media.
Clinical strain.
beit in moderate yield (24%). We then focused our program on
functionalizing the hydroxyl group with more varied chemical
functionalities as shown in Scheme 2. Saponification of 1 under
smooth conditions provided the poorly stable hydroxyl derivative
7 which could not be purified by flash chromatography. This crude
product was therefore used without further purification in the fol-
lowing steps.
Methylation of 7 with methyl iodide provided the methoxy
derivative 8 while acylation with acetic anhydride provided the
acetate 9. These two compounds were more stable than the free
hydroxyl derivative and were used as the starting point to access
more radical modifications of the natural product framework.
The imidazole derivative 10 was obtained from 9 by treatment
with ammonium acetate in refluxing acetic acid. Furthermore,
the saturated analogs of (À)-A26771B were prepared as the succi-
nate 11, free hydroxyl 12 and methoxy derivatives 13 after a se-
quence of hydrogenation, saponification and methylation.
Modification of the 4-oxygenated 2-enoic carboxyl functionality
was further investigated to address both the SAR and to improve
the poor metabolic stability of (À)-A26771B. The sterically hin-
dered methyl substituted derivative 14 was obtained from a two
step procedure implying a 1,4-addition of a methyl cuprate to
the double bond and quenching the resulting enolate with phe-
nylselenium bromide. Oxidation of the crude selenide intermediate
with hydrogen peroxide provided compound 14, albeit in low yield
(4% over two steps).
Cyclopropane 15 was obtained in low yield as a mixture of two
diastereomers using the conditions described by Taylor.4 A slightly
better yield was obtained upon epoxidation with t-butyl hydroper-
oxide to provide compound 16 as a mixture of trans-diastereomers
which were separated by flash chromatography. The extreme sen-
sitivity of the 4-oxygenated 2-enoic carboxyl functionality is illus-
trated by the very fast addition of phenyl thiol to the ene-system in
dichloromethane at rt. Thioether 17 was further oxidized to the
sulfone in the presence of m-CPBA while the trifluoroethyl thioe-
ther 19 was prepared as the free hydroxyl derivative in low yield
due principally to difficulty of isolation.
Although the synthesis of analogs of (À)-A26771B from semi-
synthesis allowed us to prepare a large number of compounds
and to better understand the SAR around the unique 16 member
ring macrolactone, the quest for metabolically more stable com-
pounds encouraged us to explore the synthesis of lactam deriva-

4770
S. Canova et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4768–4772
O
O
O
O
O
O
a)
d)
O
O
O
N
O
NH
O
OH
R
O
10
1
7, R =H
8, R =Me
9, R =Ac
b)
c)
k)
O
O
F3C
S
e)
i)
f)
O
g) or h)
O
R
19, R =H
O
O
O
O
O
O
O
O
X
X
O
O
O
O
O
O
O
O
R
R
R
R
11, R =COCH2H4CO2H
12, R =H
14, R =Me
17, R =Me, X=S
15, R =Me, X=CH2
16a, 16b, R =Me, X=O
a)
b)
j)
18, R =Me, X=SO2
13, R =Me
Scheme 2. Analog synthesis via semisynthesis. Reagents and conditions: (a) LiOH, THF/H2O, 0 °C; used as crude; (b) MeI, Ag2O, CH2Cl2, rt; 35% from 1; (c) Ac2O, pyridine,
CH2Cl2, 0 °C to rt; 46% from 1; (d) NH4OAc, AcOH, rt to 80 °C; 32% from 9; (e) H2, Pd/C, EtOAc, rt; 89%; then conditions (a) 71% for 11 and (b); 46% for 12; (f) MeLi, CuI, Et2O,
À40 °C then PhSeBr, THF, À40 °C to rt; mixture of compounds isolated in 11% yield and then submitted to H2O2, pyridine, CH2Cl2, 0 °C to rt; 38%; (g) Me3SOI, DBU, CH3CN, 0 °C;
14% from 8; (h) TBHP, Triton B, Tol. 0 °C to rt; 30% from 8; (i) PhSH, CH2Cl2, rt; 56%; (j) m-CPBA, CH2Cl2, rt; 85%; (k) CF3CH2SH, CH2Cl2, rt; 18% from 1.
tives. Preparation of lactam derivatives from a parent macrolac-
tone has been previously exemplified for an epothilone in the liter-
ature.5 However, a short procedure could not be envisioned in the
case of (À)-A26771B. Furthermore, we anticipated some chemistry
limitations due to the high reactivity of the 4-oxygenated 2-enoic
functionality. A total synthesis of macrolactam analogs was there-
fore envisaged. Our retrosynthetic analysis is highlighted in
Scheme 3. A macrolactamization comparable to the cyclization
step performed by Chang3k was chosen as the final step. We then
decided to take advantage of the elegant methodology developed
by Kobayashi3h,j to introduce the 4-oxygenated 2-enoic acid sys-
tem from a 2-substituted furan. We thought that this key furan
intermediate C could readily be accessible via a cross-metathesis6a
disconnection not previously reported for the total synthesis of
(À)-A26771B. This very concise approach would allow us to reach
the desired 16 member ring macrolactam in seven sequential
steps. The first intermediate 21 was readily prepared in enantio-
merically pure form in two steps from the commercially available
Boc-protected (R)-alaninol (Scheme 4).
to avoid homodimerization of olefin 21.7 This excess was recovered
during purification. Compound 24 was obtained as a mixture of
four stereoisomers which were hydrogenated to provide 25 as an
inseparable mixture of two isomers. Opening of the furan ring un-
der Kobayashi’s conditions3h,j followed by sodium chlorite oxida-
tion of the formed aldehyde provided the key intermediate acid
27 as a mixture of two diastereomers. The macrolactamization
was performed under dilute conditions (10À2 M) after Boc-depro-
tection, to give the expected lactam 28 in 25% non-optimized over-
all yield (Scheme 5). The two diastereomers were separated at this
stage by flash chromatography.8,9
The antibiotic activity of (À)-A26771B analogs was then as-
sessed against a panel of various bacterial strains (Table 1). As
mentioned previously, the t-butyl succinate 2 as well as the acetate
derivative 9 appeared to have a profile similar to the natural prod-
uct with a slightly more pronounced effect on the S. aureus MRSA
strain (MIC = 0.5 lg/mL). As similar results were obtained with
the free hydroxyl 7, we concluded that the succinate moiety in
the natural product had no obvious effect on the activity. This
hypothesis was confirmed by the synthesis of the methoxy deriva-
tive 8 which turned out to be slightly more active than the natural
product 1. Furthermore all our attempts to further stabilize the
molecule by modification of the sensitive 4-oxygenated 2-enoic
carboxyl functionality led to a virtually complete loss of the anti-
bacterial activity (compounds 6, 10, 13–16). It is worth noting that
Racemic compound 23 was obtained in two steps and quantita-
tive yield as described by Chang et al.3k As anticipated, cross-
metathesis of intermediates 21 and 23 afforded the desired com-
pound 24 in very good yield (83%). 15% Grubbs I catalyst6b,c in
dichloromethane at room temperature turned out to be the ideal
conditions. A fivefold excess of furan derived olefin 23 was used
R2
N
R2
N
R2
N
Cross
Metathesis
O
R2
N
PG
PG
O
+
PG
O
OR1
O
HO
OR1
OR1
OR1
O
O
R1 =MOM or Me
C
A, PG=Boc
B,
R2 =H, Me
Scheme 3. Retrosynthetic analysis for the total synthesis of macrolactam analogs of (À)-A26771B.

S. Canova et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4768–4772
4771
Boc
NH
Boc
NH
Boc
NH
a)
b)
OH
OMs
H
N
20
21
Boc
O
e)
O
OH
OMe
O
c)
d)
O
O
O
24
H
22
23
Scheme 4. Synthesis of intermediate 24 via Grubbs I catalyzed cross-metathesis. Reagents and conditions: (a) MsCl, Et3N, CH2Cl2, À10 °C; 98%; (b) CuI, C5H9MgBr, THF,
À15 °C; 48%; (c) C5H9MgBr, THF, 0 °C; quant.; (d) NaH, MeI, THF, 0 °C to rt; quant.; (e) Grubbs I (15 mmol %), CH2Cl2, rt; 83%.
H
N
H
N
H
N
Boc
O
Boc
Boc
O
a)
b)
O
O
H
O
O
O
O
26
25
24
H
N
H
N
Boc
c)
d), e)
O
HO
O
O
O
O
27
28a and 28b
(separated by flash chromatography)
Scheme 5. Synthesis of compound 28 via macrolactamization. Reagents and conditions: (a) H2, Pd/C, AcOEt, rt; 97%; (b) NBS, NaHCO3, acetone/H2O, À15 °C, 30 min then
pyridine, rt; 66%; (c) NaClO2, isopentene, t-BuOH; quant.; (d) TFA, CH2Cl2, rt; used without purification; (e) HATU, HOBt, DIPEA, DMAP, CH2Cl2, rt; 25%.
although the acylated amino derivative 5b was also inactive, the
activity was rescued by the introduction of a basic nitrogen b to
the ketone as exemplified by the N-methyl piperazine analog 4
(this result was confirmed with other sec- and tert-amine deriva-
tives: data not shown). In addition, phenylthioether 17, phenyl sul-
fone 18 and trifluoroethyl thioether 19 had an antibacterial activity
similar to the methoxy analog 8. As the four compounds 4, 17, 18,
19 share a strong sensitivity toward beta-elimination, we believe
they could act as prodrugs by their capacity to regenerate in situ
the 4-oxygenated 2-enoic carboxyl functionality which seems to
be critical to maintain the antibacterial activity. Macrolactams
28a and 28b were then evaluated against the same panel of bacte-
rial strains. Both compounds were active against a broad spectrum
of bacteria and 28b was revealed as the most active compound
identified within these series. Worth noting is the activity of 28a
and 28b on Haemophilus influenzae ATCC 31517 and H. influenzae
LS2 Efflux Knock-out while natural product (À)-A26771B was inac-
tive. Furthermore, unlike analogs derived from natural macrolac-
tones, the antibacterial activity of these two compounds was
successfully maintained in the presence of serum in the screening
assay (S. aureus ATCC25923). This important result opened encour-
aging perspectives of 16 member ring macrolactams to access bio-
logically active compounds with improved pharmacological
properties. Unfortunately, progress obtained in vitro did not trans-
late into an orally active candidate. No activity was observed after
oral administration of 28a and 28b at a dose of 50 mg/kg in a lung
infection model.
functionality in the antibiotic activity. The first synthesis of macro-
lactam analogs was achieved via a new cross-metathesis approach.
These compounds revealed a more pronounced antibacterial activ-
ity as compared to the natural product (À)-A26771B.
Acknowledgments
We would like to thank Drs. Phil Dudfield, Alfred Greiner, Jean-
Yves Ortholand and Fritz Hansske for valuable discussions. We
would like to thank Kathleen Sonck and Carole Delachaume for their
contribution to the microbiological study. We are also thankful to
BioFocus, Allschwil, for providing substantial amounts of pure natu-
ral product (À)-A26771B to complete the semisynthetic program.
And finally, we would like to thank Dr. Richard Jarvest and David
Holmes (GSK) for constructive discussions and for providing us with
the H. influenzae LS2 strain.
References and notes
1. Michel, K. H.; Demarco, P. V.; Nagarajan, R. J. Antibiot. 1977, 30, 571.
2. Cf Dictionary of Natural product on DVD version 19:1, June 2010, CRC Press.
3. (a) Hase, T. A.; Nylund, E. L. Tetrahedron Lett. 1979, 28, 2633; (b) Tatsuta, K.;
Amemiya, Y.; Kanemura, Y.; Kinoshita, M. Bull. Chem. Soc. Jpn. 1982, 55, 3248; (c)
Trost, B. M.; Brickner, S. J. J. Am. Chem. Soc. 1983, 105, 568; (d) Bestmann, H. J.;
Schobert, R. Angew. Chem. 1985, 97, 784; (e) Arai, K.; Rawlings, B. J.; Yoshisawa,
Y.; Vederas, J. C. J. Am. Chem. Soc. 1989, 111, 3391; (f) Quinkert, G.; Kueber, F.;
Knauf, W.; Wacker, M.; Koch, U.; Becker, H.; Nestler, H. P.; Duerner, G.;
Zimmermann, G. Helv. Chim. Acta 1991, 74, 1853; (g) Sinha, S. C.; Sinha-Bagchi,
A.; Keinan, E. J. Org. Chem. 1993, 58, 7789; (h) Kobayashi, Y.; Nakano, M.; Biju
Kumar, G. B.; Kishihara, K. J. Org. Chem. 1998, 63, 7505; (i) Nagarajan, M.
Tetrahedron Lett. 1999, 40, 1207; (j) Kobayashi, Y.; Okui, H. J. Org. Chem. 2000, 65,
612; (k) Lee, W. W.; Shin, H. J.; Chang, S. Tetrahedron: Asymmetry 2001, 12, 29; (l)
Gebauer, J.; Blechert, S. J. Org. Chem. 2006, 71, 2021.
To conclude, we achieved the synthesis of various analogs of
natural product (À)-A26771B. The SAR has been established and
emphasizes the role of the sensitive 4-oxygenated 2-enoic carboxyl

4772
S. Canova et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4768–4772
4. Paxton, R. J.; Taylor, R. J. K. Synlett 2007, 633.
0.72 mmol, 6.0 equiv) were added successively. The reaction mixture was
stirred at room temperature for 2 h then water was added. The 2 layers were
separated. The aqueous phase was extracted with CH2Cl2. The combined
organics were washed with a saturated aqueous solution of NaHCO3, with a
saturated aqueous solution of NH4Cl and with brine then dried over Na2SO4. The
solvent was removed under reduced pressure and the crude residue was
purified by column chromatography on silica gel (pentane/EtOAc: 6/4–5/5) to
give 28a (4 mg, 11%) and 28b (5 mg, 14%) as colourless oils.
5. Borzilleri, R. M.; Zheng, X.; Schmidt, R. J.; Johnson, J. A.; Kim, S. H.; DiMarco, J. D.;
Fairchild, C. R.; Gougoutas, J. Z.; Lee, F. Y. F.; Long, B. H.; Vite, G. D. J. Am. Chem.
Soc. 2000, 122, 8890.
6. (a) Chatterjee, A. K.; Choi, T.-L.; Sanders, D. P.; Grubbs, R. H. J. Am. Chem. Soc.
2003, 125, 11360; (b) Schwab, P.; France, M. B.; Ziller, J. W.; Grubbs, R. H. Angew.
Chem., Int. Ed. Engl. 1995, 34, 2039; (c) Schwab, P.; Grubbs, R. H.; Ziller, J. W. J.
Am. Chem. Soc. 1996, 118, 100.
7. To a solution of Grubbs I catalyst (179 mg, 0.22 mmol, 0.15 mol %) in CH2Cl2
(170 mL, degazed by N2 for 10 min) was added via canula a solution of 21
(330 mg, 1.46 mmol, 1.0 equiv) and 23 (1.30 g, 7.21 mmol, 5.0 equiv) in CH2Cl2
(100 mL, degazed by N2 for 10 min) at rt over 50 min. The reaction mixture was
stirred at rt for 24 h and more Grubbs I catalyst (70 mg, 0.085 mmol) was added.
After 4 h, the reaction mixture was concentrated under vacuum. Expected
Compound 28a: 1H NMR (CDCl3, 300 MHz): d (ppm) 7.48 (d, 1H, J = 15.5 Hz),
6.78 (d, 1H, J = 15.5 Hz), 5.47 (br d, 1H, J = 9.2 Hz), 4.24 (m, 1H), 3.76 (dd, 1H,
J = 7.6 and 4.6 Hz), 3.36 (s, 3H), 1.85–1.60 (m, 2H), 1.38–1.02 (br s, 16H), 1.20 (d,
3H, J = 6.7 Hz); 13C NMR (CDCl3, 75 MHz): d (ppm) 202.6 (s), 164.1 (s), 134.6 (d),
132.5 (d), 86.5 (d), 57.9 (q), 45.6 (d), 35.8 (t), 30.9 (t), 28.3 (t), 28.1 (t), 27.9 (t),
27.5 (t), 27.1 (t), 23.8 (t), 22.2 (t), 21.1 (q); MS (ES+) 296 (M+H), 318 (M+Na), 591
(2M+H), 613 (2M+Na); (ESÀ) 294 (MÀH), 340 (M+HCOOÀ).
product (463 mg, 83%, mixture of
4 stereoisomers) was isolated by flash
chromatography (pentane/Et2O: 9/1) as a brownish oil.
Compound 28b: 1H NMR (CDCl3, 300 MHz): d (ppm) 7.15 (d, 1H, J = 15.5 Hz),
6.92 (d, 1H, J = 15.5 Hz), 5.65 (br d, 1H, J = 9.0 Hz), 4.21 (m, 1H), 3.76 (dd, 1H,
J = 6.5 and 4.2 Hz), 3.40 (s, 3H), 1.91–1.68 (m, 2H), 1.45–1.00 (br s, 16H), 1.20 (d,
3H, J = 6.7 Hz); 13C NMR (CDCl3, 75 MHz): d (ppm) 200.0 (s), 164.0 (s), 134.8 (d),
132.7 (d), 86.3 (d), 57.9 (q), 45.7 (d), 35.7 (t), 30.1 (t), 28.1 (t), 27.8 (t), 27.8 (t),
27.2 (t), 27.1 (t), 24.0 (t), 21.5 (t), 21.2 (q); MS (ES+) 296 (M+H), 318 (M+Na), 591
(2M+H), 613 (2M+Na); (ESÀ) 294 (MÀH), 340 (M+HCOOÀ).
8. Stereochemistry of each diastereomer 28a and 28b could not be assigned by
NMR analysis.
9. A solution of 27 (50 mg, 0.12 mmol) in CH2Cl2 (1 mL) at room temperature was
treated with TFA (0.1 mL, 1.3 mmol, 12.0 equiv) and stirred for 70 min. The
reaction mixture was then concentrated under vacuum. The crude residue was
dissolved in CH2Cl2 (12 mL) and HATU (55 mg, 0.144 mmol, 1.2 equiv), HOBt
(10 mg, 0.072 mmol, 0.6 equiv), a catalytic amount of DMAP and DIPEA (126 lL,