A.L. Pietkiewicz et al. / Tetrahedron Letters xxx (2014) xxx–xxx
3
22
(1.2 equiv.)
Table 1
1. TMSD
TBTU (1 equiv.)
HATU (1 equiv.)
DIPEA (12 equiv.)
CH2Cl2 (0.1 M)
Conformational assignment of SanB-14 (4) and its analogues 6–11. Orientation of the
MeOH:benzene
13
(1:3)
(0.1 M)
H2, Pd/C (10% w/w)
EtOH (0.1 M)
quant.
OMe
NH
O
prolyl amide bond is determined by
D
dCb
.
(Data for SanB-4 were referenced from
c
OH
OMe
O
the published result.12
O
O
Br
NH
NH2
BnO
2. anisole (2 equiv.)
TFA:CH2Cl2 (1:3)
(0.1 M)
83%
OMe
BnO
BnO
Boc
OMe
dCbc13 Pro A
D
dCb
c
Pro B
Assignment
27
23
28
Compound
Conformer
D
quant. for 2 steps
SanB-412
SanB-6
5.7
9.5
4.4
7.5
7.0
9.6
4.3
6.6
9.3
9.9
9.7
9.5
15.0
10.9
14.2
7.7
2.5
8.6
5.0
trans, cis-
cis, cis
1. DAST (2 equiv.)
K2CO3 (2 equiv.)
-78 oC to rt
A
B
trans, cis
trans, cis
trans, trans
cis, trans
trans, cis
trans, trans
cis, cis
formic acid (0.1 M)
OMe
NH
OMe
Br
OMe
Br
rt to 60 o
93%
C
SanB-7
SanB-8
O
CH2Cl2 (0.1 M)
Br
OMe
N
O
N
O
HO
2. DBU (2 equiv.),
BrCCl3 (2 equiv.)
-46 oC to rt
CH2Cl2 (0.1 M)
75% over 2 steps
OMe
OMe
A
B
A
B
O
29
O
O
O
OMe
19
21
SanB-9
1. anisole (2 equiv.)
TFA:CH2Cl2 (1:3)
(0.1 M)
R2
1. KHCO3 (8 equiv.)
20 (1.2 equiv.)
SanB-10
SanB-11
14.7
14.0
7.8
O
BocHN
OMe
A
B
cis, cis
cis, trans
OMe
N
O
quant.
DME (0.05 M)
BocN
N
S
N
O
O
2. pyridine (9 equiv.),
TFAA (4 equiv.),
DME (0.05 M)
N
N
O
2. NHBoc-Amino Acid-OH (1.2
equiv.)
S
(see Supporting information for details on structure conformation.)
18
TBTU (1 equiv.)
HATU (1 equiv.)
DIPEA (12 equiv.)
CH2Cl2 (0.1 M)
30
0
oC, 2 h,
3. Et3N (2 equiv.),
0
oC to rt, 2 h,
84% over 2 steps
deprotection of 29 using formic acid gave 19, which was reacted
with Pro-thioamide derivative 20 under modified Hantzsch thia-
zole conditions to give the bisheterocycle 18. Amine deprotection
of 18 with trifluoroacetic acid (TFA) and elongation by peptide cou-
pling with NHBoc-amino acid gave the ester 30, where coupling of
-Leu, D-Leu, L-Phe, D-Phe or N6-Cbz-L-Lys generated the corre-
sponding Fragment B analogues for each derivative. Coupling
between 30 and NH-Pro-OMe yielded 13.
72% over 3 steps
R2
O
O
BocHN
OMe
1. LiOH (8 equiv.)
MeOH (0.1 M)
N
N
O
N
N
O
2. NH-Pro-OMe (1.2 equiv.)
TBTU (1 equiv,)
S
HATU (1 equiv.)
Fragment B
13
DIPEA (12 equiv.)
CH2Cl2 (0.1 M)
L
74% over 2 steps
Scheme 3. Synthesis of Fragment B, 13.
Coupling between the free amine and free acid, after respective
amine and acid deprotection reactions, yielded linear precursor 31
(Scheme 4). Subsequent acid and amine deprotection of 31, fol-
lowed by macrocyclization under dilute conditions generated all
the analogues, except 5. Compound 5 was generated by synthesiz-
ing compound 4 and removing the Cbz group from the lysine using
HBr and acetic acid (33% HBr in acetic acid) at 0.1 M.
TBTU and HATU as coupling agents, yielded Fragment A (12). Frag-
ment A required for synthesizing derivatives 7 and 8 had an -Val
incorporated in place of Lys, and derivative 6 replaced the N6-Cbz-
-Lys with an N6-Ac- -Lys.
The synthesis of Fragment B (Scheme 3), 13, was achieved by
L
L
L
Each analogue was purified by HPLC and LCMS, and the result-
ing NMR spectra showed that numerous analogues had more than
one conformation (Table 1). Identification of each conformer uti-
lized 1H and 13C NMR and 2D NMR experiments (1H–1H COSY,
1H–13C HSQC, 1H–13C HMBC), as well as HPLC, LC/MS, and HRMS
(see Supporting information).
reacting Boc-Ser(Bn)-OH (27) with TMSD to generate the ester,
which was subsequently converted into the amine 23. A coupling
reaction between bromo-ketal acid 22 and NH2-Ser(Bn)-OMe (23)
yielded bromomethoxyketal serine derivative 28, which was sub-
jected to hydrogenation yielding compound 21. Cyclization of 21
using dimethylaminosulfur trifluoride (DAST), followed by oxida-
tion with bromotrichloromethane (BrCCl3) and 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU) generated the oxazole 29. Ketone
Utilizing a gradation of temperatures in a 1H NMR analysis pro-
vided the optimal temperature at which sharp peaks were
observed in the NMR.10 Upon determining the optimal tempera-
ture for evaluation, 2D NMR data were collected at that tempera-
ture, which allowed identification of the configuration of each
prolyl amide bond present in the macrocycle. Examination of the
anisole (2 equiv.)
TFA:CH2Cl2 (1:3)
(0.1 M)
quant.
S
O
O
N
Pro Cb and C chemical shifts provides evidence of the Pro being
S
O
c
OEt
DMTMM (1 equiv.),
HATU (1 equiv.),
DIPEA (12 equiv.)
CH2Cl2 (0.1 M)
NH
R1
O
N
‘cis’ or ‘trans’. A Pro amide bond that adopts cis orientation has a
OEt
BocHN
NH
NH
NHBoc
R1
O
R2
O
Fragment A
12
larger
DdCb than a Pro with an amide bond in the trans orienta-
c
tion.13 The Pro Cb and C shifts for each conformer are shown in
Yields ranged from
33-98% over 3 steps
c
N
R2
O
BocHN
LiOH.H2O (8 equiv.)
H2O2 (3.4 equiv.)
MeOH (0.1 M)
quant.
N
N
Table 1.
N
OMe
O
S
O
N
From the data, it appeared that neither proline has a preferred
conformation. Although individual proline shifts were visible in
the 2D NMR data, which allowed us to assign the proline orienta-
tion, all but one compound (compound 11) were inseparable mix-
tures. As observed by isolating each peak of the inseparable
mixture on the LCMS and re-injecting into the LCMS, these mole-
cules were in fact oscillating between the two conformations. For
example, 6A and 6B were isolated as an interconverting mixture
of cis, cis and trans, cis, where prolyl A was rapidly oscillating
between cis and trans. Compound 8 was also isolated as two inter-
converting conformations with Pro A also oscillating between cis
and trans. Compounds 9 and 11 had Pro B oscillating between cis
and trans, where compound 11 interconverted slowly enough that
the two conformations could be isolated (Table 1).
O
31
N
O
N
N
O
S
Fragment B
13
1. LiOH.H2O (8 equiv.)
EtOH (0.1 M)
2. anisole (2 equiv.)
TFA:CH2Cl2 (1:3)
(0.1 M)
NH2
R1
=
H
N
CH3
S
O
O
O
N
R2
HN
NH
NH
R1
O
NHCbz
O
3. HATU (1 equiv.)
DMTMM (1 equiv.)
T3P (1 equiv.)
DIPEA (12 equiv.)
CH2Cl2 (0.0005 M)
10-79% over 3 steps
N
B
O
N
A
N
N
S
R2
=
O
SanB analogue
5
* for compound HBr/AcOH (33%) 0.1 M
was added to 4 which removed the Cbz group
NHCbz
The results of the biological testing show that the Cbz-Lys group
is essential for activity, since removal or modification of this resi-
Scheme 4. Cyclization and synthesis of SanB analogues.