Highly potent macrocyclic BACE-1 inhibitors incorporating a hydroxyethylamine core: Design, synthesis and X-ray crystal structures of enzyme inhibitor complexes

Article abstract of DOI:10.1016/j.bmc.2012.05.039

A series of P1-P3 linked macrocyclic BACE-1 inhibitors containing a hydroxyethylamine (HEA) isostere scaffold has been synthesized. All inhibitors comprise a toluene or N-phenylmethanesulfonamide P2 moiety. Excellent BACE-1 potencies, both in enzymatic an

Full text of DOI:10.1016/j.bmc.2012.05.039

Bioorganic & Medicinal Chemistry 20 (2012) 4377–4389
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Highly potent macrocyclic BACE-1 inhibitors incorporating a
hydroxyethylamine core: Design, synthesis and X-ray crystal structures
of enzyme inhibitor complexes
Veronica Sandgren a, Tatiana Agback b, Per-Ola Johansson b, Jimmy Lindberg b, Ingemar Kvarnström a,
a,
Bertil Samuelsson b, Oscar Belda b, , Anders Dahlgren
⇑
⇑
a Department of Chemistry, Linköping University, S-581 83 Linköping, Sweden
b Medivir AB, Lunastigen 7, S-141 44 Huddinge, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of P1–P3 linked macrocyclic BACE-1 inhibitors containing a hydroxyethylamine (HEA) isostere
scaffold has been synthesized. All inhibitors comprise a toluene or N-phenylmethanesulfonamide P2 moi-
ety. Excellent BACE-1 potencies, both in enzymatic and cell-based assays, were observed in this series of
target compounds, with the best candidates displaying cell-based IC50 values in the low nanomolar range.
As an attempt to improve potency, a phenyl substituent aiming at the S3 subpocket was introduced in the
macrocyclic ring. X-ray analyzes were performed on selected compounds, and enzyme-inhibitor interac-
tions are discussed.
Received 27 January 2012
Revised 17 April 2012
Accepted 16 May 2012
Available online 24 May 2012
Keywords:
Alzheimer’s disease
BACE-1 inhibition
Macrocycles
Ó 2012 Elsevier Ltd. All rights reserved.
Hydroxyethylamine (HEA) isostere
1. Introduction
to obtain these macrocycles have been used, such as metathesis,8–12
reductive amination,13 peptide coupling9,14 and macrolactoni-
Although it has been more than 100 years since the symptoms
of Alzheimer’s disease (AD) were first described, the underlying
pathophysiological mechanisms behind the disease remain ob-
scure. However, it is believed that the accumulation of the so called
amyloid-b peptides (Ab), leading to plaques and eventually neuro-
fibrillary tangles in the brain, has a crucial role in the pathology of
AD.1 The mechanism by which these amyloid-b peptides are
formed involves the enzymes b-secretase (BACE-1) followed by
zation.15
In this study, we describe the synthesis and biological evaluation
of a series of macrocyclic BACE-1 inhibitors containing a hydroxy-
ethylamine (HEA) isostere. The P1 and P3 moieties were connected
by ring closing metathesis (RCM) (Fig. 1). The P2 part of the target
molecules incorporated a benzene ring with a sulfonamide func-
tionality or a toluene core (see Table 1). These P2 moieties have
emerged as promising building blocks in earlier reported stud-
ies.8,9,16 Also, a phenyl substituent was introduced in the macrocy-
clic ring in an attempt to reach into the S3 subpocket (S3sp) of the
enzyme and thus improve the activity even further.
c-secretase, which sequentially cleave the amyloid precursor pro-
tein (APP), generating the Ab peptides. Furthermore, it is known
that BACE-1 catalyzes the rate-limiting step in the sequence.2 This,
together with the knowledge that BACE-1 knockout mice are viable
and fertile, makes BACE-1 a promising therapeutic target.3,4
The development of low molecular weight, brain penetrating
BACE-1 inhibitors has proven to be quite challenging.5,6 However,
it has been shown that the pharmacokinetics of standard linear
inhibitors can be significantly improved by cyclization,7 and this
approach has indeed been incorporated in the search for promising
drug candidates in the field of AD research. Different methodologies
2. Results and discussion
2.1. Chemistry
The target compounds 17 and 21–33 were prepared according
to Schemes 1–4 (see also Table 1). Synthesis of key building block
3, which was used in synthesis of all the target compounds, is out-
lined in Scheme 1. The alcohol 1, synthesized from commercially
available (ꢀ)-diethyl
D-tartrate in four steps according to literature
⇑
Corresponding authors. Tel.: +46 13 281000; fax: +46 13 281399 (A.D.); tel.: +46
8 54683100; fax: +46 8 54683199 (O.B.).
procedures,17–19 was allylated with allyl bromide and Ag2O in tol-
uene to give compound 2 in 78% yield. This reaction performed
with NaH resulted in low yields. Reduction of the azide group in
E-mail addresses:
(A. Dahlgren).
oscar.belda@medivir.com (O. Belda),
andda@ifm.liu.se
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.039

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V. Sandgren et al. / Bioorg. Med. Chem. 20 (2012) 4377–4389
S2
R2
R2
S2'
OH
H
OH
H
N
H
N
H
N
S3
O
N
O
R1
R1
HN
R3
HN
O
(CH2)x
O
O
O
(CH2)x
S1
Figure 1. General structures of the macrocyclic target compounds (see Table 1).
Table 1
BACE-1 inhibition data for compounds 17 and 21–33
Compound
Structure
IC50 enzyme (nM)
IC50 cell (nM)
O O
S
N
OH
OH
OH
H
N
H
N
21
270
560
O
O
O
O
HN
O
O
O
O
O O
S
N
H
N
H
N
17
14
2.3
HN
O
O O
S
N
H
N
H
N
22
15
4.6
HN
O
O O
S
N
OH
23a
180
73
H
N
H
N
HN
O
O
O
O
S
N
OH
H
N
H
N
24
5300
>10000
O
HN
O
O

V. Sandgren et al. / Bioorg. Med. Chem. 20 (2012) 4377–4389
4379
Table 1 (continued)
Compound
Structure
IC50 enzyme (nM)
IC50 cell (nM)
O
O
S
N
OH
H
H
N
25
26a
27
16
49
O
N
HN
O
O
O
S
O
N
OH
H
N
H
N
18
7.7
O
HN
O
O
O
O
S
N
OH
H
N
H
7.5
6.9
N
O
N
O
O
O
O
S
N
OH
H
H
N
N
O
28
400
260
HN
O
O
O
O
S
N
OH
H
N
H
N
O
29
3.2
0.15
HN
O
O
OH
OH
H
H
N
N
O
30
>10000
>10000
HN
O
O
O
H
H
N
N
O
31
500
220
HN
O
(continued on next page)

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V. Sandgren et al. / Bioorg. Med. Chem. 20 (2012) 4377–4389
Table 1 (continued)
Compound
Structure
IC50 enzyme (nM)
IC50 cell (nM)
OH
H
N
H
N
O
32
4900
2300
HN
O
O
O
OH
H
N
H
N
O
33
78
160
HN
O
a
16-membered macrocycle.
compound 2 was achieved using 1,3-propanedithiol and TEA in
methanol to provide the corresponding amine 3 in 73% yield.
Synthesis of the building blocks (R)-9, (S)-9, (R)-10, and (S)-10 is
described in Scheme 2. Commercially available (R)-(+)-styrene
oxide was treated with lithium acetylide ethylenediamine complex
to give compound (S)-4 in 95% yield. Mitsunobu conditions utiliz-
ing DPPA generated the azide (R)-5 with inversion of configuration
in 97% yield. The azide group was subsequently converted to the
corresponding amine (R)-6, using 1,3-propanedithiol and TEA in
methanol, in 92% yield. The enantiomer (S)-6 was synthesized in
the same manner from (S)-(ꢀ)-styrene oxide. The primary amines
(R)-6 and (S)-6 were thereafter coupled with either commercially
available 3-methoxycarbonyl-5-methylbenzoic acid or compound
1120 (see Scheme 3) using HCTU and DIPEA in DMF to furnish
the amides (R)-7, (R)-8, (S)-7, and (S)-8 in 73–89% yield. Finally, al-
kyne reduction using Lindlar’s catalyst afforded the four desired
compounds (R)-9, (R)-10, (S)-9, and (S)-10 in 84–99% yield.
Target compound 17 was prepared as described in Scheme 3.
Compounds 21–23 and 27–33 (Table 1) were synthesized using
the same synthetic route and with similar yields (benzylamine
was used instead of 3-isopropylbenzylamine in the final step for
compounds 21 and 30, and 3-tert-butylbenzylamine was used for
compound 22). Compound 12a was prepared in 83% yield from
compound 11 in a peptide coupling step using 3-butenylamine
hydrochloride, EDC, HOBt, and TEA. The ester functionality in 12a
was hydrolyzed using aqueous LiOH in H2O/dioxane and the corre-
sponding carboxylic acid was coupled with compound 3 using
HATU and DIPEA in DMF to afford compound 13 in 90% yield over
two steps. The acetal in 13 was then hydrolyzed (AcOH/H2O, 60 °C),
resulting in the diol 14 (quant.). The subsequent ring closing
metathesis step was achieved utilizing Hoveyda-Grubbs catalyst
2nd generation in refluxing DCM to give the desired 15-membered
macrocycle 15 in 98% yield. The cyclization resulted in exclusive
formation of the trans isomer, as confirmed by 1H-NMR analysis.
Attempts to cyclisize compound 13 were unsuccessful, tentatively
because of the more rigid structure found in 13. The primary hy-
droxyl group in 15 was selectively tosylated with p-TsCl, Bu2SnO,
and TEA in dry DCM to generate compound 16 (64%). Finally, the
tosylate 16 was reacted with 3-isopropylbenzylamine in refluxing
ethanol, furnishing target compound 17 in 37% yield. The amide
nitrogen in 12a was methylated using NaH and MeI in DMF to give
compound 12b in 100% yield. This building block was used to syn-
thesize target compound 27, which proved to be a separable mix-
ture of cis-27 (regio chemistry at the macrocyclic double bond
position) and trace amounts of a related compound that tentatively
could be identified by NMR analysis as the trans isomer. Notewor-
thy, the methylated amide functionality in 27 seems to have chan-
ged the preferred regio chemistry in the macrocycle from trans to
cis.
The synthesis of compounds 19 and 20 is outlined in Scheme 4.
Compound 18 was synthesized in the same manner as compound
15 with the exception that 4-pentenylamine hydrochloride21 was
used instead of 3-butenylamine hydrochloride in the peptide cou-
pling step (see Scheme 3). Hydrogenation of the double bond in
compound 15 and 18 was achieved in the presence of Pd-C in
methanol to give compounds 19 and 20 in 95% and 78% yield,
respectively. To obtain the three saturated macrocyclic compounds
24–26, the same synthetic route was used as for compound 17 (see
Scheme 3) with the exception that benzylamine was used instead
of 3-isopropylbenzylamine in the final step for compound 24.
2.2. Biological data and structure activity relationships
Table 1 contains all target compounds presented in this paper. All
the macrocycles are 15-membered, except 23 and 26, which are 16-
membered. The inhibitors were all collected and tested as trans iso-
mers, except for compound 27, which was tested as the cis isomer.
Encouraged by the biological results for the first synthesized target
O
O
O
O
O
N3
O
N3
O
H2N
O
i
ii
OH
1
2
3
Scheme 1. Synthesis of compound 3, employed as a building block for synthesis of
all the target compounds. Reagents and conditions: (i) allyl bromide, Ag2O, toluene,
rt, 26 h, 78%; (ii) 1,3-propanedithiol, TEA, MeOH, rt, 22 h, 73%.

V. Sandgren et al. / Bioorg. Med. Chem. 20 (2012) 4377–4389
4381
OH
N3
NH2
O
i
ii
iii
*
*
*
*
(R)
(S)
(S)-4
(R)-4
(R)-5
(S)-5
(R)-6
(S)-6
R
R
H
N
H
iv
v
O
N
O
*
*
O
O
O
O
(R)-7
(R)-9
R = Me
(R)-8 R = N(Me)SO2Me
(S)-7
R = Me
(R)-10 R = N(Me)SO2Me
(S)-9
R = Me
(S)-8 R = N(Me)SO2Me
R = Me
(S)-10 R = N(Me)SO2Me
Scheme 2. Synthesis of building blocks 9–10. Reagents and conditions: (i) lithium acetylide ethylenediamine complex, DMSO (dry), Ar, rt, 25 h, 95% and quantitative yield,
respectively; (ii) PPh3, DIAD, DPPA, THF, ꢀ12 °C to rt, 25 h, 97% and 76%, respectively; (iii) 1,3-propanedithiol, TEA, MeOH, rt, 47 h, 92% and 87%, respectively; (iv) 3-
methoxycarbonyl-5-methylbenzoic acid or compound 11, HCTU, DIPEA, DMF, rt, 4 h, 73–89%; (v) Lindlar’s catalyst, quinoline, MeOH, H2, rt, 1.5 h, 84–99%.
in this series, compound 21, further investigations were of interest.
In order to reach further into the S20 pocket of BACE-1, targets 17 and
22, containing alkyl substituents on the benzylamine moiety, were
synthesized. Both compounds showed similar IC50 values in the
enzymatic assay (14 and 15 nM, respectively), that is they were
20-fold more active than 21. Even more significant, 17 displayed
O O
O O
S
O O
S
S
N
N
N
O
i
iii
H
N
O
R
O
O
O
HO
O
N
O
HN
O
O
O
O
11
12a R = H
ii
12b R = Me
13
O O
S
O O
S
N
N
OH
OH
H
N
H
N
iv
v
vi
O
OH
O
OH
HN
O
HN
O
O
O
15
14
O O
S
O O
S
N
N
OH
OH
H
H
N
H
N
vii
O
N
OTs
O
HN
O
HN
O
O
O
17
16
Scheme 3. Synthesis of target molecule 17. Reagents and conditions: (i) 3-butenylamine hydrochloride, TEA, HOBt, EDC, DMF, 0 °C to rt, 19 h, 83%; (ii) NaH, MeI, DMF, rt, 23 h,
100%; (iii) (a) LiOH, H2O/dioxane 1:2, 70 min, (b) compound 3, DIPEA, HATU, DMF, rt, 16 h, 90% over two steps; (iv) AcOH/H2O, 60 °C, 1 h 45 min, 100%; (v) Hoveyda-Grubbs
catalyst 2nd generation, DCM (dry), N2, reflux, 20 h, 98%; (vi) p-TsCl, Bu2SnO, TEA, DCM (dry), rt, 23 h, 64%; (vii) 3-isopropylbenzylamine, EtOH 99.5%, reflux, 24 h, 37%.

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V. Sandgren et al. / Bioorg. Med. Chem. 20 (2012) 4377–4389
O O
S
O
O
S
N
N
OH
OH
H
N
i
H
N
O
OH
O
OH
HN
O
HN
O
O
O
(CH2)x
(CH2)x
15
19
X = 1
X = 1
18 X = 2
20 X = 2
Scheme 4. Synthesis of the saturated compounds 19 and 20 used to produce target compounds 24–26. Reagents and conditions: (i) Pd-C 10%, H2, MeOH, rt, 3 h, 95% and 78%,
respectively.
an IC50 of 2.3 nM in the cell-based assay, with a 240-fold improve-
ment compared to 21, whereas compound 22 showed a 120-fold in-
crease in cellular activity.
The 16-membered macrocycle 23 was synthesized to investi-
gate the way ring-size of this series affects biological activity. This
resulted in a 13-fold loss of activity in the enzymatic assay and a
30-fold loss in the cell-based assay compared to inhibitor 17. All
attempts to synthesize the corresponding 14-membered macrocy-
cle were unsuccessful.
The somewhat more flexible saturated macrocycles 24–26 were
prepared in order to see if the double bond affected potency. Com-
pound 24 lost almost all its activity in both assays, while com-
pound 25 kept the enzymatic activity but had a 20-fold drop in
the cell-based assay compared to 17. Interestingly, the 16-mem-
bered macrocycle 26 gained activities compared to its unsaturated
analogue 23 and showed nearly similar potencies as inhibitor 17.
This result may be explained by the fact that this 16-membered
macrocycle’s enhanced flexibility allowed it to mimic the confor-
mation of its 15-membered unsaturated counterpart.
Compound 27 showed similar enzyme activity as 17, which
possibly could indicate that substitution of the P2–P3 amide is
non-critical for BACE-1 activity. Furthermore, since inhibitor 27
was tested as the cis isomer and the rest of the unsaturated macro-
cyclic targets were identified as trans (see above), the configuration
of this double bond tentatively seems not to be crucial for activity
in this series of compounds.
the P1–P3 macrocyclic BACE-1 inhibitors was as anticipated for
HEA-inhibitors (Fig. 2).23–26 The protonated amino and hydroxyl
groups form hydrogen bonds with the catalytic aspartates Asp32
and Asp228, respectively. The carbonyl of Gly34 also forms a
hydrogen bond with the ionized amine. Moreover, the P20 isopro-
pyl substituted benzyl has a snug fit to the S20 pocket. The benzyl
is sandwiched between Tyr198 and the peptide backbone of the
flap, and the isopropyl extension is clamped in-between Ile126
(S20) and Val69 (flap). This firm binding is reflected in the SAR,
and underlines the importance of these additional hydrophobic
interactions for compound activity (Table 1).
The co-crystal structure of 33 in complex with BACE-1 was
solved to 1.9 Å resolution, and shows the complete P1–P3 macrocy-
cle spanning the S1-S3 subsites (Fig. 2). Similar to P1–P3 groups of
linear HEA analogues, the P1–P3 macrocycle is coordinated
between the flap and active site by a set of hydrogen bonds between
the P1–P3-amides and the protein backbone (Gln73 NH and/or
Gly230 CO). One additional hydrogen bond connecting the inhibitor
and flap was observed between the ether-oxygen of the macrocycle
and the backbone carbonyl of the flap residue, Gln73 bridged by
structural water (Fig. 3). Moreover, the P3-amide functionality of
A phenyl substituent was introduced in the macrocyclic ring in
compounds 28 and 29 in an attempt to reach into the S3sp and fur-
ther optimize potency. The (S)-isomer 28 displayed almost 30-fold
less enzyme activity and 110-fold less cellular activity compared to
17. However, the (R)-isomer 29 yielded a small improvement of the
enzymatic activity compared to inhibitor 17 and
a 15-fold
improvement of the cell activity.
Although some of the sulfonamide-based BACE-1 inhibitors dis-
played very high potencies all compounds exhibited poor cell-
based Caco-2 permeability (around or less than 1 ꢁ 10ꢀ6 cm/s).22
Other BACE-1 inhibitors with a sulfonamide group in the P2 posi-
tion have been shown to lack permeability due to for example
P-gp efflux.9 Replacement of the sulfonamide group with a less
polar methyl substituent (compounds 30–33) led merely to
decreased potencies and no permeability improvement could be
observed.
Inhibition of cathepsin D for selected compounds at an inhibitor
concentration of 10 lM: 17 (23%), 22 (10%), 28 (46%), 31 (30%), and
33 (14%).
2.3. X-ray crystallography analysis
Figure 2. Compound 33 bound to the active site of BACE-1 (PDB id: 4DPI). The
mode of binding of the compound is unambiguously shown in the electron density
calculated at 1.9 Å resolution and contoured at 1.
Compounds 28 and 33 were co-crystallized with BACE-1 (PDB
ids: 4DPF and 4DPI, respectively). The general binding mode of

V. Sandgren et al. / Bioorg. Med. Chem. 20 (2012) 4377–4389
4383
rotamer of Thr232. Similarly, a P3-keto analogue of an unsubstitut-
ed macrocycle adopts the same set of two conformational isomers
(unpublished observations). These observations point to the fact
that the energetically unfavorable binding mode of the P3-amide
is inherent to the P1–P3 macrocycle scaffold, rather than the mac-
rocycle substituent. Computer modeling suggests that replacing
the P3-amide with a secondary amine will release the strain on
the P1–P3 macrocycle and maintain the hydrogen bond to
Gly230, which may lead to better potency and permeability in this
series of compounds.
The preference for the (R)- over the (S)-isomer was apparent
from the structures of 28 and 33 with BACE-1, which is illustrated
in the structural overlay of the two compounds (Fig. 4). In the case
of the (R)-isomer 33, the phenyl protrudes from the macrocycle
into the amphiphilic S3sp gaining hydrophobic interactions,
whereas for the (S)-isomer 28 the macrocycle and phenyl adopt
an energetically unfavorable binding conformation, where the phe-
nyl protrudes on top of the S3sp facing the solvent. In order to
improve potency for the (R)-isoform, the amphiphilic nature of
the S3sp seen as a narrow polar entrance and a lipophilic interior
needs to be reflected in the macrocycle substituent. The X-ray
structure of 33 suggests extended aryls and heteroaryls as good
substituents for future chemistry design.
Figure 3. The hydrogen bond coordination of the macrocycle of compound 33
between residues of S1–S3 and the flap. Shown are also the two conformational
isomers of the compound and the two accompanying rotamers of Thr232;
equiplanar (yellow) and staggered (light blue).
3. Conclusion
In summary, a series of macrocyclic BACE-1 inhibitors contain-
ing a hydroxyethylamine (HEA) isostere scaffold have been de-
signed and synthesized, some of which displayed excellent
potency. The most potent inhibitor 29 displayed an IC50 value of
3.2 nM in the enzymatic assay and 0.15 nM in the cell-based assay.
Some additional target compounds also showed activity in the low
nanomolar range. Furthermore, good selectivity over cathepsin D
could generally be observed in this series of inhibitors. X-ray
crystallography studies concluded that the macrocycle-attached
phenyl group, for example, in inhibitor 29, did not fully reach into
the S3 subpocket of BACE-1, which indicates that there is room for
further potency optimizations.
Even though we could achieve low nanomolar BACE-1 activity
and good selectivity, the compounds synthesized suffered from
inadequate permeability tentatively preventing them from reach-
ing the CNS. Future work should be aimed at designing inhibitors
with enhanced brain permeability, while retaining the reported
potencies.
compound 33 is observed in two distinct conformations, staggered
and equiplanar with respect to the P2-phenyl plane. In solution, the
low energy state of the P3-amide is in staggered conformation. In
the X-ray structure of 33 however, the equiplanar conformation is
predominant. In this conformation, the amide NH donates a proton
to the backbone CO of Gly230 in a hydrogen bond, and the amide CO
is in range for an electrostatic interaction to the side chain of Gln73
in the flap. In the case of the staggered conformation, the amide CO
is facing the side chain of Thr232 and the amide NH points towards
Gln73 with no apparent interactions with the protein. This confor-
mation is accommodated for the protein by a shift in the side chain
4. Experimental section
4.1. BACE-1 enzyme assay
The BACE-1 assay was performed as previously described.27
4.2. BACE-1 cell assay
The cell-based assay was performed as previously described.28
4.3. Caco-2 assay
The permeability measurements were performed as previously
described.29
4.4. Cathepsin D assay
The cathepsin D enzyme assay was performed as previously
described.27
Figure 4. Structural overlay of compounds 28 (pink) and 33 (yellow) showing the
preference for the (R)-isomer in S3sp interactions.

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V. Sandgren et al. / Bioorg. Med. Chem. 20 (2012) 4377–4389
4.5. General
made basic with 1 M NaOH and extracted with Et2O. The combined
organic extracts were dried, filtered and concentrated to yield 3
+9.0 (c 0.1, MeOH). 1H NMR
(CDCl3, 300 MHz): d 1.32 (s, 3H), 1.38 (s, 3H), 2.30 (bs, 2H), 3.03–
3.09 (m, 1H), 3.39 (dd, J = 6.3, 9.3 Hz, 1H), 3.56 (dd, J = 3.6, 9.3 Hz,
1H), 3.84–3.91 (m, 1H), 3.97–4.03 (m, 4H), 5.12–5.26 (m, 2H),
5.80–5.93 (m, 1H); 13C NMR (CDCl3, 75.5 MHz): d 25.3, 26.7, 53.3,
66.3, 71.8, 72.3, 77.0, 109.0, 117.1, 134.6. MS Calcd (M+H)+:
202.1. Found 202.2.
NMR-spectra were recorded on a Varian 300 MHz instrument
using CDCl3, CD3OD or DMSO-d6 as solvent. Thin layer chromatog-
raphy (TLC) was carried out on Merck precoated 60 F254 plates
using UV-light and charring with ethanol/sulfuric acid/p-anisalde-
hyde/acetic acid 90:3:2:1, or a solution of 0.5% ninhydrin in etha-
nol for visualization. Flash column chromatography was
performed using silica gel 60 (0.040–0.063 mm, Merck). Organic
phases were dried over anhydrous magnesium sulfate. Drying of
solvents: DCM was refluxed over calcium hydride and distilled
onto 4 Å molecular sieves, DMSO was stored overnight with 4 Å
MS. Optical rotations were measured using a Perkin-Elmer 141
polarimeter at 22 °C. Gradient LC–MS was performed on a Gilson
(82 mg, 73%) as a colorless oil. ½aꢂD
4.7.3. (S)-1-Phenylbut-3-yn-1-ol ((S)-4)
Lithium
acetylide
ethylenediamine
complex
(2.24 g,
21.9 mmol) was added to dry DMSO (14 mL) and the solution
was stirred for 30 min under Ar atmosphere. (R)-(+)-Styrene oxide
(1.0 mL, 8.76 mmol) was added and the mixture was stirred for
25 h at room temperature under Ar atmosphere. The mixture
was quenched with H2O and extracted with Et2O. The combined
organic extracts were dried, filtered and concentrated. Purification
using flash column chromatography (toluene/EtOAc 39:1) gave (S)-
system (column: Phenomenex C18, 100 ꢁ 21 mm, 5
lm for pre-
parative runs and xBridge™ C18, 50 ꢁ 4.6 mm, 2.5 m for analyti-
l
cal runs; pump: Gilson gradient pump 322; UV/vis-detector:
Gilson 152; MS detector: Thermo Finnigan Surveyor MSQ; Gilson
Fraction Collector FC204) using acetonitrile with 0.05% formic acid
and deionized water with 0.05% formic acid as mobile phases. High
resolution mass spectra (HRMS) were recorded on a Waters Synapt
HDMS instrument equipped with an electrospray interface.
4 (1.22 g, 95%) as a colorless oil. ½a D
ꢂ
ꢀ12.1 (c 0.14, MeOH). 1H NMR
(CDCl3, 300 MHz): d 2.10 (t, J = 2.4 Hz, 1H), 2.65 (dd, J = 2.5, 6.3 Hz,
2H), 4.84 (t, J = 6.3 Hz, 1H), 7.32–7.44 (m, 5H); 13C NMR (CDCl3,
75.5 MHz): d 29.4, 71.0, 72.4, 80.8, 125.8, 128.0, 128.5, 142.6. MS
Calcd (M+H)+: 147.1. Found 147.2.
4.6. LC–MS purity measurements
4.6.1. Chromatography system A
4.7.4. (R)-1-Phenylbut-3-yn-1-ol ((R)-4)
Column: Phenomenex C18, 50 ꢁ 4.6 mm, 3
l
m; pump: Gilson
Compound (R)-4 (340 mg, quant.) was synthesized from (S)-
(ꢀ)-styrene oxide in the same manner as (S)-4 and was collected
gradient pump 322; UV/vis-detector: Gilson 152; MS detector:
Thermo Finnigan Surveyor MSQ; Software: Gilson Unipoint 4.0
and Xcalibur 1.3. Mobile phase A: 10 mM NH4OAc in water; mobile
phase B: 10 mM NH4OAc in 90% acetonitrile; gradient: 20–100% B
over 6 min at 1 mL/min followed by 100% of B for 3 min at 1 mL/
min. Peaks were detected at 254 nM.
as a slightly yellow oil. ½a D
ꢂ
+12.8 (c 0.15, MeOH). 1H NMR (CDCl3,
300 MHz): d 2.07 (t, J = 2.6 Hz, 1H), 2.62 (dd, J = 2.5, 6.3 Hz, 2H),
2.80 (bs), 4.83 (t, J = 6.3 Hz, 1H), 7.29–7.39 (m, 5H); 13C NMR
(CDCl3, 75.5 MHz): d 29.3, 71.0, 72.3, 80.8, 125.8, 127.9, 128.4,
142.5. MS Calcd (M+H)+: 147.1. Found 147.2.
4.6.2. Chromatography system B
4.7.5. (R)-(1-Azidobut-3-yn-1-yl)benzene ((R)-5)
Column: XBridge C8, 50 ꢁ 4.6 mm, 2.5
l
m; pump: Gilson gradi-
Compound (S)-4 (1.04 g, 7.09 mmol) and PPh3 (2.79 g,
10.6 mmol) were dissolved in THF (25 mL) and the mixture was
cooled to ꢀ12 °C. DIAD (2.1 mL, 10.7 mmol) was added and the
mixture was stirred at ꢀ12 °C for 10 min. DPPA (2.3 mL, 10.7 mmol)
was added and the solution was left to slowly reach room temper-
ature for 25 h. The solution was then concentrated and purified
using flash column chromatography (hexane/EtOAc 39:1). Com-
pound (R)-5 (1.18 g, 97%) was collected as a colorless oil. 1H NMR
(CDCl3, 300 MHz): d 2.03–2.07 (m, 1H), 2.57–2.74 (m, 2H), 4.66 (t,
J = 6.9 Hz, 1H), 7.31–7.42 (m, 5H); 13C NMR (CDCl3, 75.5 MHz): d
26.9, 64.5, 71.2, 80.0, 126.9, 128.8, 129.0, 138.3. MS Calcd (M+H)+:
172.1. Found 172.0.
ent pump 322; UV/vis-detector: Gilson 152; MS detector: Thermo
Finnigan Surveyor MSQ; Software: Gilson Unipoint 4.0 and Xcali-
bur 1.3. Mobile phase A: 10 mM NH4OAc in water; mobile phase
B: 10 mM NH4OAc in 90% acetonitrile; gradient: 35–100% B over
7 min at 1 mL/min followed by 100% of B for 3 min at 1 mL/min.
Peaks were detected at 254 nM.
4.7. Synthesis
4.7.1. (S)-4-((S)-2-(Allyloxy)-1-azidoethyl)-2,2-dimethyl-1,3-
dioxolane (2)
Compound 1 (129 mg, 0.69 mmol) was dissolved in toluene
(2.5 mL). Ag2O (326 mg, 1.4 mmol) and allyl bromide (350
lL,
4.7.6. (S)-(1-Azidobut-3-yn-1-yl)benzene ((S)-5)
4.1 mmol) were added and the reaction mixture was stirred at
room temperature for 26 h. The solution was filtered, concentrated
and the crude remainder was purified using flash column chroma-
tography (hexane/EtOAc 39:1) to give compound 2 (117 mg, 78%)
as a colorless oil. 1H NMR (CDCl3, 300 MHz): d 1.32 (s, 3H), 1.42
(s, 3H), 3.49–3.55 (m, 1H), 3.61–3.70 (m, 2H), 3.88–3.93 (m, 1H),
4.00–4.07 (m, 4H), 5.16–5.31 (m, 2H), 5.82–5.95 (m, 1H); 13C
NMR (CDCl3, 75.5 MHz): d 25.4, 26.7, 63.1, 66.7, 70.2, 72.5, 75.2,
109.9, 117.5, 134.3. MS Calcd (M+H)+: 228.1. Found 228.4.
Compound (S)-5 (299 mg, 76%) was synthesized from (R)-4 in
the same manner as (R)-5 and was collected as a colorless oil. 1H
NMR (CDCl3, 300 MHz): d 2.06 (t, J = 2.7 Hz, 1H), 2.59–2.75 (m,
2H), 4.67 (t, J = 6.9 Hz, 1H), 7.32–7.44 (m, 5H); 13C NMR (CDCl3,
75.5 MHz): d 27.0, 64.6, 71.3, 80.0, 127.0, 128.8, 129.0, 128.3. MS
Calcd (M+H)+: 172.1. Found 172.2.
4.7.7. (R)-1-Phenylbut-3-yn-1-amine ((R)-6)
1,3-Propanedithiol (3.5 mL, 34.8 mmol) was added to a solution
of (R)-5 (1.2 g, 6.9 mmol) and TEA (4.8 mL, 34.7 mmol) in MeOH
(15 mL). The solution was stirred at room temperature for 47 h,
after which the reaction mixture was diluted with water and
MeOH, acidified with 1 M HCl and washed with DCM. Thereafter,
the water phase was made basic with 1 M NaOH and extracted
with DCM. The combined organic extracts were dried, filtered
and concentrated to give (R)-6 (930 mg, 92%) as a colorless oil.
1H NMR (CDCl3, 300 MHz): d 2.00 (bs, 2H), 2.04 (t, J = 2.4 Hz, 1H),
4.7.2. (S)-2-(Allyloxy)-1-((S)-2,2-dimethyl-1,3-dioxolan-4-
yl)ethanamine (3)
Compound 2 (126 mg, 0.55 mmol) was dissolved in MeOH
(3 mL). TEA (386 lL, 2.77 mmol) and 1,3-propanedithiol
(0.278 mL, 2.77 mmol) were added. The solution was stirred at
room temperature for 22 h, diluted with water and MeOH, acidi-
fied with 1 M HCl and washed with Et2O. The water phase was

V. Sandgren et al. / Bioorg. Med. Chem. 20 (2012) 4377–4389
4385
2.49 (ddd, J = 2.7, 7.8, 16.5 Hz, 1H), 2.60 (ddd, J = 2.7, 5.1, 16.8, Hz,
1H), 4.16 (dd, J = 5.1, 7.8 Hz, 1H), 7.21–7.36 (m, 5H); 13C NMR
(CDCl3, 75.5 MHz): d 29.7, 54.8, 70.6, 81.8, 126.3, 127.6, 128.6,
144.3. MS Calcd (M+H)+: 146.1. Found 146.5.
(m, 2H), 7.42–7.45 (m, 2H), 8.06–8.07 (m, 1H), 8.14–8.15 (m,
1H), 8.28–8.29 (m, 1H); 13C NMR (CDCl3, 75.5 MHz): d 25.6, 35.9,
38.0, 52.1, 52.8, 72.0, 79.9, 126.1, 126.8, 128.1, 128.9, 129.2,
130.0, 131.8, 135.9, 140.2, 142.4, 164.9, 165.6. MS Calcd (M+H)+:
415.1. Found 415.4.
4.7.8. (S)-1-Phenylbut-3-yn-1-amine ((S)-6)
Compound (S)-6 (905 mg, 87%) was synthesized from (S)-5 in
the same manner as compound (R)-6 and was collected as a color-
less oil. 1H NMR (CDCl3, 300 MHz): d 1.79 (s, 2H), 2.03 (t, J = 2.7 Hz,
1H), 2.44 (ddd, J = 2.7, 7.8, 16.5 Hz, 1H), 2.55 (ddd, J = 2.7, 5.1,
16.5 Hz, 1H), 4.11 (dd, J = 5.1, 7.8 Hz, 1H), 7.21–7.26 (m, 1H),
7.28–7.37 (m, 4H); 13C NMR (CDCl3, 75.5 MHz): d 29.5, 54.5, 70.5,
81.6, 126.1, 127.3, 128.3, 144.2. MS Calcd (M+H)+: 146.1. Found
145.8.
4.7.13. (R)-Methyl 3-methyl-5-((1-phenylbut-3-en-1-yl)carbam
oyl)benzoate ((R)-9)
Compound (R)-7 (27 mg, 0.08 mmol) was dissolved in MeOH
(7 mL). Lindlar’s catalyst (4 mg, ꢃ5% Pd on CaCO3) and quinoline
(20 lL, 0.17 mmol) were added and the mixture was stirred at
room temperature for 1.5 h under H2 (1 atm). The mixture was di-
luted with Et2O and washed with H2O. The organic phase was
dried, filtered through Celite and concentrated. Compound (R)-9
(27 mg, 99%) was collected as a white solid. 1H NMR (CDCl3,
300 MHz): d 2.42 (s, 3H), 2.70 (t, J = 6.9 Hz, 2H), 3.91 (s, 3H),
5.10–5.20 (m, 2H), 5.26–5.33 (m, 1H), 5.70–5.84 (m, 1H), 6.50
(bs, 1H), 7.24–7.29 (m, 1H), 7.34–7.36 (m, 4H), 7.83 (s, 1H), 7.96
(s, 1H), 8.14 (s, 1H); 13C NMR (CDCl3, 75.5 MHz): d 21.3, 40.6,
52.4, 53.1, 118.6, 124.7, 126.7, 127.6, 128.8, 130.5, 132.8, 133.2,
134.1, 135.0, 139.2, 141.6, 166.0, 166.7. MS Calcd (M+H)+: 324.2.
Found 324.3.
4.7.9. (R)-Methyl 3-methyl-5-((1-phenylbut-3-yn-1-yl)carbam
oyl)benzoate ((R)-7)
Compound (R)-6 (270 mg, 1.86 mmol) and 3-methoxycarbonyl-
5-methylbenzoic acid (506 mg, 1.76 mmol) were dissolved in DMF
(8 mL). DIPEA (0.92 mL, 5.3 mmol) and HCTU (1.09 g, 2.6 mmol)
were added and the mixture was stirred at room temperature for
4 h. The solution was diluted with brine and H2O and extracted
with EtOAc. The combined organic extracts were dried, filtered
and co-evaporated with toluene. The residue was purified using
flash column chromatography (toluene/EtOAc 9:1) to give (R)-7
(650 mg, 89%) as a white solid. 1H NMR (DMSO-d6, 300 MHz): d
2.45 (s, 3H), 2.52–2.53 (m, 1H), 2.70–2.79 (m, 1H), 2.81–2.88
(m, 1H), 3.90 (s, 3H), 5.21–5.28 (m, 1H), 7.25–7.30 (m, 1H), 7.36
(t, J = 7.2 Hz, 2H), 7.46–7.48 (m, 2H), 7.95 (s, 1H), 7.99 (s, 1H),
8.30 (s, 1H), 9.11 (d, J = 8.4 Hz, 1H); 13C NMR (DMSO-d6,
75.5 MHz): d 20.7, 25.3, 52.2, 52.4, 72.5, 81.7, 125.3, 126.6, 127.1,
128.2, 129.7, 132.1, 132.6, 134.8, 138.5, 142.0, 164.9, 165.9. MS
Calcd (M+H)+: 322.1. Found 322.4.
4.7.14. (S)-Methyl 3-methyl-5-((1-phenylbut-3-en-1-
yl)carbamoyl)benzoate ((S)-9)
Compound (S)-9 (213 mg, 99%) was synthesized from (S)-7 in
the same manner as (R)-9 and was collected as a white powder.
1H NMR (CDCl3, 300 MHz): d 2.36 (s, 3H), 2.66–2.71 (m, 2H), 3.88
(s, 3H), 5.08–519 (m, 2H), 5.28 (m, 1H), 5.69–5.83 (m, 1H), 6.80
(d, J = 8.1 Hz, 1H), 7.24–7.29 (m, 1H), 7.31–7.37 (m, 4H), 7.80
(s, 1H), 7.92 (s, 1H), 8.16 (s, 1H); 13C NMR (CDCl3, 75.5 MHz): d
21.2, 40.4, 52.3, 53.1, 118.3, 124.8, 126.6, 127.5, 128.7, 130.4,
132.7, 133.0, 134.2, 134.9, 139.0, 141.6, 166.0, 166.6. MS Calcd
(M+H)+: 324.2. Found 324.4.
4.7.10. (S)-Methyl 3-methyl-5-((1-phenylbut-3-yn-1-yl)carbam
oyl)benzoate ((S)-7)
4.7.15. (R)-Methyl 3-(N-methylmethylsulfonamido)-5-((1-
phenylbut-3-en-1-yl)carbamoyl)benzoate ((R)-10)
Compound (S)-7 (219 mg, 73%) was synthesized from (S)-6 in
the same manner as compound (R)-7 and was collected as a white
solid. 1H NMR (DMSO-d6, 300 MHz): d 2.40 (s, 3H), 2.67–2.84 (m,
3H), 3.86 (s, 3H), 5.21 (m, 1H), 7.21–7.26 (m, 1H), 7.30–7.35 (m,
2H), 7.42–7.45 (m, 2H), 7.91 (s, 1H), 7.95 (s, 1H), 8.27 (s, 1H),
9.09 (d, J = 8.4 Hz, 1H); 13C NMR (DMSO-d6, 75.5 MHz): d 20.7,
25.4, 52.2, 52.5, 72.5, 81.8, 125.3, 126.7, 128.2, 129.7, 132.1,
132.6, 134.8, 138.5, 142.0, 165.0, 165.9. MS Calcd (M+H)+: 322.1.
Found 322.3.
Compound (R)-10 (326 mg, 84%) was synthesized from (R)-8 in
the same manner as (R)-9 and was collected as a white solid. 1H
NMR (CDCl3, 300 MHz): d 2.63–2.72 (m, 2H), 2.87 (s, 3H), 3.32
(s, 3H), 3.89 (s, 3H), 5.08–5.29 (m, 3H), 5.70–5.83 (m, 1H), 6.87
(d, J = 8.0 Hz, 1H), 7.22–7.35 (m, 5H), 8.04 (m, 1H), 8.12 (m, 1H),
8.24 (m, 1H); 13C NMR (CDCl3, 75.5 MHz): d 35.8, 37.9, 40.4, 52.7,
53.5, 118.6, 126.0, 126.7, 127.6, 128.7, 129.1, 130.0, 131.6, 134.1,
136.1, 141.4, 142.4, 164.7, 165.6. MS Calcd (M+H)+: 417.1. Found
417.4.
4.7.11. (R)-Methyl 3-(N-methylmethylsulfonamido)-5-((1-
phenylbut-3-yn-1-yl)carbamoyl)benzoate ((R)-8)
4.7.16. (S)-Methyl 3-(N-methylmethylsulfonamido)-5-((1-
phenylbut-3-en-1-yl)carbamoyl)benzoate ((S)-10)
Compound (R)-8 (649 mg, 89%) was synthesized from (R)-6 in
the same manner as compound (R)-7, using compound 11 instead
of 3-methoxycarbonyl-5-methylbenzoic acid, and was collected as
a white powder. 1H NMR (CDCl3, 300 MHz): d 2.06–2.08 (m, 1H),
2.81–2.88 (m, 2H), 2.86 (s, overlap, 3H), 3.32 (s, 3H), 3.89 (s, 3H),
5.31–5.42 (m, 1H), 7.15–7.44 (m, 5H), 8.08 (s, 1H), 8.13 (s, 1H),
8.30 (s, 1H); 13C NMR (CDCl3, 75.5 MHz): d 25.6, 35.8, 37.9, 52.2,
52.7, 71.8, 80.0, 126.1, 126.7, 128.0, 128.8, 129.2, 130.0, 131.7,
135.9, 140.3, 142.4, 164.9, 165.6. MS Calcd (M+H)+: 415.1. Found
415.4.
Compound (S)-10 (1.5 g, 92%) was synthesized from (S)-8 in the
same manner as (R)-9 and was collected as a white solid. 1H NMR
(CDCl3, 300 MHz): d 2.67–2.73 (m, 2H), 2.87 (s, 3H), 3.33 (s, 3H),
3.90 (s, 3H), 5.10–5.22 (m, 2H), 5.23–5.30 (m, 1H), 5.70–5.84
(m, 1H), 6.87 (d, J = 7.8 Hz, 1H), 7.22–7.38 (m, 5H), 8.06–8.07 (m,
1H), 8.12 (bs, 1H), 8.26 (bs, 1H); 13C NMR (CDCl3, 75.5 MHz):
d 35.8, 38.0, 40.4, 52.8, 53.6, 118.6, 125.4, 126.0, 126.7, 127.7,
128.8, 129.2, 130.1, 131.6, 134.1, 136.1, 141.4, 142.4, 164.8,
165.7. MS Calcd (M+H)+: 417.1. Found 417.3.
4.7.12. (S)-Methyl 3-(N-methylmethylsulfonamido)-5-((1-
phenylbut-3-yn-1-yl)carbamoyl)benzoate ((S)-8)
4.7.17. Methyl 3-(but-3-en-1-ylcarbamoyl)-5-(N-methylmethyl-
sulfonamido)benzoate (12a)
Compound (S)-8 (381 mg, 89%) was synthesized from (S)-6 in
Compound 11 (414 mg, 1.44 mmol) was dissolved in DMF
(5 mL). 3-Butenylamine hydrochloride (186 mg, 1.73 mmol), TEA
the same manner as (R)-8 and was collected as a white solid. 1H
NMR (CDCl3, 300 MHz):
d
2.08 (t, J = 2.6 Hz, 1H), 2.79–2.96
(500 lL, 3.60 mmol) and HOBt (234 mg, 1.73 mmol) were added
(m, 2H), 2.87 (s, overlap, 3H), 3.34 (s, 3H), 3.91 (s, 3H), 5.35–5.42
and the solution was cooled to 0 °C. EDC (332 mg, 1.73 mmol)
(m, 1H), 7.01 (d, J = 8.1 Hz, 1H), 7.26–7.32 (m, 1H), 7.34–7.39
was added and the reaction mixture was allowed to attain room

4386
V. Sandgren et al. / Bioorg. Med. Chem. 20 (2012) 4377–4389
temperature and was stirred for 19 hours. Brine was added and the
solution was extracted with EtOAc. The combined organic extracts
were dried, filtered and concentrated. The residue was purified
using flash column chromatography (toluene/EtOAc 3:1) to give
12a (406 mg, 83%) as a white solid. 1H NMR (CDCl3, 300 MHz): d
2.40 (quartet, J = 6.9 Hz, 2H), 2.88 (s, 3H), 3.37 (s, 3H), 3.51–3.58
(m, 2H), 3.94 (s, 3H), 5.11–5.19 (m, 2H), 5.76–5.90 (m, 1H), 6.38
(bs, 1H), 8.03 (t, J = 1.8 Hz, 1H), 8.14–8.15 (m, 1H), 8.24–8.25 (m,
1H); 13C NMR (CDCl3, 75.5 MHz): d 33.8, 35.9, 38.0, 39.3, 52.8,
117.7, 126.1, 129.1, 129.6, 131.9, 135.1, 136.4, 142.4, 165.5,
165.7. MS Calcd (M+H)+: 341.1. Found 341.3.
(s, 2H), 8.03 (s, 1H); 13C NMR (CDCl3, 75.5 MHz): d 33.7, 35.7,
38.0, 39.5, 52.0, 63.2, 68.5, 71.2, 72.6, 117.6, 118.2, 124.1, 127.5,
127.9, 134.1, 135.1, 136.1, 142.3, 165.5, 166.7. MS Calcd (M+H)+:
470.2. Found 470.4.
4.7.21. N-[(E)-(S)-4-((S)-1,2-Dihydroxy-ethyl)-2,13-dioxo-6-oxa-
3,12-diaza-bicyclo[12.3.1]-octadeca-1(18),8,14,16-tetraen-16-
yl]-N-methyl-methanesulfonamide (15)
Compound 14 (119 mg, 0.25 mmol) was dissolved in dry DCM
(200 mL) and the solution was degassed with N2. Hoveyda-Grubbs
catalyst 2nd generation (37 mg, 0.06 mmol) was added and the
reaction mixture was refluxed overnight under N2 atmosphere.
The solution was subsequently concentrated and the crude mate-
rial was purified using flash column chromatography (MeOH/
EtOAc 1:12). Compound 15 (109 mg, 98%) was collected as a white
powder. 1H NMR (CDCl3/CD3OD, 300 MHz): d 2.27–2.37 (m, 2H),
2.86 (s, 3H), 3.24–3.26 (m, 1H), 3.28 (s, 3H), 3.51–3.55 (m, 1H),
3.58–3.64 (m, 3H), 3.77 (d, J = 4.8 Hz, 2H), 3.94–4.00 (m, 3H),
5.65 (dt, J = 6.9, 15.3 Hz, 1H), 5.87 (dt, J = 6.9, 15.3 Hz, 1H), 7.62
(bs, 1H), 7.80 (s, 2H), 7.93 (s, 1H); 13C NMR (CDCl3/CD3OD,
75.5 MHz): d 32.3, 35.1, 37.2, 37.4, 52.2, 63.3, 68.9, 71.3, 71.5,
126.3, 127.2, 127.3, 130.4, 131.7, 136.0, 136.4, 142.5, 167.4,
169.2. MS Calcd (M+H)+: 442.2. Found 442.4.
4.7.18. Methyl 3-(but-3-en-1-yl(methyl)carbamoyl)-5-(N-
methylmethyl-sulfonamido)benzoate (12b)
Compound 12a (429 mg, 1.3 mmol) was dissolved in DMF
(6 mL). NaH (60%) (66 mg, 1.7 mmol) and MeI (237 lL, 3.8 mmol)
were added and the mixture was stirred at room temperature for
23 h. The reaction was quenched with water and extracted with
EtOAc three times. The organic phases were dried, filtered and
evaporated. This gave compound 12b (448 mg, 100%) as a slightly
yellow powder. 1H NMR (CDCl3, 300 MHz): d 2.23–2.36 (m, 2H),
2.82 (s, 3H), 2.88 (s, 3H), 3.23–3.27 (m, 1H), 3.29 (s, 3H), 3.53–
3.54 (m, 1H), 3.87 (s, 3H), 4.96–5.12 (m, 2H), 5.51–5.79 (m, 1H),
7.58 (s, 1H), 7.91 (s, 1H), 7.96 (s, 1H); 13C NMR (CDCl3,
75.5 MHz): d 29.6, 35.7, 37.8, 46.9, 50.7, 52.5, 117.0, 117.9, 126.6,
129.5, 131.6, 135.0, 138.1, 141.9, 162.5, 165.5. MS Calcd (M+H)+:
355.1. Found 355.3.
4.7.22. Toluene-4-sulfonic acid (S)-2-hydroxy-2-[(E)-(S)-16-
(methanesulfonyl-methyl-amino)-2,13-dioxo-6-oxa-3,12-
diaza-bicyclo[12.3.1]octadeca-1(18),8,14,16-tetraen-4-yl]-ethyl
ester (16)
Compound 15 (145 mg, 0.33 mmol) was dissolved in dry DCM
(5 mL). p-TsCl (75 mg, 0.39 mmol), Bu2SnO (2 mg, 0.007 mmol)
4.7.19. N1-((S)-2-Allyloxy-1-((S)-2,2-dimethyl-1,3-dioxolan-4-
yl)-ethyl)-N3-but-3-enyl-5-(methanesulfonyl-methyl-amino)-
isophthalamide (13)
Compound 12a (429 mg, 1.26 mmol) was dissolved in H2O
(8 mL) and dioxane (16 mL). Aqueous LiOH (1.5 mL, 40 mg/ml,
60 mg, 2.5 mmol) was added and the reaction mixture was stirred
at room temperature for 1 h and 10 min. The solution was co-evap-
orated with toluene and the crude material was re-dissolved in
and TEA (55 lL, 0.39 mmol) were added and the mixture was stir-
red at room temperature for 23 hours. DCM was added and the
solution was washed with brine three times. The organic phase
was dried, filtered and concentrated. The crude product was puri-
fied using flash column chromatography (toluene/EtOAc 1:2 to 1:9)
to give compound 16 (125 mg, 64%) as a colorless oil. 1H NMR
(CDCl3, 300 MHz): d 2.28–2.37 (m, 2H), 2.41 (s, 3H), 2.82 (s, 3H),
3.16–3.23 (m, 1H), 3.27 (s, 3H), 3.55–3.66 (m, 2H), 3.75–3.80 (m,
1H), 3.91–3.95 (m, 3H), 4.00 (d, J = 6.6 Hz, 2H), 4.05–4.11 (m,
1H), 5.63–5.73 (m, 1H), 5.82–5.91 (m, 1H), 6.74 (bs, 1H), 7.15 (d,
J = 6.9 Hz, 1H), 7.31 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H),
7.79 (bs, 2H), 7.81 (bs, 1H); 13C NMR (CDCl3, 75.5 MHz): d 21.7,
32.6, 36.4, 37.5, 37.9, 53.3, 68.0, 69.4, 71.6, 71.9, 125.4, 127.0,
128.0, 130.1, 131.1, 132.2, 132.3, 136.4, 136.7, 142.7, 145.3,
166.1, 168.4. MS Calcd (M+H)+: 596.2. Found 596.5.
DMF (12 mL). Compound 3 (250 mg, 1.24 mmol), DIPEA (658 lL,
3.78 mmol) and HATU (719 mg, 1.89 mmol) were added and the
reaction mixture was stirred at room temperature for 16 h. H2O
was added and the solution was extracted with EtOAc. The com-
bined organic phases were washed with brine, dried, filtered and
concentrated. The residue was purified using flash column chroma-
tography (toluene/EtOAc 1:1). Compound 13 (571 mg, 90% over
two steps) was collected as an orange oil. 1H NMR (CD3OD,
300 MHz): d 1.32 (s, 3H), 1.41 (s, 3H), 2.33–2.40 (m, 2H), 2.95 (s,
3H), 3.34 (s, 3H), 3.44 (t, J = 6.9 Hz, 2H), 3.71 (d, J = 4.5 Hz, 2H),
3.90 (dd, J = 5.4, 8.7 Hz, 1H), 4.00–4.03 (m, 2H), 4.06 (dd, J = 6.0,
8.7 Hz, 1H), 4.24–4.37 (m, 2H), 5.02–5.30 (m, 4H), 5.78–5.97 (m,
2H), 7.99 (bs, 2H), 8.18 (m, 1H); 13C NMR (CD3OD, 75.5 MHz): d
25.6, 27.1, 34.7, 35.9, 38.3, 40.5, 53.9, 68.1, 70.1, 73.1, 76.2,
110.8, 117.1, 117.4, 125.8, 128.9, 129.0, 135.9, 136.6, 137.2,
137.3, 143.7, 168.2, 168.5. MS Calcd (M+H)+: 510.2. Found 510.5.
4.7.23. N-{(E)-(S)-4-[(R)-1-Hydroxy-2-(3-isopropyl-
benzylamino)-ethyl]-2,13-dioxo-6-oxa-3,12-diaza-
bicyclo[12.3.1]octadeca-1(18),8,14,16-tetraen-16-yl}-N-methyl-
methanesulfonamide (17)
Compound 16 (80 mg, 0.14 mmol) and 3-isopropylbenzylamine
(57 mg, 0.38 mmol) were dissolved in EtOH (99.5%, 5 mL) and the
reaction mixture was refluxed for 24 hours. The mixture was con-
centrated and the crude product was purified using flash column
chromatography (MeOH/EtOAc 1:9 + 1% TEA). Compound 17
4.7.20. N1-((2S,3S)-1-(Allyloxy)-3,4-dihydroxybutan-2-yl)-N3-
(but-3-en-1-yl)-5-(N-
methylmethylsulfonamido)isophthalamide (14)
(29 mg, 37%) was collected as a white powder. ½a D
ꢂ
+40.0 (c 0.1,
Compound 13 (42 mg, 0.08 mmol) was dissolved in AcOH
(1.3 mL) and H2O (0.5 mL) and heated to 60 °C. The reaction mix-
ture was stirred for 1 h and 45 min, co-evaporated with toluene
and concentrated. The residue was purified using flash column
chromatography (MeOH/EtOAc 1:12) to give 14 (39 mg, 100%) as
MeOH). 1H NMR (CDCl3, 300 MHz): d 1.24 (d, J = 6.60 Hz, 6H),
2.12–2.46 (m, 5H), 2.90 (s, 3H), 3.35 (s, 3H), 3.52–3.55 (m, 1H),
3.61–3.82 (m, 3H), 3.86 (s, 2H), 3.94–4.23 (m, 4H), 5.71 (m, 1H),
5.88–6.01 (m, 1H), 6.40 (bs, 1H), 7.00–7.07 (m, 1H), 7.11–7.14
(m, 2H), 7.17 (s, 1H), 7.24–7.26 (m, 1H), 7.84 (s, 1H), 7.90–7.96
(m, 2H); 13C NMR (CDCl3, 75.5 MHz): d 23.8, 23.9, 34.1, 36.3,
37.6, 38.0, 44.0, 45.9, 52.8, 52.9, 63.2, 69.3, 71.8, 125.6, 126.4,
127.4, 127.7, 128.4, 129.2, 129.8, 131.1, 132.5, 132.7, 136.2,
137.0, 137.1, 150.1, 162.1, 166.2. HRMS Calcd (M+H)+: 573.2741.
a
yellow oil. 1H NMR (CDCl3, 300 MHz):
d 2.36 (quartet,
J = 6.9 Hz, 2H), 2.89 (s, 3H), 3.33 (s, 3H), 3.46–3.52 (m, 2H), 3.66–
3.70 (m, 3H), 3.75–3.77 (m, 1H), 3.90 (dd, J = 3.9, 9.9 Hz, 1H),
4.04 (d, J = 5.7 Hz, 2H), 4.15–4.22 (m, 1H), 5.07–5.31 (m, 4H),
5.73–5.94 (m, 2H), 6.82 (bs, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.90

V. Sandgren et al. / Bioorg. Med. Chem. 20 (2012) 4377–4389
4387
Found 573.2728. LC–MS purity system A: tR = 4.58 min, 100%; sys-
tem B: tR = 1.64 min, 100%.
(M+H)+: 531.2272. Found 531.2293. LC–MS purity system A:
tR = 3.38 min, 96%; system B: tR = 0.72 min, 96%.
4.7.24. N-[(E)-(S)-4-((S)-1,2-Dihydroxy-ethyl)-2,14-dioxo-6-oxa-
3,13-diaza-bicyclo[13.3.1]nonadeca-1(19),8,15,17-tetraen-17-
yl]-N-methyl-methanesulfonamide (18)
4.7.28. N-{(E)-(S)-4-[(R)-2-(3-tert-Butyl-benzylamino)-1-
hydroxy-ethyl]-2,13-dioxo-6-oxa-3,12-diaza-
bicyclo[12.3.1]octadeca-1(17),8,14(18),15-tetraen-16-yl}-N-
methyl-methanesulfonamide (22)
Compound 22 was synthesized according to the synthetic route
for 17, using 3-tert-butylbenzylamine in the final step, and was col-
Compound 18 was synthesized according to the synthetic route
for 15 using 4-pentenylamine hydrochloride instead of 3-butenyl-
amine hydrochloride in the peptide coupling step and was collected
as a white powder. 1H NMR (CDCl3/CD3OD, 300 MHz): d 1.81–1.93
(m, 2H), 2.31–2.39 (m, 2H), 2.96 (s, 3H), 3.38 (s, 3H), 3.40–3.41 (m,
2H), 3.60 (dd, J = 4.2, 9.6 Hz, 1H), 3.66–3.73 (m, 2H), 3.77–3.83 (m,
1H), 3.88–3.94 (m, 1H), 4.03 (dd, J = 3.9, 9.6 Hz, 1H), 4.09–4.16 (m,
2H), 5.73–5.86 (m, 1H), 5.98 (dt, J = 6.9, 15.6 Hz, 1H), 7.89–7.90 (m,
1H), 7.95 (s, 1H), 8.01–8.02 (m, 1H); 13C NMR (CDCl3/CD3OD,
75.5 MHz): d 27.6, 31.8, 35.0, 37.1, 41.2, 51.4, 63.3, 68.2, 70.4,
71.7, 123.9, 126.5, 127.4, 127.5, 133.9, 135.4, 135.9, 142.5, 166.2,
167.9. MS Calcd (M+H)+: 456.4. Found 456.4.
lected as a white powder (34% for the final step). ½a D
ꢂ
+8.0 (c 0.1,
MeOH). 1H NMR (CDCl3, 300 MHz): d 1.30 (s, 9H), 2.28–2.49 (m,
2H), 2.72–2.87 (m, 2H), 3.23 (s, 2H), 3.30–3.38 (m, 4H), 3.42–
3.53 (m, 3H), 3.57–3.62 (m, 2H), 3.81–3.87 (m, 1H), 3.93–4.00
(m, 1H), 4.02–4.22 (m, 2H), 4.44 (bs, 1H), 5.60–5.74 (m, 1H),
5.85–5.95 (m, 1H), 6.45 (bs, 1H), 7.01 (bs, 1H), 7.10–7.17 (m, 1H),
7.23–7.32 (m, 3H), 7.50–7.53 (m, 1H), 7.81 (s, 1H), 7.91 (s, 1H);
13C NMR (CDCl3, 75.5 MHz): d 31.5, 34.8, 36.1, 37.8, 46.9, 53.7,
54.3, 57.5, 62.5, 69.3, 71.4, 73.0, 123.9, 124.2, 124.9, 125.4, 125.8,
128.3, 129.0, 131.9, 132.1, 133.2, 134.9, 137.4, 142.2, 151.5,
161.4, 165.2. HRMS Calcd (M+H)+: 587.2898. Found 587.2929.
LC–MS purity system A: tR = 3.89 min, 96%; system B: tR = 2.40 min,
95%.
4.7.25. N-[(S)-4-((S)-1,2-Dihydroxy-ethyl)-2,13-dioxo-6-oxa-
3,12-diaza-bicyclo[12.3.1]octadeca-1(18),14,16-trien-16-yl]-N-
methyl-methanesulfonamide (19)
Compound 15 (113 mg, 0.26 mmol) was dissolved in MeOH
(8 mL) and Pd-C 10% (40 mg) was added. The mixture was stirred
at room temperature under H2 (1 atm) for 3 h. The solution was
then filtered through Celite and concentrated. The crude material
was purified using flash column chromatography (EtOAc to
EtOAc/MeOH 12:1) to yield compound 19 (108 mg, 95%) as a color-
less oil. 1H NMR (CDCl3/CD3OD, 300 MHz): d 0.89–0.97 (m, 2H),
1.12–1.18 (m, 2H), 1.36–1.41 (m, 2H), 1.72 (bs, 2H), 2.96 (s, 3H),
3.34–3.36 (m, 2H), 3.38 (s, 3H), 3.39–3.41 (m, 2H), 3.45–3.56 (m,
2H), 3.67–3.70 (m, 3H), 3.81–3.97 (m, 1H), 7.85 (s, 1H), 7.92 (s,
1H), 8.00–8.02 (m, 1H), 8.08 (s, 1H) 8.21–8.22 (m, 1H); 13C NMR
(CDCl3/CD3OD, 75.5 MHz): d 21.4, 28.3, 29.1, 34.8, 37.0, 39.2,
51.9, 63.2, 68.8, 70.0, 70.9, 126.7, 127.6, 135.5, 137.2, 142.6,
168.1, 168.9. MS Calcd (M+H)+: 444.2. Found 444.4.
4.7.29. N-{(E)-(S)-4-[(R)-1-Hydroxy-2-(3-isopropyl-
benzylamino)-ethyl]-2,14-dioxo-6-oxa-3,13-diaza-
bicyclo[13.3.1]nonadeca-1(18),8,15(19),16-tetraen-17-yl}-N-
methyl-methanesulfonamide (23)
Compound 23 was synthesized according to the synthetic route
for 17, using 4-pentenylamine hydrochloride in the first peptide
coupling step, and was collected as a white powder (13% for the fi-
nal step). ½a D
ꢂ
ꢀ2.2 (c 0.1, MeOH). 1H NMR (CDCl3, 300 MHz): d 1.22
(s, 3H), 1.26 (s, 3H), 1.83 (bs, 2H), 2.33 (bs, 2H), 2.85–2.94 (m, 4H),
3.32–3.37 (m, 5H), 3.45–3.53 (m, 2H), 3.57–3.67 (m, 3H), 3.78 (s,
2H), 3.84–3.89 (m, 2H), 3.98–4.15 (m, 1H), 5.70–6.00 (m, 2H),
7.10–7.13 (m, 2H), 7.17 (s, 1H), 7.20–7.23 (m, 1H), 7.52 (s, 1H),
7.63 (s, 1H), 7.90 (s, 1H), 7.94 (s, 1H), 8.00 (s, 1H); 13C NMR (CDCl3,
75.5 MHz): d 23.9, 24.0, 28.0, 29.7, 32.3, 36.3, 37.9, 41.9, 51.7, 60.5,
63.3, 68.9, 71.6, 72.0, 122.3, 124.9, 125.4, 125.7, 126.6, 127.3,
128.3, 128.7, 128.9, 134.7, 135.8, 136.0, 143.3, 149.8, 165.0,
167.5. HRMS Calcd (M+H)+: 587.2898. Found 587.2910. LC–MS
purity system A: tR = 4.21 min, 98%; system B: tR = 2.76 min, 97%.
4.7.26. N-[(S)-4-((S)-1,2-Dihydroxy-ethyl)-2,14-dioxo-6-oxa-
3,13-diaza-bicyclo[13.3.1]nonadeca-1(19),15,17-trien-17-yl]-N-
methyl-methanesulfonamide (20)
Compound 20 (106 mg, 78%) was synthesized from 18 in the
same manner as 19 and was collected as white crystals. 1H NMR
(CD3OD, 300 MHz): d 1.55–1.65 (m, 4H), 1.66–1.76 (m, 4H), 2.95
(s, 3H), 3.35–3.38 (m, 2H), 3.36 (s, overlapped, 3H), 3.40–3.50 (m,
2H), 3.52–3.60 (m, 2H), 3.63 (d, J = 3.3 Hz, 1H), 3.65–3.71 (m,
1H), 3.75–3.81 (m, 1H), 3.90–3.94 (m, 1H), 7.90–7.93 (m, 2H),
7.95–7.96 (m, 1H); 13C NMR (CD3OD, 75.5 MHz): d 30.0, 30.2,
30.4, 32.1, 35.9, 38.3, 42.3, 53.1, 65.0, 70.3, 72.1, 73.0, 126.4,
128.6, 128.7, 137.2, 138.2, 144.5, 169.0, 169.2. MS Calcd (M+H)+:
458.2. Found 458.5.
4.7.30. N-[(S)-4-((R)-2-Benzylamino-1-hydroxy-ethyl)-2,13-
dioxo-6-oxa-3,12-diaza-bicyclo[12.3.1]octadeca-1(18),14,16-
trien-16-yl]-N-methyl-methanesulfonamide (24)
Compound 24 was synthesized from 19 according to the syn-
thetic route for 17, using benzylamine in the final step, and was
collected as a colorless oil (51% for the final step). ½a D ꢀ9.0 (c
ꢂ
0.1, MeOH). 1H NMR (CDCl3/CD3OD, 300 MHz): d 1.06–1.14 (m,
2H), 1.24–1.40 (m, 4H), 1.66 (bs, 2H), 2.93 (s, 3H), 3.15–3.22 (m,
1H), 3.29–3.34 (m, 5H), 3.38–3.47 (m, 2H), 3.51–3.62 (m, 5H),
3.74–3.82 (m, 2 H), 7.21–7.35 (m, 5H), 7.81–7.87 (m, 2H), 8.01 (s,
1H); 13C NMR (CDCl3/CD3OD, 75.5 MHz): d 22.8, 29.2, 35.1, 37.2,
39.3, 43.4, 51.4, 53.2, 67.6, 68.7, 70.1, 72.1, 126.8, 127.0, 127.3,
127.3, 127.6, 127.9, 128.0, 128.2, 128.2, 128.3, 135.8, 137.3,
142.6, 167.6, 168.9. HRMS Calcd (M+H)+: 533.2428. Found
533.2437. LC–MS purity system A: tR = 3.53 min, 96%; system B:
tR = 0.93 min, 97%.
4.7.27. N-[(E)-(S)-4-((R)-2-Benzylamino-1-hydroxy-ethyl)-2,13-
dioxo-6-oxa-3,12-diaza-bicyclo[12.3.1]octadeca-
1(17),8,14(18),15-tetraen-16-yl]-N-methyl-
methanesulfonamide (21)
Compound 21 was synthesized according to the synthetic route
for 17, using benzylamine in the final step, and was collected as a
colorless oil (42% for the final step). ½a D
ꢂ
+54.3 (c 0.1, MeOH). 1H
NMR (CDCl3, 300 MHz): d 2.22–2.52 (m, 2H), 2.73–2.78 (m, 1H),
2.87 (s, 3H), 3.26–3.31 (m, 2H), 3.33 (s, 3H), 3.45–3.52 (m, 1H),
3.60–3.74 (m, 3H), 3.84 (d, J = 8.7 Hz, 2H), 3.87–4.11 (m, 3H),
5.68–5.75 (m, 1H), 5.86–5.94 (m, 1H), 6.58 (bs, 1H), 7.04 (d,
J = 7.5 Hz, 1H), 7.19–7.33 (m, 5H), 7.84 (s, 1H), 7.89 (s, 2H); 13C
NMR (CDCl3, 75.5 MHz): d 32.7, 36.5, 37.5, 38.0, 51.0, 53.6, 54.0,
69.2, 71.9, 124.8, 127.1, 127.3, 127.4, 127.6, 128.5, 128.6, 128.7,
129.0, 131.4, 132.4, 136.9, 138.6, 143.2, 165.8, 168.0. HRMS Calcd
4.7.31. N-{(S)-4-[(R)-1-Hydroxy-2-(3-isopropyl-benzylamino)-
ethyl]-2,13-dioxo-6-oxa-3,12-diaza-bicyclo[12.3.1]octadeca-
1(18),14,16-trien-16-yl}-N-methyl-methanesulfonamide (25)
Compound 25 was synthesized from 19 according to the syn-
thetic route for 17 and was collected as a colorless oil (77% for
the final step). ½a D
ꢂ
+10.0 (c 0.1, MeOH). 1H NMR (CDCl3/CD3OD,
300 MHz): d 1.23–1.27 (m, 12H), 1.70 (bs, 2H), 2.76–2.80 (m,

4388
V. Sandgren et al. / Bioorg. Med. Chem. 20 (2012) 4377–4389
1H), 2.86–2.93 (m, 2H), 2.96 (s, 3H), 3.35–3.37 (m, 1H), 3.38 (s, 3H),
3.45–3.62 (m, 3H), 3.66–3.74 (m, 2H), 3.78–3.84 (m, 3H), 3.86–3.90
(m 1H), 7.14–7.20 (m, 2H), 7.23–7.29 (m, 2H), 7.85 (s, 1H), 7.92 (s,
1H), 8.07 (s, 1H); 13C NMR (CDCl3/CD3OD, 75.5 MHz): d 22.7, 23.2,
28.4, 29.1, 33.7, 35.0, 37.2, 39.2, 45.1, 51.5, 53.4, 68.7, 70.0, 71.8,
124.3, 124.8, 125.0, 125.2, 127.0, 128.0, 128.1, 135.8, 137.2,
138.4, 142.6, 148.9, 167.6, 168.9. HRMS Calcd (M+H)+: 575.2898.
Found 575.2899. LC–MS purity system A: tR = 4.54 min, 97%; sys-
tem B: tR = 2.07 min, 97%.
d 1.26 (d, J = 6.9 Hz, 6H), 1.95 (bs, 1H), 2.59–2.97 (m, 5H), 3.34 (s,
3H), 3.62–3.66 (m, 2H), 3.67–3.73 (m, 1H), 3.82–3.89 (m, 1H),
3.85 (s, overlapped, 3H), 3.90–3.96 (m, 1H), 4.00–4.08 (m, 1H),
4.11–4.14 (m, 1H), 4.19–4.25 (m, 1H), 5.14–5.20 (m, 1H), 5.73–
5.83 (m, 1H), 5.99–6.09 (m, 1H), 6.82 (d, J = 6.9 Hz, 1H), 7.06–7.33
(m, 10H), 7.88 (s, 1H), 7.95 (s, 1H), 8.01 (s, 1H); 13C NMR (CDCl3,
75.5 MHz): d 24.0, 34.1, 36.4, 37.9, 40.4, 46.4, 52.2, 52.7, 53.3,
68.3, 68.9, 71.9, 123.7, 124.6, 125.0, 125.3, 126.0, 126.1, 126.9,
127.4, 128.6, 128.7, 130.6, 132.0, 136.5, 141.4, 143.4, 149.3, 165.1,
167.2. HRMS Calcd (M+H)+: 649.3054. Found 649.47. LC–MS purity
system A: tR = 5.75 min, 97%; system B: tR = 4.13 min, 96%.
4.7.32. N-{(S)-4-[(R)-1-Hydroxy-2-(3-isopropyl-benzylamino)-
ethyl]-2,14-dioxo-6-oxa-3,13-diaza-bicyclo[13.3.1]nonadeca-
1(19),15,17-trien-17-yl}-N-methyl-methanesulfonamide (26)
Compound 26 was synthesized from 20 according to the syn-
thetic route for 17 and was collected as a colorless oil (17% for
4.7.36. (E)-(S)-4-((R)-2-Benzylamino-1-hydroxy-ethyl)-16-
methyl-6-oxa-3,12-diaza-bicyclo[12.3.1]octadeca-1(18),8,14,16-
tetraene-2,13-dione (30)
the final step).
½
a D
ꢂ
+31.1 (c 0.1, MeOH). 1H NMR (CD3OD,
Compound 30 was synthesized from 3-methoxycarbonyl-5-
methylbenzoic acid according to the synthetic route for 17, using
benzylamine in the final step, and was collected as a colorless oil
300 MHz): d 1.11–1.15 (m, 2H), 1.24 (d, J = 6.9 Hz, 6H), 1.52–1.76
(m, 8H), 2.77–2.90 (m, 2H), 2.91–2.97 (m, 1H), 2.96 (s, overlapped,
3H), 3.35–3.38 (m, 1H), 3.37 (s, overlapped, 3H), 3.44–3.55 (m, 4H),
3.63–3.71 (m, 2H), 3.86–3.92 (m, 2H), 3.94–4.06 (m, 1H), 7.16–7.19
(m, 2H), 7.24–7.31 (m, 2H), 7.90–7.92 (m, 2H), 7.94–7.96 (m, 1H);
13C NMR (CD3OD, 75.5 MHz): d 24.4, 30.1, 30.2, 30.3, 32.1, 35.3,
36.0, 38.3, 42.3, 52.6, 54.0, 54.6, 69.2, 70.2, 73.0, 126.4, 127.1,
127.5, 128.2, 128.6, 129.0, 129.8, 137.0, 138.3, 144.6, 150.7,
153.9, 169.0, 169.3. HRMS Calcd (M+H)+: 589.3054. Found
589.3075. LC–MS purity system A: tR = 5.07 min, 96%; system B:
tR = 2.86 min, 97%.
(48% for the final step). ½a D
ꢂ
+31.1 (c 0.1, MeOH). 1H NMR (CDCl3,
300 MHz): d 2.28–2.51 (m, overlapped, 3H), 2.44 (s, overlapped,
3H), 2.72 (dd, J = 3.6, 12.4 Hz, 1H), 2.89 (dd, J = 5.2, 12.4 Hz, 1H),
3.30–3.48 (m, 2H), 3.53–3.65 (m, 3H), 3.69–3.74 (m, 1H), 3.85 (d,
J = 11.8 Hz, 2H), 3.95–4.00 (m, 1H), 4.14–4.21 (m, 1H), 5.68–5.78
(m, 1H), 5.92–6.01 (m, 1H), 6.20 (bs, 1H), 6.75 (d, J = 7.1 Hz, 1H),
7.22–7.34 (m, 5H), 7.66 (s, 1H), 7.74 (s, 1H), 7.81 (s, 1H); 13C
NMR (CDCl3, 75.5 MHz): d 21.5, 32.7, 37.4, 50.9, 53.1, 54.1, 69.1,
69.4, 71.9, 122.6, 127.2, 127.3, 128.4, 128.6, 131.4, 131.7, 132.5,
135.6, 140.6, 166.8, 168.6. HRMS Calcd (M+H)+: 438.2393. Found
438.2404. LC–MS purity system A: tR = 3.67 min, 97%; system B:
tR = 0.85 min, 98%.
4.7.33. N-{(Z)-(S)-4-[(R)-1-Hydroxy-2-(3-isopropyl-
benzylamino)-ethyl]-12-methyl-2,13-dioxo-6-oxa-3,12-diaza-
bicyclo[12.3.1]octadeca-1(18),8,14,16-tetraen-16-yl}-N-methyl-
methanesulfonamide (27)
4.7.37. (E)-(S)-4-[(R)-1-Hydroxy-2-(3-isopropyl-benzylamino)-
ethyl]-16-methyl-6-oxa-3,12-diaza-bicyclo[12.3.1]octadeca-
1(18),8,14,16-tetraene-2,13-dione (31)
Compound 31 was synthesized from 3-methoxycarbonyl-5-
methylbenzoic acid according to the synthetic route for 17 and
Compound 27 was synthesized from 12b according to the syn-
thetic route for 17 and was collected as a colorless oil (5% for the
final step). 1H NMR (CDCl3, 500 MHz): d 1.25 (bs, 6H), 2.34 (bs,
1H), 2.52 (bs, 1H), 2.77–2.79 (m, 1H), 2.82–3.06 (m, 5H), 3.08 (bs,
3H), 3.33 (s, 3H), 3.65–3.79 (m, 5H), 3.82 (s, 2H), 3.88–4.13 (m,
3H), 5.81–5.84 (m, 2H), 6.64 (bs, 1H), 7.11–7.20 (m, 3H), 7.27 (bs,
1H), 7.54 (bs, 1H), 7.67 (s, 1H), 7.87 (bs, 1H). HRMS Calcd
(M+H)+: 587.2898. Found 587.2916.
was collected as a white solid (66% for the final step). ½a D
ꢂ
+82.0
(c 0.1, MeOH). 1H NMR (CDCl3, 300 MHz): d 1.23 (d, J = 7.1 Hz,
6H), 2.30–2.46 (m, 2H), 2.37 (s, 3H), 2.76 (dd, J = 3.8, 12.6 Hz,
1H), 2.84–2.95 (m, 2H), 3.16–3.76 (m, 7H), 3.79 (d, J = 13.2 Hz,
1H), 3.86 (d, J = 13.2 Hz, 1H), 3.90–4.05 (m, 2H), 4.06–4.18 (m,
1H), 5.70 (dt, J = 7.5, 14.9 Hz, 1H), 5.94 (dt, J = 7.5, 14.9 Hz, 1H),
6.46 (t, J = 4.9 Hz, 1H), 6.93 (d, J = 7.4 Hz, 1H), 7.09–7.30 (m, 4H),
7.61 (s, 1H), 7.64 (s, 1H), 7.72 (s, 1H); 13C NMR (CDCl3,
75.5 MHz): d 21.4, 24.1, 24.2, 32.7, 34.2, 37.5, 51.1, 53.3, 54.0,
69.1, 69.4, 71.9, 123.1, 125.5, 125.9, 126.7, 128.6, 131.2, 131.3,
131.5, 132.5, 135.4, 135.7, 139.0, 140.2, 149.3, 167.1, 169.0. HRMS
Calcd (M+H)+: 480.2857. Found 480.2872. LC–MS purity system A:
tR = 4.62 min, 96%; system B: tR = 3.97 min, 95%.
4.7.34. N-{(E)-(4S,11S)-4-[(R)-1-Hydroxy-2-(3-isopropyl-
benzylamino)-ethyl]-2,13-dioxo-11-phenyl-6-oxa-3,12-diaza-
bicyclo[12.3.1]octadeca-1(18),8,14,16-tetraen-16-yl}-N-methyl-
methanesulfonamide (28)
Compound 28 was synthesized from (S)-10 according to the
synthetic route for 17 and was collected as a white solid (34% for
the final step).
½
a D
+52.7 (c 0.1, MeOH). 1H NMR (CDCl3,
ꢂ
300 MHz): d 1.25 (d, J = 6.9 Hz, 6H), 2.34 (bs, 1H), 2.71 (s, 3H),
2.75–2.95 (m, 5H), 3.17 (s, 3H), 3.47–3.79 (m 3H), 3.84 (s, 2H),
3.92–4.13 (m, 3H), 4.99–5.06 (m, 1H), 5.79–5.89 (m, 1H), 6.05–
6.13 (m, 1H), 7.10–7.35 (m, 11H), 7.63 (s, 1H), 7.73 (s, 1H), 7.84
(s, 1H); 13C NMR (CDCl3, 75.5 MHz): d 24.1, 34.2, 36.3, 40.7, 46.6,
53.2, 53.5, 53.9, 63.5, 69.7, 71.8, 72.2, 124.7, 125.0, 125.4, 125.9,
126.0, 126.2, 126.6, 126.9, 127.6, 128.6, 128.8, 131.6, 136.9,
139.1, 142.0, 143.1, 149.3, 166.2, 170.0. HRMS Calcd (M+H)+:
649.3054. Found 649.3037. LC–MS purity system A: tR = 5.66 min,
96%; system B: tR = 3.92 min, 97%.
4.7.38. (E)-(4S,11S)-4-[(R)-1-Hydroxy-2-(3-isopropyl-
benzylamino)-ethyl]-16-methyl-11-phenyl-6-oxa-3,12-diaza-
bicyclo[12.3.1]octadeca-1(18),8,14,16-tetraene-2,13-dione (32)
Compound 32 was synthesized from (S)-9 according to the syn-
thetic route for 17 and was collected as a white solid (61% for the
final step). ½a D
ꢂ
+101.4 (c 0.1, MeOH). 1H NMR (CD3OD, 300 MHz): d
1.23 (d, J = 7.1 Hz, 3H), 1.25 (d, J = 6.9 Hz, 3H), 2.33 (s, 3H), 2.46–
2.72 (m, 2H), 2.75–2.98 (m, 3H), 3.58–3.90 (m, 4H), 3.92–4.09
(m, 3H), 4.13–4.24 (m, 1H), 4.97 (dd, J = 4.4, 11.4 Hz, 1H), 5.79–
5.94 (m, 1H), 6.00–6.15 (m, 1H), 7.05–7.50 (m, 11H), 7.91 (s, 1H);
13C NMR (CD3OD, 75.5 MHz): d 21.3, 24.5, 35.3, 41.9, 53.2, 54.4,
55.0, 55.8, 70.9, 71.8, 73.3, 126.5, 126.9, 127.2, 127.4, 127.8,
128.1, 129.5, 129.6, 130.9, 131.0, 132.2, 133.0, 137.6, 137.8,
139.6, 140.3, 144.4, 150.4, 170.9, 173.0. HRMS Calcd (M+H)+:
556.3170. Found 556.3190. LC–MS purity system A: tR = 5.85 min,
96%; system B: tR = 4.21 min, 95%.
4.7.35. N-{(E)-(4S,11R)-4-[(R)-1-Hydroxy-2-(3-isopropyl-
benzylamino)-ethyl]-2,13-dioxo-11-phenyl-6-oxa-3,12-diaza-
bicyclo[12.3.1]octadeca-1(18),8,14,16-tetraen-16-yl}-N-methyl-
methanesulfonamide (29)
Compound 29 was synthesized from (R)-10 according to the
synthetic route for 17 and was collected as a white solid (91% for
the final step). ½a D
ꢂ
+8.9 (c 0.1, MeOH). 1H NMR (CDCl3, 300 MHz):

V. Sandgren et al. / Bioorg. Med. Chem. 20 (2012) 4377–4389
4389
13. Huang, Y.; Strobel, E. D.; Ho, C. Y.; Reynolds, C. H.; Conway, K. A.; Piesvaux, J. A.;
Brenneman, D. E.; Yohrling, G. J.; Moore Arnold, H.; Rosenthal, D.; Alexander, R.
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Baxter, E. W. Bioorg. Med. Chem. Lett. 2010, 20, 3158.
14. Lindsley, S. R.; Moore, K. P.; Rajapakse, H. A.; Selnick, H. G.; Young, M. B.; Zhu,
H.; Munshi, S.; Kuo, L.; McGaughey, G. B.; Colussi, D.; Crouthamel, M. C.; Lai, M.
T.; Pietrak, B.; Price, E. A.; Sankaranarayanan, S.; Simon, A. J.; Seabrook, G. R.;
Hazuda, D. J.; Pudvah, N. T.; Hochman, J. H.; Graham, S. L.; Vacca, J. P.;
Nantermet, P. G. Bioorg. Med. Chem. Lett. 2007, 17, 4057.
15. Moore, K. P.; Zhu, H.; Rajapakse, H. A.; McGaughey, G. B.; Colussi, D.; Price, E.
A.; Sankaranarayanan, S.; Simon, A. J.; Pudvah, N. T.; Hochman, J. H.; Allison, T.;
Munshi, S. K.; Graham, S. L.; Vacca, J. P.; Nantermet, P. G. Bioorg. Med. Chem.
Lett. 2007, 17, 5831.
16. Maillard, M. C.; Hom, R. K.; Benson, T. E.; Moon, J. B.; Mamo, S.; Bienkowski, M.;
Tomasselli, A. G.; Woods, D. D.; Prince, D. B.; Paddock, D. J.; Emmons, T. L.;
Tucker, J. A.; Dappen, M. S.; Brogley, L.; Thorsett, E. D.; Jewett, N.; Sinha, S.;
John, V. J. Med. Chem. 2007, 50, 776.
4.7.39. (E)-(4S,11R)-4-[(R)-1-Hydroxy-2-(3-isopropyl-
benzylamino)-ethyl]-16-methyl-11-phenyl-6-oxa-3,12-diaza-
bicyclo[12.3.1]octadeca-1(18),8,14,16-tetraene-2,13-dione (33)
Compound 33 was synthesized from (R)-9 according to the syn-
thetic route for 17 and was collected as a white solid (30% for the
final step). ½a D
ꢂ
+28.9 (c 0.1, MeOH). 1H NMR (CDCl3, 300 MHz): d
1.24 (d, J = 7.1 Hz, 3H), 1.25 (d, J = 6.9 Hz, 3H), 2.42 (s, 3H), 2.54–
2.67 (m, 1H), 2.67–2.82 (m, 2H), 2.83–2.97 (m, 2H), 3.37 (s, 1H),
3.61 (dd, J = 4.1, 8.5 Hz, 1H), 3.64–3.72 (m, 1H), 3.82 (s, 2H), 3.85–
4.05 (m, 2H), 4.07–4.16 (m, 1H), 4.17–4.26 (m, 1H), 5.12–5.24 (m,
1H), 5.76 (dt, J = 7.3, 14.6 Hz, 1H), 6.03 (dt, J = 7.3, 14.6 Hz, 1H),
6.70 (d, J = 7.1 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 7.07–7.38 (m, 10H),
7.74 (s, 1H), 7.81 (s, 2H); 13C NMR (CDCl3, 75.5 MHz): d 21.5, 24.2,
24.3, 34.2, 40.7, 51.0, 52.0, 52.6, 54.2, 68.7, 69.3, 72.1, 122.2,
125.3, 125.8, 126.0, 126.5, 127.4, 128.6, 128.8, 130.8, 131.8, 132.0,
132.2, 135.4, 135.5, 139.9, 140.7, 141.7, 149.3, 166.4, 167.8. HRMS
Calcd (M+H)+: 556.3170. Found 556.3167. LC–MS purity system
A: tR = 5.96 min, 97%; system B: tR = 4.37 min, 96%.
17. He, L.; Byun, H.; Bittman, R. Tetrahedron Lett. 1998, 39, 2071.
18. Calderon, F.; Doyaguez, E. G.; Fernandez-Mayoralas, A. J. Org. Chem. 2006, 71,
6258.
19. Mori, K.; Kinsho, T. Liebigs Ann. Chem. 1991, 1991, 1309.
20. Stachel, S. J.; Coburn, C. A.; Steele, T. G.; Jones, K. G.; Loutzenhiser, E. F.; Gregro,
A. R.; Rajapakse, H. A.; Lai, M. T.; Crouthamel, M. C.; Xu, M.; Tugusheva, K.;
Lineberger, J. E.; Pietrak, B. L.; Espeseth, A. S.; Shi, X. P.; Chen-Dodson, E.;
Holloway, M. K.; Munshi, S.; Simon, A. J.; Kuo, L.; Vacca, J. P. J. Med. Chem. 2004,
47, 6447.
Acknowledgments
21. 4-Pentenylamine hydrochloride was synthesized according to a published
procedure: Bäck, M.; Johansson, P. O.; Wångsell, F.; Thorstensson, F.;
Kvarnström, I.; Ayesa, S.; Wahling, H.; Pelcman, M.; Jansson, K.; Lindström,
S.; Wallberg, H.; Classon, B.; Rydergård, C.; Vrang, L.; Hamelink, E.; Hallberg, A.;
Rosenquist, Å.; Samuelsson, B. Bioorg. Med. Chem. 2007, 15, 7184. 4-Penten-1-ol
(1.20 mL, 11.61 mmol), Et3N (4.9 mL, 34.83 mmol) and DMAP (0.142 g,
1.16 mmol) were dissolved in DCM (40 mL) and the solution was cooled to
0 °C. Methanesulfonyl chloride (1.35 mL, 17.42 mmol) was added and the
solution was stirred at 0 °C for two hours. The reaction mixture was washed
with water and the water phase was extracted with EtOAc. The organic phases
were pooled, dried and evaporated carefully (volatile product) which gave the
crude compound as an orange oil. The crude intermediate was dissolved in
MeOH (90 mL) and conc. NH3 (103 mL) was added. The solution was stirred at
room temperature for 48 h. After careful evaporation to eliminate the
ammonia, more water was added and the solution was washed with EtOAc
twice. The water phase was acidified with HCl (1 M) and the solution was
evaporated. This gave 4-pentenylamine hydrochloride (1.41 g, 100% over two
steps) as a white salt. 1H NMR (D2O, 300 MHz): d 1.51–1.59 (m, 2H), 1.87–1.96
(m, 2H), 2.78 (t, J = 7.28 Hz, 2H), 4.81–4.92 (m, 2H), 5.59–5.68 (m, 1H); 13C
NMR (D2O, 75.5 MHz): d 25.9, 29.9, 38.8, 115.9, 137.6.
We gratefully thank the following personnel at Medivir AB: Kurt
Benkestock for helping with the HRMS measurements, Elizabeth
Hamelink and Cathrine Åhgren for providing the inhibition data.
Finally, we would like to acknowledge Medivir AB for performing
the X-ray crystallography work and for financial support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.039.
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