Iminolactones as tools for inversion of the absolute configuration of α-amino acids and as inhibitors of cancer cell proliferation

Article abstract of DOI:10.1016/j.ejmech.2016.02.037

A library of iminolactones was prepared by esterification of several 2-hydroxyketones with a number of N-protected d- and l-α-amino acids. Some of the hydroxyketones were of terpenoid origin while others were obtained via synthesis. After N-deprotection of the intermediate esters, the free amines spontaneously underwent condensation with the ketone to form iminolactones. Esters of (1S,2S,5S)-2-hydroxypinan-3-one with both d- and l-α-amino acids were partially epimerized at the α-carbon atom to give a diastereomeric ester mixture. Only iminolactones of l-amino acids were formed after cyclization of (1S,2S,5S)-2-hydroxypinan-3-one, and correspondingly only d-amino acid iminolactones were formed after reaction with (1R,2R,5R)-2-hydroxypinan-3-one. The protocol thus enables inversion of the absolute configuration of amino acids. Some members of the prepared library of iminolactones displayed significant anti-proliferative effects toward three cancer cell lines (EL4, MCF7, PC3) with insignificant effect on non-malign cell lines (McCoy, MCF10A, NIH3T3). Thus, iminolactones appear to be potential lead structures for preparation of drugs selectively affecting proliferation of malign cell lines.

Full text of DOI:10.1016/j.ejmech.2016.02.037

European Journal of Medicinal Chemistry 114 (2016) 118e133
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Iminolactones as tools for inversion of the absolute configuration of
-amino acids and as inhibitors of cancer cell proliferation
a
Christina Mernøe Jensen a, Hsiao-Qing Chow a, Ming Chen b, Lin Zhai a,
,
*
Karla Frydenvang a, Huizhen Liu a, Henrik Franzyk a, Søren Brøgger Christensen a
a Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark
b Department of Clinical Microbiology 7602, Rigshospitalet, Tagensvej 20, DK-2200 Copenhagen N, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of iminolactones was prepared by esterification of several 2-hydroxyketones with a number of
N-protected - and -amino acids. Some of the hydroxyketones were of terpenoid origin while others
were obtained via synthesis. After N-deprotection of the intermediate esters, the free amines sponta-
Received 19 January 2016
Received in revised form
14 February 2016
Accepted 15 February 2016
Available online 23 February 2016
D
L-a
neously underwent condensation with the ketone to form iminolactones. Esters of (1S,2S,5S)-2-
hydroxypinan-3-one with both
to give a diastereomeric ester mixture. Only iminolactones of
of (1S,2S,5S)-2-hydroxypinan-3-one, and correspondingly only
D
- and
L
-
a
-amino acids were partially epimerized at the
-amino acids were formed after cyclization
-amino acid iminolactones were formed
a-carbon atom
L
D
Keywords:
after reaction with (1R,2R,5R)-2-hydroxypinan-3-one. The protocol thus enables inversion of the absolute
configuration of amino acids. Some members of the prepared library of iminolactones displayed sig-
nificant anti-proliferative effects toward three cancer cell lines (EL4, MCF7, PC3) with insignificant effect
on non-malign cell lines (McCoy, MCF10A, NIH3T3). Thus, iminolactones appear to be potential lead
structures for preparation of drugs selectively affecting proliferation of malign cell lines.
© 2016 Elsevier Masson SAS. All rights reserved.
Iminolactones
Inversion of amino acids
Anti-proliferative effects
Selective inhibition
InChIKey:
JNUXNNIJITXLIQ-IZHRTLMJSA-N
1. Introduction
Encouraged by these ideas we performed a screening of the fruiting
bodies of Danish basidomycetes for activity toward cancer cells, and
Natural products constitute an important source of chemo-
therapeutics [1e3], important examples being vincristine,
vinblastine, doxorubicin and paclitaxel, for which the actual drug is
the original natural product. For other therapeutics like podo-
phyllotoxin the registered drugs are derivatives of natural product
leads, e.g. etoposide and teniposide [1,4,5]. These natural products
are all isolated from higher plants or bacteria. In contrast no
registered chemotherapeutics have been isolated from basidomy-
cetes even though fungi have been sources for important drugs like
the penicillins, cephalosporins and griseofulvins [5e7]. Basidomy-
cetes have only to a very limited extend been used in traditional
Western medicine, whereas they have been used intensively in
Asian traditional medicine [8e10]. Although no drugs originating
from basidomycetes have been registered, several compounds with
significant biological effects are known, suggesting that basido-
mycetes have been disregarded as sources of new drugs [6,11e13].
by this an extract of Schizophyllum commune was found to reduce
proliferation of cancer cells far more efficiently than it inhibited
growth of non-malign cells [14]. A bioguided fractionation of this
fungal extract afforded three iminolactones schizine A and B (i.e. 1a
and 1b) and epischizine A (2a), of which the latter probably is an
artifact having the configuration at C-20 inverted during the isola-
tion procedure. The schizines (Scheme 1) constitute a group of
unprecedented natural products that appear to be formed by
esterification of an amino acid with 2-hydroxy-1-keto-mniopetal
followed by a condensation to give the iminolactone or vice versa
[14].
In cell growth assays schizine A and B (1a, b) displayed
enhanced growth inhibition of cancer cell lines in the low
range, whereas epischizine A (2a) did not show any inhibitory ef-
fects up to a concentration of 200 M [14]. In contrast to most other
mM
m
cytotoxic agents no significant activity was seen against non-
malignant human cell lines when using the SRB assay [15].
Iminolactones of terpenoids have been intensively investigated
as chiral auxiliaries in stereoselective synthesis of amino acids
[16e18]. Nevertheless biological testing of this type of compounds
* Corresponding author.
E-mail address: soren.christensen@sund.ku.dk (S.B. Christensen).
http://dx.doi.org/10.1016/j.ejmech.2016.02.037
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
119
Scheme 1. Hypothetic late stages in biosynthesis of schizines (1a,b) via two suggested intermediates I and II. Lg depicts a leaving group.
has not received much attention.
2. Results and discussion
2.1. Chemistry
after esterification of 3R with Z-L-Phe-OH (4La) is the enantiomer
of 5SLa, as evidenced by the triplet at 2.82 ppm originating from H-
1 in 5RDa, which will be at the same shift value as that of H-1 in
5Sla. A signal corresponding to only a trace amount of 6RLa was
present as a triplet at 2.78 ppm originating from its H-1. In the
spectrum of the product mixture from reaction of 3S with racemic
Z-Phe (i.e. 4a) a low-intensity triplet could be seen at 2.78 ppm in
addition to the major signal at 2.82 ppm, revealing only a minor
degree of isomerization. By analogy, even in the reaction mixture of
2.1.1. Iminolactones of
one
a-amino acids and 2-exo-hydroxypinan-3-
Initially a series of iminolactones was prepared by using Na-
benzyloxycarbonyl-protected amino acids and 2-exo-hydroxy-
pinanones as the starting materials (Scheme 2).
3S with Z-
D
-Phe-OH (4Da) the major product was 5SLa indicating
-form.
that a major part of the D-Phe moiety had isomerized to the
L
These results constitute an elaboration of a previous observation
[19]. Previously, only racemic amino acids were used as starting
materials for iminolactones of (1S,2S,5S)-2-hydroxypinan-3-one
(3S) or the enantiomer. Since the yields in previous work were
below 50% the possibility that an enantiospecific iminolactone
formation took place could not be excluded. We have observed that
As previously found, an optimal yield of Steglich esterification of
(1S,2S,5S)-2-hydroxypinan-3-one (3S) with Na-benzyloxycarbonyl-
protected -phenylalanine (4La) was obtained when dicyclohex-
L
ylcarbodiimide (DCC) was used as coupling reagent with 4-
dimethylaminopyridine (DMAP) as catalyst to give 5SLa [19].
Somewhat unexpectedly, we observed that esterification of 3S with
a true isomerization of the
a-atom of the amino acid takes place
Z-(
6SDa, while subsequent hydrogenation only afforded iminolactone
7SLa, inferring that an isomerization at C- in the amino acid
moiety of 6SDa had occurred. To ensure that only the SL-con-
figurated product was formed, irrespectively of whether - or
amino amino acid derivatives or the racemic mixture were used as
starting materials, an X-ray structural analysis of such a product
was performed. The crystalline nature of the Boc-protected Lys
derivative encouraged us to examine 7SLc. Unequivocal proof of the
identity of this iminolactone as 7SLc, independently of the absolute
configuration of the starting amino acid, was obtained by X-ray
D
-Phe)-OH (4Da) afforded a mixture of the epimers 5SLa and
when an Na-protected
D
-amino acid is reacted with a (1S,2S,5S)-2-
hydroxypinan-3-one (3S) to give the iminolactone of the
acid (7SL) and vice versa.
L-amino
a
The investigation was expanded to include Na-Z-protected
alanine (4b) and Na-Z-protected lysine (4c) and ornithine (4d) with
Nε-Boc-protected side chains. In all cases the reaction runs ana-
loguous to the reaction of 2-hydroxypinan-3-one with phenylala-
nine. A similar in-depth analysis of the removal of the protecting
group by hydrogenation and the concomitant spontaneous cycli-
zation to the iminolactone was performed (Fig. 3). Thus, after hy-
drogenation of 5SLa for 24 h, the product mixture only contained
trace amounts of 6SDa, while no signals from the Z protecting
group could be detected (purple graph in Fig. 3). In contrast, the
spectrum of the product mixtures obtained from 3S to 4Da showed
that significant amounts of 5SLa and 6SDa were present after hy-
drogenation for 24 h (Fig. 3 red graph), and thus hydrogenation was
extended to 48 h to reach complete conversion (green graph in
Fig. 3), after which almost pure iminolactone 7SLa was seen.
D
L-
analysis of the products obtained from the conversions involving
and -forms of Z-Lys(Boc)-OH (i.e. 4Dc or 4Lc). In both cases the
product was 7S c (Fig. 1.). Since iminolactones can be hydrolyzed in
hydrochloric acid to yield the hydroxyketone and the amino acid
D-
L
L
[16,20], this procedure presents a formal protocol for inversion of
amino acids.
a-
To understand the reaction path in details NMR spectra of the
reaction mixtures were recorded (Fig. 2). The prevalent product

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C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
Scheme 2. Conditions: (a) DCC, DMAP, (b) H2, Pd/C. In the scheme only one of the enantiomers is depicted, and consequently 7RDa is the enantiomer of 7SLa.
Hence, irrespective of the absolute configuration of the
a
-amino
method for converting D-amino acids into L-amino acids and vice
acid, esterification of (1S,2S,5S)-2-hydroxypinan-3-one followed by
hydrogenation and cyclization affords only iminolactones 7SL. By
using this procedure the four possible iminolactones from 3S and
the four amino acids 4a-d as well as the four possible isomers
employing 3R and the same four amino acids were prepared to give
7SLa-d and 7RDa-d, respectively. In addition, iminolactones 7SLe-i
were prepared. Since hydrolysis of the iminolactones with hydro-
chloric acid to afford the amino acids is described this is a formal
versa [16].
The protecting group from 7SLe was removed by using tri-
fluoroacetic acid in dichloromethane to give 7SLf. However,
removal of the protecting group from 7SLc and 7SLd lead to the free
amine which turned out to be unstable.
2.1.2. Iminolactones of
hydroxycamphan-3-one
2-exo-Hydroxycamphan-3-one (8) was esterified with glycine
and -phenylalanine to give 11a and 11b by using a procedure very
a-L-amino acids and (1R,2S,4S)-exo-
L
similar to that employed for preparation of the iminolactones of 2-
exo-hydroxypinan-3-one (Scheme 3). No isomerization was
observed when (1R,2S,4S)-2-exo-hydroxycamphan-3-one was
reacted with
L
-phenylalanine. Compound 11b has previously been
prepared by benzylation of 11a [16].
2.1.3. Iminolactones of a-amino acids and (2S,4aS,8aR)-2-hydroxy-
8a-methyltransdecalin-1-one
(4aS,8aR)-8a-Methyltransdecalin-1-one (12) was prepared as
previously described [22].
a
-Bromination of ketone 12 with pyridinium bromide per-
bromide (PBB) afforded selectively bromide 13 in almost quanti-
tative yield (Scheme 4). The equatorial position of the bromine
substituent was established by the 3J coupling constants between
H-2 and Hax-3/Heq-3 of 13 Hz and 6 Hz, respectively. Treatment of
13 with a diluted solution of sodium hydroxide afforded 14. A likely
explanation for retention of the stereochemistry at C-2 is that an
SN2 reaction is followed by base-catalyzed epimerization [23,24].
Again the stereochemistry of the 2-hydroxy substituent was veri-
fied by the characteristic values of the two 3J coupling constants of
12.0 Hz and 7.6 Hz. Esterification and subsequent hydrogenation to
give iminolactones 16a-d were performed by using the procedures
described above for the series based on 2-exo-hydroxy-pinanones.
While the pinane-derived iminolactones 7Sa-d only could be
Fig. 1. Perspective drawing (ORTEP-3) [21] of compound 7SLc. Displacement ellipsoids
of the non-hydrogen atoms are shown at the 50% probability level. Hydrogen atoms
have been shown as spheres of arbitrary size. Nitrogen atoms are shown in blue, while
oxygen atoms are colored red. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
formed as R,D or S,L isomers both epimers of the decalin-based

C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
121
Fig. 2. 1H NMR spectra illustrating the reaction sequence starting with 3S and Z-Phe-OH (4a); blue entry: 3S, red entry: product from acylation of 3S with 4Da, green entry: product
mixture from acylation of 3S with racemic 4a, and purple entry: product mixture from acylation of 3S with 4La. Blue entry is numbered according to that of 3S. The red arrow
indicates the signal from H-1 in the terpenoid moiety in 5SLa while the blue arrow indicates the signal from H-1 in 6Sa. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
iminolactones, e.g. 1c and 16d, were obtained. However, the decalin
iminolactones generally appeared to be less stable than the pinane-
based iminolactones. This is illustrated by the isolation and iden-
tification of the two byproducts 17 and 18 (Fig. 4) from the reaction
mixture obtained in the preparation of 16a.
active compounds were only found among derivatives of
(1R,2R,5R)- and (1S,2S,5S)-2-hydroxypinan-3-one (3R and 3S,
respectively) and (4aS,8aR)-8a-methyl-trans-decalin-1-one (12).
2.3. Conclusion
2.1.4. Iminolactones of 2-hydroxytetral-1-ones
In conclusion preparation of iminolactones of (1S,2S,5S)-2-
The bromine in 2-bromotetralin-1-one (19) was substituted
hydroxypinan-3-one with -amino acids or (1R,2R,5R)-2-
hydroxypinan-3-one with L-a-amino acids offers a possibility for
D
-
a
with the carboxylate of Na-Boc-protected
L
-phenylalanine or
L-
tryptophan (Scheme 5). After removal of the Boc group the free
amine spontaneously formed the iminolactone. The two pairs of
epimeric iminolactones (21a and 22a, 21b and 22b) were separated
by HPLC. The relative configuration of 21a was established by the
presence of a NOE correlation between H-2 and H-4a, proving a cis-
disposition of these protons. No correlation between H-2 and H-4a
was found in 22a.
inversion of the absolute configuration of the amino acids. In
addition iminolactones appear to be promising starting points for
development of agents exhibiting selective anti-proliferative effect
against cancer cell lines leaving non-malignant cell lines unaf-
fected. Significant differences in the activities of stereoisomers
indicate a selective interaction with an at present unknown target
characteristic for cancerous cell lines. Future investigations may
reveal the target and thus allow for optimization of iminolactones
to give drug leads.
2.2. Biological testing
In order to verify the hypothesis that the cell growth-inhibiting
activity of the natural schizines could be attributed to the presence
of an iminolactone functionality the prepared iminolactones were
tested in the SRB assay [15]. The results are presented in Tables 1e4.
Inspection of the data in Tables 1e4 reveals that six of the 27
iminolactones (i.e. 7RDa, 7RDc, 7SLc, 7SLd, 16a, and 16d) exhibit
IC50 values in the low micromolar range. Remarkable selectivity
toward cancer cell lines is observed for these compounds, since
they did not show significant anti-proliferative effects on non-
3. Experimental
3.1. Chemistry
All reactions requiring anhydrous conditions were carried out
under an atmosphere of argon or nitrogen in dry solvents according
to standard procedures. Reagents were purchased at the highest
commercial quality and solvents were HPLC grade. Reagents and
solvents were used without further purification unless otherwise
stated. Reactions were followed by TLC using silica-coated
aluminum plates (Merck, 60 F254) and visualized with UV light
and/or spraying with a ceric ammonium molybdate solution fol-
lowed by heating. Flash column chromatography was performed
malignant cell lines up to 900 mM or even higher concentrations.
Comparison of the activities for enantiomeric pairs show that for
most analogues significant differences in potency are found for
enantiomers except for 7RDc and 7SLc, which are equipotent.
Among the tested amino acids only Gly, Phe, Lys and Orn gave rise
to potent iminolactones. Among the selected skeletons highly
with silica gel (35e75
mm, Fisher Scientific). All new compounds
were characterized by 1H NMR, 13C NMR, COSY, HSQC, and HMBC in

122
C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
Scheme 4. Conditions: (a) pyridinium hydrobromide perbromide (PBB); (b) 6% NaOH;
(c) Z- -L-Amino acid, DCC, and DMAP; (d) H2 and Pd/C.
a
CD3CN (1.94 ppm and 118.26 ppm) or CD3OD (3.31 ppm and
49.00 ppm). Coupling constants (J) are given in Hertz (Hz). HRMS
were recorded on a Bruker micrOTOF-Q instrument using electro-
spray ionization (ESI). Low-resolution mass spectra were recorded
on a Bruker microflex MALDI-TOF mass spectrometer. Optical ro-
tations were measured on a Bellingham þ Stanley ADP410 polar-
imeter. Melting points were measured on a Mettler Toledo MP70
Melting Point System. Analytical HPLC was performed on a Shi-
madzu HPLC system consisting of an SCL-10A VP controller, an SIL-
10AD VP auto injector, an LC-10AT VP Pump, a diode array detector
SPDM10A VP DAD, and a CTO-10AC VP column oven, using a Phe-
Fig. 3. 1H NMR spectra of the product mixture obtained when a 3S is reacted with 4La
to give a mixture of 90% of 5SLa and 10% of 6SDa (blue entry) b: crude product after
hydrogenation for 24 h of a 60:40 mixture of 5SLa and 6SDa (red entry); c: crude
product after continued hydrogenation for 48 h of the 60:40 mixture of 5SLa and 6SDa
(green entry); and d: crude product after hydrogenation for 24 h of a 90:10 mixture of
5SLa and 6SDa (purple entry). Numbering is according to 7. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
this article.)
nomenex Luna C18(2) column (150 ꢀ 4.6 mm; 3
m
m) eluted at a rate
addition to HRMS. NMR spectra were recorded on Bruker Avance
400 MHz and 600 MHz, or Varian Mercury 300 MHz spectrometers.
The chemical shifts (d) are given in parts per million (ppm) relative
of 0.8 mL/min. Injection volumes were 5e10
m
L of a 1 mg/mL so-
lution and separations were performed at 40 ꢁC. The system was
controlled by Class VP 6 software. Eluents A (H2O/MeCN/TFA)
95:5:0.1) and B (MeCN/H2O/TFA 95:5:0.1) were employed for linear
gradient elution. Preparative HPLC was performed on an Agilent
1100 Series system with a multiple-wavelength UV detector using a
to residual signals of the solvent: CDCl3 (7.26 ppm and 77.16 ppm),
Phenomex Luna C18(2) column (250 ꢀ 21.2 mm; 5
mm) with gra-
dients of eluent A and B as above and a flow rate of 20 mL/min.
3.1.1. (5R,8S,8aR)-8,9,9-trimethyl-3,5,6,7,8,8a-hexahydro-2H-5,8-
methanobenzo[b][1,4]oxazin-2-one (11a) was prepared according
to Xu et al. [16].
3.1.1.1. (1S,2R,4R)-1,7,7-Trimethyl-3-oxobicyclo[2.2.1]heptan-2-yl
(S)-2-((benzyloxycarbonyl)amino)-3-oxo-3-phenylpropanoate (11b).
Z-Phenylalanine (213.5 mg, 1.2 equiv.) was stirred with DMAP
(72.6 mg, 1 equiv.) and DCC (159.4 mg, 1.3 equiv.) in dry THF (2 mL)
at 0 ꢁC for 15 min. A solution of 9 (100 mg, 594
mmol) in THF
(0.5 mL) was added dropwise to the reaction mixture and the
mixture was stirred at 0 ꢁC for 2 h and then at room temperature for
20 h. After filtration though a celite filter the filtrate was concen-
trated in vacuo to give 11b as a crude product (190.6 mg,
Scheme 3. Conditions: (a) DCC, DMAP; (b) H2, Pd/C.
339.2 mmol, 77%), which was used without further purification.

C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
123
Schlenk tube was charged with Pd/C (5e10 mg, 10%), and the tube
was purged with argon and then hydrogen. A solution of crude 10b
(150 mg, 267 mmol) in EtOH (99.9%, 2 mL) was added to the Schlenk
tube, and the flask was purged with hydrogen. A hydrogen-filled
balloon was attached and the mixture was stirred overnight. The
reaction mixture was filtered through a pad of Celite, and the
filtrate was concentrated and purified by flash chromatography
using heptane-EtOAc (4:1) to give 11b (38.1 mg, 128
Spectral data were in agreement with those published [16].
1H NMR (300 MHz, CDCl3)
ppm 7.11e7.40 (m, 5H, Ph), 4.27 (d,
mmol, 48%).
d
Fig. 4. Structures of the side products isolated after hydrogenation of 16a.
Scheme 5. Conditions: (a) Boc-Amino acid and DIPEA, (b) TFA, and then Na2CO3. The reaction sequence is shown for the L-form of the amino acids.
Spectral data are in agreement with those published [16].
J ¼ 1.2 Hz, 1H, H-8a), 3.96 (ddd, J ¼ 8.6, 4.6, 1.5 Hz, 1H, H-3endo), 3.56
(dd,J¼ 14.7, 4.4Hz,1H,CH2-Ph), 3.23(dd, J¼14.4, 8.5Hz,1H,CH2-Ph),
2.44(d, J ¼ 4.4Hz,1H,H-5),1.77e2.04(m, 2H,H-6, H-7),1.29e1.58(m,
2H, H-6, H-7), 1.08 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.80 (s, 3H, CH3).
3.1.1.2. (3S,5R,8S,8aR)-3-Benzyl-8,9,9-trimethyl-3,5,6,7,8,8a-hexahy-
dro-2H-5,8-methanobenzo[b][1,4]oxazin-2-one (11b). A flame-dried
Table 1
In vitro anti-proliferative effect (as IC50 values in
lines: McCoy, MCF10A, and NIH 3T3.
m
M) of iminolactones of 3R with
EL4
D
-amino acids on three cancer cell lines: EL4, MCF7, and PC3 and on three non-malignant cell
MCF7
PC3
McCoy
MCF10A
NIH 3T3
R1
R2
7Rg
H
H
H
H
H
>300
>300
>300
>2000
>1500
>1800
>1300
>2000
>1500
>1800
>1300
>2000
>1500
>1800
>1300
7RDa
7RDb
7RDc
CH2-Ph
CH3
7.7 1.7
27 11
14.5 9.3
8.4 4.4
38 16
15.3 9.3
9.4 4.7
42 17
15.9 8.7
7RDd
7RDi
H
H
48 10
56 10
60 12
>1500
>1300
>1500
>1300
>1500
>1300
139 13
142 17
147 17
7RDe
7RDf
7RDh
H
51 12
30 9.8
149 19
54 10
34 10
162 20
55 14
38 13
166 19
>1200
>1200
>2000
>1200
>1200
>2000
>1200
>1200
>2000
H
CH3
CH3
Data are given as mean SD from three experiments. EL4: Leukemic cell line, MCF7: Human breast cancer cell line, and PC3: Human prostate cancer cell line, McCoy: synovival
cell line (murine), MCF10A: Epithelial cells from mammary gland (human), NIH 3T3: Embryonic fibroblasts (murine).

124
C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
Table 2
In vitro anti-proliferative effect (as IC50 values in
lines: McCoy, MCF10A, and NIH 3T3.
mM) of iminolactones of 3S with
L-amino acids on three cancer cell lines: EL4, MCF7, and PC3 and on three non-malignant cell
EL4
MCF7
PC3
McCoy
MCF10A
NIH 3T3
R1
R2
7Sg
H
H
H
H
H
19 0.8
>300
145 25
22 0.2
>300
154 31
43 0.01
>300
149 24
>900
>1000
>2000
>800
>900
>1000
>2000
>800
>900
>1000
>2000
>800
7SLa
7SLb
7SLc
CH2-Ph
CH3
15
4
17
7
18
8
7SLd
7SLe
7SLg
H
H
H
4.1 6.6
36 8.4
36 9.3
6.3 6.6
40 10
6.6 4.9
42 7.5
42 12
>1200
>1000
>1200
>1200
>1000
>1200
>1200
>1000
>1200
38
9
Data are given as mean SD from three experiments. EL4: Leukemic cell line, MCF7: Human breast cancer cell line, and PC3: Human prostate cancer cell line, McCoy: synovival
cell line (murine), MCF10A: Epithelial cells from mammary gland (human), NIH 3T3: Embryonic fibroblasts (murine).
3.1.2. General procedure for esterification of 2-exo-
hydroxypinanone (3) with Z-protected amino acids (4)
Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl (benzyloxycarbonyl)-
nylalaninate (6RLa). The mixture of 5RDa and 6RLa was prepared
from 2-exo-hydroxy-(R)-pinanone (3R) and -phenylalanine us-
L-phe-
A
solution of Na-benzyloxycarbonyl-protected amino acid
D-a
(1.5 mmol), 2-exo-hydroxy-(R)-pinanone (3R) (200 mg, 1.19 mmol),
and DMAP (0.2 mmol) in dry THF (5 mL) was stirred at 0 ꢁC for
15 min. After addition of a solution of DCC (1.5 equiv) in dry THF
(1 mL), the mixture was stirred at 0 ꢁC for 2 h, and then at room
temperature for 17 h. The reaction mixture was concentrated. The
residue was suspended in cold EtOAc (5 ꢁC) and 1,3-
dicyclohexylurea (DCU) was removed by filtration through a pad
of celite. The filtrate (about 15 mL) was washed with saturated
aqueous NaHCO3 (2 ꢀ 5 mL), H2O (2 ꢀ 5 mL), and brine (5 mL),
dried over MgSO4, filtered and concentrated in vacuo. The crude
product was purified by flash chromatography over silica gel
ing the general procedure for acylation. The product was purified
by using heptane-EtOAc with increasing amounts of EtOAc in the
eluent yielding a colorless oil (76.0%). Compounds 5RDa and 6RLa
were formed in a 90:10 ratio. Compound 5RDa: 1H NMR (400 MHz,
CDCl3) d ppm 7.12e7.39 (m, 10H), 5.02e5.17 (m, 2H), 4.50e4.60 (m,
1H), 3.12 (dd, J ¼ 14, 5.8 Hz, 1H), 3.02 (dd, J ¼ 14, 5.8 Hz, 1H), 2.90 (t,
J ¼ 6.1 Hz, 1H), 2.60e2.78 (m, 2H), 2.39 (d, J ¼ 2.9 Hz,1H), 2.06e2.15
(m, 1H), 1.57 (s, 3H), 1.51 (d, J ¼ 11.0 Hz, 1H), 1.35 (s, 3H), 0.86 (s, 3H).
The ratios between the formed diastereomers are based on the
relative integrals of the respective H-1 signals. The general NMR
assignment of the acylation products is shown for 6RDb.
15e40
mm).
3.1.2.2. (1R,2R,5R)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl
3.1.2.1. (1R,2R,5R)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl
(benzyloxycarbonyl)-
Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl
L
-alaninate (6RLb) and (1R,2R,5R)-2,6,6-
(benzyloxycarbonyl)-D-
(benzyloxycarbonyl)-
D
-phenylalaninate (5RDa) and (1R,2R,5R)-2,6,6-
Table 3
In vitro anti-proliferative effect (as IC50 values in mM) of iminolactones of (4aS,8aR)-8a-methyltransdecalin-1-one (13) with amino acids on three cancer cell lines: EL4, MCF7,
and PC3 and on three non-malignant cell lines: McCoy, MCF10A, and NIH 3T3.
EL4
MCF7
PC3
McCoy
MCF10A
NIH 3T3
R1
R2
16a
16b
16c
16d
16e
18
H
H
H
CH3
H
CH2-Ph
H
7.7 15
53 24
50 21
12 3.5
71 10
114 37
11 10
57 23
53 14
12 4.2
77 10
119 25
13 11
>2000
>2000
>1700
>1200
>1500
>2000
>2000
>2000
>1700
>1200
>1500
>2000
>2000
>2000
>1700
>1200
>1500
>2000
57 15
51 11
12 3.9
83 10
119 21
CH2-Ph
H
CH2eIn
Data are given as mean SD IC50
(mM) from three experiments. EL4: Leukemic cell line, MCF7: Human breast cancer cell line, and PC3: Human prostate cancer cell line, McCoy:
synovival cell line (murine), MCF10A: Epithelial cells from mammary gland (human), NIH 3T3: Embryonic fibroblast (murine).

C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
125
Table 4
In vitro anti-proliferative effect (as IC50 values in mM) of iminolactones of (1R,2S,4S)-exo-hydroxycamphan-3-one with a-L-amino acids and 2-hydroxytetralin-3-ones with a-L-
amino acids on three cancer cell lines: EL4, MCF7, and PC3 and on three non-malignant cell lines: McCoy, MCF10A, and NIH 3T3.
Compound
Mean IC50
EL4
(mM)
MCF7
PC3
McCoy
MCF10A
NIH 3T3
11a
11b
R ¼ H
82.0 17
ND
135 17
ND
145 21
160
˃900
ND
˃900
ND
˃900
ND
R ¼ CH2Ph
21a
21b
R ¼ CH2Ph
R ¼ CH2In
39 17
36 11
41 14
39 13
45 14
39 12
˃1000
˃1000
˃1000
˃1000
˃1000
˃1000
22a
22b
R ¼ CH2Ph
R ¼ CH2In
24 15
27 11
25 12
30 12
26 11
˃1000
˃1000
˃1000
˃1000
˃1000
˃1000
22
8
Data are given as mean SD from three experiments. EL4: Leukemic cell line, MCF7: Human breast cancer cell line, and PC3: Human prostate cancer cell line, McCoy: synovival
cell line (murine), MCF10A: Epithelial cells from mammary gland (human), NIH 3T3: Embryonic fibroblasts (murine).
alaninate (5RDb). The mixture of 6RLb and 5RDb was prepared
according to the general procedure for acylation using 2-exo-hy-
When the reaction was run using 2-exo-hydroxy-(R)-pinanone
and Na-Z-Nε-Boc-
-lysine as starting materials the yield was 66%
D
droxy-(R)-pinanone (3R) and Z-
L
-alanine as starting materials. The
and the ratio between 6RLc and 5RDc was 3:97.
product was purified by using toluene-DCM-EtOAc (13:1:1) as the
eluent to yield 6RLb and 5RDb (66:34) as an oil (43.0%). Compound
3.1.2.4. (1R,2R,5R)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl
6RLb: 1H NMR (300 MHz, CDCl3)
d ppm 7.30e7.40 (m, 5H), 5.18 (d,
Na-(benzyloxycarbonyl)-Nd-(tert-butoxycarbonyl)-
L
-ornithinate
(6RLd) and (1R,2R,5R)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-
yl-Na-(benzyloxycarbonyl)-Nd-(tert-butoxycarbonyl)-
-ornithinate
(5RDd). The mixture of 6RLd and 5RDd was prepared from 2-exo-
hydroxy-(R)-pinanone (3R) and Na-Z-Nd-Boc-
-ornithine using the
J ¼ 8.5 Hz, 1H), 5.06e5.13 (m, 2H), 4.21e4.37 (m, 1H), 2.90 (t,
J ¼ 6.2 Hz, 1H), 2.65e2.74 (m, 2H), 2.37e2.50 (m, 1H), 2.08e2.18 (m,
1H), 1.46e1.68 (m, 1H), 1.60 (s, 3H), 1.34e1.42 (s, 3H), 1.36 (s, 3H),
D
0.87 (s, 3H). Compound 5RDb: 1H NMR (400 MHz, CDCl3)
d
ppm
7.27e7.38 (m, 5H, Z-Ph), 5.24 (d, J ¼ 6.5 Hz, 1H, NH), 5.09 (dd,
), 2.95 (t,
L
general procedure for acylation. The product was purified by using
heptane-EtOAc with increasing amounts of EtOAc as the eluents to
give a colorless oil (35.0%) consisting of 6RLd and 5RDd in a 57:43
J ¼ 17.3, 12.0 Hz, 2H, Z-CH2), 4.19e4.33 (m, 1H, H-
a
J ¼ 6.0 Hz, 1H, Heq-1), 2.59e2.80 (m, 2H, H-4), 2.37e2.49 (m, 1H, H-
7ax), 2.07e2.17 (m, 1H, Heq-5), 1.52e1.61 (m, 1H, Heq-7), 1.59 (s, 3H,
H-10), 1.32e1.39 (m, 3H, Ala-CH3), 1.36 (m, 3H, H-9), 0.86 (s, 3H, H-
ratio. Compound 6RLd: 1H NMR (600 MHz, CDCl3)
d ppm 7.30e7.38
(m, 5H), 5.74 (br. s, 1H), 5.06e5.26 (m, 3H), 4.51e4.61 (br.s, 1H),
4.22e4.34 (m,1H), 3.96e4.03 (m,1H), 3.50e3.56 (m,1H), 3.09e3.13
(m, 2H), 2.85 (t, J ¼ 6.1 Hz, 1H), 2.71e2.78 (m, 1H), 2.61e2.69 (m,
1H), 2.39e2.47 (m, 1H), 2.09e2.16 (m, 1H), 1.71e1.99 (m, 1H),
1.55e1.61 (m, 4H),1.40e1.46 (m, 9H),1.36 (s, 3H),1.23e1.32 (m, 2H),
8), 13C NMR (101 MHz, CDCl3)
d
ppm 205.8 (C-3), 171.8 (OeC]O),
155.6 (N-(C]O)eO) 136.4 (Z-Ph), 128.6 (Z-Ph), 128.3 (Z-Ph), 128.2
(Z-Ph), 87.6 (C-2), 70.0 (Z-CH2), 50.1 (C- ), 48.9 (C-1), 43.4 (C-4),
a
39.3 (C-6), 38.3 (C-5), 27.9 (C-7), 27.5 (C-9), 22.6 (C-8), 21.0 (C-10),
18.6 (Ala-CH3).
0.87 (s, 3H). Compound 5RDd: 1H NMR (400 MHz, CDCl3)
d ppm
If the reaction was run using 2-exo-hydroxy-(R)-pinanone and
D-Z- alanine as starting materials the yield was 66% and the ratio
6RLb and 5RDb was 13:87.
7.28e7.39 (m, 5H), 5.07e5.27 (m, 3H), 4.52e4.63 (br.s, 1H),
4.19e4.29 (m, 1H), 3.95e4.03 (m, 1H), 3.48e3.57 (m, 1H), 3.07e3.16
(m, 1H), 2.93 (t, J ¼ 6.1 Hz, 1H), 2.61e2.80 (m, 2H), 2.09e2.17 (m,
1H), 1.70e1.87 (m, 1H), 1.56e1.67 (m, 4H), 1.43 (s, 9H), 1.36 (s, 3H),
1.23e1.30 (m, 2H), 0.87 (s, 3H).
3.1.2.3. (1R,2R,5R)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl
Na-(benzyloxycarbonyl)-Nε-(tert-butoxycarbonyl)-
L
-lysinate (6RLc)
and (1R,2R,5R)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl Na-
(benzyloxycarbonyl)-Nε-(tert-butoxycarbonyl)-
-lysinate (5RDc).
If the reaction was run using 2-exo-hydroxy-(R)-pinanone
and Na-Z-Nd-Boc-
D-ornithine as starting materials the yield was
66% and the ratio between 6RLd and 5RDd was 1:99. The spectrum
of 5RDd was identical to that of 5SLd.
D
The mixture of 6RLc and 5RDc was prepared from 2-exo-hydroxy-
(R)-pinanone (3R) and Na-Z-Nε-L-Boc-lysine. The product was pu-
rified by using heptane-EtOAc with increasing amounts of EtOAc
aseluents yielding a colorless oil (75.3%) consisting of 5RDc and
6RLc in a 58:42 ratio. Compound 6RLc: 1H NMR (400 MHz, CDCl3)
3.1.2.5. (1R,2R,5R)-2,6,6-trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl 2-
((benzyloxycarbonyl)amino)-2-methylpropanoate
Compound 5Rh was prepared from 2-exo-hydroxy-(R)-pinanone
(3R) and -alanine using the general procedure for
-methyl-Na-Z-
(5Rh).
a
L
d
ppm 7.29e7.37 (m, 5H), 4.99e5.38 (m, 3H), 4.51e4.68 (m, 1H),
acylation. The product was purified by using heptane-EtOAc with
increasing amounts of EtOAc as the eluents to give 5Rh (37.1%) as a
colorless oil.
4.16e4.32 (m, 1H), 3.01e3.15 (m, 2H), 2.86 (t, J ¼ 6.1 Hz, 1H),
2.60e2.79 (m, 1H), 2.37e2.48 (m, 1H), 2.09e2.17 (m, 1H), 1.69e1.89
(m, 3H), 1.61 - 1.52 (m, 4H), 1.53 - 1.11 (m, 2H), 1.42 (s, 9H), 1.36 (m,
3H), 0.86 (s, 3H). Compound 5RDc:. 1H NMR (600 MHz, CDCl3)
d
ppm 7.29e7.38 (m, 5H), 4.99e5.38 (m, 3H), 4.60e4.68 (m, 1H),
3.1.2.6. (1R,2R,5R)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl
(benzyloxycarbonyl)glycinate (5Rg). Compound 5Rg was made ac-
cording to the general procedure for acylation using 2-exo-hy-
droxy-(R)-pinanone (3R) and Na-Z-glycine as starting material. The
4.20e4.26 (m, 1H), 3.03e3.15 (m, 2H), 2.95 (t, J ¼ 6.1 Hz, 1H),
2.61e2.78 (m, 2H), 2.40e2.47 (m, 1H), 2.10e2.16 (m, 1H), 1.22e1.83
(m, 6H), 1.60 (s, 3H), 1.42 (s, 9H), 1.36 (s, 3H), 0.87 (s, 3H).

126
C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
product was purified by using heptane-EtOAc with increasing
amounts of EtOAc as the eluents to yield 5Rg as a colorless oil
3.1.2.11. (1S,2S,5S)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl
Na-(benzyloxycarbonyl)-Nε-(tert-butoxycarbonyl)-
-lysinate (5SLc)
and (1S,2S,5S)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl Na-
(benzyloxycarbonyl)-Nε-(tert-butoxycarbonyl)-
-lysinate (6SDc).
Compound 5SLc was prepared from 2-exo-hydroxy-(S)-pinanone
(3S) and Na-Z-Nε-Boc-
-lysine using the general procedure for
L
(23.6%). 1H NMR (600 MHz, CDCl3)
d ppm 7.29e7.38 (m, 5H), 5.12
(dd, J ¼ 16.1, 12.1 Hz, 3H), 3.91 (dd, J ¼ 5.5, 3.3 Hz, 2H), 2.94 (t,
J ¼ 6.1 Hz, 1H), 2.74 (s, 1H), 2.66 (dt, J ¼ 19.1, 2.9 Hz, 1H), 2.42e2.48
(m, 1H), 2.12e2.16 (m, 1H), 1.62 (s, 3H), 1.51e1.56 (m, 1H), 1.37 (s,
3H), 0.87 (s, 3H).
D
L
acylation. The product was purified by using heptane-EtOAc with
increasing amounts of EtOAc as eluents to give 5SLc as a colorless
oil (69.1%). 1H NMR data were identical to those of 5RDc.
3.1.2.7. 5-(tert-Butyl) 1-((1R,2R,5R)-2,6,6-trimethyl-3-oxobicyclo
When Na-Z-Nε-Boc-
Delysine was used as a starting material a
[3.1.1]heptan-2-yl) (benzyloxycarbonyl)-
(tert-Butyl) 1-((1R,2R,5R)-2,6,6-trimethyl-3-oxobicyclo[3.1.1]heptan-
2-yl) (benzyloxycarbonyl)- -glutamate (5RDd). The mixture of 6RLd
and the epimer 5RDd was prepared from 2-exo-hydroxy-(R)-
pinanone (3R) and Na-Z-
-(tert-butyl)-glutaminate using the gen-
L
-glutamate (6RLd) and 5-
mixture of 5SLc and 6SDc (50:50) was obtained.
D
3.1.2.12. (1S,2S,5S)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl
Na-(benzyloxycarbonyl)-Nd-(tert-butoxycarbonyl)-
L
-ornithinate
(5SLd) and (1S,2S,5S)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl
Na-(benzyloxycarbonyl)-Nd-(tert-butoxycarbonyl)-
-ornithinate
(6SDd). Compound 5SLd was prepared from 2-exo-hydroxy-(S)-
pinanone (3S) and Na-Z-Nd-Boc-
-ornithine using the general pro-
d
eral procedure for acylation. The product was purified by using
toluene-EtOAc with increasing amounts of EtOAc as the eluents to
give a colorless oil consisting of 6RLd and 5RDd (45.5%) in a 71:29
D
ratio. Compound 6RLd: 1H NMR (300 MHz, CDCl3)
d ppm 7.29e7.40
L
(m, 5H), 5.24e5.35 (m, 1H), 5.09 (s, 2H), 4.21e4.36 (m, 1H), 2.87 (t,
J ¼ 6.2 Hz,1H), 2.58e2.80 (m, 2H), 2.37e2.48 (m,1H), 2.23e2.35 (m,
2H), 2.05e2.17 (m, 2H), 1.80e1.97 (m, 1H), 1.60 (s, 3H), 1.51e1.59 (m,
1H), 1.40e1.44 (m, 9H), 1.36 (m, 3H), 0.86 (s, 3H). The NMR spec-
trum of the epimer was identical to that of 5RDd.
cedure for acylation. The product was purified by using toluene-
DCM -EtOAc (13:1:1) as the eluent to give 5SLd as a colorless oil
(46.9%). 1H NMR data identical to those of 5RDd.
When Na-Z-Nd-Boc-
D-ornithine was used as a starting material a
mixture of 6SDd and 5SLd (55%) was obtained in a ratio of 46:54.
3.1.2.8. (1R,2R,5R)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl
3.1.2.13. 5-(tert-Butyl) 1-((1S,2S,5S)-2,6,6-trimethyl-3-oxobicyclo
Nd-Boc-Na-Z-
L
-histidinate (6RLi) and (1R,2R,5R)-2,6,6-Trimethyl-3-
oxobicyclo[3.1.1]heptan-2-yl -histidinate (5RDi).
Nd-Boc-Na-Z-
The mixture of 6RLi and the epimer was prepared from 2-exo-hy-
droxy-(R)-pinanone (3R) and Nd-Boc-Na-Z-
-histidine using the
[3.1.1]heptan-2-yl) Na-(benzyloxycarbonyl)-
L
-glutamate (5SLe) and
5-(tert-butyl) 1-((1S,2S,5S)-2,6,6-trimethyl-3-oxobicyclo[3.1.1]hep-
tan-2-yl) Na-(benzyloxycarbonyl)-
-glutamate (6SDe). The mixture
of compound 5SLe and its epimer was prepared from 2-exo-hy-
droxy-(S)-pinanone (3S) and Na-Z-5-tbutyl
-glutaminate using the
D
D
L
general procedure for acylation The product was purified by using
toluene-EtOAc with increasing amounts of EtOAc as the eluents to
L
general procedure for acylation. The crude product was purified by
using toluene with increasing amounts of EtOAc as the eluents to
give a colorless oil (71.9%) consisting of 5SLe and 6SDe in a ratio of
give a colorless oil consisting of 6RLi and its
D
-epimer 5RDi (55.0%)
in a 37:63 ratio. Compound 6RLi:1H NMR (300 MHz, CDCl3)
d
ppm
7.95 (s, 1H), 7.27e7.37 (m, 5H), 7.12e7.19 (m, 1H), 6.04 (d, J ¼ 8.5 Hz,
1H), 5.03e5.15 (m, 2H), 4.46e4.58 (m, 1H), 2.96e3.09 (m, 2H), 2.85
(t, J ¼ 6.2 Hz, 1H), 2.74 (dd, J ¼ 19.3, 2.1 Hz, 1H), 2.59e2.68 (m, 1H),
2.28e2.44 (m, 1H), 2.09 (dtt, J ¼ 12.2, 6.1, 6.1, 2.9, 2.9 Hz, 1H), 1.68
(dt, J ¼ 13.5, 3.8 Hz, 1H), 1.59e1.63 (m, 9H), 1.53 (s, 3H), 1.33 (s, 3H),
0.83 (s, 3H). The spectrum of 5RDi was identical to that of 6SLi.
81:18. Compound 6SDe 1H NMR (300 MHz, CDCl3)
d ppm 7.27e7.39
(m, 5H), 5.48 (d, J ¼ 8.2 Hz, 1H), 5.09 (dd, J ¼ 15.2, 12.3 Hz, 2H), 4.25
(td, J ¼ 8.4, 4.8 Hz, 1H), 2.94 (t, J ¼ 6.2 Hz, 1H), 2.59e2.80 (m, 2H),
2.38e2.49 (m, 1H), 2.25e2.36 (m, 2H), 2.03e2.17 (m, 2H), 1.80e1.97
(m, 1H), 1.54e1.62 (m, 1H), 1.60 (s, 3H), 1.41e1.45 (m, 9H), 1.36 (s,
3H), 0.86 (s, 3H). The NMR spectrum of the epimer 6SDe was
identical to that of compound 6RLe.
3.1.2.9. (1S,2S,5S)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl Na-
(benzyloxycarbonyl)-
L
-phenylalaninate (5SLa) and (1S,2S,5S)-2,6,6-
Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl Na-(benzyloxycarbonyl)-
D-
3.1.2.14. (1S,2S,5S)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl
Na-(benzyloxycarbonyl)glycinate (5Sg). Compound 5Sg was pre-
pared from 2-exo-hydroxy-(S)-pinanone (3S) and Na-Z-glycine us-
ing the general procedure for acylation. The product was purified
by using heptane-EtOAc with increasing amounts of EtOAc as the
eluents to give 5Sg (75%) as a colorless oil. 1H NMR data were
identical to those of 5Rg.
phenylalaninate (6SDa). The mixture of compound 5SLa and 6SDa
was prepared from 2-exo-hydroxy-(S)-pinanone (3S) and Na-Z-
Lephenylalanine using the general procedure for acylation. The
product was purified by using heptane-EtOAc with increasing
amounts of EtOAc as an eluent to give a colorless oil (66.4%) con-
sisting of 5SLa and the epimer 6SDa in a ratio of 38:62. The 1H NMR
data were identical to those of 5RDa and 6RLa, respectively.
3.1.2.15. tert-Butyl 4-((S)-2-(((benzyloxy)carbonyl)amino)-3-oxo-3-
(((1S,2S,5S)-2,6,6-trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl)oxy)pro-
pyl)-1H-imidazole-1-carboxylate (5SLi). Compound 5SLi was pre-
3.1.2.10. (1S,2S,5S)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl
Na-(benzyloxycarbonyl)-
L
-alaninate (5SLb) and (1S,2S,5S)-2,6,6-
Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl Na-(benzyloxycarbonyl)-
alaninate (6SDb).. The mixture of compound 5SLb and 6SDb was
prepared from 2-exo-hydroxy-(S)-pinanone (3S) and Na-Z-
D-
pared from 2-exo-hydroxy-(S)-pinanone (3S) and Na-Z-Nd-Boc-
L-
histidine using the general procedure for acylation. The crude
product was purified by using toluene with increasing amounts of
EtOAC as the eluents to give 5SLi as a colorless oil (26.8%). 1H NMR
L-
alanine using the general procedure for acylation. The product was
purified by using heptane-EtOAc with increasing amounts of EtOAc
as the eluents to give a colorless oil (70.4%) consisting of 5SLb and
6SDb in a 78:22 ratio. NMR data were identical to those of 5RDb
and 6RLb, respectively.
(300 MHz, CDCl3)
d
ppm 7.96 (d, J ¼ 1.2 Hz, 1H), 7.27e7.37 (m, 5H),
7.18 (br. s, 1H), 6.15 (d, J ¼ 8.2 Hz, 1H), 5.07e5.12 (m, 2H), 4.52 (dt,
J ¼ 8.2, 5.1 Hz, 1H), 3.01 (dd, J ¼ 6.7, 5.6 Hz, 2H), 2.92 (t, J ¼ 6.2 Hz,
1H), 2.73 (dd, J ¼ 19.0, 2.3 Hz, 1H), 2.63 (dt, J ¼ 18.8, 2.8 Hz, 1H),
2.36e2.44 (m, 1H), 2.10 (tt, J ¼ 6.0, 2.8 Hz, 1H), 1.88e1.97 (m, 1H),
1.60 (s, 9H), 1.53 (s, 3H), 1.34 (s, 3H), 0.84 (s, 3H).
When Na-Z-
ratio of 5SLb to 6SDb was 39:61.
D-alanine was used as starting material the obtained

C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
127
3.1.3. General procedure for cyclization of Na-Z-protected esters-(5)
of amino acids with 2-exo-hydroxypinanone to give oxazinols (7)
In a 50 mL flame-dried Schlenk flask was added 10% Pd/C
(20 mg) and then the flask was purged with argon. A solution of the
ester (350 mg) in EtOAc (5 mL) was added, and then the flask was
purged with hydrogen. A balloon filled with hydrogen was attached
to the flask, and the mixture was stirred overnight. The reaction
mixture was filtered through a pad of Celite, washing the Celite
with EtOAc, and the resulting filtrate was concentrated. Chroma-
NMR (151 MHz, CDCl3)
(NH-(C]O)eO), 85.29 (C-8a), 79.13 (OeC-CH3)3), 59.03 (C-3), 50.55
(C-8), 40.55 (CLys-ε), 39.73 (C-7), 39.48 (C-6), 37.13 (C-5), 31.78
d
ppm 173.05 (C-4a), 171.99 (C-2), 156.11
(CLys
ꢃ
b
), 30.09 (CLys g ), 28.60 (C(CH3)3), 27.75 (C-9), 27.57 (C-11),
ꢃ
ꢃ
23.21 (CLys-d), 23.14 (C-10), 22.27 (C-12). HRMS m/z 379.2587
[MþH]þ (379.2591, calc. for C21H35N2Oþ4 ).
3.1.3.4. tert-Butyl
(3-((3R,6R,8R,8aR)-7,7,8a-trimethyl-2-oxo-
3,5,6,7,8,8a-hexahydro-2H-6,8-methanobenzo[b][1,4]oxazin-3-yl)
propyl)carbamate (7RDd). Compound 7RDd was obtained from
mixture 5RDd and 6RLd (87:13) using the general procedure for
hydrogenation followed by flash chromatography using heptane
with increasing amounts of EtOAc as the eluents to give 7RDc as
colourless crystals (49.3%). mp 96e98 ꢁC. ½aꢂD25 ¼ 163ꢁ (c 0.33). 1H
tography was performed over silica gel (15e40
mensions 2 ꢀ 5 cm).
mm, column di-
3.1.3.1. (3R,6R,8R,8aR)-3-Benzyl-7,7,8a-trimethyl-3,5,6,7,8,8a-hex-
ahydro-2H-6,8-methanobenzo[b][1,4]oxazin-2-one (7RDa).
Compound 7RDa was obtained from 5RDa and 6RLa (87:13) using
the general procedure for hydrogenation followed by flash chro-
matography using heptane with increasing amounts of EtOAc as the
eluents to give 7RDa as a colorless oil (80.4%). ½aꢂ2D5 ¼ 237ꢁ (c 0.24).
NMR (400 MHz, CDCl3)
d ppm 4.85 (br.s.,1H, NH), 4.00e4.06 (m,1H,
Hendo-3), 3.16e3.27 (m, 2H, HOrn
-d), 2.87 (ddt, J ¼ 17.6, 3.7, 1.8,
1.8 Hz, 1H, Hexo-5), 2.75 (ddd, J ¼ 17.6, 4.2, 2.4 Hz, 1H, Hendo-5), 2.37
(dtd, J ¼ 11.2, 5.7, 5.7, 2.4 Hz, 1H, Hexo-9), 2.20 (t, J ¼ 5.7 Hz, 1H, H-8),
1H NMR (600 MHz, CDCl3)
d
ppm 7.39 (d, J ¼ 7.7 Hz, 2H, Ho-Ph), 7.31
2.16e2.24 (m, 1H, HOrn
7.5 Hz,1H, HOrn
(m, 9H, C(CH3)3), 1.40 (s, 3H, H-11), 1.15 (d, J ¼ 11.5 Hz, 1H, Hendo-9),
1.06 (s, 3H, H-10), 13C NMR (151 MHz, CDCl3)
ppm 173.37 (C-4a),
171.85 (C-2), 156.18 (NH-(C]O)eO), 85.38 (C-8a), 79.12
(OeC(CH3)3), 58.74 (C-3), 50.55 (C-8), 40.46 (COrn ), 39.76 (C-7),
39.47 (C-6), 37.10 (C-5), 29.32 (COrn ), 28.61 (C(CH3)3), 27.78 (C-9),
-
b
), 2.10e2.14 (m, 1H, H-6), 1.97 (td, J ¼ 14.4,
(t, J ¼ 7.5 Hz, 2H, Hm-Ph), 7.23 (t, J ¼ 7.3 Hz 1H, Hp-Ph), 4.25 (dtd,
J ¼ 8.3, 4.3, 4.3, 2.2 Hz, 1H, H-3endo), 3.59 (dd, J ¼ 13.9, 4.4 Hz, 1H,
CH2-Ph), 3.19 (dd, J ¼ 14.3, 8.4 Hz, 1H, CH2-Ph), 2.84 (ddt, J ¼ 17.6,
-b), 1.72e1.80 (m, 2H, HOrn-g), 1.63 (s, 3H, H-12),1.44
d
3.7, 2.2, 2.2 Hz,1H, Hend ꢃ5), 2.73 (ddd, J ¼ 17.6, 4.4, 2.2 Hz,1H, Hexo
-
5), 2.34 (dtd, J ¼ 11.2, 5.6, 5.6, 2.2 Hz, 1H, Hexo-9), 2.19 (t, J ¼ 5.7 Hz,
1H, H-8), 2.08e2.12 (m, 1H, H-6), 1.60 (s, 3H, H-12), 1.38 (s, 3H, H-
11), 1.12 (d, J ¼ 11.1 Hz, 1H, Hendo-9), 1.02 (s, 3H, H-10). 13C NMR
-d
-b
27.56 (C-11), 26.21 (COrn-g), 23.15 (C-10), 22.25 (C-12). HRMS m/z
(151 MHz, CDCl3)
d
ppm 172.65 (C-4a), 171.80 (C-2), 139.01 (CPh-1),
365.2431 [MþH]þ (365.2435, calc. for C20H33N2Oþ4 ).
129.99 (CPh-2 and 6), 128.37 (CPh-3 and 5), 126.57 (CPh-4), 85.42 (C-
8a), 60.82 (C-3), 50.57 (C-8), 39.72 (C-7), 39.49 (C-6), 38.37 (CH2-
Ph), 37.22 (C-5), 27.70 (C-9), 27.55 (C-11), 23.09 (C-10), 22.31 (C-12).
HRMS m/z 298.1808 [MþH]þ (298.1802, calc. for C19H24NOþ2 ).
3.1.3.5. tert-Butyl
3-((3R,6R,8R,8aR)-7,7,8a-trimethyl-2-oxo-
3,5,6,7,8,8a-hexahydro-2H-6,8-methanobenzo[b][1,4]oxazin-3-yl)
propanoate (7RDd). Compound 7RDd was obtained from the
mixture 5RDd and the epimer 6RLi (87:13) using the general pro-
cedure for hydrogenation followed by flash chromatography using
toluene with increasing amounts of EtOAc as the eluents to give
7RDd (58.0%) as a colourless amorphous powder. ½aꢂ2D5 ¼ 188ꢁ (c
3.1.3.2. (3R,6R,8R,8aR)-3,7,7,8a-Tetramethyl-3,5,6,7,8,8a-hexahydro-
2H-6,8-methanobenzo[b][1,4]oxazin-2-one
(7RDb). Compound
7RDb was obtained from the mixture 5RDb and 6RSb (87:13) using
the general procedure for hydrogenation followed by flash chro-
matography using heptane with increasing amounts of EtOAc as the
eluents to give 7RDb as a colorless amorphous powder (83.6%).
0.32). 1H NMR (300 MHz, CDCl3)
d
ppm 4.06e4.19 (m, 1H, Hendo-3),
2.84 (d, J ¼ 17.6 Hz, 1H, Hexo-5), 2.69 (ddd, J ¼ 17.3, 3.8, 1.8 Hz, 1H,
endo-5), 2.44e2.52 (m, 2H, HGlu ), 2.28e2.43 (m, 2H, Hexo-9, HGlu
), 1.59
H
-g
-
mp ¼ 82 ꢁC. ½aꢂ2D5 ¼ 254ꢁ (c 0.31). 1H NMR (300 MHz, CDCl3)
d
ppm
b
), 2.17 (t, J ¼ 5.9 Hz, 1H, H-8), 2.05e2.23 (m, 2H, H-6, HGlu
-b
4.13 (qdd, J ¼ 6.9, 6.9, 6.9, 4.2, 2.5 Hz, 1H, Hendo-3), 2.85 (ddt,
J ¼ 17.6, 3.5, 2.1, 2.1, Hz, 1H, Hexo-5), 2.70 (dddd, J ¼ 17.6, 3.8, 2.6,
(s, 3H, H-12), 1.41 (s, 9H, OeC(CH3)3), 1.36 (s, 3H, H-11), 1.11 (d,
J ¼ 11.4 Hz,1H, H-9endo),1.02 (s, 3H, H-10). 13C NMR (75 MHz, CDCl3)
1.2 Hz, 1H, Hendo-5), 2.35 (dtdd, J ¼ 11.1, 5.6, 5.6, 2.1, 1.2H z, 1H, Hexo
-
d
ppm 172.91 (C-4a),172.56 (OeC ¼ OGlu),171.50 (C-2), 85.35 (C-8a),
9), 2.18 (t, J ¼ 3.6, 1H, H-8), 2.06e2.13 (m, 1H, H-6), 1.65 (dd, J ¼ 7.0,
1.5 Hz, 3H, H-13), 1.63e1.61 (m, 3H, H-12), 1.37 (s, 3H, H-11), 1.15 (d,
J ¼ 11.4 Hz,1H, Hendo-9),1.03 (s, 3H, H-10). 13C NMR (75 MHz, CDCl3)
80.25 (OeC(CH3)3), 57.89 (C-3), 50.42 (C-8), 39.69 (C-7), 39.38 (C-
6), 37.06 (C-5), 31.47 (CGlu ), 28.27 (OeC(CH3)3), 27.70 (C-9), 27.53
-
g
(C-11), 27.40 (CGlu-b), 23.14 (C-10), 22.18 (C-12). HRMS m/z 358.1970
d
ppm 172.86 (C-4a), 172.60 (C-2), 85.49 (C-8a), 55.01 (C-3), 50.52
[MþH]þ (358.1989, calc. for C19H30NOþ4 ).
(C-8), 39.70 (C-7), 39.43 (C-6), 37.07 (C-5), 27.71 (C-9), 27.55 (C-11),
23.16 (C-10), 22.23 (C-12), 18.46 (C-13). HRMS m/z 222.1509
[MþH]þ (222.1489, calc. for C13H20NOþ2 ).
3.1.3.6. 3-((3R,6R,8R,8aR)-7,7,8a-Trimethyl-2-oxo-3,5,6,7,8,8a-hex-
ahydro-2H-6,8-methanobenzo[b][1,4]oxazin-3-yl)propanoic
(7RDf). TFA (0.5 mL) was added dropwise to a cooled solution of
7RDd (50 mg, 149 mol) in DCM (1 mL), and then the reaction
acid
3.1.3.3. tert-Butyl
(4-((3R,6R,8R,8aR)-7,7,8a-trimethyl-2-oxo-
m
3,5,6,7,8,8a-hexahydro-2H-6,8-methanobenzo[b][1,4]oxazin-3-yl)
butyl)carbamate (7RDc).. Compound 7RDc was obtained from
5RDc and 6RLc (87:13) using the general procedure for hydroge-
nation followed by flash chromatography using heptane with
increasing amounts of EtOAc as the eluents to give 7RDc. The
product was crystallized from EtOAc and heptane (68%).
mp ¼ 80e81 ꢁC. ½aꢂD25 ¼ 162ꢁ (c 0.28). 1H NMR (600 MHz, CDCl3)
mixture was stirred at rt for 2 h. Concentration of the reaction
mixture followed by flash chromatography starting elution
toluene-Et2O-acetic acid (80:20:0.5) continuing with increasing
amounts of Et2O in the elutent to yield 7RDf as a colourless
amorphous powder (32.8 mg, 78.8%). ½aꢂ2D5 ¼ 191ꢁ (c 0.26). 1H NMR
(300 MHz, CDCl3)
d ppm 4.14e4.22 (m, 1H, Hendo-3), 2.88 (ddt,
J ¼ 17.6, 3.8,1.8,1.8 Hz,1H, Hexo-5), 2.74 (ddd, J ¼ 17.9, 4.1, 2.3 Hz,1H,
endo-5), 2.67 (t, J ¼ 6.7, 2H, HGlu ), 2.43e2.56 (m, 1H, HGlu , 2.38
(dtd, J ¼ 11.1, 5.8, 5.8, 2.1 Hz,1H, Hexo-9), 2.26 (dt, J ¼ 14.4, 7.0 Hz,1H,
HGlu
), 2.21 (t, J ¼ 5.7 Hz, 1H, H-8), 2.08e2.16 (m, 1H, H-6), 1.64 (s,
3H, H-12), 1.39 (s, 3H, H-11), 1.14 (d, J ¼ 11.1 Hz, 1H, H-9endo), 1.05 (s,
3H, H-10). 13C NMR (75 MHz, CDCl3)
ppm 177.97 (HOeC]O),
d
ppm 4.61 (br. s, 1H, NH), 3.99 (dtd, J ¼ 7.2, 4.4, 4.4, 2.2 Hz, 1H, H-
endo), 3.12e3.20 (m, 2H, HLys-ε), 2.87 (ddt, J ¼ 17.6, 4.0, 1.8, 1.8 Hz,
H
-g
-b
3
1H, Hexo-5), 2.75 (ddd, J ¼ 17.6, 4.4, 2.2 Hz, 1H, Hendo-5), 2.37 (dtd,
J ¼ 11.3, 5.5, 5.5, 2.2 Hz, 1H, Hexo-9), 2.20 (t, J ¼ 5.7 Hz, 1H, H-8),
-b
2.10e2.18 (m, 2H, HLys
-b
, H-6), 1.92e1.99 (m, 1H, HLys
-b
), 1.62 (s, 3H,
d
H-12), 1.50e1.61 (m, 4H, HLys
-
g, HLys
-
d
), 1.44 (s, 9H, C(CH3)3) 1.39 (s,
174.41 (C-4a), 171.03 (C-2), 85.61 (C-8a), 57.98 (C-3), 50.44 (C-8),
39.80 (C-7), 39.33 (C-6), 36.97 (C-5), 30.72 (CgꢃGlu), 27.79 (C-9),
3H, H-11), 1.16 (d, J ¼ 11.4 Hz, 1H, Hendo-9), 1.06 (s, 3H, H-10). 13
C

128
C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
27.53 (C-11), 26.68 (CbꢃGlu), 23.16 (C-10), 22.21 (C-12). HRMS m/z
280.1532 [MþH]þ (280.1544, calc. for C15H22NOþ4 ).
(m, J ¼ 7.3 Hz, 1H, HPh-4), 4.54 (dtd, J ¼ 7.7, 4.4, 4.4, 2.2 Hz, 1H,
H
endo-3), 3.50 (dd, J ¼ 14.3, 4.8 Hz, 1H, CH2-Ph), 3.14 (dd, J ¼ 14.3,
7.7 Hz, 1H, CH2-Ph), 2.89 (ddt, J ¼ 17.5, 3.9, 2.0, 2.0 Hz, 1H, Hexo-5),
2.69 (ddd, J ¼ 17.6, 4.4, 2.2 Hz, 1H, Hendo-5), 2.38 (dtd, J ¼ 11.2, 5.6,
5.6, 2.2 Hz,1H, Hexo-9), 2.16 (t, J ¼ 5.7 Hz,1H, H-8), 2.07e2.11 (m,1H,
H-6), 1.66 (s, 3H, H-12), 1.39 (s, 3H, H-11), 1.06 (s, 3H, Hendo-9), 0.99
3.1.3.7. (6R,8R,8aR)-7,7,8a-Trimethyl-3,5,6,7,8,8a-hexahydro-2H-
6,8-methanobenzo[b][1,4]oxazin-2-one (7Rg). Compound 7Rg was
obtained from of 5Rg using the general procedure for hydrogena-
tion to give 7Rg as a colorless amorphous powder (94.8%).
(d, J ¼ 11.0 Hz, 1H, H-10). 13C NMR (75 MHz, CDCl3)
d ppm 172.51 (C-
½aꢂ2D5 ¼ 253ꢁ (c 0.29). 1H NMR (400 MHz, CDCl3)
d
ppm 4.63 (d,
4a), 171.64 (C-2), 138.82 (CPh-1), 129.84 (CPh-2 and 6), 128.25 (CPh-3
and 5), 126.47 (CPh-4), 85.41 (C-8a), 60.80 (C-3), 50.53 (C-8), 39.77
(C-7), 39.48 (C-6), 38.37 (CH2-Phe), 37.26 (C-5), 27.76 (C-9), 27.61
(C-11), 23.18 (C-10), 22.36 (C-12). HRMS m/z 298.1808 [MþH]þ
(298.1802, calc. for C19H24NOþ2 ).
J ¼ 19.8 Hz, 1H, Hexo-3), 4.17 (ddd, J ¼ 19.6, 4.2, 2.4 Hz, 1H, Hendo-3),
2.89 (m, 1H, Hexo-5), 2.75 (ddd, J ¼ 17.6, 4.2, 2.4 Hz, 1H, Hendo-5),
2.40 (dtd, J ¼ 11.3, 5.7, 5.7, 2.1 Hz, 1H, Hexo-9), 2.21 (t, J ¼ 5.7 Hz, 1H,
H-8), 2.13 (q, J ¼ 5.7 Hz, 1H, H-6), 1.62 (s, 3H, H-12), 1.40 (s, 3H, H-
11), 1.21 (d, J ¼ 11.2 Hz, 1H, Hendo-9), 1.06 (s, 3H, H-10). 13C NMR
(101 MHz, CDCl3)
d ppm 173.72 (C-4a), 170.20 (C-2), 85.03 (C-8a),
3.1.3.11. (1S,2S,5S)-2,6,6-Trimethyl-3-oxobicyclo[3.1.1]heptan-2-yl
phenylalaninate. A small amount of the non-cyclized free amine
could be isolated from the reaction mixture for preparing 7SLa
affording 5SLa not protected at the nitrogen atom. 1H NMR
51.74 (C-3), 50.42 (C-8), 39.84 (C-7), 39.42 (C-6), 37.11 (C-5), 27.79
(C-9), 27.60 (C-11), 23.16 (C-10), 22.39 (C-12). HRMS m/z 208.1354
[MþH]þ (208.1332, calc. for C12H18NOþ2 ).
(600 MHz, CD3OD)
1H, H-
d
ppm 7.16e7.35 (m, 5H, HPhe), 3.63 (t, J ¼ 7.0 Hz,
3.1.3.8. (6R,8R,8aR)-3,3,7,7,8a-Pentamethyl-3,5,6,7,8,8a-hexahydro-
2H-6,8-methanobenzo[b][1,4]oxazin-2-one (7Rh). Compound 7Rh
was obtained from 5Rh using the general procedure for hydroge-
nation followed by flash chromatography using heptane with
increasing amounts of EtOAc as the eluents to give 7Rh as a col-
ourless amorphous powder (29.6%). ½aꢂ2D5 ¼ 128ꢁ (c 0.13). 1H NMR
a
), 2.97 (dd, J ¼ 13.8, 7.2 Hz, 1H, CH2-Ph), 2.85 (dd, J ¼ 13.6,
7.0 Hz, 1H, CH2-Ph), 2.78 (t, J ¼ 6.2 Hz, 1H, H-1), 2.74 (dd, J ¼ 19.1,
2.2 Hz, 1H, Hexo-4), 2.61 (dt, J ¼ 18.7, 3.1 Hz, 1H, Hendo-4), 2.34 (dtd,
J ¼ 11.3, 6.1, 6.1, 2.9 Hz, 1H, Hexo-7), 2.08 (tt, J ¼ 6.1, 2.9 Hz, 1H, H-5),
1.50 (s, 3H, H-10), 1.42 (d, J ¼ 11.0 Hz, 1H, H-7endo), 1.36 (s, 3H, H-9),
0.86 (s, 3H, H-8).
(600 MHz, CDCl3)
d
ppm 2.90 (d, J ¼ 16.5 Hz, 1H, Hexo-5), 2.69 (dd,
J ¼ 16.5 Hz, 1H, Hendo-5), 2.39 (dtd, J ¼ 11.0, 5.4, 5.4, 2.0 Hz, 1H, Hexo
-
3.1.3.12. (3S,6S,8S,8aS)-3,7,7,8a-Tetramethyl-3,5,6,7,8,8a-hexahydro-
2H-6,8-methanobenzo[b][1,4]oxazin-2-one (7SLb). Compound 7SLb
was obtained from the mixture of 5SLb and 6SDb (78:22) using the
general procedure for hydrogenation followed by flash chroma-
tography using toluene with increasing amounts of EtOAc as elu-
ents to yield 7SLb as a colourless crystals (52.5%), mp ¼ 80e81 ꢁC.
9), 2.11e2.18 (m, 1H, H-6, H-8), 1.66 (s, 3H, H-12), 1.62 (s, 3H, CH3),
1.53 (s, 3H, CH3), 1.39 (s, 3H, H-11), 1.22 (d, J ¼ 11.4 Hz, 1H, Hendo-9),
1.09 (s, 3H, H-10). 13C NMR (151 MHz, CDCl3)
d ppm 176.12 (C-4a),
168.76 (C-2), 85.62 (C-8a), 59.07 (C-3), 51.73 (C-8), 40.36 (C-7),
40.03 (C-6), 37.37 (C-5), 31.29 (CH3), 28.03 (C-9), 27.80 (C-11), 27.51
(CH3), 27.34 (C-12), 23.15 (C-10). HRMS m/z 236.1663 [MþH]þ
(236.1645, calc. for C14H22NOþ2 ).
½aꢂD25 ¼ ꢃ244ꢁ (c. 0.26). 1H NMR (600 MHz, CDCl3)
d ppm 4.15 (qdd,
J ¼ 7.0, 7.0, 7.0, 4.4, 2.2 Hz, 1H, Hendo-3), 2.87 (ddt, J ¼ 17.6, 4.0, 2.2,
2.2 Hz, 1H, Hexo-5), 2.74 (ddd, J ¼ 17.6, 4.0, 2.2 Hz, 1H, Hendo-5), 2.38
(dtd, J ¼ 11.3, 5.6, 5.6, 2.4 Hz,1H, Hexo-9), 2.21 (t, J ¼ 5.6 Hz,1H, H-8),
2.12 (tdd, J ¼ 5.9, 5.9, 4.4, 1.5 Hz, 1H, H-6), 1.68 (d, J ¼ 7.0 Hz, 3H, H-
13), 1.64 (s, 3H, H-12), 1.40 (s, 3H, H-11), 1.19 (d, J ¼ 11.4 Hz, 1H,
3.1.3.9. tert-Butyl
4-(((3R,6R,8R,8aR)-7,7,8a-trimethyl-2-oxo-
3,5,6,7,8,8a-hexahydro-2H-6,8-methanobenzo[b][1,4]oxazin-3-yl)
methyl)-1H-imidazole-1-carboxylate (7RDi). Compound 7RDi was
obtained from the mixture 5RDi and the epimer 6RLi (87:13) using
the general procedure for hydrogenation followed by flash chro-
matography using toluene with increasing amounts of EtOAc as
eluents to give 7RDi as a colourless amorphous powder (47.9%).
H
endo-9), 1.06 (s, 3H, H-10). 13C NMR (151 MHz, CDCl3)
d ppm 173.08
(C-4a), 172.79 (C-2), 85.56 (C-8a), 55.09 (C-3), 50.60 (C-8), 39.73 (C-
7), 39.50 (C-6), 37.09 (C-5), 27.72 (C-9), 27.55 (C-11), 23.16 (C-10),
22.24 (C-12), 18.45 (C-13). HRMS m/z 222.1492 [MþH]þ (222.1489,
calc. for C13H20NO)2.
½aꢂ2D5 ¼ 122ꢁ (c 0.14). 1H NMR (600 MHz, CDCl3)
d ppm 8.01 (d,
J ¼ 1.47 Hz,1H, N]CHeN), 7.17 (s,1H, C]CHeN), 4.54 (dtd, J ¼ 8.30,
4.38, 4.38, 2.38 Hz, 1H, Hendo-3), 3.51 (ddd, J ¼ 15.22, 4.59, 1.10 Hz,
1H, CH2-Im), 3.10 (dd, J ¼ 15.04, 8.44 Hz, 1H, CH2-Im), 2.85 (ddt,
J ¼ 17.61, 3.85, 2.11, 2.11 Hz, 1H, Hexo-5), 2.72 (ddd, J ¼ 17.6, 4.4,
2.2 Hz, 1H, Hendo-5), 2.36 (dtd, J ¼ 11.2, 5.6, 5.6, 2.4 Hz, 1H, Hexo-9),
2.20 (t, J ¼ 5.7 Hz, 1H, H-8), 2.10 (tdd, J ¼ 5.5, 4.4, 1.5 Hz, 1H, H-6),
1.65 (s, 3H, H-12), 1.59 (s, 9H, C(CH3)3), 1.38 (s, 3H, H-11), 1.17 (d,
J ¼ 11.3 Hz,1H, Hendo-9),1.03 (s, 3H, H-10). 13C NMR (75 MHz, CDCl3)
3.1.3.13. tert-Butyl
(4-((3S,6S,8S,8aS)-7,7,8a-trimethyl-2-oxo-
3,5,6,7,8,8a-hexahydro-2H-6,8-methanobenzo[b][1,4]oxazin-3-yl)
butyl)carbamate (7SLc). Compound 7SLc was obtained from 5SLc
using the general procedure for hydrogenation followed by flash
chromatography using heptane to which increasing amounts of
EtOAc were added as an eluent yielded 7SLc as an oil which was
crystallized from ethyl acetate-heptane (86.5%). Mp 81e82 ꢁC.
d
ppm 173.01 (C-4a),171.51 (C-2), 147.04 (O]CeOeC(CH3)3), 140.36
(CH]C]N), 136.55 (N]CHeN), 114.66 (C]CH]N), 85.47 (C-8a),
85.26 (O]CeOeC(CH3)3), 58.74 (C-3), 50.53 (C-8), 39.67 (C-7),
39.43 (C-6), 37.21 (C-5), 31.25 (CH2-Im), 28.03 (O]CeOeC(CH3)3),
27.73 (C-9), 27.52 (C-11), 23.10 (C-10), 22.29 (C-12). HRMS m/z
388.2243 [MþH]þ (388.2231, calc. for C21H30N3Oþ4 ).
½aꢂD25 ¼ ꢃ157ꢁ (c. 0.28). 1H NMR (600 MHz, CDCl3)
d ppm 4.62 (br. s.,
1H, NH), 3.99 (dtd, J ¼ 7.3, 4.4, 4.4, 2.2 Hz, 1H, Hendo-3), 3.11e3.20
(m, 2H, HLys-ε), 2.87 (ddt, J ¼ 17.6, 3.8, 1.8, 1.8 Hz, 1H, Hexo-5), 2.75
(ddd, J ¼ 17.6, 4.4, 2.2 Hz, 1H, Hendo-5), 2.37 (dtd, J ¼ 11.2, 5.7, 5.7,
2.4 Hz, 1H, Hexo-9), 2.20 (t, J ¼ 5.7 Hz, 1H, H-8), 2.10e2.18 (m, 2H, H-
beLys, H-6), 1.92e1.99 (m, 1H, HLys-b), 1.62 (s, 3H, H-12), 1.49e1.65
3.1.3.10. (3S,6S,8S,8aS)-3-Benzyl-7,7,8a-trimethyl-3,5,6,7,8,8a-hex-
(m, 4H, HLys-g, HLys-d), 1.44 (s, 9H, C(CH3)3), 1.39 (s, 3H, H-11), 1.16
ahydro-2H-6,8-methanobenzo[b][1,4]oxazin-2-one
(7SLa).
(d, J ¼ 11.4 Hz, 1H, Hendo-9), 1.05 (s, 3H, H-10). 13C NMR (151 MHz,
Compound 7SLa was obtained from a mixture of 5SLa and 6SLa
(90:10) using the general procedure for hydrogenation followed by
flash chromatography using heptane with increasing amounts of
EtOAc as eluents to yield 7SLa as colourless oil (94.2%).
CDCl3) d ppm 173.15 (C-4a), 171.96 (C-2), 156.11 (NH-(C]O)eO),
85.30 (C-8a), 79.12 (OeC(CH3)3), 59.02 (C-3), 50.55 (C-8), 40.53
(CLys-ε), 39.73 (C-7), 39.47 (C-6), 37.11 (C-5), 31.75 (C-bLys), 30.07 (C-
gLys), 28.59 (C(CH3)3), 27.75 (C-9), 27.56 (C-11), 23.19 (C-dLys), 23.13
(C-10), 22.25 (C-12). HRMS m/z 379.2587 [MþH]þ (379.2591, calc.
for C21H35N2Oþ4 ).
½aꢂD25 ¼ ꢃ233ꢁ (c 0.29). 1H NMR (600 MHz, CD3OD)
d ppm 7.34 (d,
J ¼ 7.0 Hz, 2H, HPhe-2 and 6), 7.26 (t, J ¼ 7.7 Hz, 2H, HPh-3 and 5), 7.18

C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
129
3.1.3.14. tert-Butyl
(3-((3S,6S,8S,8aS)-7,7,8a-trimethyl-2-oxo-
increasing amounts of EtOAc as eluents to give 7Sg as a colourless
amorphous powder (74.4%). ½aꢂ2D5 ¼ ꢃ225ꢁ (c 0.32). 1H NMR
3,5,6,7,8,8a-hexahydro-2H-6,8-methanobenzo[b][1,4]oxazin-3-yl)
propyl)carbamate (7SLd). Compound 7SLd was obtained from 5SLd
using the general procedure for hydrogenation followed by flash
chromatography using heptane with increasing amounts of EtOAc
as eluents to yield 7SLd as an oil, which was crystallized from i-
PrOH (50.3%). Mp 114e116 ꢁC. ½aꢂD25 ¼ ꢃ168ꢁ (c 0.30). 1H NMR
(300 MHz, CDCl3)
d
ppm 4.60 (d, J ¼ 19.6 Hz, 1H, Hexo-3), 4.16 (ddd,
J ¼ 19.9, 4.4, 2.6 Hz, 1H, Hendo-3), 2.86 (dquin, J ¼ 17.6, 2.3, 2.3, 2.3,
2.3 Hz, 1H, Hexo-5), 2.72 (ddd, J ¼ 17.6, 4.1, 2.3 Hz, 1H, Hendo-5), 2.37
(dtd, J ¼ 11.1, 5.6, 5.6, 2.3 Hz, 1H, Hexo-9), 2.18 (t, J ¼ 5.9 Hz, 1H, H-8),
2.11 (m, J ¼ 3.9, 3.9, 2.2 Hz,1H, H-6),1.59 (s, 3H, H-12),1.38 (s, 3H, H-
11), 1.17 (d, J ¼ 11.4 Hz, 1H, Hendo-9), 1.04 (s, 3H, H-10). 13C NMR
(400 MHz, CDCl3)
d
ppm 3.99e4.06 (m, 1H, Hendo-3), 3.21 (m, 2H,
HOrn
-
d
), 2.87 (ddt, J ¼ 17.6, 3.9, 1.7, 1.7 Hz, 1H, Hexo-5), 2.75 (ddd,
(75 MHz, CDCl3) d ppm 173.41 (C-4a), 170.03 (C-2), 85.12 (C-8a),
J ¼ 17.6, 4.2, 2.2 Hz, 1H, Hendo-5), 2.37 (dtd, J ¼ 11.2, 5.6, 5.6, 2.2 Hz,
51.68 (C-3), 50.28 (C-8), 39.80 (C-7), 39.32 (C-6), 37.08 (C-5), 27.77
(C-9), 27.58 (C-11), 23.17 (C-10), 22.37 (C-12). HRMS m/z 208.1371
[MþH]þ (208.1332, calc. for C12H18NOþ2 ).
1H, Hexo-9), 2.20 (t, J ¼ 5.7 Hz, 1H, H-8), 2.16e2.26 (m, 1H, HOrn
2.09e2.15 (m, 1H, H-6), 1.97 (td, J ¼ 14.4, 7.5 Hz, 1H, HOrn
-
-
b
b
),
),
1.70e1.80 (m, 2H, HOrn-g), 1.63 (s, 3H, H-12), 1.44 (m, 9H, C(CH3)3),
1.39 (s, 3H, H-11), 1.15 (d, J ¼ 11.5 Hz, 1H, H-9endo), 1.06 (s, 3H, H-10).
13C NMR (151 MHz, CDCl3)
ppm 173.19 (C-4a), 171.92 (C-2), 156.17
(NH-(C]O)eO), 85.37 (C-8a), 79.11 (OeC(CH3)3), 58.75 (C-3), 50.55
(C-8), 40.46 (COrn ), 39.75 (C-7), 39.47 (C-6), 37.12 (C-5), 29.36
(COrn-b), 28.60 (C(CH3)3), 27.76 (C-9), 27.56 (C-11), 26.20 (COrn-g),
3.1.4. (2S,4aS,8aR)-2-Bromo-8a-methyloctahydronaphthalen-
1(2H)-one (13)
d
Pyridinium bromide perbromide (PBB, 14.99 g 42 mmol, 90%)
was added to a solution of ketone 12 (6.98 g, 42 mmol) in acetic acid
(350 ml), and then the mixture was stirred for 3 h at rt. The mixture
was poured onto ice (200 mL) and extracted with Et2O (400 mL).
The organic phase was washed with saturated aqueous NaHCO3
until effervescence ceased. The combined organic phases were
dried (MgSO4), filtered, and concentrated to give 13 as a white solid
(10.29 g, 42.0 mmol, 97% purity, quant.) which was used without
further purification. The product was contaminated with a trace
-d
23.14 (C-10), 22.24 (C-12). HRMS m/z 365.2441 [MþH]þ (365.2435,
calc. for C20H33N2Oþ4 ).
3.1.3.15. tert-Butyl
3-((3S,6S,8S,8aS)-7,7,8a-trimethyl-2-oxo-
3,5,6,7,8,8a-hexahydro-2H-6,8-methanobenzo[b][1,4]oxazin-3-yl)
propanoate (7SLe). Compound 7SLe was obtained from the mixture
of 5SLe and its
D
-epimer 6SDe (82:18) using the general procedure
amount of the starting material. 1H NMR (600 MHz, CDCl3)
d ppm
for hydrogenation followed by flash chromatography using
toluene-Et2O with increasing amounts of Et2O as eluents to give
7SLe as a colourless amorphous solid (78.1%). ½aꢂ2D5 ¼ ꢃ190ꢁ (c 0.28).
5.03 (dd, Jaa ¼ 13.2 Hz, Jae ¼ 6.5 Hz, Hax-2), 2.62 (dddd, J ¼ 13.2, 6.5,
3.9, 2.8 Hz, Heq-3), 2.08 (qd, J ¼ 13.2, 4.8 Hz, Hax-3), 1.66e1.78 (m,
3H, H-4, H-5, H-8), 1.59e1.65 (m, 1H, H-7), 1.49e1.59 (m, 3H, H-4,
H-4a, H-8), 1.44e1.49 (m, 1H, H-6), 1.41 (tt, J ¼ 13.6, 4.0 Hz, 1H, H-7),
1.32e1.40 (m, 1H, H-6), 1.18 (qt, J ¼ 13.2, 4.3 Hz, 1H, H-5), 1.13 (s, 3H,
1H NMR (300 MHz, CDCl3)
d ppm 4.11e4.19 (m, 1H, Hendo-3), 2.87
(ddt, J ¼ 17.6, 3.8, 1.8, 1.8 Hz, 1H, Hexo-5), 2.72 (ddd, J ¼ 17.6, 3.8,
1.8 Hz, 1H, Hendo-5), 2.47e2.56 (m, 2H, HGlu ), 2.39e2.47 (m, 1H,
HGlu
), 2.36 (ddd, J ¼ 11.2, 5.9, 2.1 Hz,1H, Hexo-9), 2.07e2.26 (m,3H,
H-6, H-8, HGlu
-g
H-9). 13C NMR (151 MHz, CDCl3)
d ppm 205.67 (C-1), 54.62 (C-2),
-b
49.25 (C-8a), 46.09 (C-4a), 39.11 (C-3), 33.09 (C-8), 29.19 (C-4),
27.35 (C-6), 25.76 (C-5), 20.76 (C-7), 15.62 (C-9). HRMS m/z
245.0361 [MþH]þ (245.0536, calc. for C11H19BrOþ).
-b), 1.62 (s, 3H, H-12), 1.44 (s, 11H, OeC(CH3)3), 1.39 (s,
3H, H-11), 1.14 (d, J ¼ 11.2 Hz, 1H, Hendo-9), 1.05 (s, 3H, H-10). 13
C
NMR (151 MHz, CDCl3)
d
ppm 173.12 (C-4a), 172.81 (OeC ¼ OGlu),
171.72 (C-2), 85.43 (C-8a), 80.35 (OeC(CH3)3), 58.01 (C-3), 50.55 (C-
8), 39.74 (C-7), 39.48 (C-6), 37.12 (C-5), 31.56 (CGlu ), 28.31
), 23.14 (C-10),
3.1.4.1. (2S,4aS,8aR)-2-Hydroxy-8a-methyloctahydronaphthalen-
1(2H)-one (14). To a solution of the bromide 13 (10.55 g, 42 mmol)
in DMF (150 ml) aqueous NaOH (36.27 ml, 6%) was slowly added at
rt, and then the mixture was stirred for 2 h before neutralization at
-g
(OeC(CH3)3), 27.75 (C-9), 27.56 (C-11), 27.49 (CGlu-b
22.22 (C-12). HRMS m/z 358.1975 [MþH]þ (358.1989, calc. for
C
19H30NOþ4 ).
0
ꢁC by addition of 1% HCl. The mixture was extracted with Et2O,
and the organic phase washed with brine, dried (MgSO4), and
concentrated to yield a crude product (16.2 g), which was purified
by flash chromatography using heptane-acetic acid (100:1) with
increasing amounts of EtOAc to give 14 as a colourless oil (6.61 g,
36.3 mmol, 87%). ½aꢂD22 þ63ꢁ (c 0.29). 1H NMR (600 MHz, CDCl3)
3.1.3.16. 3-((3S,6S,8S,8aS)-7,7,8a-Trimethyl-2-oxo-3,5,6,7,8,8a-hex-
ahydro-2H-6,8-methanobenzo[b][1,4]oxazin-3-yl)propanoic acid
(7SLf). TFA (0.5 mL) was added dropwise to a cooled solution of
7SLf (103 mg, 307 mol) in DCM (1 mL) and the mixture was stirred
at rt for 2 h. Concentration of the reaction mixture followed by flash
chromatography using toluene-Et2O (4:1) with increasing amounts
of Et2O as eluents yielded 7SLf as colourless amorphous solid
(72.7 mg, 84.8%). ½aꢂD25 ¼ ꢃ187ꢁ (c 0.31). 1H NMR (300 MHz, CDCl3)
m
d
ppm 4.46 (dddd, J ¼ 12.0, 7.6, 3.8, 1.7 Hz, 1H, H-2), 3.65 (d,
J ¼ 3.8 Hz, 1H, OH), 2.37e2.46 (m, 1H, Heq-3), 1.65e1.73 (m, 3H, H-4,
H-5, H-8), 1.59e1.65 (m, 1H, H-7), 1.52e1.59 (m, 1H, H-8), 1.38e1.52
(m, 6H, Hax-3, H-4, H-4a, H-6, H-7), 1.14e1.24 (m, 1H, H-5), 1.11e1.14
d
ppm 4.15e4.23 (m, 1H, H endo-3), 2.92 (ddt, J ¼ 17.9, 3.8, 2.1, 2.1 Hz,
(m, 3H, H-9). 13C NMR (151 MHz, CDCl3)
d ppm 215.94 (C-1), 71.34
1H, Hexo-5), 2.78 (ddd, J ¼ 17.6, 4.1, 2.6 Hz, 1H, Hendo-5), 2.70 (t,
J ¼ 6.4, 2H, HGlu ), 2.47e2.59 (m,1H, HGlu ), 2.42 (dtd, J ¼ 11.4, 5.6,
5.6, 2.3 Hz, 1H, Hexo-9), 2.27e2.37 (m, 1H, HGlu
), 2.24 (t, J ¼ 5.7 Hz,
(C-2), 47.51 (C-8a), 47.02 (C-4a), 36.49 (C-3), 32.30 (C-8), 27.43 (C-
6), 26.22 (C-4), 25.89 (C-5), 20.47 (C-7), 15.52 (C-9). HRMS m/z
165.1278 [M þ H e H2O]þ (165.1274, calc. for C11H18Oþ).
-g
-b
-
b
1H, H-8), 2.12e2.19 (m, 1H, H-6), 1.67 (s, 3H, H-12), 1.41 (s, 3H, H-
11), 1.16 (d, J ¼ 11.1 Hz, 1H, H-9endo), 1.07 (s, 3H, H-10). 13C NMR
3.1.5. General procedure for acylation of 14
(151 MHz, CDCl3)
d
ppm 175.75 (HOeC]O), 175.25 (C-4a), 170.58
(2S,4aS,8aR)-2-Hydroxy-8a-methyloctahydronaphthalen-
1(2H)-one (14) (200 mg) was acylated according to the general
procedure for acylating hydroxy-pinan-2-ones. The crude product
(C-2), 85.72 (C-8a), 58.45 (C-3), 50.50 (C-8), 39.83 (C-7), 39.29 (C-6),
36.95 (C-5), 31.47 (CGlu-g), 27.83 (C-9), 27.46 (C-11), 26.33 (CGlu-b),
23.11 (C-10), 22.18 (C-12). HRMS m/z 280.1528 [MþH]þ (280.1544,
was purified by flash chromatography over silica gel (15e40 mm).
calc. for C15H22NOþ4 ).
3.1.5.1. (2S,4aS,8aR)-8a-Methyl-1-oxodecahydronaphthalen-2-yl 2-
3.1.3.17. (6S,8S,8aS)-7,7,8a-Trimethyl-3,5,6,7,8,8a-hexahydro-2H-
6,8-methanobenzo[b][1,4]oxazin-2-one (7Sg). Compound 7Sg was
obtained from 5Sg by using the general procedure for hydrogena-
tion followed by flash chromatography using toluene with
((benzyloxycarbonyl)-glycinate (15a). Compound 15a was prepared
from
(2S,4aS,8aR)-2-hydroxy-8a-methyloctahydronaphthalen-
1(2H)-one (14) and Na-Z-glycine using the general acylation pro-
cedure followed by flash chromatography using heptane with

130
C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
increasing amounts of EtOAc as eluents to yield 15a as a colorless oil
(77.7%). 1H NMR (600 MHz, CDCl3)
ppm 7.29e7.37 (m, 5H, Ph),
5.58 (dd, J ¼ 12.3, 7.2 Hz,1H, H-2), 5.24 (br s.,1H, NH), 5.09e5.16 (m,
2H, CH2-Ph), 4.16 (dd, J ¼ 18.3, 6.2 Hz, 1H, H- ), 4.07 (dd, J ¼ 18.1,
4.8 Hz, 1H, H- ), 2.28 (br s., 1H, Heq-3), 1.67e1.78 (m, 3H, H-5, H-4,
ax-3), 1.57e1.66 (m, 3H, H-8, H-7, H-4), 1.34e1.54 (m, 5H, H-8, H-7,
H-4a, H-2 H-6), 1.13e1.22 (m, 1H, H-5), 1.17 (s, 3H CH3). 13C NMR
(151 MHz, CDCl3) ppm 208.19 (C-1), 169.41 (C]O), 156.40 (CPh-1),
136.42 (CPh-4), 128.65 and 128.28 (CPh-2 and CPh-6), 128.22 (CPhe-3
and CPhe-5), 74.65 (C-2), 67.20 (C- ), 48.50 (C-8a), 46.41 (C-4a),
J ¼ 11.9, 7.2 Hz, 1H), 5.18 (d, J ¼ 8.4 Hz, 1H), 5.07 (d, J ¼ 12.1 Hz, 1H),
5.03 (t, J ¼ 12.1 Hz, 1H), 4.78 (dt, J ¼ 7.7, 5.9 Hz, 1H), 3.54 (dd,
J ¼ 15.0, 5.1 Hz, 1H), 3.35 (dd, J ¼ 15.0, 6.2 Hz, 1H), 2.21e2.27 (m,
1H), 1.55e1.80 (m, 6H), 1.35e1.53 (m, 4H), 1.15e1.33 (m, 2H), 1.19 (s,
d
a
a
3H). 13C NMR (151 MHz, CDCl3)
d ppm 208.70, 171.45, 156.09,
H
136.49, 136.22, 128.59, 128.20, 123.81, 122.19, 119.77, 118.81, 111.26,
110.01, 74.37, 66.97, 54.63, 48.62, 46.47, 32.42, 27.82, 27.56, 26.61,
26.06, 22.84, 20.63, 15.20. For assignment see 15a.
d
a
3.1.6. General procedure for oxazinonization of 15
42.78 (C-13), 32.30 (C-3), 32.21 (C-8), 27.52 (C-6), 26.52 (C-4), 26.01
(C-5), 20.56 (C-7), 15.09 (CH3).
The general procedure for oxazinonization of 15 (200 mg) to
give compounds 16 is the same as used for preparation of the 2-
hydroxy-pinan-3-one derived iminolactones 7.
3.1.5.2. (2S,4aS,8aR)-8a-Methyl-1-oxodecahydronaphthalen-2-yl 2-
((benzyloxycarbonyl)-
pared from (2S,4aS,8aR)-hydroxy-8a-methyloctahydronaphthalen-
1(2H)-one (14) and Na-Z-
-alanine using the general acylation
D
-alaninate (15b). Compound 15b was pre-
3.1.6.1. (4aS,6aS,10aR)-10a-Methyl-2,4a,5,6,6a,7,8,9,10,10a-decahy-
dro-3H-naphtho[2,1-b][1,4]oxazin-3-one (16a). Compound 16a was
prepared from 15a by using the general hydrogenation procedure
followed by flash chromatography using heptane with increasing
amounts of EtOAc as eluents to yield an oil which precipitated from
EtOAc-heptane to give 16a as a colorless amorphous powder
(49.0 mg, 25%). ½aꢂD25 ¼ 15.2ꢁ (c 0.18). 1H NMR (600 MHz, CD3CN)
D
procedure followed by flash chromatography using heptane with
increasing amounts of EtOAc to yield 15b as a colorless oil (78.0%).
1H NMR (600 MHz, CDCl3)
d ppm 7.29e7.38 (s, 5H), 5.53 (dd,
J ¼ 11.6, 7.5 Hz, 1H), 5.35 (d, J ¼ 6.6 Hz, 1H, NH), 5.18e5.16 (m, 2H),
4.51 (quint, J ¼ 7.1 Hz, 1H), 2.21e2.27 (m, 1H), 1.67e1.78 (m, 3H),
1.56e1.67 (m, 3H), 1.34e1.53 (m, 5H), 1.48 (d, J ¼ 7.3 Hz, 3H, Ala-
Me), 1.18 (qt, J ¼ 13.2, 4.0 Hz, 1H), 1.17 (s, 3H). 13C NMR (151 MHz,
d
ppm 5.21 (dddd, J ¼ 12.2, 6.3, 3.9, 2.2 Hz, 1H, H-4a), 4.29 (dd,
J ¼ 21.7, 2.1 Hz, 1H, H-2), 4.10 (dd, J ¼ 21.7, 4.0 Hz, 1H, H-20), 2.29 (m,
J ¼ 3.7 Hz, 1H, H-5), 1.40e1.75 (m, 9H, H-50, H-6, H-7, 2H-8, 2H-9,
2H-10), 1.32e1.39 (m, 2H H-7, H-6a), 1.17e1.26 (m, 1H, H-60), 1.04 (s,
CDCl3)
d ppm 208.00, 172.02, 155.66, 136.55, 128.65, 128.26, 74.49,
66.98, 50.00, 48.52, 46.39, 32.32, 32.14, 27.55, 26.53, 26.04, 20.59,
3H, CH3). 13C NMR (151 MHz, CD3CN)
d ppm 173.75 (C-10b), 166.92
19.01 (Ala), 15.10. For assignment see 15a.
(C-3), 77.65 (C-4a), 49.81 (C-2), 46.05 (C-6a), 44.46 (C-10a), 35.47
(C-10), 34.94 (C-5), 28.49 (C-7), 27.07 (C-6), 26.93 (C-8), 21.75 (C-9),
16.86 (CH3). HRMS m/z 240.1622 [M þ H3O]þ (240.1594, calc. for
3.1.5.3. (S)-(2S,4aS,8aR)-8a-Methyl-1-oxodecahydronaphthalen-2-yl
2-((benzyloxycarbonyl)-
was prepared from (2S,4aS,8aR)-hydroxy-8a-methyloctahy-
dronaphthalen-1(2H)-one (14) and Na-Z-
-phenylalanine using the
L
-phenylalaninate (15c). Compound 15c
C
13H20NOþ2 ).
L
3.1.6.2. (2S,4aS,6aS,10aR)-2,10a-Dimethyl-2,4a,5,6,6a,7,8,9,10,10a-
decahydro-3H-naphtho[2,1-b][1,4]oxazin-3-one (16b).
general acylation procedure followed by flash chromatography
using heptane with increasing amounts of EtOAc as eluents to yield
Compound 16b was prepared from 15b by using the general hy-
drogenation procedure to give 16b (97.3%) as a colorless oil.
15c as a colorless oil (73.9%). 1H NMR (600 MHz, CDCl3)
d ppm
7.21e7.35 (m, 10H), 5.59 (dd, J ¼ 12.3, 7.2 Hz, 1H), 5.10 (d, J ¼ 8.4 Hz,
1H), 5.00e5.08 (m, 2H), 4.70 (td, J ¼ 8.1, 5.5 Hz, 1H, Phe), 3.41 (dd,
J ¼ 14.3, 5.1 Hz, 1H, Phe), 3.09 (dd, J ¼ 14.3, 8.1 Hz, 1H, Phe),
2.23e2.32 (m, 1H), 1.56e1.80 (m, 6H), 1.35e1.54 (m, 5H), 1.14e1.24
½aꢂD25 þ41.8ꢁ (c 0.50). 1H NMR (600 MHz, CD3CN)
d ppm 5.23 (ddd,
J ¼ 11.8, 6.7, 1.7 Hz, 1H, H-4a), 4.29 (qd, J ¼ 7.4, 1.8 Hz, 1H, H-2),
2.26e2.31 (m, 1H, Heq-5), 1.74e1.78 (m, 1H, H-10), 1.67e1.72 (m, 1H,
H-8), 1.31e1.67 (m, 9H, H-5ax, H-6, H-6a, 2H-7, H-8, 2H-9, H-10),
1.34 (d, J ¼ 7.4 Hz, 3H, Ala-Me), 1.14e1.26 (m, 1H, H-6), 1.02 (s, 3H,
(m,1H),1.19 (s, 3H), 13C NMR (151 MHz, CDCl3)
d ppm 208.42,171.27,
155.95, 136.40, 136.22, 129.59, 128.67, 128.63, 128.25, 128.18, 127.08,
74.42, 67.06, 54.77, 48.59, 46.45, 38.32 (Phe), 32.37, 27.56, 26.59,
26.05, 22.85, 20.62, 15.16. For assignment see 15a.
CH3). 13C NMR (151 MHz, CD3CN)
d ppm 172.68 (C-10b), 170.26 (C-
3), 77.17 (C-4a), 54.43 (C-2), 45.91 (C-6a), 44.23 (C-10a), 35.49 (C-
10), 34.96 (C-5), 28.55 (C-7), 27.00 (C-6), 26.94 (C-8), 21.77 (C-9),
19.96 (CAla
-
b
), 17.03 (CH3). HRMS m/z 236.1651 [MþH]þ (236.1645,
3.1.5.4. (2S,4aS,8aR)-8a-Methyl-1-oxodecahydronaphthalen-2-yl 2-
calc. for C14H22NOþ2 ).
((benzyloxycarbonyl)-
prepared from (2S,4aS,8aR)-hydroxy-8a-methyloctahydronaph-
thalen-1(2H)-one (14) and Na-Z-
-phenylalanine using the general
D
-phenylalaninate (15d). Compound 15d was
3 . 1. 6 . 3 . ( 2 S , 4 a S , 6 a S , 10 a R ) - 2 - B e n z y l - 1 0 a - m e t h y l -
2,4a,5,6,6a,7,8,9,10,10a-decahydro-3H-naphtho[2,1-b][1,4]oxazin-3-
one (16c). Compound 16c was prepared from 15c using the general
hydrogenation procedure to give 16c in quantitative yield as a
colorless oil. ½aꢂD25 ¼ 39.2ꢁ (c 0.26). 1H NMR (600 MHz, CD3CN)
D
acylation procedure followed by flash chromatography using hep-
tane with increasing amounts of EtOAc as eluents to yield 15d as a
colorless oil (71.7%). 1H NMR (400 MHz, CDCl3)
d ppm 7.11e7.39 (m,
10H), 5.51 (dd, J ¼ 11.7, 6.6 Hz,1H), 5.27 (d, J ¼ 8.1 Hz,1H), 5.05e5.13
(m, 2H), 4.77 (q, J ¼ 6.8 Hz, 1H), 3.23 (dd, J ¼ 14.1, 5.6 Hz, 1H), 3.13
(dd, J ¼ 14.1, 6.6 Hz, 1H), 2.13e2.22 (m, 1H), 1.32e1.78 (m, 11H),
1.11e1.24 (m, 1H), 1.15 (s, 3H). For assignment see 15a.
d ppm 7.25e7.32 (m, 2H, HPhe-2 and 6), 7.20e7.23 (m, 1H, HPh-4),
7.11e7.15 (m, 2H, HPh-3 and 5), 4.99 (ddd, J ¼ 12.6, 6.3, 3.5 Hz,1H, H-
4a), 4.47 (td, J ¼ 4.9, 3.5 Hz, 1H, H-2), 3.31 (dd, J ¼ 13.2, 5.1 Hz, 1H,
CH2-Ph), 3.22 (dd, J ¼ 13.2, 4.8 Hz, 1H, CH2-Ph), 1.83 (dddd, J ¼ 12.1,
6.4, 3.8, 2.8 Hz, 1H H-5eq), 1.59e1.71 (m, 4H, H-8, 2H-10, H-9),
1.43e1.53 (m, 1H, H-90), 1.38 (tdd, J ¼ 13.9, 13.9, 12.5, 3.5 Hz, 1H, H-
6), 1.16e1.34 (m, 4H, H2-7, H-8, H-60), 1.05e1.11 (m, 1H, H-6a), 0.98
(s, 3H, CH3), 0.23 (dtd, J ¼ 13.6, 12.1, 12.1, 4.8 Hz, 1H, Hax-5). 13C NMR
3.1.5.5. (2S,4aS,8aR)-8a-Methyl-1-oxodecahydronaphthalen-2-yl 2-
((benzyloxycarbonyl)-
prepared from (2S,4aS,8aR)-hydroxy-8a-methyloctahydronaph-
thalen-1(2H)-one (14) and Na-Z-
-tryptophan using the general
L
-tryptophanate (15e). Compound 15e was
L
(151 MHz, CD3CN) d ppm 172.85 (C-10b), 168.60 (C-3), 137.78 (CPh-
acylation procedure followed by flash chromatography using hep-
tane with increasing amounts of EtOAc as eluents to yield 15e as a
1), 131.50 (CPh-2 and 6), 128.94 (CPh-3 and 5), 127.74 (CPh-4), 77.99
(C-4a), 59.45 (C-2), 45.96 (C-6a), 44.39 (C-10a), 40.52 (CH2-Ph),
35.44 (C-10), 34.91 (C-5), 28.46 (C-7), 27.10 (C-6), 27.02 (C-8), 21.81
(C-9), 16.52 (CH3). HRMS m/z 312.1974 [MþH]þ (312.1958, calc. for
white powder (24.5%). 1H NMR (600 MHz, CDCl3)
d ppm 8.04 (br. s.,
1H, NH), 7.59 (d, J ¼ 8.1 Hz, 1H, HIn-4), 7.27e7.37 (m, 6H, HIn-7), 7.18
(t, J ¼ 7.3 Hz, 1H, HIn-2), 7.08 (t, J ¼ 7.7 Hz, 1H, HIn-6), 5.58 (dd,
C
20H26NOþ2 ).

C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
131
3.1.6.4. (6aS,10aR)-2-Benzyl-10a-methyl-5,6,6a,7,8,9,10,10a-octahy-
dro-3H-naphtho[2,1-b][1,4]oxazin-3-one (17). When compound 16c
was adsorbed on celite and eluted as described for 15c degradation
was observed to give compound 17 and 18. Compound 17: 1H NMR
H
ax-5). 13C NMR (151 MHz, CD3CN)
d
ppm 172.41 (C-10b), 169.39 (C-
3), 136.89 (CIn-7a), 129.10 (CIn-3a), 125.08 (CIn-2), 122.32 (CIn-6),
120.75 (CIn-4), 119.77 (CIn-5), 111.97 (CIn-7), 110.92 (CIn-3), 77.88 (C-
4a), 60.03 (C-2), 45.14 (C-6a), 44.20 (C-10a), 34.94 (C-5), 34.82 (C-
10), 30.22 (CH2eIn), 28.43 (C-7), 27.15 (C-6), 26.72 (C-8), 21.82 (C-
9), 16.38 (C-11). HRMS m/z 351.2079 [MþH]þ (351.2067, calc. for
(600 MHz, CDCl3)
d
ppm 7.36 (d, J ¼ 7.7 Hz, 2H, HPhe-2 and 6), 7.29
(t, J ¼ 7.5 Hz, 2H, HPhe-3 and 5), 7.22 (t, J ¼ 7.7 Hz, 1H, HPhe-4), 4.02
(dd, J ¼ 18.3, 13.9 Hz, 2H, CH2-Phe), 2.59 (ddd, J ¼ 18.7, 11.0, 7.7 Hz,
1H, H-5), 2.45 (dd, J ¼ 18.7, 6.6 Hz, 1H, H-5), 2.28 (d, J ¼ 13.2 Hz, 1H,
C
22H27N2Oþ2 ).
H
eq-10), 1.78 (d, J ¼ 12.5 Hz, 1H, H-8), 1.51e1.68 (m, 4H, H-6 and H-
3.1.7. 2-Bromo-3,4-dihydronaphthalen-1(2H)-one (19)
9), 1.41e1.47 (m, 2H, H-6a, H-7), 1.37 (qd, J ¼ 12.8, 3.3 Hz, 1H, H-7),
1.29 (qt, J ¼ 12.8, 4.4 Hz, 2H, H-8), 1.14 (td, J ¼ 13.3, 4.2 Hz, 1H, Hax
10), 1.06 (s, 3H, H-11). 13C NMR (151 MHz, CDCl3)
ppm 154.89 (C-
Freshly recrystallized N-bromosuccinimide (0.91 g, 5.1 mmol)
was added to a solution of p-toluenesulfonic acid (1.53 g, 8.1 mmol)
and 1-tetralone (0.742 g, 5.1 mmol) in acetonitrile (60 mL), and
then the solution was refluxed for 22 h. After concentration, the
residue was dissolved in DCM and washed with water. The organic
phase was concentrated and 19 was isolated from the residue by
flash chromatography using heptane-toluene (1:1 with increasing
amounts of toluene) as eluent to give 19 (0.941 g, 82%). 1H NMR
-
d
3), 152.75 (C-2) 147.83 (C-4a), 136.62 (CPhe-1 and C-10b), 129.59
(CPhe-2 and 6) 128.55 (CPhe-3 and 5) 126.82 (CPhe-4), 43.07 (C-6a),
40.00 (CH2-Phe), 37.07 (C-10a), 35.25 (C-10), 28.00 (C-7), 26.80 (C-
8), 26.60 (C-5), 24.34 (C-6), 21.46 (C-9), 18.71 (C-11). HRMS m/z
310.1821 [MþH]þ (310.1802, calc. for C20H24NOþ2 ).
(300 MHz, CDCl3)
d
ppm 8.10 (dd, J ¼ 7.8, 1.5 Hz, 1H, H-8), 7.53 (td,
3.1.6.5. (4aS,8aR)-2-Hydroxy-8a-methyl-4a,5,6,7,8,8a-hexahy-
dronaphthalen-1(4H)-one (18). When compound 16c was adsorbed
on celite and eluted as describedfor 15c degradation was observed
J ¼ 7.6, 1.5 Hz, 1H, H-6), 7.36 (ddd, J ¼ 7.8, 7.6, 1.0 Hz, 1H, H-7), 7.29
(dd, J ¼ 7.6, 1.0 Hz, 1H, H-5), 4.74 (t, J ¼ 4.1 Hz, 1H, H-2), 3.33 (ddd,
J ¼ 17.0, 10.0, 5.0 Hz, 1H, H-3), 2.93 (dt, J ¼ 17.0, 4.4 Hz, H-30),
2.41e2.60 (m, 2H, H-4).
to give 17 and 18. Compound 18: 1H NMR (600 MHz, CD3CN)
d ppm
6.15 (s, 1H, OH), 5.94e5.96 (m, 1H, H-3), 2.10e2.15 (m, 2H, H-4, H-
5), 1.83e1.91 (m, 2H, H-8, H-4a), 1.66e1.71 (m, 1H, H-6), 1.58e1.63
(m, 1H, H-7), 1.41e1.51 (m, 2H, H-4, H-5, H-7), 1.29 (td, J ¼ 13.7,
3.9 Hz, 1H, H-8), 1.17e1.26 (m, 1H, H-6), 1.03 (s, 3H, H-9). 13C NMR
3.1.7.1. 1-Oxo-1,2,3,4-tetrahydronaphthalen-2-yl
ycarbonyl)- -phenylalaninate (20a). A solution of 19 (0.566 g,
2.5 mmol), Boc-protected -Phe (798 mg, 3.0 mmol) and diisopro-
(tert-butox-
L
L
(151 MHz, CD3CN)
d
ppm 202.52 (C-1), 146.16 (C-2), 116.59 (C-3),
pylethylamine (456 mg, 3.5 mmol) in MeCN (13 mL) was refluxed
for 23 h. After concentration the residue was dissolved in DCM and
washed with water. The organic phase was concentrated and the
residue was purified by flash chromatography using heptane-EtOAc
(1:7 with increasing amounts of EtOAc) to give the epimer mixture
20a (0.55 g, 54%) with the two epimers in the ratio 1:1. 1H NMR
44.74 (C-8a), 42.81 (C-4a), 33.07 (C-8), 28.55 (C-4), 28.37 (C-5),
26.43 (C-6), 21.88 (C-7), 14.88 (CH3). HRMS m/z 163.1117 [M þ H e
H2O]þ (163.1069, calc. for C11H17Oþ2 ).
3 . 1. 6 . 6 . ( 2 R , 4 a S , 6 a S , 10 a R ) - 2 - B e n z y l - 10 a - m e t h y l -
2,4a,5,6,6a,7,8,9,10,10a-decahydro-3H-naphtho[2,1-b][1,4]oxazin-3-
one (16d). Compound 16d was prepared from 15d by using the
general hydrogenation procedure to give 16d (quant.) as a colorless
(300 MHz, CDCl3)
d
ppm 8.05 (dd, J ¼ 7.4, 1.2 Hz, 1H, H-8), 8.02 (dd,
J ¼ 7.4, 1.4 Hz, 1H H-80), 7.53 (td, J ¼ 7.4, 1.4 Hz, 1H, H-6), 7.52 (td,
J ¼ 7.4, 1.4 Hz, 2H H-7 and H-60), 7.21 e 7-39 (m, 14H), 5.62 (dd,
J ¼ 13.2, 5.3 Hz, 1H, H-2), 5.53 (dd, J ¼ 12.0, 6.2, 1H, H-20), 5.08 (d,
J ¼ 8.2 Hz,1H, NH), 4.95 (d, J ¼ 8.8 Hz,1H, NH), 4.65e4.80 (m, 2H, H-
oil. ½aꢂ2D5 ¼ 53.7ꢁ (c 0.34). 1H NMR (400 MHz, CD3CN)
d ppm
7.16e7.35 (m, 3H, HPh-2, 6 and 4), 7.08e7.13 (m, 2H, HPh-3 and 5),
4.61 (td, J ¼ 5.3, 2.0 Hz, 1H, H-2), 4.41 (ddd, J ¼ 11.7, 6.7, 1.8 Hz, 1H,
a
and H-
a
0), 3.39 (dd, J ¼ 14.1, 5.2 Hz, 1H, H-
b), 3.02e3.31 (m, 7H),
H
ax-4a), 3.22 (dd, J ¼ 13.4, 5.6 Hz, 1H, CH2-Ph), 3.11 (dd, J ¼ 13.4,
2.19e2.48 (m, 4H), 1.42 (s, 9H, (CH3)3), 1.40 (s, 9H, (CH3)3).
4.9 Hz, 1H, CH2-Ph), 2.09e2.16 (m, 1H, Heq-5), 1.75e1.81 (m, 1H, H-
10ax), 1.63e1.70 (m, 1H, H-8), 1.56e1.62 (m, 1H, H-9), 1.33e1.55 (m,
6H, H-5, H-6, H-7, H-8, H-9, Heq-10), 1.25e1.33 (m, 2H, Hax-6a, H-7),
1.12e1.20 (m, 1H, H-6) 0.83 (s, 3H, H-11). 13C NMR (101 MHz,
3.1.7.2. 1-Oxo-1,2,3,4-tetrahydronaphthalen-2-yl
ycarbonyl)- -tryptophanate (20b). A solution of 19 (366 mg,
1.6 mmol), Boc-protected -Trp (612 mg, 2.0 mmol) and diisopro-
(tert-butox-
L
L
CD3CN)
d
ppm 173.42 (C-10b), 168.84 (C-3), 137.53 (CPh-1), 131.02
pylethylamine (305 mg, 1.4 mmol) in MeCN (8 mL) was refluxed for
23 h. After concentration the residue was dissolved in DCM and
washed with water. The organic phase was concentrated and the
residue was purified by flash chromatography using heptane-EtOAc
(1:7 with increasing amounts of EtOAc) as elunt to give the epimer
mixture 20b (0.48 g, 66%) with the two epimers in the ratio 1:1. 1H
(CPh-2 and 6), 129.15 (CPh-3 and 5), 127.80 (CPh-4), 77.25 (C-4a),
59.77 (C-2), 45.91 (C-6a), 44.32 (C-10a), 40.45 (CH2-Ph), 35.73 (C-
10), 35.19 (C-5), 28.43 (C-7), 26.93 (C-6), 26.90 (C-8), 21.76 (C-9),
16.92 (C-11). HRMS m/z 312.1973 [MþH]þ (312.1958, calc. for
C
20H26NOþ2 ).
NMR (300 MHz, CDCl3)
d
ppm 8.12 (br.s., 2H, NH) 8.05 (dd, J ¼ 7.9,
3.1.6.7. (2S,4aS,6aS,10aR)-2-((1H-Indol-3-yl)methyl)-10a-methyl-
2,4a,5,6,6a,7,8,9,10,10a-decahydro-3H-naphtho[2,1-b][1,4]oxazin-3-
one (16e). Compound 16e was prepared from 15e by using the
general hydrogenation procedure to give 16e in quantitative yield
as a colorless powder. ½aꢂ2D5 ¼ ꢃ25.5ꢁ (c 0.24). 1H NMR (400 MHz,
1.0 Hz, 1H, H-8), 8.01 (dd, J ¼ 7.9, 1.0 Hz, 1H, H-80), 7.65 (d, J ¼ 7.6 Hz,
1H, H-6). 7.63 (d, J ¼ 7.6 Hz, H-60), 7 53 (td, J ¼ 7.4, 1.0 Hz, 1H, H-7),
7.51 (td, J ¼ 7.4, 1.4 Hz, 2H H-6 and H-60), 7.09 e 7-45 (m, 12H), 5.62
(dd, J ¼ 12.8, 5.8 Hz, 1H, H-2), 5.48 (dd, J ¼ 12.0, 6.4, 1H, H-20), 5.17
(d, J ¼ 8.0 Hz,1H, NH), 5.05 (d, J ¼ 8.0 Hz,1H, NH), 4.73e4.84 (m, 2H,
CD3CN)
d
ppm 9.14 (br s., 1H, NH), 7.56 (d, J ¼ 7.8 Hz, 1H, HIn-4), 7.34
H-
a
and H-
a
0), 3.55 (dd, J ¼ 15.0, 5.2 Hz,1H, H-
b), 3.31e3.49 (m, 3H),
(dt, J ¼ 7.8,1.0 Hz,1H, HIn-7), 7.09 (ddd, J ¼ 7.8, 7.1,1.5 Hz,1H, HIm-6),
7.03 (ddd, J ¼ 7.8, 7.1,1.2 Hz,1H, HIn-5), 6.97 (d, J ¼ 2.4 Hz,1H, HIn-2),
4.92 (ddd, J ¼ 12.5, 6.3, 3.4 Hz, 1H, Hax-4a), 4.48 (td, J ¼ 4.4, 3.4 Hz,
1H, H-2), 3.48 (ddd, J ¼ 14.2, 4.5, 0.6 Hz, 1H, CH2eIn), 3.38 (ddd,
J ¼ 14.2, 4.4, 0.5 Hz, 1H, CH2eIn), 1.65e1.79 (m, 2H, Heq-5, H-10),
1.52e1.64 (m, 3H, H-8, H-9, H-10), 1.41e1.49 (m, 1H, H-9), 1.26 (qd,
J ¼ 13.4, 3.9 Hz, 1H, H-6) 1.09e1.21 (m, 3H, 2 H-7, H-8) 0.94e0.99
(m, 1H, H-6), 0.93 (d, J ¼ 0.7 Hz, 3H, H-11), 0.50 (dddd, J ¼ 15.4, 6.8,
5.7, 3.3 Hz, 1H, Hax-6a), ꢃ0.03 (dtd, J ¼ 13.2, 12.2, 12.2, 4.9 Hz, 1H,
2.97e3.26 (m, 4H), 2.13e2.42 (m, 4H), 1.49 (s, 9H, (CH3)3), 1.41 (s,
9H, (CH3)3).
3.1.7.3. (2S,4aR)-2-benzyl-2,4a,5,6-tetrahydro-3H-naphtho[2,1-b]
[1,4]oxazin-3-one (22a) and (2S,4aS)-2-benzyl-2,4a,5,6-tetrahydro-
3H-naphtho[2,1-b][1,4]oxazin-3-one (21a). TFA (3 mL) was added to
a solution of 20a (520 mg, 1.3 mmol) in DCM (3 mL), and then the
mixture was stirred for 30 min. After concentration the residue was
dissolved in DCM and the solution washed with saturated aq.

132
C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
Na2CO3. The organic phase was dried (MgSO4) and concentrated in
vacuo. The residue (391 mg) was purified by HPLC (20 ml/min,
gradient: 40% MeCN 60% water to 80% MeCN over 30 min) afforded
21a (154 mg, 20%) as a colorless powder and 22a (150 mg, 20%) as a
colorless powder. 22a ½aꢂ2D5 ¼ ꢃ10.5ꢁ (c 0.17). 1H NMR (300 MHz,
0.7 Hz, 1H, CH2In), 2.96e2.85 (overlaid, 2H, H-6), 2.44 (ddt, J ¼ 12.3,
5.9, 3.8, 1H, H-5), 1.84 (dddd, J ¼ 12.4, 12.2, 12.3, 5.5, Hz, 1H, H-50).
13C NMR (100 MHz, CD3CN)
d ppm 171.5 (C-3), 165.0 (C-10b), 141.3
(C-6a),137.1 (CIn-7a), 132.1 (C-8), 131.7 (C-10a),129.5 (C-7), 127.7 (C-
9),126.1 (C-10),129.1 (CIn-3a),124.8 (CIn-2),122.2 (CIn-6),120.2 (CIn-
4), 119.6 (CIn-5), 112.9 (CIn-3), 112.1 (CIn-7), 76.0 (C-4a), 62.1 (C-2),
29.4 (C-5), 28.1. (CH2eIn), 27.3 (C-6). HRMS m/z 331.1441 [MþH]þ
(331.1441, calc. for C21H19N2Oþ2 ).
CD3CN)
d
ppm 8.11 (dd, J ¼ 7.9, 1.3 Hz, 1H, H-10), 7.41e7.46 (m, 2H,
HPh-2 and 6), 7.19e7.40 (m, 5H, H-7-H-10, HPh- 3 and 5, HPh-4), 5.10
(ddd, J ¼ 12.2, 5.9, 3.0 Hz, 1H, H-4a), 4.32 (ddd, J ¼ 8.2, 4.5, 3.2, 1H,
H-2), 3.53 (dd, J ¼ 14.1, 4.5 Hz, 1H, CH2-Ph), 3.24 (dd, J ¼ 14.1, 8.2 Hz,
1H, CH02-Ph), 2.93 (ddd, J ¼ 16.5, 5.0, 3.5 Hz, 1H, H-6), 2.88 (ddd,
J ¼ 16.5, 12.6, 3.8 Hz, 1H, H-60), 2.47 (dddd, J ¼ 12.3, 5.9, 3.8, 3.5 Hz,
1H, H-5), 1.90 (dddd, J ¼ 12.6, 12.3, 12.2, 5.2, Hz, 1H, H-50). 13C NMR
3.2. X-ray crystallography
Single crystals of 7SLc suitable for X-ray diffraction studies were
grown from a solution in ethyl acetate and heptane. Data were
(75 MHz, CD3CN)
d
ppm 171.3 (C-3), 165.2 (C-1), 141.4 (C-6a), 140.0
(CPh-1), 132.3 (C-8), 131.6 (C-10a), 131.0 (CPh-2 and 6), 129.5 (C-7),
129.0 (CPh-3 and 5), 127.8 (CPh-4), 127.2 (C-9), 126.0 (C-10), 76.0 (C-
4a), 62.2 (C-2), 38.3 (C-Ph), 29.3 (C-5), 27.2 (C-6). HRMS m/z
292.1325 [MþH]þ (292.1332, calc. for C19H19NOþ2 ).
collected (T ¼ 123(1)K), using graphite-monochromated MoK
a
radiation (
l
¼ 0.71073 Å) on a Bruker D8 Venture diffractometer.
Data collection and cell refinement were performed using the
Bruker Apex2 Suite software [25]. Data reduction using SAINT [26]
and multi-scan correction for absorption using SADABS-2012/1 [27]
were performed within the Apex2 Suite. The crystal data, data
collection and the refinement data are given in Table S1 (Supple-
mentary data).
22a ½aꢂ2D5 ¼ ꢃ16.0ꢁ (c 0.09). 1H NMR (300 MHz, CD3CN)
d ppm
8.18 (dd, J ¼ 8.0, 1.2 Hz, 1H, H-10), 7.42 (td, J ¼ 7.4, 1.3, 1H, H-8), 7.32
(ddd, J ¼ 8.0, 7.4, 1.1, 1H, H-9), 7.25e7.19 (m, 6H, H-7, Ph), 4.96 (ddd,
J ¼ 12.2, 5.9, 3.0 Hz, 1H, H-2), 4.07 (ddd, J ¼ 13.0, 5.2, 1.4, 1H, H-4a),
3.28 (dd, J ¼ 13.5, 6.2 Hz, 1H, CH2-Ph), 3.16 (dd, J ¼ 13.5, 5.5 Hz, 1H,
CH2-Ph), 2.93 (ddd, J ¼ 17.0, 5.0, 2.6 Hz, 1H, H-6), 2.79 (ddd, J ¼ 17.0,
13.0, 4.4 Hz, 1H, H-60), 2.25 (dddd, J ¼ 12.2, 5.2, 4.4, 2.6 Hz, 1H, H-5),
1.90 (tdd, J ¼ 13.0, 12.2, 5.0, Hz, 1H, H-50). 13C NMR (75 MHz, CD3CN)
3.3. Structure solution and refinement
Positions of all non-hydrogen atoms were found by direct
methods (SHELXS97) [28]. Full-matrix least-squares refinements
(SHELXL97) [28] were performed on F2, minimizing Sw(Fo2 e kFc2)2.
The position of hydrogen atoms were included in calculated posi-
tion with fixed isotropic displacement parameters (Uiso ¼ 1.2Ueq for
CH, and CH2 and Uiso ¼ 1.5Ueq for CH3), except for the hydrogen
atoms bonded to chiral CH or NH. They were refined with fixed
isotropic displacement parameters (Uiso ¼ 1.2Ueq). Refinement (263
parameters, 4585 unique reflections) converged at RF ¼ 0.0372,
d
ppm 170.0 (C-3), 163.0 (C-10b), 137.5 (CPh-1), 132.3 (C-8), 131.6 (C-
10a), 130.8 (CPh-2 and 6), 129.6 (C-7), 129.3. (CPh-3 and 5), 128.0
(CPh-4), 127.7 (C-9), 126.4 (C-10), 76.8 (C-4a), 62.5 (C-2), 39.5 (CH2-
Ph), 30.4 (C-5), 27.8 (C-6). HRMS m/z 292.1328 [MþH]þ (292.1332,
calc. for C19H19NOþ2 ).
3.1.7.4. (2S,4aR)-2-[(1H-indol-3-yl)methyl]-5,6-dihydro-2H-naphto
[2,1-b][1,4]oxazin-3(4aH)-one (22b) and (2S,4aS)-2-[(1H-indol-3-yl)
methyl]-5,6-dihydro-2H-naphto[2,1-b][1,4]oxazin-3(4aH)-one (21b)..
TFA (2.3 mL) was added to a solution of 20b (482 mg, 1.1 mmol) in
DCM (2.3 mL) and stirred for 30 min. After concentration the res-
idue was dissolved in DCM and the solution washed with a satu-
rated aqueous solution of Na2CO3. The organic phase was dried
(MgSO4) and concentrated in vacuum. Preparative HPLC of the
residue (355 mg) (20 ml/min, gradient: 40% MeCN 60% water to 80%
MeCN over 30 min) afforded 21b (54 mg, 15%) as a colorless powder
and 22b (72 mg, 20%)) as a colorless powder.
wR2F
¼
0.0766 [4015 reflections with Fo
>
4s(Fo);
wꢃ1 ¼ (
s
2(Fo2) þ (0.0300P)2 þ 0.1854P), where P ¼ (Fo2 þ 2F2c)/3;
S ¼ 1.044]. The residual electron density varied between ꢃ0.18 and
0.22 e Åꢃ3. Non-centrosymmetric space group is assigned, but the
absolute configuration cannot be determined (Flack ¼ 0.0(9) [29]).
However the chirality for three chiral centers is known, and the
fourth center is assigned relative to those. Complex scattering
factors for neutral atoms were taken from International Tables for
Crystallography as incorporated in SHELXL97 [28,30]. Crystallo-
graphic data for compound 7SLc have been deposited with the
Cambridge Crystallographic Data Centre (deposition number CCDC
1404090). Copies of the data can be obtained, free of charge, on
application to the director, CCDC, 12 Union Road, Cambridge
CB21EZ, UK (fax: þ44 1223 336033 or e-mail: deposit@ccdc.cam.ac.
uk).
22b ½aꢂ2D5 ¼ þ8ꢁ (c 0.09). 1H NMR (400 MHz, CD3CN)
d ppm 9.16
(br.s., 1H, NH), 8.22 (dd, J ¼ 7.9, 1.2 Hz, 1H, H-10), 7.49 (dd, J ¼ 8.1,
0.5 Hz, 1H, HIn-4) 7.39 (ddd, J ¼ 7.6, 7.3, 1.2 Hz, 1H, H-8), 7.34 (dd,
J ¼ 8.2, 0.8 Hz, 1H, HIn-7), 7.32 (ddd, J ¼ 7.9, 7.3, 0.6, 1H, H-9), 7.17
(dd, J ¼ 7.6, 0.5 Hz, 1H, H-7), 7.05 (ddd, J ¼ 8.0, 7.2, 0.6, 1H, HIn-6),
6.97 (d, J ¼ 2.3, 1H, HIn-2), 4.99 (ddd, J ¼ 5.5, 5.1, 1.4 Hz, 1H, H-2),
3.93 (ddd, J ¼ 13.1, 5.1, 1.2, 1H, H-4a), 3.50 (dd, J ¼ 14.5, 5.5 Hz, 1H,
CH2eIn), 3.32 (dd, J ¼ 14.5, 5.1 Hz, 1H, CH2eIn), 2.85 (ddd, J ¼ 17.1,
4.5, 2.5 Hz, 1H, H-6), 2.64 (ddd, J ¼ 17.1, 13.2, 4.4 Hz, 1H, H-60), 2.14
(dddd, J ¼ 12.4, 5.1, 4.4, 2.5 Hz, 1H, H-5), 1.84 (dddd, J ¼ 13.2, 13.1,
3.4. Anti-proliferative assay
Three different cancer cell lines, murine leukemic cell line (EL4),
human breast cancer cell line (MCF7), and human prostate cancer
cell line (PC3) purchased from National Cancer Institute, were
tested by a standard high-flux anticancer-drug screening method
[15]. Briefly, cancer cells were incubated with the test compound in
different concentrations at 37 ꢁC for 48 h. Cultures were fixed with
trichloroacetic acid, then stained with sulforhodamine B and read
at 490 nm by ELISA reader.
12.4, 4.6, Hz, 1H, H-50). 13C NMR (100 MHz, CD3CN)
d ppm 170.7 (C-
3), 162.6 (C-10b), 140.7 (C-6a), 137.1 (CIne7a), 132.1 (C-8), 131.6 (C-
10a), 129.5 (C-7), 127.5 (C-9), 126.7 (C-10), 128.2 (CIn-3a), 125.2 (CIn-
2), 122.5 (CIn-6), 119.9 (CIn-4), 119.8 (CIn-5), 112.2 (CIn-7), 110.5 (CIn-
3), 76.8 (C-4a), 62.4 (C-2), 30.4 (C-5), 29.4 (CeIn), 27.7 (C-6). HRMS
m/z 331.1431 [MþH]þ (331.1441, calc. for C21H19N2Oþ2 ).
21b ½aꢂ2D5 ¼ ꢃ99ꢁ (c 0.09). 1H NMR (400 MHz, CD3CN)
d ppm 9.10
(br.s, 1H, NH), 8.12 (dd, J ¼ 7.8, 1.2 Hz, 1H, H-10), 7.75 (dddd, J ¼ 7.9,
1.2, 0.8, 0.6 Hz, 1H, HIn-4) 7.42e7.40 (overlaid, 1H, H-8), 7.34e7.31
(overlaid, 1H, HIn-7), 7.23 (dd, J ¼ 7.8, 0.5 Hz, 1H, H-7), 7.22 (d,
J ¼ 2.4, 1H, HIn-2), 7.11 (ddd, J ¼ 8.0, 7.0, 1.2, 1H, HIn-6), 5.08 (ddd,
J ¼ 12.2, 5.8, 3.1 Hz,1H, H-4a), 4.33 (ddd, J ¼ 7.5, 4.4, 3.1 Hz,1H, H-2),
3.64 (ddd, J ¼ 14.7, 4.4, 0.9 Hz, 1H, CH2In), 3.40 (ddd, J ¼ 14.7, 7.5,
Acknowledgements
Protech Investment Ltd, Hong Kong has supported the project.
The technical assistance of Mr. Niels Vissing Holst and Dr. Anders Ø.
Madsen, Department of Chemistry, University of Copenhagen, with

C.M. Jensen et al. / European Journal of Medicinal Chemistry 114 (2016) 118e133
1165e1168.
133
collecting X-ray data and data reduction is gratefully
acknowledged.
[15] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. Mcmahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity
assay for anticancer-drug screening, J. Natl. Cancer Inst. 82 (1990) 1107e1112.
[16] P.F. Xu, Y.S. Chen, S.I. Lin, T.J. Lu, Chiral tricyclic iminolactone derived from
(1R)-(þ)-camphor as a glycine equivalent for the asymmetric synthesis of a-
amino acids, J. Org. Chem. 67 (2002) 2309e2314.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.02.037.
[17] P.F. Xu, S. Li, T.J. Lu, C.C. Wu, B.T. Fan, G. Golfis, Asymmetric synthesis of a,a-
disubstituted a-amino acids by diastereoselective alkylation of camphor-
based tricyclic iminolactone, J. Org. Chem. 71 (2006) 4364e4373.
[18] H.F. Wang, G.H. Ma, S.B. Yang, R.G. Han, P.F. Xu, A concise synthesis of
(þ)-conagenin and its isomer using chiral tricyclic iminolactones, Tetrahedron
Asymmetry 19 (2008) 1630e1635.
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