Synthesis and Biological Evaluation of (?) and (+)-Spiroleucettadine and Analogues

Article abstract of DOI:10.1002/cmdc.202000954

A second-generation enantiospecific synthesis of spiroleucettadine is described. The original reported antibacterial activity was not observed when the experiment was repeated on the synthetic samples; however, significant anti-proliferative activity was uncovered for both enantiomers of spiroleucettadine. Comparison of the optical rotational data and ORD-CD spectra of both enantiomers and the reported spectrum from the natural source have not provided a definitive answer regarding the absolute stereochemistry of naturally occurring spiroleucettadine. Efforts then focussed on alteration at the C-4 and C-5 positions of the slightly more active (?)-spiroleucettadine. Ten analogues were synthesised, with three analogues found to possess similar anti-proliferative profiles to spiroleucettadine against the H522 lung cancer cell line.

Full text of DOI:10.1002/cmdc.202000954

Accepted Article
Title: Synthesis and biological evaluation of (-) and (+)-
spiroleucettadine and analogues
Authors: Michael Badart, Emma M Barnes, Andrew P Cording, Selena
Gilmer, Ian D Billinghurst, Veera Edupuganti, Guillaume
Lessene, Abigail Bland, Rebekah Bower, Zohaib Rana,
Scott Ferguson, Helen Opel Reading, Greg Cook, Rhonda
Rosengren, Kurt Krause, Allan Gamble, John Ashton, and Bill
C. Hawkins
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000954
Link to VoR: https://doi.org/10.1002/cmdc.202000954
01/2020

10.1002/cmdc.202000954
ChemMedChem
FULL PAPER
Synthesis and biological evaluation of (-) and (+)-
spiroleucettadine and analogues
Michael P. Badart,[a] Emma M. Barnes,[a] Andrew P. Cording, [a] Selena C. L. Gilmer, [a] Ian D.
Billinghurst, [a] Veera V. Shivaji R. Edupuganti,[b] Guillaume Lessene, [c][d][e] Abigail R. Bland, [f] Rebekah
L. Bower, [f] Zohaib Rana, [f] Scott A. Ferguson, [g] Helen K. Opel Reading, [h] Gregory M. Cook,[g]
Rhonda J. Rosengren,[f] Kurt L. Krause, [h] Allan B. Gamble, [b] John C. Ashton,[f] and Dr. Bill C.
Hawkins*[a]
[a]
Michael P. Badart, Emma M. Barnes, Andrew P. Cording, Ian Billinghurst, Selena Gilmer, Dr. Bill C. Hawkins.
Department of Chemistry
University of Otago
Dunedin 9054 (New Zealand)
E-mail: bhawkins@chemistry.otago.ac.nz
Veera V. Shivaji R. Edupuganti, Dr. Allan B. Gamble.
School of Pharmacy
University of Otago
Dunedin 9054 (New Zealand)
[b]
[c]
Prof. Guillaume Lessene
ACRF Chemical Biology Division
The Walter and Eliza Hall Institute of Medical Research
1G Royal Parade, Parkville, 3052 (Australia)
Prof. Guillaume Lessene
Department of Medical Biology
The University of Melbourne, Parkville, VIC 3050 (Australia)
Prof. Guillaume Lessene
Department of Pharmacology and Therapeutics
The University of Melbourne, Parkville, VIC 3050 (Australia)
Abigail R. Bland, Rebekah L. Bower, Zohaib Rana, Prof. Rhonda J. Rosengren, Assoc. Prof. John C. Ashton
Department of Pharmacology and Toxicology
University of Otago
[d]
[e]
[f]
Dunedin 9054 (New Zealand)
[g]
[h]
Scott A. Ferguson, Prof. Gregory M. Cook
Department of Microbiology and Immunology
University of Otago
Dunedin 9054 (New Zealand)
Helen K. Opel Reading, Prof. Kurt L. Krause
Department of Biochemistry
University of Otago
Dunedin 9054 (New Zealand)
Supporting information for this article is given via a link at the end of the document.
Abstract:
A
second generation enantiospecific synthesis of
The Leucetta genus of calcareous sea-sponges is a rich source
of bioactive alkaloids.[1] Alkaloids produced as secondary
metabolites from Leucetta chagosensis were first outlined in 1987
and included naamidine A (1). The naamidine alkaloid isolated
from this organism has been shown to possess inhibitory activity
against the epidermal growth factor receptor (EGF), as well as
anti-proliferative effects on tumours which rely on this mitogen for
growth.[2] Following this discovery in 1987 more than 70 alkaloids,
including spiro-fused alkaloids and napthimidazoles, which vary
in complexity and biological relevance, have been isolated from
sea-sponges in the Leucetta genus.[3] Since then more heavily
oxygenated alkaloids like (+)-spironaamidine (2) and (-)-
spiroleucettadine (3) have been reported (Figure 1).[4]
spiroleucettadine is described. The original reported antibacterial
activity was not observed when repeated on the synthetic samples,
however, significant anti-proliferative activity was uncovered for both
enantiomers of spiroleucettadine. Comparison of the optical rotational
data and ORD-CD spectra of both enantiomers and the reported
spectrum from the natural source has not provided a definitive answer
regarding the absolute stereochemistry of naturally occurring
spiroleucettadine. Efforts then focussed on alteration at the C-4 and
C-5 position of the slightly more active (-)-spiroleucettadine. Ten
analogues were synthesised, with three analogues found to possess
similar anti-proliferative profiles to spiroleucettadine against the H522
lung cancer cell line.
Introduction
1
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000954
ChemMedChem
FULL PAPER
properties, a synthetic sample of spiroleucettadine (3) was sent to
the National Cancer Institute (NCI) to be tested against the NCI-
60 cancer cell lines. From this, spiroleucettadine was found to
possess anti-cancer activity against several cancer cell lines in
the nanomolar range.[6] The highest anti-proliferative activity was
reported against the non-small cell lung carcinoma line NCI-H522
(IC50 0.37 µM).[6] Beyond the significant potency of
spiroleucettadine, the range of activity observed also suggests a
degree of selectivity between particular cancer cell types that
warrants further investigation.
Figure 1. The structures of naamidine A (1), (+)-spironaamidine (2) and (-)-
spiroleucettadine (3).
Table 1. Minimum Inhibitory Concentration (MIC)
Compound
E. coli
S. aureus
E. faecalis
Discovered by the Crews group in 2004,[4a, 4b] spiroleucettadine
(3) represents one of the most structurally complex molecules to
be isolated from the Leucetta sponge (Figure 1). Beyond the
interesting structural features, spiroleucettadine was also
reported to possess good anti-bacterial activity against
Enterococcus durans with a minimum inhibitory concentration
(MIC) of less than 6.25 μg mL-1.[4a] Recently, we reported the first
and, to date, only synthesis of spiroleucettadine (reported as (-)
spiroleucettadine, [훼]퐷25 = -2.6°, c 0.25, MeOH).[5] Whilst the
synthesis provided access to spiroleucettadine, it suffered from a
low yielding reaction sequence (Scheme 1) which hampered the
ability to readily produce meaningful quantities of
spiroleucettadine and analogues. Herein, we report a second-
generation synthesis which has greatly enhanced access to
spiroleucettadine itself and facilitated the rapid synthesis of
analogues. We also revise the optical rotational data associated
with spiroleucettadine derived from L-tyrosine and report its
potent biological activity against lung cancer cells.
(-) spiroleu. (3)
(µM)
>256
256
256
(+) spiroleu. (11)
(µM)
>512
512
-
Penicillin G (µg/mL)
-
0.06
-
-
Gentamycin
(µg/mL)
2
Ampicillin
4
0.0625
1
MIC values were determined by broth microdilution in cation-adjusted Mueller-
Hinton broth. The initial inoculum of bacterial cells was 5105 CFU mL -1. The
MIC was determined after 16-24 hour at 352C according to the CLSI
guidelines.[7]
Inspired by the promising screening results we re-evaluated the
original synthetic pathway, in particular its ability to provide rapid
access to analogues. The main set back in the synthesis, from an
analogue generation point-of-view, was the low yielding urea
formation/oxidative spirocyclization sequence, along with limited
opportunities for the incorporation of structural diversity. With
these limitations in mind we designed a second generation
synthesis (Scheme 2). The hydantoin 8 was accessed by
treatment of the protected tyrosine derivative 7 with N-methyl
carbamoylimidazole (5) in the presence of triethylamine to yield
the acyclic urea (not shown), which was not isolated but rather
immediately used in the next reaction. The addition of freshly
prepared sodium ethoxide to the crude residue provided the
hydantoin 8 in an 89% yield over two steps.[8] Subsequent
treatment with p-methoxybenzyl magnesium chloride afforded the
acid-sensitive tertiary hemiaminal 9, after trituration with cold
ether, in a 68% yield. Unsurprisingly, this compound readily
dehydrated in the presence of acid to form the corresponding 2-
imidazolone 12, even with low concentrations of HCl such as
those found in CDCl3 (Scheme 2). Given the propensity of the
alcohol to dehydrate, benzyl ether hydrogenolysis was performed
under buffered conditions using 10% wt. Pd(OH)2/C to afford the
phenol 10, with the spectra recorded in base washed deuterated
chloroform.
Scheme 1. Original strategy used to access the spirocyclic
cyclohexadienone core of spiroleucettadine.[5a]
Results and Discussion
The original synthesis of spiroleucettadine allowed for
a
preliminary evaluation of its biological activity. Surprisingly,
despite screening against a range of bacterial genera including
Gram-negative and Gram-positive bacteria, only Staphylococcus
aureus was inhibited by very high concentrations of (-) and (+)-
spiroleucettadine. Enterococcus faecalis was inhibited by (+)-
spiroleucettadine at high concentrations (Table 1). Neither (-) or
(+)-spiroleucettadine had any effect on the growth of Escherichia
coli. Gentamicin and ampicillin were potent inhibitors of E. coli
(positive control) as was penicillin G against S. aureus and
ampicillin against E. faecalis (Table 1).
Upon reaction with PIDA,[9] the phenol 10 underwent oxidative
spirocyclization to afford the spirocycle 6. From here,
spiroleucettadine (3) was accessed in a similar fashion to that
described in our original synthesis with an overall yield of 16.8%
(c.f 5.3% for original route).[5a]
Given that other alkaloids isolated from the Leucetta sponge, such
as naamidine
A (1, Figure 1), exhibited anti-proliferative
2
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000954
ChemMedChem
FULL PAPER
occurred for either generation of syntheses.[6] This suggests our
original measurement of optical rotation[5a] and potentially the
4b]
measurements reported in the isolation papers[4a,
were
inaccurate. In an effort to obtain reproducible data we found
chloroform to be a more reliable solvent for recording optical
rotations of spiroleucettadine: (3) [훼]2퐷0 -23° c 1.23, CHCl3, 98%
ee and (11) [훼]2퐷0 +17.9° c 0.34, CHCl3, 95% ee.
Given the relatively small magnitude, and irreproducibility (in
methanol), of rotation of plane polarized light and limited access
to the natural source, unambiguous determination of the absolute
configuration of the spiroleucettadine remained elusive.
Interestingly, the structurally similar spironaamidine (2, Figure 1,
[훼]1퐷4 = +7.1°, c 0.85, MeOH) was reported to be dextrorotatory.[4c]
Further attempts to confirm the absolute configuration of the
natural product by comparison of the ORD and CD spectra of
synthesised (-) and (+)-spiroleucettadine to the data from the
original isolation paper was not enlightening.[6]
At this point, (+)-spiroleucettadine (11) (derived from D-tyrosine)
was tested against several bacterial strains with no significant
antibacterial activity observed (Table 1). In addition, (+)
spiroleucettadine (11) was also tested against the same cancer
cell lines as (-)-spiroleucettadine (3) and found to be
approximately half as potent (11 IC50 = 0.69 µM vs 3 IC50 = 0.28
µM, Scheme 2).[6]
In order to ascertain which features of the more potent (-)-
spiroleucettadine were important for biological activity, and
keeping synthetic tractability in mind, we began with the
modification of the methylamine and the methoxybenzyl side
chain. With the desmethylamino analogue 6 already in hand,
efforts were turned towards the synthesis of the other deletion
analogues 15 and 17 (Scheme 3). To this end, Grignard addition
of benzyl magnesium chloride to the hydantoin 8 afforded the
tertiary alcohol 13, after trituration with cold ether, in a 59% yield
(Scheme 3). Benzyl ether hydrogenolysis under pH buffered
reaction conditions then provided the phenol 14 in excellent yield
(92%). The phenol 14 was taken up in 2,2,2-trifluoroethanol and
treated with PIDA at 0 °C to afford the desmethylamino-
desmethoxy analogue 15 in a 51% yield (Scheme 3). Following
this, installation of the methyl amine group was achieved using
the same chemistry described earlier to provide the desmethoxy
analogue 17, in a 46% yield over two steps (Scheme 3).
Scheme 2. The second-generation synthesis and bioactivity against H522
cancer cells of (-)-spiroleucettadine (3) and (+)-spiroleucettadine (11).
Prior to this second-generation synthesis, it was difficult to obtain
meaningful quantities of the spiroleucettadine enantiomers.
However, the new synthetic route allowed for the rapid, high
yielding, enantiospecific preparation of both spiroleucettadine (3,
starting from L-tyrosine) and spiroleucettadine (11, starting from
D-tyrosine). Upon measuring the optical rotation for
spiroleucettadine (11, derived from D-tyrosine), we were
surprised to find that, presumably due to the small magnitude of
rotation, the values obtained were inconsistent and varied
between [훼]2퐷5 +5 to -5° when recorded in methanol. Intriguingly,
varying sample concentration and temperatures appeared to
have little to no effect on the rotational value obtained.[6] This
prompted us to re-measure the rotation of spiroleucettadine (3),
derived from L-tyrosine, from both the original and new syntheses
and again both sign and magnitude of light rotation was varying.
Notably, Crews and co-workers reported significant variation in
optical rotational values between isolated samples which they
attributed to isolation of scalemic mixtures of spiroleucettadine
([훼]퐷 -27.1° c 0.38, MeOH,[4a] [훼]퐷 -5.1° c 0.56, MeOH[4b], no
temperature provided for either measurement). Furthermore,
HPLC studies on the samples indicated no racemisation had
3
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000954
ChemMedChem
FULL PAPER
aromatic system. However, at stages in the study the para-
methoxybenzyl group will be reintroduced at the C-4 position to
re-test this hypothesis. Given that the methylamino deletion
analogue 6 is the least active, this position needed to be probed
further to determine whether or not this moiety was required for
the biological activity of spiroleucettadine. Desmethylamino-
spiroleucettadine 6 was synthesised to probe whether the N-
methylamine side chain was potentially involved in these
interactions (Figure 2).
Synthesis of analogues with variation at C-5
Given the ability of the acetate 16 to undergo nucleophilic
substitution with methylamine we decided to focus attention on
the role of the N-methylamine side chain in spiroleucettadine’s
bioactivity. Therefore, acetate 16 was separately treated with
piperidine and morpholine to afford tertiary amine adducts 18
(cLogP 4.44) and 19 (cLogP 3.37), respectively, in excellent yield
(Table 2 entries 1 and 2). To examine the effect of having a
hydrophobic amine side chain at C5, substitution of the acetate of
the para-methoxybenzyl analogue 20 with n-butylamine afforded
21, while treatment of benzyl acetate 16 with n-butyl amine
afforded 22. With the end goal of protein/enzyme pull-down
studies in mind, substitution with propargyl amine gave access to
23 in excellent yield (Table 2). Finally, in order to screen for an
additional pi-stacking interaction, benzyl amine was chosen for
the nucleophilic substitution providing the N-benzyl analogue 24,
in good yield.
Biological activity of N-methylamine side chain analogues
Spiroleucettadine (3, cLogP 3.10) features a secondary amine,
which can act as both a hydrogen bond donor and acceptor. Like
secondary amines, tertiary amines are basic and can change
ionisation status, however, they can only act as hydrogen bond
acceptors. Furthermore, tertiary amines are often used in drug
design because they are metabolised to less toxic metabolites
than the equivalent primary and secondary amines, and hence,
exhibit an improved safety profile.[10] Replacement of the
methylamine side chain with the relatively hydrophobic cyclic
tertiary piperidine based analogue (cLogP = 4.44, IC50 0.50 M,
Table 2, entry 1) did not appear to adversely affect biological
activity. Likewise, the more hydrophilic morpholine derivative also
maintained a similar level of activity (cLogP = 3.37, Entry 2 IC50
0.47 M.)
Scheme 3. Synthesis of the C-4 benzyl deletion analogues 15 and 17.
The IC50 of 6 against the NCI-H522 cancer cell line was found to
be 3.93 µM, compared to that of spiroleucettadine 3 (IC50 0.28
µM). In contrast, testing found desmethoxy-spiroleucettadine 17
to be less active than 3 (IC50 1.78 µM c.f. 0.28 µM). In addition to
this, desmethylamino-desmethoxy-spiroleucettadine 15 was
synthesised to investigate the effect of deleting both functional
groups. Interestingly, 15 was the most active of the three
analogues with an IC50 of 0.61 µM.
Table 2. SN1 substitution with various amines provided the analogues 18, 19
and 21-24.23 and their IC50 values against the NCI-H522 cancer cell line.
Figure 2. The deletion analogues 5, 15 and 17, with the IC50 values against the
NCI-H522 lung cancer cell line.
Entry
#
Compound
18
R
R’
Yield
(%)
IC50 (M)a
0.50 ±
0.06
Given that the analogues 15 and 17 possessed greater activity
than the desmethoxy analogue 6, it was postulated that the
methoxy group was not essential for the activity of
spiroleucettadine 3. Thereby, simplifying the structure while also
decreasing the metabolic liability posed by an electron rich
1
2
piperidine
H
96
79
0.47±
0.04
19
morpholine
H
4
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000954
ChemMedChem
FULL PAPER
Thin layer chromatography (TLC) was performed on ALUGRAM®
aluminium-backed UV254 silica gel 60 (0.20 mm) plates. Compounds
were initially detected using UV light, and developed with either p-
anisaldehyde, basic permanganate, vanillin, or phosphomolybdic acid with
heating. Column chromatography was performed using silica gel 60.
Infrared spectra were recorded on a Bruker Optics Alpha ATR FT-IR
spectrometer. No sample preparation was required. High resolution mass-
spectra were recorded on a Bruker microTOFQ mass spectrometer or
Shimadzu LCMS-9030 mass spectrometer using an electrospray
0.33 ±
0.03
3
4
5
6
21
22
23
24
butylamine
OCH3
47
78
83
68
0.38 ±
0.03
butylamine
H
H
H
0.61 ±
0.08
propargylamine
benzylamine
1
0.28 ±
0.02
ionisation (ESI) source in either the positive or negative modes. H NMR
spectra were recorded at either 400 MHz on a Varian 400-MR NMR
system or at 500 MHz on a Varian 500 MHz AR premium shielded
spectrometer. Spectra were recorded from samples in the designated
deuterated solvent at 25 °C in 5 mm NMR tubes. 13C NMR spectra were
recorded at either 101 MHz on a Varian 400-MR NMR system or at 125
MHz on a Varian 500 MHz AR premium shielded spectrometer under the
same conditions as the 1H NMR spectra. Chemical shifts were reported in
parts per million and referenced to the appropriate solvent peak. Melting
points were measured on a DigiMelt MPA 161 apparatus. The optical
rotation of chiral compounds were recorded on a Rudolph Research
Analytical AUTOPOL® IV automatic polarimeter. Dichloromethane
(CH2Cl2), diethyl ether (Et2O), tetrahydrofuran (THF), methanol (MeOH),
and acetonitrile (MeCN) were dried using the PURE SOLV MD-6 solvent
purification system. All other solvents and reagents were used as received.
Analytical HPLC was carried out on a Shimadzu Prominence HLPC
system using a Shimadzu ELSD-LT II detector and a Diacel CHIRALPAK
IC-3 chiral column, eluting with acetonitrile.
[a] Assay was performed in triplicate (n = 3), see Supporting Information for
dose response curves.
Hydrophobic interactions have long been recognised as an
important biological phenomena, particularly in drug design and
drug activity.[11] They are one of the governing interactions in the
binding of drugs to receptors, and hence, are important in
determining binding affinities and drug efficacy.[11b] However,
there is a delicate balance between the hydrophobic and
hydrophilic nature of a molecule. If molecules are too hydrophobic,
they can become trapped in membranes and not reach their site
of action.[11]
The para-methoxybenzyl N-butyl analogue 21 was found to
possess comparable activity to (-)-spiroleucettadine (cLogP 4.31,
Table 2, entry 3, IC50 0.33 µM). The simplified benzyl N-butyl
analogue 22 was also a potent inhibitor (cLogP 4.56, IC50 0.38
µM) which is consistent with the working hypothesis that the
methoxy group is not essential for the biological activity of 3.
Interestingly, the N-propargyl analogue 23 was approximately half
as potent as 3 against the H522 cell line (entry 5, IC50 0.61 µM).
The N-benzyl analogue was found to possess equipotent activity
to (-)-spiroleucettadine (3) (entry 6, IC50 0.28 µM).
Biological assay: H522 cells (10 x 103cells/well) were seeded in 96-well
plates for 24 h at 37 °C in RPMI growth medium supplemented with 5%
FBS and 1% penicillin/streptomycin. Cells were treated with the respective
compound for 72 h, using DMSO as the vehicle control. To determine cell
viability, the sulforhodamine B (SRB) assay was used. Briefly cells were
fixed in 10% trichloroacetic acid (TCA) for 30 min at 4 °C. To remove the
TCA, plates were washed twice in distilled water and dried for 30 min. SRB
(0.4% in 1% acetic acid) was added to the wells for 10 min, followed by
three 1% acetic acid washes to remove unbound dye. The dye was
solubilized in 10 mM Tris/HCl (pH 10.5) and the absorbance read at 510
nm on a BioRad Microplate Spectrophotometer. Cell viability was
calculated as a percentage of the DMSO control and EC50values were
determined using non-linear regression (equation: log(inhibitor) vs.
response – variable slope (four parameters)) using GraphPad Prism 8.
Antimicrobial Susceptibility Testing: To determine the minimum
inhibitory concentration (MIC) of spiroleucettadine (3) or (11) against S.
aureus ATCC 6538 or E. coli ATCC 10536, cells were grown in cation
adjusted Mueller Hinton (CAMHB) broth at 37 °C with shaking (200 rpm).
E. faecalis JH2-2 was grown under the same conditions without shaking.
The MIC assay was performed by microbroth dilution following the CLSI
guidelines.[7] Whereby a polystyrene 96-well plate was set up such that
100 L of CAHMB media was added to column 1 (A-H) and 50 L of media
was added to the remaining wells. Spiroleucettadine (3) or (11) was added
to column 1, to yield a final concentration of 512 g mL-1, and then serially
diluted 2-fold (50 L transfer) into the neighbouring wells, resulting in a
serial dilution of each compound from 512 g mL-1 to 0.25 g mL-1.
Overnight cultures of bacteria were diluted in fresh CAMHB before adding
50 L of culture to each well of the plate to achieve a uniform CFU mL -1
(final) of approximately 5 × 105 in the MIC plate. The plates were incubated
at 37°C with shaking as appropriate for each bacteria for 16-24 h before
determining the MIC. MICs were determined as the lowest concentration
at which growth did not occur. Ampicillin, penicillin G, and gentamycin were
used as positive controls.
Whilst the precise molecular attributes necessary for biological
activity are yet to be elucidated, it is clear that substitution of the
methyl amine side chain (C-5) does not adversely affect activity.
Thereby, providing a possible opportunity for proteomic studies.
This will be the focus of future work and will be reported in due
course.
Conclusion
In conclusion, we have reported a second-generation synthesis
which allowed for the rapid, enantiospecific preparation of
spiroleucettadine and analogues. With larger quantities of both
(-)- and (+)-spiroleucettadine we have revised our original optical
rotation data and performed ORD-CD on both enantiomers of
spiroleucettadine. Our results suggest that spiroleucettadine does
not possess significant antibacterial properties, however, it does
possess potent (nanomolar) anti-proliferative activity against
H522 (lung) cancer cells. Through the synthesis of analogues, we
have established that modification of the methyl amine side chain
is possible without significant loss of biological activity. This
information will facilitate further studies centred around
determining the mode of action.
5‐{[4‐(benzyloxy)phenyl]methyl}‐3‐methylimidazolidine‐2,4‐dione (8).
Et3N (2.32 mL, 16.7 mmol) was added to a suspension of HCl salt of 7
(4.40 g, 13.9 mmol) in THF (50 mL), followed by N-methyl
carbamoylimidazole (2.10 g, 16.7 mmol), and the reaction was heated to
reflux for 16 h. The solution was cooled to room temperature and
partitioned between water (100 mL) and EtOAc (100 mL). The aqueous
layer was extracted with EtOAc (3 × 50 mL), and the combined organic
Experimental Section
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000954
ChemMedChem
FULL PAPER
phases were washed with brine (50 mL), dried over MgSO4, and
concentrated in vacuo. The crude residue was then dissolved in EtOH (70
mL); Na (384 mg, 16.7 mmol) was added to the solution, and a white
precipitate appeared. The reaction was then stirred at room temperature
for a further 30 h then concentrated in vacuo to half volume. The mixture
was then diluted with water (200 mL) and extracted with
EtOAc/isopropanol (4:1) (3 × 100 mL). The combined organic phases were
washed with brine (50 mL), dried over MgSO4, and concentrated in vacuo.
The crude product was then triturated with cold Et2O to yield the title
compound as a white crystalline solid (3.34 g, 77% over two steps). m.p:
over MgSO4 and concentrated in vacuo. The crude residue was triturated
with Et2O to afford the title compound (191 mg, 59%) as a white solid. m.p.
166–167 °C. 1H NMR (400 MHz, DMSO) δ 7.46 – 7.41 (m, 2H), 7.41 –
7.35 (m, 2H), 7.34 – 7.28 (m, 1H), 7.27 – 7.17 (m, 3H), 7.05 – 6.96 (m, 4H),
6.91 (d, J = 8.8 Hz, 2H), 6.25 (d, J = 2.0 Hz, 1H), 5.91 (s, 1H), 5.07 (s, 2H),
3.40 (ddd, J = 7.5, 5.6, 1.9 Hz, 1H), 2.92 (d, J = 13.7 Hz, 1H), 2.72 – 2.65
(m, 4H), 2.54 (dd, J = 13.0, 6.1 Hz, 1H), 2.44 (dd, J = 13.8, 8.2 Hz, 1H).
13C NMR (101 MHz, DMSO) δ 159.9, 156.7, 137.2, 136.3, 130.9, 130.3,
130.3, 128.4, 128.0, 127.7, 127.6, 126.4, 114.6, 88.9, 69.1, 57.7, 42.1,
34.9, 24.2. HRMS-ESI (m/z): calcd for C25H26N2NaO3 [M + Na]+, 425.1836;
found, 425.1820. vmax (ATR-IR) cm-1 3294, 2924, 2854, 1704. [훼]퐷22 = -276
(c = 0.077, MeOH).
1
166−167 °C. H NMR (400 MHz, DMSO) δ 8.18 (s, 1H), 7.49 – 7.28 (m,
5H), 7.07 (d, J = 8.6 Hz, 2H), 6.91 (d, J = 8.7 Hz, 2H), 5.05 (s, 2H), 4.31 (t,
J = 5.1 Hz, 1H), 2.91 (dd, J = 14.1, 4.8 Hz, 1H), 2.86 (dd, J = 14.1, 5.3 Hz,
1H), 2.65 (s, 3H). 13C NMR (101 MHz, DMSO) δ 174.1, 157.6, 157.2, 137.6,
131.1, 128.8, 128.2, 128.1, 128.0, 114.8, 69.5, 57.9, 36.1, 24.2. HRMS-
ESI (m/z): calcd for C18H18N2NaO3 [M + Na+], 333.1210; found, 333.1180.
νmax (ATR-IR), cm−1: 3316, 2924, 1774, 1708, 1600, 1511. [훼]2퐷2 = -51.0 (c
= 0.32, pyridine).
3'a‐[(4‐methoxyphenyl)methyl]‐3'‐methyl‐6',6'a‐dihydro‐1'H‐
spiro[cyclohexane‐1,5'‐furo[2,3‐d]imidazole]‐2,5‐diene‐2',4‐dione (6).
The aminol 14 (122 mg, 0.356 mmol) was taken up in 2,2,2-trifluoroethanol
(2.1 mL) and cooled to 0 °C. PIDA (126 mg, 0.392 mmol) was added and
the solution was stirred at 0 °C for 10 min. The reaction mixture was
quenched with Na2S2O3 (1 M, 5 mL) and partitioned between EtOAc (15
mL) and H2O (15 mL). The aqueous layer was extracted with EtOAc (3 x
15 mL) and the combined organic extracts were washed with brine (30 mL),
dried over MgSO4 and concentrated in vacuo. Column chromatography
using EtOAc as the eluent yielded the title compound (51 mg, 42%) as a
white crystalline solid. m.p. 122-123 °C. 1H NMR (500 MHz, CDCl3) δ 7.19
(d, J = 8.5 Hz, 2H), 6.89 (d, J = 8.5 Hz, 2H), 6.84 (dd, J = 10.3, 3.0 Hz, 1H),
6.34 (dd, J = 10.1, 3.0 Hz, 1H), 6.12 (dd, J = 10.1, 3 Hz, 1H), 6.09 (br s,
1H), 6.06 (dd, J = 10.1, 3.0 Hz, 1H), 4.24 (dd, J = 6.5, 1.5 Hz, 1H), 3.82 (s,
3H), 3.36 and 2.84 (abq, J = 14.1, 2H), 2.93 (s, 3H), 1.83 (d, J = 13.8 Hz,
1H), 1.45 (dd, J = 13.8, 6.8 Hz, 1H). 13C NMR (126 MHz, CDCl3) δ 185.0,
159.5, 159.1, 149.2, 148.8, 131.8, 129.0, 127.3, 126.1, 113.9, 102.7, 79.7,
58.3, 55.4, 44.8, 41.1, 25.1. HRMS-ESI (m/z): calcd for C19H20N2NaO4 [M
+ Na]+, 363.1315; found 363.1342. νmax (ATR-IR) cm-1 3277, 2956, 2851,
1697, 1670, 1631, 1513, 1440, 1400, 1302, 1172, 1067, 1030, 943, 827.
[훼]2퐷4 = -37.3 (c = 0.53, MeOH).
4‐{[4‐(benzyloxy)phenyl]methyl}‐5‐hydroxy‐5‐[(4‐
methoxyphenyl)methyl]‐1-methylimidazolidin‐2‐one (9). To a solution
of the hydantoin 8 (1.50 g, 4.83 mmol) in THF (10 mL) was added a
solution of para-methoxybenzyl magnesium chloride[12] in THF (56.8 mL,
10.63 mmol) at 0 °C. The reaction was then allowed to warm to room
temperature and stirred overnight. The reaction mixture was quenched
with saturated aqueous NaHCO3 (10 mL) and partitioned between water
(50 mL) and EtOAc (50 mL). The aqueous layer was washed with EtOAc
(3 x 50 mL) and the combined organic layers were washed with brine (100
mL), dried over MgSO4 and concentrated in vacuo to afford the crude
compound as a yellow oil. This was triturated with Et2O to afford the title
compound (1.41 g, 68%) as a white solid. m.p: 157–158 °C. 1H NMR (400
MHz, DMSO) δ 7.47 – 7.41 (m, 2H), 7.41 – 7.34 (m, 2H), 7.35 – 7.28 (m,
1H), 7.07 – 6.99 (m, 2H), 6.95 – 6.86 (m, 4H), 6.83 – 6.76 (m, 2H), 6.23 (d,
J = 2.0 Hz, 1H), 5.87 (s, 1H), 5.08 (s, 2H), 3.70 (s, 3H), 3.39 (ddd, J = 7.8,
5.7, 1.8 Hz, 1H), 2.85 (d, J = 14.1 Hz, 1H), 2.67 (s, 3H), 2.62 – 2.55 (m,
2H), 2.45 (dd, J = 14.0, 7.5 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 159.9,
157.9, 156.7, 137.3, 131.3, 130.9, 130.3, 128.4, 128.1, 127.7, 127.6, 114.6,
113.4, 89.0, 69.1, 57.6, 54.9, 41.1, 34.9, 24.2. HRMS-ESI (m/z): calcd for
C26H28N2NaO4 [M + Na]+, 455.1941; found, 455.1927. νmax (ATR-IR) cm-1
3380, 3253, 2920, 2835, 1695, 1613, 1512, 1248. [훼]2퐷0 = +1.8 (c = 0.10,
MeOH).
5‐benzyl‐5‐hydroxy‐4‐[(4‐hydroxyphenyl)methyl]‐1‐
methylimidazolidin‐2‐one (14). The benzyl ether 13 (191 mg, 0.475
mmol) and K2HPO4 (82.7 mg, 0.475 mmol) were suspended in MeOH (8
mL). To this was added Pd(OH)2/C (19 mg, 10% wt.) and the system was
purged with H2. The reaction mixture was stirred at room temperature for
90 min. Following this, it was diluted with EtOAc (50 mL), filtered through
a plug of celite and concentrated in vacuo to yield the title compound solid
(136 mg, 92%) as a white solid. m.p. 164–165 °C. 1H NMR (400 MHz,
DMSO) δ 9.17 (s, 1H), 7.33 – 7.13 (m, 3H), 7.00 (d, J = 6.8 Hz, 2H), 6.89
(d, J = 8.5 Hz, 2H), 6.66 (d, J = 8.4 Hz, 2H), 6.20 (s, 1H), 5.89 (s, 1H), 3.38
(td, J = 6.0, 2.9 Hz, 1H), 2.92 (d, J = 13.6 Hz, 1H), 2.71 – 2.64 (m, 4H),
2.54 – 2.51 (m, 1H), 2.39 (dd, J = 13.9, 7.8 Hz, 1H). 13C NMR (101 MHz,
DMSO) δ 159.9, 155.6, 136.4, 130.4, 130.2, 128.7, 127.9, 126.4, 115.0,
88.9, 57.8, 42.1, 34.9, 24.3. HRMS-ESI (m/z): calcd for C18H20N2NaO3 [M
+ Na]+, 335.1366; found, 335.1366. νmax (ATR-IR) cm-1 3375, 3033, 2920,
1693, 1679, 1589, 1493, 1048. [훼]2퐷0 = +27.5 (c = 1.01, MeOH).
5‐hydroxy‐4‐[(4‐hydroxyphenyl)methyl]‐5‐[(4‐
methoxyphenyl)methyl]‐1‐methylimidazolidin‐2‐one (10). The benzyl
ether 9 (200 mg, 0.462 mmol) and K2HPO4 (80.6 mg, 0.462 mmol) were
taken up in MeOH (8 mL). To this was added Pd(OH)2/C (20 mg, 10% wt.)
and the system was purged with H2. The reaction mixture was stirred at
room temperature for 90 min. This was then diluted with EtOAc (50 mL),
filtered through a plug of celite and concentrated in vacuo to afford the
crude residue. Given the propensity to eliminate, this was used without
further purification. m.p. 135-136 °C. 1H NMR (400 MHz, DMSO) δ 9.16 (s,
1H), 6.95 – 6.86 (m, 4H), 6.80 (d, J = 8.8 Hz, 2H), 6.66 (d, J = 8.6 Hz, 2H),
6.18 (d, J = 1.8 Hz, 1H), 5.84 (s, 1H), 3.70 (s, 3H), 3.37 (td, J = 5.7, 1.5 Hz,
1H), 2.85 (d, J = 13.8 Hz, 1H), 2.67 (s, 3H), 2.62 – 2.52 (m, 2H), 2.40 (dd,
J = 13.6, 7.6 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 160.1, 158.0, 155.7,
131.4, 130.3, 128.9, 128.2, 115.1, 113.5, 89.2, 57.7, 55.0, 41.2, 34.9, 24.3.
HRMS-ESI (m/z): calcd for C19H22N2NaO4 [M + Na]+, 365.14718; found,
365.14692. vmax (ATR-IR) cm-1 3320, 3222, 2943, 2828, 1673, 1514, 1244.
[훼]2퐷2 = +16.7 (c = 0.25, MeOH).
3'a‐benzyl‐3'‐methyl‐6',6'a‐dihydro‐1'H‐spiro[cyclohexane‐1,5'‐
furo[2,3‐d]imidazole]‐2,5‐diene‐2',4‐dione (15). The aminol 14 (146 mg,
0.467 mmol) was taken up in 2,2,2-trifluoroethanol (3 mL) and cooled to
0 °C. PIDA (166 mg, 0.514 mmol) was added and the solution was stirred
at 0 °C for 30 min. The reaction mixture was quenched with Na2S2O3 (1 M,
5 mL) and partitioned between EtOAc (15 mL) and H2O (15 mL). The
aqueous layer was extracted with EtOAc (3 x 15 mL) and the combined
organic extracts were washed with brine (30 mL), dried over MgSO4 and
concentrated in vacuo. Column chromatography using EtOAc as the
eluent yielded the title compound (72.7 mg, 51%) as an orange oil. 1H NMR
(400 MHz, CDCl3) δ 7.41 – 7.26 (m, 5H), 6.84 (dd, J = 10.3, 3.1 Hz, 1H),
6.30 (dd, J = 10.0, 3.0 Hz, 1H), 6.11 (dd, J = 10.1, 2.0 Hz, 1H), 6.07 (dd, J
= 10.3, 2.0 Hz, 1H), 5.81 (s, 1H), 4.26 (d, J = 6.5 Hz, 1H), 3.43 (d, J = 13.7
Hz, 1H), 2.96 (s, 3H), 2.92 (d, J = 13.7 Hz, 1H), 1.86 (d, J = 13.8 Hz, 1H),
1.41 (dd, J = 13.8, 6.5 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 185.0, 159.3,
149.1, 148.7, 134.2, 130.8, 129.0, 128.6, 127.8, 127.4, 102.6, 79.7, 58.3,
5‐benzyl‐4‐{[4‐(benzyloxy)phenyl]methyl}‐5‐hydroxy‐1‐
methylimidazolidin‐2‐one (13). To a solution of the hydantoin 8 (250 mg,
0.806 mmol) in THF (7 mL) was added benzylmagnesium chloride[12]
dropwise (6.91 mL, 2.42 mmol) at 0 °C. This was warmed to room
temperature and stirred overnight. The reaction mixture was quenched
with aqueous NaHCO3 (10 mL) and partitioned between H2O (20mL) and
EtOAc (20 mL). The aqueous layer was washed with EtOAc (3 x 20 mL)
and the combined organic layers were washed with brine (50 mL), dried
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000954
ChemMedChem
FULL PAPER
44.8, 42.1, 25.1. HRMS-ESI (m/z): calcd for C18H18N2NaO3 [M + Na]+,
333.1210; found, 333.1192. νmax (ATR-IR) cm-1 2925, 2853, 1698, 1667,
1627, 1435, 1397, 1242. [훼]2퐷0 = +6.2 (c = 0.11, MeOH).
µL) at room temperature was added morpholine (4.7 µL, 0.054 mmol). The
reaction was stirred at room temperature for 3.5 h before an additional
aliquot of morpholine (2.4 µL, 0.027 mmol) was added. The reaction was
stirred for 1.5 h before a final aliquot of morpholine (2.4 µL, 0.027 mmol)
was added. The reaction was then stirred a further 1.5 h before being
quenched with water (5 mL). The aqueous layer was then extracted with
EtOAc (3 x 5 mL) and the combined organic layers washed with brine (10
mL), dried over Na2SO4, and concentrated in vacuo. The crude residue
was purified using gradient eluted column chromatography, eluting with
3:2 EtOAc to PE to neat EtOAc to yield the title compound (8.5 mg, 79%)
as a yellow-orange waxy solid. 1H NMR (400 MHz, CDCl3) δ 7.68 – 7.60
(m, 2H), 7.41 – 7.32 (m, 3H), 7.04 (dd, J = 10.4, 3.1 Hz, 1H), 6.85 (s, 1H),
6.00 (dd, J = 10.3, 2.0 Hz, 1H), 5.87 (dd, J = 10.2, 2.0 Hz, 1H), 5.19 (dd, J
= 10.1, 3.0 Hz, 1H), 3.88 (br s, 4H), 3.22 (s, 2H), 2.93 (s, 3H), 2.78 (br s,
2H), 2.66 (br s, 2H), 2.09 (d, J = 12.9 Hz, 1H), 1.99 (d, J = 12.9 Hz, 1H).
13C NMR (101 MHz, CDCl3) δ 185.0, 160.0, 149.5, 135.5, 132.7, 128.3,
128.1, 127.5, 127.1, 103.0, 84.9, 77.4, 67.4, 66.4, 48.8, 46.8, 46.5, 37.9,
29.8, 26.0. HRMS-ESI (m/z): calcd for C22H25N3NaO4 [M + Na]+, 418.1737;
found, 418.1709. νmax (ATR-IR) cm-1 2951, 2858, 1719, 1695, 1666, 1618,
1512, 1425, 1249, 1091. [훼]2퐷0 = +36.6 (c = 0.16, MeOH).
3'a‐benzyl‐3'‐methyl‐2',4‐dioxo‐1',6'‐dihydrospiro[cyclohexane‐1,5'‐
furo[2,3‐d]imidazole]‐2,5‐dien‐6'a‐yl acetate (16). To a solution of
spirocycle 15 (56 mg, 0.181 mmol) in fluorobenzene (3.5 mL) was added
Dess-Martin periodinane (192 mg, 0.454 mmol) at room temperature. The
reaction was then heated to 60 °C for 75 min. The reaction mixture was
then cooled to room temperature and quenched with solid NaHCO3,
filtered through a plug of silica, and concentrated in vacuo. Column
chromatography eluting with 1:1 EtOAc to PE afforded the title compound
(54 mg, 81%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.42 – 7.28 (m,
4H), 6.85 (dd, J = 10.2, 3.0 Hz, 1H), 6.30 (s, 1H), 6.08 (dd, J = 10.2, 2.0
Hz, 1H), 6.04 (dd, J = 10.0, 2.0 Hz, 1H), 5.94 (dd, J = 10.0, 3.0 Hz, 1H),
3.40 (d, J = 14.4 Hz, 1H), 3.21 (d, J = 14.4 Hz, 1H), 2.93 (s, 3H), 2.81 (d,
J = 13.7 Hz, 1H), 2.20 (s, 3H), 1.86 (d, J = 13.7 Hz, 1H). 13C NMR (101
MHz, CDCl3) δ 184.6, 170.2, 156.7, 148.0, 147.4, 134.5, 132.0, 129.2,
128.1, 127.8, 127.5, 101.9, 95.1, 78.0, 47.8, 39.0, 25.5, 21.6. HRMS-ESI
(m/z): calcd for C20H20N2NaO5 [M + Na]+, 391.1264; found, 391.1226. νmax
(ATR-IR) cm-1 2930, 1714, 1670, 1632, 1435. 1396, 1370, 1236. [훼]2퐷0
=
+30.4 (c = 0.32, MeOH).
3'a‐benzyl‐6'a‐(butylamino)‐3'‐methyl‐1',6'‐
dihydrospiro[cyclohexane‐1,5'‐furo[2,3‐d]imidazole]‐2,5‐diene‐2',4‐
dione (21). To a solution of acetate 16 (10 mg, 0.027 mmol) in THF (300
µL) at room temperature was added butylamine (5.4 µL, 0.054 mmol). The
reaction was stirred at room temperature for 3.5 h before being quenched
with water (5 mL). The aqueous layer was then extracted with EtOAc (3 x
5 mL) and the combined organic layers washed with brine (10 mL), dried
over Na2SO4, and concentrated in vacuo. The crude residue was purified
using column chromatography, eluting with 3:2 EtOAc to PE to yield the
title compound (8.1 mg, 78%) as a pale yellow oil. 1H NMR (400 MHz,
CDCl3) δ 7.54 – 7.47 (m, 2H), 7.39 – 7.29 (m, 3H), 6.99 (dd, J = 10.3, 3.1
Hz, 1H), 6.04 (dd, J = 10.3, 2.1 Hz, 1H), 5.98 (dd, J = 10.1, 2.0 Hz, 1H),
5.81 – 5.72 (m, 2H), 3.22 (ABq, J = 14.4 Hz, 2H), 2.91 (s, 3H), 2.74 – 2.65
(m, 1H), 2.65 – 2.56 (m, 1H), 2.16 (d, J = 13.1 Hz, 1H), 2.01 (d, J = 13.1
Hz, 1H), 1.57 – 1.47 (m, 2H), 1.44 – 1.34 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H).
13C NMR (101 MHz, CDCl3) δ 185.1, 158.6, 149.5, 149.1, 135.4, 131.9,
128.4, 128.2, 127.4, 127.3, 102.6, 82.5, 77.5, 48.8, 42.6, 38.7, 32.9, 25.9,
20.6, 14.1. HRMS-ESI (m/z): calcd for C22H27N3NaO3 [M + Na]+, 404.1945;
found, 404.1928. νmax (ATR-IR) cm-1 2958, 2928, 2858, 1699, 1670, 1435,
1394, 1258. [훼]2퐷0 = +22.6 (c = 0.22, MeOH).
3'a‐benzyl‐3'‐methyl‐6'a‐(methylamino)‐1',6'‐
dihydrospiro[cyclohexane‐1,5'‐furo[2,3‐d]imidazole]‐2,5‐diene‐2',4‐
dione (17). To a solution of acetate 16 (10 mg, 0.027 mmol) in THF (300
µL) at room temperature was added methylamine (2 M, 27 µL, 0.054 mmol).
The reaction was stirred at room temperature for 3 h before being
quenched with water (5 mL). The aqueous layer was then extracted with
EtOAc (3 x 5 mL) and the combined organic layers washed with brine (10
mL), dried over Na2SO4, and concentrated in vacuo. The crude residue
was purified using column chromatography, eluting with EtOAc to yield the
title compound (5.2 mg, 57%) as a pale yellow oil. 1H NMR (500 MHz,
CDCl3) δ 7.53 – 7.45 (m, 2H), 7.39 – 7.29 (m, 3H), 7.00 (dd, J = 10.3, 3.0
Hz, 1H), 6.05 (dd, J = 10.3, 2.0 Hz, 1H), 5.99 (dd, J = 10.1, 2.0 Hz, 1H),
5.82 (dd, J = 10.1, 3.1 Hz, 1H), 5.80 (br s, 1H), 3.23 (ABq, J = 14.6 Hz,
2H), 2.90 (s, 3H), 2.49 (s, 3H), 2.15 (d, J = 13.2 Hz, 1H), 1.96 (d, J = 13.2
Hz, 1H). 13C NMR (126 MHz, CDCl3) δ 185.0, 158.6, 149.4, 148.9, 135.3,
131.8, 128.6, 128.3, 127.4, 127.3, 102.3, 82.9, 77.3, 48.6, 38.8, 29.2, 25.9.
HRMS-ESI (m/z): calcd for C19H21N3NaO3 [M + Na]+, 362.1475; found,
362.1476. νmax (ATR-IR) cm-1 3062, 2929, 1696, 1668, 1629, 1438, 1395,
1091. [훼]2퐷0 = +6.7 (c = 0.23, MeOH).
6'a‐(butylamino)‐3'a‐[(4‐methoxyphenyl)methyl]‐3'‐methyl‐1',6'‐
dihydrospiro[cyclohexane‐1,5'‐furo[2,3‐d]imidazole]‐2,5‐diene‐2',4‐
dione (22). To a solution of the acetate derivative 20 (30.2 mg, 0.0758
mmol) in THF (3 mL) was added butylamine (25.0 µL, 0.252 mmol). This
was stirred at room temperature for 2 h, then concentrated in vacuo. The
crude residue was purified using column chromatography with EtOAc to
afford the title compound (14.6 mg, 47%) as an orange solid. 1H NMR (500
MHz, CDCl3) δ 7.44 – 7.37 (m, 2H), 6.98 (dd, J = 10.3, 3.1 Hz, 1H), 6.90 –
6.84 (m, 2H), 6.06 – 5.99 (m, 2H), 5.93 (s, 1H), 5.90 (dd, J = 10.0, 3.1 Hz,
1H), 3.82 (s, 3H), 3.14 (ABq, J = 14.5 Hz, 2H), 2.89 (s, 3H), 2.72 – 2.65
(m, 1H), 2.64 – 2.56 (m, 1H), 2.17 (d, J = 13.2 Hz, 1H), 2.00 (d, J = 13.1
Hz, 1H), 1.55 – 1.47 (m, 2H), 1.44 – 1.33 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H).
13C NMR (126 MHz, CDCl3) δ 185.1, 158.9, 158.8, 149.7, 149.1, 132.9,
128.4, 127.3, 127.2, 113.5, 102.6, 82.6, 77.4, 55.4, 48.8, 42.7, 37.9, 32.9,
25.9, 20.6, 14.1. HRMS-ESI (m/z): calcd for C23H29N3NaO4 [M + Na]+,
434.2050; found, 434.2045. νmax (ATR-IR) cm-1 2957, 2929, 2857, 1702,
1665, 1511, 1438, 1392, 1245, 1084. [훼]2퐷0 = +21.3 (c = 0.22, MeOH).
3'a‐benzyl‐3'‐methyl‐6'a‐(piperidin‐1‐yl)‐1',6'‐
dihydrospiro[cyclohexane‐1,5'‐furo[2,3‐d]imidazole]‐2,5‐diene‐2',4‐
dione (18). To a solution of acetate 16 (10 mg, 0.027 mmol) in THF (300
µL) at room temperature was added piperidine (5.4 µL, 0.054 mmol). The
reaction was stirred at room temperature for 3 h before being quenched
with water (5 mL). The aqueous layer was then extracted with EtOAc (3 x
5 mL) and the combined organic layers washed with brine (10 mL), dried
over Na2SO4, and concentrated in vacuo. The crude residue was purified
using gradient eluted column chromatography, eluting with 1:1 EtOAc to
PE to 3:2 EtOAc to PE to yield the title compound (10 mg, 96%) as an
orange wax. 1H NMR (500 MHz, CDCl3) δ 7.68 – 7.60 (m, 2H), 7.39 – 7.30
(m, 3H), 7.05 (dd, J = 10.3, 3.0 Hz, 1H), 6.16 (s, 1H), 5.98 (dd, J = 10.3,
2.1 Hz, 1H), 5.84 (dd, J = 10.1, 2.0 Hz, 1H), 5.15 (dd, J = 10.1, 3.0 Hz, 1H),
3.21 (ABq, J = 14.0 Hz, 2H), 2.99 (br s, 1H), 2.94 (s, 3H), 2.84 (br s, 1H),
2.48 – 2.22 (br m, 1H), 2.07 (d, J = 12.9 Hz, 1H), 1.97 (d, J = 12.9 Hz, 1H),
1.84 (br s, 2H), 1.82 – 1.69 (br m, 4H), 1.41 – 1.21 (br s, 1H). 13C NMR
(126 MHz, CDCl3) δ 185.0, 158.6, 149.4, 148.9, 135.3, 131.8, 128.6, 128.3,
127.4, 127.3, 102.3, 82.9, 77.3, 48.6, 38.8, 29.2, 25.9. HRMS-ESI (m/z):
calcd for C23H28N3O3 [M + H]+, 394.21252; found, 394.21470. νmax (ATR-
3'a‐benzyl‐3'‐methyl‐6'a‐(prop‐2‐yn‐1‐ylamino)‐1',6'‐
dihydrospiro[cyclohexane‐1,5'‐furo[2,3‐d]imidazole]‐2,5‐diene‐2',4‐
dione (23). To a solution of acetate 16 (6.0 mg, 0.016 mmol) in THF (2
mL) was added propargyl amine (17 µL, 0.265 mmol). The reaction was
stirred at room temperature for 3 h before being quenched with water (3
mL). The aqueous layer was extracted with EtOAc (3 x 3 mL) and the
combined organic extracts were washed with brine (3 mL), dried over
Na2SO4 and the solvent was removed under reduced pressure. The crude
IR) cm-1 2932, 2852, 1762, 1691, 1631, 1452, 1444, 1395, 1240. [훼]2퐷0
+17.8 (c = 0.27, MeOH).
=
3'a‐benzyl‐3'‐methyl‐6'a‐(morpholin‐4‐yl)‐1',6'‐
dihydrospiro[cyclohexane‐1,5'‐furo[2,3‐d]imidazole]‐2,5‐diene‐2',4‐
dione (19). To a solution of acetate 16 (10 mg, 0.027 mmol) in THF (300
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000954
ChemMedChem
FULL PAPER
[6]
[7]
See Supporting Information for details.
residue was purified using column chromatography, eluting with EtOAc to
yield the title compound as a pale orange solid (4.8 mg, 83%). 1H NMR
(400 MHz, CDCl3) δ 7.46 (m, 1H), 7.38 – 7.29 (m, 3H), 6.99 (dd, J = 10.3,
3.1 Hz, 1H), 6.06 (dd, J = 10.4, 2.0 Hz, 1H), 6.00 (dd, J = 10.1, 2.0 Hz, 1H),
5.83 (s, 1H), 5.79 (dd, J = 10.0, 3.1 Hz, 1H), 3.66 (dd, J = 17.8, 2.5 Hz,
1H), 3.47 (dd, J = 17.8, 2.3 Hz, 1H), 3.22 (ABq, J = 14.4 2H), 2.91 (s, 3H),
2.37 (t, J = 2.4 Hz, 1H), 2.29 (d, J = 13.2 Hz, 1H), 2.00 (d, J = 13.3 Hz, 1H).
13C NMR (101 MHz, CDCl3) δ 184.8, 157.8, 149.1, 148.6, 135.0, 131.8,
128.5, 128.1, 127.3, 127.2, 103.0, 82.3, 77.4, 73.3, 47.3, 38.7, 32.8, 29.7,
25.8. HRMS-ESI (m/z): calcd for C21H21N3NaO3 [M+Na]+, 386.14751;
found 386.14534. vmax (ATR-IR) cm-1 3304, 3297, 2954, 2923, 2852, 1703,
1671, 1453, 1395, 1257. [훼]2퐷3 = +27.9 (c = 0.14, MeOH).
P. Wayne, M100 Performance Standards for Antimicrobial
Susceptibility Testing, 27th ed., CLSI, 2017.
R. A. Lamb, N. T. Lucas, G. Lessene, B. C. Hawkins, J.
Org. Chem. 2018, 83, 10120-10133.
Y. Tamura, T. Yakura, J. Haruta, Y. Kita, J. Org. Chem.
1987, 52, 3927-3930.
L. Verna, J. Whysner, G. M. Williams, Pharmacol. Ther.
1996, 71, 83-105.
a) E. E. Meyer, K. J. Rosenberg, J. Israelachvili, Proc.
Natl. Acad. Sci. U.S.A. 2006, 103, 15739-15746; b) A.
Sarkar, G. E. Kellogg, Curr. Top. Med. Chem. 2010, 10,
67-83.
[8]
[9]
[10]
[11]
[12]
W. Liu, H. Li, P.-J. Cai, Z. Wang, Z.-X. Yu, X. Lei, Angew.
Chem. Int. Ed. 2016, 55, 3112-3116.
3'a‐benzyl‐6'a‐(benzylamino)‐3'‐methyl‐1',6'‐
dihydrospiro[cyclohexane‐1,5'‐furo[2,3‐d]imidazole]‐2,5‐diene‐2',4‐
dione (24). To a solution of acetate 16 (6.0 mg, 0.016 mmol) in THF (200
µL) at room temperature was added benzylamine (3.6 µL, 0.033 mmol).
The reaction was stirred at room temperature for 1.5 h before being
quenched with water (5 mL). The aqueous layer was then extracted with
EtOAc (3 x 5 mL) and the combined organic layers washed with brine (10
mL), dried over Na2SO4, and concentrated in vacuo. The crude residue
was purified using column chromatography, eluting with 3:2 EtOAc to PE
to yield the title compound (4.6 mg, 68%) as a pale yellow oil. 1H NMR
(500 MHz, CDCl3) δ 7.54 – 7.46 (m, 2H), 7.42 – 7.27 (m, 8H), 7.00 (dd, J
= 10.3, 3.1 Hz, 1H), 6.05 (dd, J = 10.3, 2.0 Hz, 1H), 6.00 (dd, J = 10.1, 2.1
Hz, 1H), 5.81 (dd, J = 10.1, 3.1 Hz, 1H), 5.40 (s, 1H), 3.87 (br s, 2H), 3.28
(ABq, J = 14.4 Hz, 2H), 2.93 (s, 3H), 2.22 (d, J = 13.4 Hz, 1H), 2.08 (d, J
= 13.2 Hz, 1H), 1.89 (br s, 1H). 13C NMR (126 MHz, CDCl3) δ 185.0, 158.3,
149.4, 148.9, 139.1, 135.3, 131.9, 129.1, 128.6, 128.2, 128.1, 127.9, 127.5,
127.4, 102.7, 82.5, 77.3, 48.8, 47.5, 38.9, 26.0. HRMS-ESI (m/z): calcd for
C25H25N3NaO3 [M + Na]+, 438.17881; found, 438.17697. vmax (ATR-IR) cm-
3375, 3253, 2955, 2856, 1700, 1248, 697. [훼]2퐷2 = +14.3 (c = 0.14,
1
MeOH).
Acknowledgements
We acknowledge the University of Otago for a Research grant
(UORG). MPB and APC gratefully acknowledge the University of
Otago for the provision of PhD scholarships. We thank the
National Cancer Institute (NCI) for the preliminary testing of
spiroleucettadine against the NCI-60 panel of cancer cells.
Keywords: total synthesis • alkaloids • oxidative spirocyclization
• hypervalent iodine • medicinal chemistry
[1]
[2]
a) S. Carmelya, M. Ilanb, Y. Kashmana, Tetrahedron
1989, 45, 2193-2200; b) P. B. Koswatta, C. J. Lovely, Nat.
Prod. Rep. 2011, 28, 511-528.
a) B. R. Copp, C. R. Fairchild, L. Cornell, A. M. Casazza,
S. Robinson, C. M. Ireland, J. Med. Chem. 1998, 41,
3909-3911; b) D. V. LaBarbera, K. Modzelewska, A. I.
Glazar, P. D. Gray, M. Kaur, T. Liu, D. Grossman, M. K.
Harper, S. K. Kuwada, N. Moghal, C. M. Ireland, Anti-
Cancer Drugs 2009, 20, 425-436.
[3]
[4]
R. Singh, C. Lovely, 2019, pp. 43-79.
a) P. Ralifo, P. Crews, J. Org. Chem. 2004, 69, 9025-
9029; b) K. N. White, T. Amagata, A. G. Oliver, K. Tenney,
P. J. Wenzel, P. Crews, J. Org. Chem. 2008, 73, 8719-
8722; c) Y. Nagasawa, H. Kato, H. Rotinsulu, R. E. P.
Mangindaan, N. J. de Voogd, S. Tsukamoto, Tetrahedron
Lett. 2011, 52, 5342-5344.
[5]
a) R. A. Lamb, N. S. Aberle, N. T. Lucas, G. Lessene, B.
C. Hawkins, Angew. Chem. Int. Ed. 2017, 56, 14663-
14666; b) R. A. Lamb, G. Lessene, B. C. Hawkins, Synlett
2018, 29, 1125-1130.
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000954
ChemMedChem
FULL PAPER
Entry for the Table of Contents
The second-generation synthesis of spiroleucettadine is described along with its unexpected anti-proliferative properties. This
improved synthesis enabled access to large quantities of both enantiomers of spiroleucettadine and structural analogues. Three
spiroleucettadine analogues were found to possess similar potency as spirolecuettadine against lung cancer cells.
Institute and/or researcher Twitter usernames: @HawkinsLab4
9
This article is protected by copyright. All rights reserved.