Synthesis of cytotoxic spirocyclic imides from a biomass-derived oxanorbornene

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Stefan.B. Lawrenson, Amanda.K. Pearce, Sam Hart, Adrian.C. Whitwood, Rachel.K.

O’Reilly, Michael North

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S0040-4020(20)30988-1

https://doi.org/10.1016/j.tet.2020.131754

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To appear in: Tetrahedron

Received Date: 8 October 2020

Revised Date: 5 November 2020

Accepted Date: 6 November 2020

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Synthesis of cytotoxic spirocyclic imides

from a biomass-derived oxanorbornene

Leave this area blank for abstract info.

Stefan B. Lawrenson, Amanda K. Pearce, Sam Hart, Adrian C. Whitwood, Rachel K. O’Reilly and Michael

North

Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK; School of Chemistry,

University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

1

Tetrahedron

journal homepage: www.elsevier.com

Synthesis of cytotoxic spirocyclic imides from a biomass-derived oxanorbornene

Stefan B. Lawrenson^ab, Amanda K. Pearce^b, Sam Hart^a, Adrian C. Whitwood^a, Rachel K. O’Reilly^band

Michael North^a

^aDepartment of Chemistry, University of York, Heslington, York, YO10 5DD, UK.

^bSchool of Chemistry, University of Birmingham, Edgbaston, B15 2TT, UK.

Dedicated to Professor Richard J. K. Taylor in appreciation of his tenure as editor in chief of Tetrahedron.

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

N-Substituted derivatives of cantharimide and norcantharimide represent a promising but

underutilized motif for therapeutic applications. Herein, we report a divergent strategy for the

preparation of secondary amides and norcantharimide resembling spirocyclic imides from a

biomass-derived oxanorbornene and assess their biological activity. Computational modelling

suggests these compounds fall perfectly within lead-like chemical space (200 Da < RMM < 350

Da, -1 < AlogP < 3), with the spirocyclic imides preferred due to their lack of reactive

functionalities. Biological analysis of the spirocyclic imides revealed that the compounds

displayed antiproliferative activity against a range of human cancer cells (A549, HCT 116,

OVCAR-3, MDA-MB-231, MCF7 and PC-3) with the N-octyl derivative displaying the greatest

potential as a potent broad-spectrum anticancer drug. Dose-response curves for the N-octyl

spirocyclic imide found EC₅₀values of 56–95 µM dependent on the cell line, with highest

activity against human colorectal carcinoma cells (HCT 116).

Available online

Keywords:

Cytotoxicity

Drug design

Active surfactants

Cantharidin

Spirocyclic

.

———

a

Michael North. Tel.: +44(0)1904 324 545; e-mail: michael.north@york.ac.uk.

b

Rachel K. O’Reilly. Tel.: +44(0)1214 147 757; e-mail: r.oreilly@bham.ac.uk.

2

Tetrahedron

1. Introduction

substituted with either

D- or L-histidine are more potent than

norcantharidin 7 and equipotent to cantharidin 5 for the inhibition

of PP1 and PP2A.¹⁴Finally, Gasser et al. has shown that N-

substituted norcantharimides display unique potential as

Cyclic imides represent an important structural motif that has

found widespread use across biological, medicinal and polymer

chemistry.^1,2They are prevalent in natural products and

therapeutics that have a wide range of biological activities;

finding potential as sedatives,³hypnotics,⁴anxiolytics,⁵anti-

inflammatories,⁶antivirals,⁷antibacterials,⁸antimicrobials⁹and

carcinostatics.¹⁰Notable therapeutic examples include

thalidomide 1, phensuximide 2, lenalidomide 3 and fluorouracil 4

(Figure 1).

Cantharidin 5, exo,exo-2,3,-dimethyl-7-oxabicyclo[2.2.1]

heptane-2,3,dicarboxylic acid anhydride, and the cyclic imide

analogue cantharimide 6 are natural products found within the

Mylabris genus of the Meloidae family of Chinese blister

beetles;¹¹the dried bodies of which have been widely utilized

within Chinese traditional medicine for over 2000 years for a

range of ailments.¹²Of particular interest is the cytotoxicity of

cantharidin 5, first reported in 1264,¹²which has since been

found to be active against a number of human cancer cell lines,

particularly liver and esophagus carcinoma.¹³Cantharidin has

also been identified to induce haematopoiesis in both humans and

animals, as well as to be a potent and selective inhibitor of the

serine/threonine protein phosphatases PP1 and PP2A.^14,15

Unfortunately, cantharidin is also known to possess a number of

severe side effects including hepato- and nephrotoxicity, thus

limiting its application to date to a topical treatment for

molluscum contagiosum.^16,17

nematocides for the treatment of Haemonchus contortus.³³We

therefore believe substantial scope exists for exploring the

potential of novel cyclic imides based on N-substituted

cantharimide

applications.

6

and norcantharimide

8

for therapeutic

Ourselves, along with Pehere et al., have previously reported,

independently and simultaneously, the synthesis of 2-

[(1S*,5S*,7S*)-4-oxo-3,10-dioxatricyclo-[5.2.1.0^1,5]dec-8-en-5-

yl] acetic acid 12, through the tandem Diels-Alder cycloaddition

and lactonization reaction of furfuryl alcohol 10 and itaconic

anhydride 9 (Scheme 1).^36,37Oxanorbornene 12 is isolated as a

single species, due to the lactonization step only being favorable

when the [4+2] cycloaddition yields the prox-exo intermediate

11, which drives ring-opening of the anhydride and subsequent

crystallization of 12 from the dynamic reaction mixture as a

racemic mixture.³⁷This direct and atom economical reaction

Figure 1. Chemical structures of thalidomide 1, phensuximide 2,

lenalidomide 3, fluorouracil 4 and the cantharidin family 5–8, cantharidin 5,

cantharimide 6, norcantharidin 7, and norcantharimide 8.

In comparison, the demethylated species norcantharidin 7 also

demonstrates similar anticancer activity to the parent compound,

however without the associated toxicity.¹⁶Its synthesis is

likewise readily accessible through the Diels-Alder [4+2]

cycloaddition between furan and maleic anhydride, followed by

subsequent reduction of the alkene.¹⁸This has led to numerous

investigations into synthetic analogues of cantharidin 5, with

several groups reporting structure-activity relationships (SAR)

for cytotoxicity and PP1/PP2A inhibition following modification

of the 7-oxabicylo[2.2.1]heptyl ring system^19–23or ring-opening

the anhydride.^24–29However, these synthetic analogues have

seldom shown cytotoxicity comparable to cantharidin 5,

suggesting only limited modification of the parent compound is

tolerated.

therefore presents

a highly efficient route to yield a

functionalized oxanorbornene. Furthermore, itaconic anhydride 9

and furfuryl alcohol 10 are readily available from biomass; the

former through the simultaneous decarboxylation and

dehydration of citric acid (ca. 1,000,000 tons per annum),³⁸the

latter by hydrogenation of furfural, which is produced through

the acid-catalyzed dehydration of pentoses (ca. 200,000 tons per

annum).³⁹

Scheme 1. Preparation of oxanorbornene 12 via the [4+2] cycloaddition

Rather surprisingly, derivatization of cantharidin

5 or

and intramolecular lactonization of furfuryl alcohol 10 and itaconic anhydride

9.

norcantharidin 7 to the corresponding N-substituted imides,

through condensation of the anhydride with an amine, has seen

minimal investigation, despite a few encouraging reports.^13–

15,23,30–35

For example, potential for broad spectrum cytotoxicity

against a range of cell lines has been highlighted a number of

times,^{13,15,31,32,34}with Lin et al. demonstrating that N-

thiazolylcantharimides were more potent than cantharidin against

the human hepatocellular carcinoma cell lines SK-Hep-1 and Hep

3B.³⁰McCluskey et al. have also found that norcantharimides

3

converted to the acid chloride intermediate, using oxalyl

chloride and catalytic DMF (Scheme 3). The acid chloride was

easily isolated, redissolved in fresh anhydrous CH₂Cl₂, and

reacted with N-butylamine in the presence of two equivalents of

triethylamine, to account for the formation of the hydrochloric

acid by-product. This yielded the desired secondary amide 20a

along with spirocyclic imide 22. Compounds 20a and 22 were

separable by flash column chromatography, allowing them to be

studied independently.

Scheme 3. Synthesis of N-butyl secondary amide 20a and spirocyclic

imide 22 by the amidation of 12 by oxalyl chloride and N-butylamine.

The respective structures of 20a and 22 were determined using

a combination of electrospray ionization-mass spectroscopy

(ESI-MS), FTIR and 2D ¹H-¹³C heteronuclear multiple bond

correlation NMR spectroscopy (Figure 2). ESI-MS showed that

the two species had identical molecular weights, suggesting they

were structural isomers. FTIR then clearly identified a sharp peak

at 3396 cm^-1in 22, which was absent in 20a, most likely

corresponding to the O-H stretch of an alcohol. This strongly

implied that compound 22 was the result of an intramolecular

cyclization, which led to the ring-opening of the lactone moiety.

Finally, ¹H-¹³C HMBC NMR spectroscopy of the two

compounds showed that the butyl protons alpha to the nitrogen

(yellow dot) had a clear correlation to both carbonyl groups (grey

dot) in 22 (Figure 2 spectrum B), rather than just the one as

observed in the secondary amide 20a (Figure 2 spectrum A).

This confirmed the spirocyclic imide structure for 22, which can

be formed by an intramolecular 5-exo-trig cyclization of

secondary amide 20a. The structures of both N-butyl secondary

amide 20a and spirocyclic imide 22 were then subsequently

Scheme 2. A. Previous work on the synthesis of bio-based ROMP

polymers from oxanorbornene lactone 12 and oxanorbornene lactam 15. B.

This work on the divergent synthesis of secondary amides and spirocyclic

imides from oxanorbornene 12.

Subsequently, we have been interested in exploring

derivatives of oxanorbornene 12 as novel reactive monomers for

the synthesis of sustainable polymers via ring-opening metathesis

polymerization (ROMP). We have reported a number of

examples utilizing ester and tertiary amide derivatives 13 for the

synthesis of various homo- and copolymers 14 derived from

biomass using Grubbs 2^ndgeneration catalyst (Scheme 2).^36,40,41

These polymerizations were found to be well-controlled, but

slow, which was attributed to the endo-orientation of the

carboxylic acid derivative within 13. In more recent work,

structurally similar N-substituted lactams 15 possessing an exo-

orientated carboxylic acid were found to display well-controlled

and extremely rapid polymerizations in the presence of Grubbs

3^rdgeneration catalyst.⁴²During the course of these

investigations, whilst attempting to produce secondary amide

derivatives of 12 for ROMP, we observed a cyclization that

yielded a mixture of two species; the desired secondary amide

and a spirocyclic imide (Scheme 2). Inspired by the structural

similarity between the spirocyclic imide and the aforementioned

cantharimide 6 and norcantharimide 8, we were motivated to

explore this avenue further. Herein, we present a divergent

strategy for the selective preparation of secondary amides 19a–e

(pathway A) and spirocyclic imides 21a–e (pathway B) based on

the 7-oxabicylo[2.2.1]heptyl ring system, from oxanorbornene

12. A computational study of these compounds by lead-likeness

and molecular analysis (LLAMA) suggested that spirocyclic

imides 21a–e represent highly novel and lead-like compounds,

and as such they were subsequently investigated for their

biological activity against a range of human cancer cell lines.

confirmed by X-ray crystallography (CCDC 2000921

2000922).

&

Intrigued by the unusual nature of spirocyclic compound 22,

we were encouraged to investigate this transformation further in

an effort to selectively produce either compound and study their

applications. Furthermore, the synthesis of cyclic imides is

traditionally performed through the dehydrative condensation of

an anhydride with an amine or the cyclisation of an amic-acid,

which usually requires either high temperature, a Lewis acid

catalyst or the use of stoichiometric acidic reagents.⁴³More

recently, there has been great interest in direct routes to cyclic

imides using readily available starting materials due to the

therapeutic potential of this class of compounds.^4,43–45The

intramolecular 5-exo-trig cyclization of a secondary amide onto a

lactone under homogeneous basic conditions, as reported here,

provides a mild and highly efficient method for the synthesis of

cyclic imides.⁴⁶

2. Results and Discussion

Initially, amide synthesis was performed in a similar manner

to that reported previously, with oxanorbornene 12 first

4

Tetrahedron

Figure 2. ¹H-¹³C HMBC NMR spectroscopy experiments for A. N-butyl secondary amide 20a and B. N-butyl spirocyclic imide 22, and associated X-ray crystal

structures.

In an effort to selectively synthesize either the N-butyl

secondary amide 20a or the spirocyclic imide 22, the quantity of

triethylamine was adjusted during the amidation step of the

reaction (Figure S101). When varying the quantity of

triethylamine from 0.8–4.0 equivalents relative to oxanorbornene

12, it was found by ¹H NMR spectroscopy of the reaction

mixture that either species could be formed, with 0.8 equivalents

giving exclusively the N-butyl secondary amide 20a whilst 4.0

equivalents produced exclusively the N-butyl spirocyclic imide

22. This was rationalized on the basis that amides are typically

poor nucleophiles for N-attack due to delocalization of the lone

pair, which results in O-attack being preferred.⁴⁷In contrast,

ambident amide nucleophiles have been shown to prefer N-attack

over O-attack.⁴⁸Deprotonation of the amide would therefore

favor N-attack through the now nucleophilic nitrogen leading to

formation of the spirocyclic product 22, whilst under neutral or

acidic conditions ring-opening of the lactone is disfavored,

leading to formation of the secondary amide 20a. The N-butyl

secondary amide 20a was then exposed to 2.0 equivalents of

triethylamine to see if it could promote formation of the imide 22

(Figure S102). It was observed that 20a was indeed converted to

22, with optimal formation of the imide found after 7 hours (20a

4%; 22 85%; 10 and 23 11%), and complete consumption of the

amide observed after 24 hours (20a 0%; 22 41%; 10 and 23

59%). This experiment reaffirmed our hypothesis that

deprotonation of the amide is necessary to form imide 22 whilst

also highlighting its inherent instability in solution, with clear

degradation products observed to form over the course of the

reaction. The degradation products were subsequently identified

to be furfuryl alcohol 10 and 1-butyl-3-methylene-pyrrolidine-

2,5,-dione 23, formed through a retro-Diels-Alder reaction, which

is possible following ring-opening of the lactone moiety (Scheme

4).

Scheme 4. Intramolecular 5-exo-trig cyclization of 20a under basic

conditions to form 22 and subsequent retro-Diels-Alder, which yields the

degradation products furfuryl alcohol 10 and 1-butyl-3-methylene-

pyrrolidine-2,5-dione 23.

On the basis of the above studies, we realized that

hydrogenation of the oxanorbornene unit within structures 20a

and 22 would prevent the retro-Diels-Alder reaction and, in the

case of the spirocyclic imide, provide a motif that bears a striking

resemblance to the natural products cantharimide

6 and

norcantharimide 8, suggesting a potential therapeutic application.

Buoyed by this we set about investigating derivatization of

oxanorbornene 12 by amidation and hydrogenation (Scheme 5).

O

N

i) 5-exo-trig

cyclization

ⁿBu

O

ii) H⁺

N

O

ⁿBu

O

HO

O

22

retro-Diels-Alder

reaction

O

OH +

N

ⁿBu

O

10

23

5

This can be done by either first hydrogenating 12 to produce the

oxanorbornane 18 which can then be functionalized with the

desired amine 19a–e (pathway A), or by producing the desired

secondary amide 20a–e derivatives which are then hydrogenated

21a–e (pathway B). Rather surprisingly, we found that it was

possible to selectively form either the secondary amides 19a–e or

the spirocyclic imides 21a–e depending on the method used,

providing a simple and divergent route to either species from

oxanorbornene 12. In the case of pathway A, oxanorbornene 12

was first hydrogenated using 10 wt% palladium on carbon,

yielding the corresponding oxanorbornane 18 in an excellent

93% yield. Amidation was then performed using a series of alkyl

and aryl amines according to the procedure described previously,

producing secondary amides 19a–e in moderate to excellent

yields (33–77%). Notably, pyridine was used as the base rather

than triethylamine due to its lower basicity (pK_aHpyridine: 5.23;

NEt₃: 10.65, both in H₂O) and less than two equivalents were

used to prevent formation of spirocyclic species.⁴⁹Alternatively,

by pathway B, amidation of oxanorbornene 12 could be used to

selectively produce the secondary amides 20a–e in moderate to

excellent yields (31–86%). Hydrogenation of 20a–e then resulted

in a tandem cyclisation to produce spirocyclic imides 21a–e in

excellent yields (76–98%). A possible explanation for this is that

the hydrogenation conditions result in the formation of palladium

hydrides which are sufficiently basic to deprotonate the amide,

facilitating the cyclisation. As described previously, spirocyclic

imides 21a–e could be easily differentiated from secondary

whether these compounds held potential as lead-like targets for

therapeutic applications. Lead-likeness and molecular analysis

(LLAMA) is an open-access, web-based tool developed by the

University of Leeds for the decoration and assessment of small

molecule scaffolds for their lead-likeness and novelty.⁵⁰

Analyzing secondary amides 19a–e and spirocyclic imides 21a–e

by LLAMA, it was found that all compounds lay within lead-like

space on the basis of their relative molecular mass (RMM) (200

Da < RMM < 350 Da) and predicted lipophilicity (-1 < AlogP

< 3) as shown in Figure 3. However, whilst spirocyclic imides

21a–e were assigned minimal lead-likeness penalties (0–1: due to

the absence of an aromatic ring in 21d,e), secondary amides 19a–

e were assigned higher penalties (5–6) due to the presence of the

undesirable lactone functionality and the absence of an aromatic

ring in 19d,e. The compounds were then analyzed for novelty

against the ZINC database, a repository of over twenty million

commercially available molecules.⁵¹In both cases, the secondary

amides 19a–e and the spirocyclic imides 21a–e were found to

possess a 0.0% likeness to a random 2% of the database. Finally,

PMI analysis found that the majority of the compounds lie along

the rod-like to disc-like axis. Overall, these results strongly

suggest that the spirocyclic imides 21a–e represent highly novel

and promising lead-like compounds for further consideration as

therapeutics.

Figure 3. A. Lead-likeness and B. PMI analysis plots for secondary

amides 19a–e and spirocyclic imides 21a–e. Blue region represents the 3D

region of PMI space where I1 + I2 > 1.2.

amides 19a–e through

a combination of ESI-MS, FTIR

spectroscopy and ¹H-¹³C HMBC NMR spectroscopy (see

supporting information), and the structure of 21d was confirmed

by X-ray crystallography (CCDC 2000923).

Previously, cantharidin and norcantharidin analogues have

shown promising anticancer activities against a range of cancer

cell types, both in vitro and in vivo.^{14,29,32,52,53}Based on this, we

screened the racemic spirocyclic imides 21a–e, for their activity

against six human cancer cell lines. For this we selected A549

human lung carcinoma cells, HCT 116 colorectal carcinoma

cells, OVCAR-3 human ovarian adenocarcinoma cells, MDA-

MB-231 human breast adenocarcinoma cells, MCF7 human

breast adenocarcinoma cells and PC-3 human prostate

adenocarcinoma cells to represent a range of cell types. For the

initial investigation, we performed a broad assessment of the

potential of all five molecules to inhibit cancer cell growth at two

moderate fixed concentrations (100 µM and 200 µM) and over

three time points: 24, 48 and 72h. Cell growth was monitored as

a function of relative cell viability, and the results are shown in

Table 1, Figure 4A and Figures S103–S108.

Scheme 5. Synthesis of secondary amides 19a–e and spirocyclic imides

21a–e by divergent amidation/hydrogenation strategy from oxanorbornene 12

and the X-ray crystal structure of 21d.

In view of the resemblance of these compounds to

cantharimide 6 and norcantharimide 8, we were intrigued as to

The data in Table 1 shows that after 72h of exposure,

promisingly, all compounds had a negative impact on the growth

of all studied cell lines. Figures S103–S108 confirm that these

results have both a concentration- and time-dependent effect. In

particular, HCT 116, A549, OVCAR-3 and MCF7 cells were

most susceptible to compounds 21a-e after 72h of exposure,

while PC-3 and MDA-MB-231 cells showed more resistance.

Furthermore, it was apparent that compound 21c consistently

exhibited the highest potency from all the compounds assayed. In

HCT 116 cells, at 200 µM, almost complete cell death was

achieved, with similar efficacy in A549 and MCF7 cells,

suggesting that compound 21c has potential as a potent, broad-

spectrum anticancer agent. When analyzing the biological results

in comparison with the chemical structures of each compound,

whereby 21c followed by 21b were the most toxic, it was

apparent that the presence of a long alkyl chain (C8 and C6

chains respectively) increased the anticancer effect. This was

unexpected, with 21a–c in fact receiving penalties during the

6

Tetrahedron

LLAMA simulation as a result of the prediction placing

importance on the presence of aromatic moieties. Instead, the

three less active compounds had either a short alkyl chain (21a,

C4) or aryl/aryloxy side-groups (21d,e).

Table 1. Cytotoxicity of compounds 21a–e in the cancer cell panel. Cytotoxicity is expressed as the percentage of relative metabolic activity (compared to

untreated cells) at 100 μM and 200 μM drug concentration after 72 h of continuous drug exposure, presented as mean ± S.D (n=5)

HCT 116

A549

PC-3

OVCAR-3

MCF7

MDA-MB-231

100 µM

200 µM

27 ± 5

23 ± 2

100 µM

200 µM

46 ± 7

43 ± 5

18 ± 7

36 ± 7

46 ± 6

100 µM

82 ± 8

69 ± 6

61 ± 4

72 ± 9

83 ± 5

200 µM

62 ± 5

55 ± 9

40 ± 9

63 ± 8

73 ± 7

100 µM

200 µM

59 ± 9

47 ± 9

16 ± 3

61 ± 2

57 ± 10

100 µM

200 µM

46 ± 5

35 ± 7

10 ± 5

48 ± 5

39 ± 4

100 µM

200 µM

64 ± 6

59 ± 3

40 ± 11

65 ± 5

63 ± 8

21a

21b

21c

21d

21e

79 ± 2

61 ± 8

31 ± 4

74 ± 3

72 ± 4

83 ± 8

74 ± 5

33 ± 5

78 ± 2

78 ± 5

69 ± 5

64 ± 7

35 ± 2

77 ± 5

63 ± 9

68 ± 12

51 ± 10

29 ± 9

72 ± 4

77 ± 4

62 ± 5

75 ± 4

76 ± 9

5

± 2

42 ± 2

49 ± 6

71 ± 12

67 ± 10

Figure 4. A. Representative cytotoxicity profiles of compounds 21a–e in HCT 116 cells over 24, 48 and 72h as assessed by the PrestoBlue metabolic assay,

B. Dose-response curves of compound 21c in all cell lines. Data are presented as mean ± S.D (n=4). C. DLS traces of compounds 21a–e showing the presence of

well-defined nanoaggregates in milliQ water (concentration 125 µg/mL).

Given the structural similarity of the two most active

compounds to traditional surfactant molecules, i.e. the presence

of a polar head group and aliphatic sidechain, it was hypothesized

that this could be the cause of the enhanced cytotoxicity of 21b-c

over the other lead-like compounds. Many surfactants have

toxicological effects on cells as a result of hydrocarbon chain

insertion into lipid bilayers, causing perturbation of cell

indicating that no cell membrane lysis was occurring. To further

verify this result, as it is reported that surfactant toxicity is

significantly heightened at concentrations lower than the

CMC/CAC, the propensity of compounds 21a–e to first self-

assemble, and then the concentration at which this occurred, was

assessed using a dynamic light scattering (DLS) method.⁵⁶In

accordance with our hypothesis, all compounds were confirmed

to form nanoaggregates in aqueous solution of a size range 100–

120 nm (Figure 4C), and with CAC values dependent upon

sidechain chemistry (Figure S110, Table S1). The calculated

CAC values confirmed that the LDH assay had been performed

both above and below CAC values, and therefore that there was

no influence of this parameter on targeted toxicity against cell

membranes. Interestingly, given the important role of surfactants

for the formulation of poorly soluble drugs,⁵⁷these findings

crucially highlight the potential of these compounds as “active”

membranes and lysis, which is typically

a function of

hydrocarbon chain length and critical micelle/aggregation

concentration (CMC/CAC).^54,55The lactate dehydrogenase

(LDH) cytotoxicity colorimetric assay was employed to assess

this, as a reliable method for measuring release of the cytosolic

enzyme LDH into the cell culture medium upon any damage to

the cell plasma membrane. Surprisingly, no LDH release was

observed after 48h incubation at the highest concentration of 200

µM in A549 cells as a representative experiment (Figure S109),

7

surfactants for formulation of a second drug, in order to achieve

combination therapies, target different mechanisms of action and

prevent drug resistance.⁵⁸

backed plates pre-coated (0.25 mm) with Merck KGaA silica

gel 60 F₂₅₄. Compounds were visualized by exposure to UV-light

(254 nm) and stained using KMNO₄or phosphomolybdic acid

followed by heating. Flash column chromatography was

performed using Fluorochem silica gel LC60A (40–60 µm). All

mixed solvent eluents are reported as v/v solutions. Brine refers

to a saturated solution of aqueous sodium chloride.

Finally, on the basis of these positive observations and due to

the potency of 21c, further biological analysis was performed to

determine the EC₅₀concentration in all the tested cell lines. EC₅₀

values were determined after 48h incubation and the results are

shown in Figure 4, Figures S111–S116 and Table 2. All values

were less than 100 µM and followed the same trend as the initial

cytotoxicity assays, whereby the lowest EC₅₀values were

observed in HCT 116 cells (56 µM). Overall, the biological

assays, in agreement with the LLAMA predictions, confirmed

that molecules based on the spirocyclic imide scaffold described

here show potential as active agents or excipients for anticancer

applications. While the final EC₅₀values were modest in

comparison to traditional anticancer drugs (typical EC₅₀<10

µM), the compounds assessed in this work can be considered as

unoptimized and preliminary candidates in terms of their

introduced functionalities.⁵⁹Future work will endeavor to further

explore the possibility to introduce different sidechain substituent

chemistries in an effort to enhance the biological activity of these

interesting compounds, assess their range of therapeutic potential

such as within antimicrobial applications, as well as investigating

their potential for the codelivery of other active agents by

exploiting their inherent propensity to self-assemble into

nanoparticles.

¹H and ¹³C-Nuclear magnetic resonance (NMR) spectra were

acquired using either a JEOL ECS 400 MHz spectrometer or a

Bruker DPX-400 400 MHz spectrometer. ¹H spectra were

referenced internally to the residual protic solvent resonance

(CHCl₃= 7.27 ppm, DMSO-d₆= 2.50 ppm and CH₃OH = 3.31

ppm). ¹³C-Spectra were referenced internally to the solvent

resonance (CDCl₃= 77.16 ppm, CD₃OD = 49.00 ppm and

DMSO-d₆= 39.52 ppm). ¹H-NMR coupling constants are

reported in Hertz (Hz). Coupling constants are reported using the

following notation, or combination of; s = singlet, br = broad, d =

doublet, t = triplet, q = quartet, quin = quintet, sex = sextet, sept =

septet, oct = octet, non = nonet and m = multiplet. Assignment of

signals in ¹H and ¹³C-spectra was determined using ¹H-¹H COSY,

DEPT-135, ¹H-¹³C HMQC and HMBC experiments where

appropriate.

High resolution mass spectra (HRMS) were recorded using

electrospray ionization (ESI) on a Bruker micrOTOF mass

spectrometer in tandem with an Agilent series 1200 liquid

chromatography system or on a Waters Xevo G2-XS Quadrupole

time-of-flight mass spectrometer. All Infra-Red (IR) data was

obtained using a Perkin-Elmer Spectrum Two or Spectrum 400

FT-IR spectrometer or an Agilent Technologies Cary 630 FTIR

spectrometer. Absorbances are reported using the following

notation; w = weak, m = medium and s = strong. Melting points

were determined using either a Stuart SMP3, SMP20 or an SRS

OptiMelt MPA100 hot stage apparatus and were not corrected.

Dynamic light scattering measurements were recorded using a

Malvern Panalytical Zetasizer Nano S fitted with a 633 nm He-

Ne laser operating at 4 mW. Measurements were performed at 25

°C using a scattering angle of 173° (backscattering), and the

results analyzed using Malvern Panalytical Zetasizer software

version 7.13.

Table 2. Calculated EC₅₀values of compound 21c.

HCT

116

A549

PC-3

OVCAR-

3

MCF7

MDA-MS-

231

EC50

(µM)

56±3

83±3

95±5

79±14

79±8

80±22

3. Conclusion

Functionalization of biomass derived oxanorbornene 12 has

led to a divergent strategy for the preparation of a range of new

spirocyclic imides 21a–e and secondary amides 19a–e.

Computational modelling identified that these compounds

represent highly novel and lead-like structures for the

development of clinical candidates, with the spirocyclic imides

21a–e preferred due to their lack of reactive functionalities.

Accordingly, the spirocyclic imides 21a–e were analyzed for

their biological activity using cytotoxicity assays against a range

of human cancer cell lines. Overall, all compounds showed some

degree of antiproliferative effect, with 21c presenting the greatest

potential as a potent broad spectrum anticancer agent. The

compounds demonstrated a propensity to self-assemble into

nanoaggregates, indicating promising application potential as

“active” surfactants for formulation of a second drug. This study

justifies further investigation into the therapeutic potential of

these unusual species.

Diffraction data were collected at 110 K (195 K for 20a) on an

Oxford Diffraction SuperNova diffractometer with Cu-Kα

radiation (λ = 1.54184 Å) using an EOS CCD camera. The

crystal was cooled with an Oxford Instruments Cryojet.

Diffractometer control, data collection, initial unit cell

determination, frame integration and unit-cell refinement was

carried out with “Crysalis”. Face-indexed absorption corrections

were applied using spherical harmonics, implemented in

SCALE3 ABSPACK scaling algorithm. OLEX2 was used for

overall structure solution, refinement and preparation of

computer graphics and publication data. Within OLEX2, the

algorithm used for structure solution was SHELXT, charge-

flipping. Refinement by full-matrix least-squares used the

SHELXT algorithm within OLEX2. Hydrogen atoms were

placed using a “riding-mode” and included in the refinement at

calculated positions. Structures for 22, 20a and 21d were

deposited in the CCDC (CCDC 2000921-3).⁶⁰

4. Experimental Section

4.1. General Experimental

LLAMA was used to determine if compounds resided in a

region of chemical space within which they have a greater

potential for development into clinical candidates, as defined by

Nadin et al.⁶¹Structural novelty was then assessed against the

Murcko framework, where all side chain functionality was

removed reducing the molecule to its ring systems and the linkers

between them, with and without the alpha atoms.⁶²These

frameworks were then assessed against a random 2% of the

All reagents and solvents were purchased from commercial

suppliers and used as sold, excluding furfuryl alcohol which was

distilled prior to use. Anhydrous CH₂Cl₂was obtained from an

Inert Technologies PureSolv EN 1-4 enclosed solvent

purification system. All air- and water-sensitive reactions were

carried out in oven-dried glassware under either a nitrogen or

argon atmosphere. Analytical TLC was performed on aluminum

8

Tetrahedron

ZINC database.⁵¹Finally, the molecules were analyzed by

coupled according to the general procedure for the synthesis of

secondary amides. The crude material was then purified by flash

column chromatography (80:20, EtOAc:PE) to give the target

compound 20a as a white solid (1.7 g, 86%). Crystals suitable for

X-ray analysis were grown by slow evaporation of an acetone

solution. R_F= 0.18 (80:20, EtOAc:PE); m.p. 83.2–83.7 °C; IR

(Neat) υ_max3240 (m), 3078 (w), 2961 (m), 2876 (w), 1768 (s),

1631 (s) and 1562 (s) cm^-1; ¹H NMR (400 MHz, CDCl₃): δ = 6.60

(d, J = 5.9 Hz, 1H, 2-H), 6.50 (dd, J = 5.9, 1.8 Hz, 1H, 1-H), 6.34

(br, 1H, N-H), 5.01 (dd, J = 4.6, 1.7 Hz, 1H, 6-H), 4.84 (d, J =

10.7 Hz, 1H, 7-H), 4.58 (d, J = 10.7 Hz, 1H, 7-H), 3.18 (dt, J =

7.1, 2.5 Hz, 2H, 11-H), 2.49 (dd, J = 11.9, 4.8 Hz, 1H, 5-H), 2.33

(d, J = 14.3 Hz, 1H, 9-H), 2.25 (d, J = 14.3 Hz, 1H, 9-H), 1.50–

1.40 (m, 2H, 12-H), 1.37–1.27 (m, 2H, 1×5-H/2×13-H), 0.89 (t, J

= 7.26, 3H, 14-H); ¹³C NMR (100 MHz, CDCl₃): δ = 179.3 (8),

168.5 (10), 137.3 (1), 131.5 (2), 94.5 (3), 78.5 (6), 69.7 (7), 52.8

(4), 42.7 (9), 39.6 (11), 36.6 (5), 31.4 (12), 20.1 (13), 13.8 (14);

HRMS (ESI) m/z calculated for C₁₄H₁₉NNaO₄288.1202

(M+Na)⁺, found 288.1206, 1.5 ppm error.

principal moments of inertia (PMI) analysis, in order to

determine shape distribution. Thus, the system randomly

generated a number of 3D conformers of each molecule and

minimized their energy, selecting the lowest energy conformer

for further analysis. The moments of inertia were then calculated

along the X, Y, and Z-axis, with the I1 coordinates calculated by

dividing inertia(X) by inertia(Z) and I2 by dividing inertia(Y) by

inertia(Z). The resulting PMI plot of I1 against I2 provides an

indication of whether the molecule is rod (I1=1.0, I2=0.0), disc

(I1=0.5, I2=0.5) or sphere (I1=1.0, I2=1.0)-like in nature (Figure

3).

4.2. Compound Data

2-[(1S*,5S*,7S*)-4-Oxo-3,10-dioxatricyclo-[5.2.1.0^1,5]dec-8-

en-5-yl] acetic acid (12): Acid 12 was prepared from itaconic

anhydride and furfuryl alcohol using either of the procedures

described below. Solvent Free: Itaconic anhydride (3.0 g, 27

mmol, 1.0 eq.) was suspended in furfuryl alcohol (2.3 mL, 27

mmol, 1.0 eq.), the slurry obtained was allowed to stir at ambient

temperature. After circa 5 h, the suspension had thickened to a

paste could no longer be stirred. The reaction mixture was left for

a further 19 h until a tan solid had formed. The crude material

was then purified by recrystallization from acetone to give the

target acid 12 as an off-white crystalline solid (3.8 g, 68%).

Reaction solvent: Itaconic anhydride (25.0 g, 223 mmol, 1.0 eq.)

and furfuryl alcohol (19.4 mL, 223 mmol, 1.0 eq.) were

suspended in acetonitrile (12 mL) and the slurry allowed to stir at

ambient temperature, after 24 h a white suspension had formed.

The solid was removed by filtration and the filtrate was

concentrated in vacuo. The concentrated filtrate was suspended in

EtOAc (100 mL) and filtered. The obtained solids were

combined and recrystallized from acetone, to give the target acid

12 as an off-white crystalline solid (20.0 g, 43%). m.p. 130.9–

131.5 °C; IR (Neat) υ_max3100 (m), 1776 (s) and 1733 (s) cm^-1; ¹H

NMR (400 MHz, CD₃OD): δ = 6.59 (dd, J = 5.9, 1.7 Hz, 1H, 1-

H), 6.55 (d, J = 5.9 Hz, 1H, 2-H), 5.02 (dd, J = 4.7, 1.6 Hz, 1H,

6-H), 4.92 (d, J = 10.8 Hz, 1H, 7-H), 4.54 (d, J = 10.8 Hz, 1H, 7-

H), 2.44–2.35 (m, 3H, 5/9-H), 1.55 (d, J = 12.3 Hz, 1H, 5-H); ¹³C

NMR (100 MHz, CD₃OD): δ = 180.0 (8), 173.0 (10), 139.3 (1),

131.5 (2), 95.6 (3), 80.1 (6), 70.0 (7), 53.3 (4), 40.7 (9), 37.7 (5);

HRMS (ESI) m/z calculated for C₁₀H₁₀NaO₅233.0420 (M+Na)⁺,

found 233.0417, 1.2 ppm error. Data is consistent with

previously reported characterization data.³⁶

N-Hexyl-2-[(1S*,5S*,7S*))-4-oxo-3,10-dioxatricyclo

[5.2.1.0^1,5]dec-8-en-5-yl]acetamide (20b): Acid 12 (2.0 g, 9.5

mmol, 1.0 eq.) and hexylamine (1.3 mL, 9.5 mmol, 1.0 eq.) were

coupled according to the general procedure for the synthesis of

secondary amides. The crude material was then purified by flash

column chromatography (70:30–100:0, EtOAc:PE) to give the

target compound 20b as a yellow crystalline solid (0.87 g, 31%).

R_F= 0.19 (70:30, EtOAc:PE); m.p. 61.6–62.4 °C; IR (Neat) υ_max

3303 (m), 2927 (m), 2855 (m), 1767 (s), 1669 (m), 1649 (s) and

1541 (s) cm^-1; H NMR (400 MHz, CDCl₃): δ = 6.60 (d, J = 5.7

1

Hz, 1H, 2-H), 5.67 (dd, J = 5.7, 1.6 Hz, 1H, 1-H), 6.39–6.32 (br,

1H, N-H), 5.01 (dd, J = 4.6, 1.6 Hz, 1H, 6-H), 4.84 (d, J = 10.5

Hz, 1H, 7-H), 4.57 (d, J = 10.5 Hz, 1H, 7-H), 3.18 (dt, J = 7.1,

6.8 Hz, 2H, 11-H), 2.49 (dd, J = 12.1, 4.6 Hz, 1H, 5-H), 2.32 (d,

J = 14.6 Hz, 1H, 9-H), 2.25 (d, J = 14.6, 1H, 9-H), 1.52–1.41 (m,

2H, 12-H), 1.33–1.20 (m, 7H, 5-H/13-H/14-H/15-H), 0.86 (t, J =

7.2 Hz, 3H, 16-H); ¹³C NMR (100 MHz, CDCl₃): δ = 179.3 (8),

168.5 (10), 137.3 (1), 131.5 (2), 94.5 (3), 78.5 (6), 69.7 (7), 52.8

(4), 42.7 (9), 39.9 (11), 36.6 (5), 31.5 (15), 29.3 (12), 26.6 (13),

22.6 (14), 14.1 (16); HRMS (ESI) m/z calculated for C₁₆H_23-

NNaO₄316.1519 (M+Na)⁺, found 316.1525, -1.9 ppm error.

N-Octyl-2-[(1S*,5S*,7S*))-4-oxo-3,10-dioxatricyclo

[5.2.1.0^1,5]dec-8-en-5-yl]acetamide (20c): Acid 12 (2.0 g, 9.5

mmol, 1.0 eq.) and octylamine (1.6 mL, 9.5 mmol, 1.0 eq.) were

coupled according to the general procedure for the synthesis of

secondary amides. The crude material was then purified by flash

column chromatography (90:10, EtOAc:PE) to give the target

compound 20c as a pale yellow crystalline solid (2.1 g, 69%). R_F

= 0.3 (90:10, EtOAc:PE); m.p. 75.8–76.2 °C; IR (Neat) υ_max3302

(m), 2918 (m), 2851 (m), 1767 (s), 1669 (m), 1649 (s) and 1543

General procedure for the synthesis of secondary amides

from 12. Acid 12 (2.0 g, 9.5 mmol, 1.0 eq.) was suspended in

anhydrous CH₂Cl₂(5 mL) under an atmosphere of nitrogen. The

suspension was cooled to 0 °C and oxalyl chloride 2.0 M solution

in CH₂Cl₂(12 mL, 24 mmol, 2.5 eq.) was added dropwise over

10 minutes, followed by DMF (4 drops). The suspension was

stirred at ambient temperature until a solution was obtained, after

which it was concentrated in vacuo, to yield the crude acid

chloride as a light brown solid. The crude material was

redissolved in fresh anhydrous CH₂Cl₂(10 mL) and cooled to 0

°C. Pyridine (0.77 mL, 9.5 mmol, 1.0 eq.) was then added

dropwise, followed by the primary amine (9.5 mmol, 1.0 eq.) in

CH₂Cl₂(10 mL). The solution was allowed to stir at ambient

temperature overnight before diluting with CH₂Cl₂(30 mL) and

H₂O (50 mL). The organic layer was separated and further

washed with 1M HCl_(aq)(50 mL), H₂O (50 mL) and brine (50

mL). The organic layer was then dried (MgSO₄), filtered and

concentrated in vacuo.

(s) cm^-1; H NMR (400 MHz, CDCl₃): δ = 6.60 (d, J = 6.1 Hz,

1

1H, 2-H), 6.50 (dd, J = 6.1, 1.9 Hz, 1H, 1-H), 6.37–6.28 (br, 1H,

N-H), 5.01 (dd, J = 4.8, 1.6 Hz, 1H, 6-H), 4.84 (d, J = 10.8 Hz,

1H, 7-H), 4.57 (d, J = 10.8 Hz, 1H, 7-H), 3.18 (dt, J = 12.3, 6.3

Hz, 2H, 11-H), 2.49 (dd, J = 12.3, 4.8 Hz, 1H, 5-H), 2.33 (d, J =

14.6 Hz, 1H, 9-H), 2.24 (d, J = 14.6 Hz, 1H, 9-H), 1.51–1.40 (m,

2H, 12-H), 1.34–1.17 (m, 11H, 5-H/13-H/14-H/15-H/16-H/17-

H), 0.86 (t, J = 7.1 Hz, 3H, 18-H); ¹³C NMR (100 MHz, CDCl₃):

δ = 179.3 (8), 168.5 (10), 137.3 (1), 131.6 (2), 94.5 (3), 78.5 (6),

69.7 (7), 52.8 (4), 42.7 (9), 40.0 (11), 36.6 (5), 31.9 (12), 29.4

(13), 29.3 (14/15), 26.9 (16), 22.7 (17), 14.2 (18); HRMS (ESI)

m/z calculated for C₁₈H₂₇NNaO₄344.1832 (M+Na)⁺, found

344.1837, -0.9 ppm error.

N-Butyl-2-[(1S*,5S*,7S*))-4-oxo-3,10-dioxatricyclo

N-Benzyl-2-[(1S*,5S*,7S*))-4-oxo-3,10-

[5.2.1.0^1,5]dec-8-en-5-yl]acetamide (20a): Acid 12 (2.0 g, 9.5

mmol, 1.0 eq.) and butylamine (0.94 mL, 9.5 mmol, 1.0 eq.) were

dioxatricyclo[5.2.1.0^1,5]dec-8-en-5-yl]acetamide (20d): Acid 12

9

(2 g, 9.52 mmol, 1.0 eq.) and benzylamine (1.04 mL, 9.52 mmol,

1.0 eq.) were coupled according to the general procedure for the

synthesis of secondary amides. The crude material was then

purified by flash column chromatography (70:30–90:10,

EtOAc:PE) to give the target compound 20d as a white

crystalline solid (1.67 g, 58%). R_F= 0.3 (80:20, EtOAc:PE); m.p.

106.8–107.4 °C; IR (Neat) υ_max3286 (m), 1765 (s), 1636 (s) and

3396 (m), 3024 (w), 2946 (w), 2874 (m), 1767 (m), 1674 (s)

and 1685 (s) cm^-1; ¹H NMR (400 MHz, CDCl₃): δ = 6.65 (dd, J =

5.7, 1.7 Hz, 1H, 1-H), 6.48 (d, J = 5.7 Hz, 1H, 2-H), 5.13 (dd, J =

4.8, 1.7 Hz, 1H, 6-H), 4.02 (d, J = 11.6 Hz, 1H, 7-H), 3.90 (dd, J

= 11.6, 4.1 Hz, 1H, 7-H), 3.50 (t, J = 7.4 Hz, 2H, 11-H), 2.72–

2.61 (m, 2H, 5-H/9-H), 2.47–2.37 (m, 2H, OH/9-H), 1.59–1.48

(m, 3H, 12-H/5-H), 1.37–1.25 (m, 2H, 13-H), 0.91 (t, J = 7.3 Hz,

3H, 14-H); ¹³C NMR (100 MHz, CDCl₃): δ = 180.4 (8), 176.2

(10), 140.3 (1), 135.1 (2), 92.8 (3), 79.1 (6), 60.3 (7), 49.9 (4),

44.0 (5), 40.1 (9), 39.0 (11), 29.7 (12), 20.1 (13), 13.7 (14);

HRMS (ESI) m/z calculated for C₁₄H₁₉NNaO₄288.1214

(M+Na)⁺, found 288.1206, -2.7 ppm error.

1

1547 (s) cm^-1; H NMR (400 MHz, CDCl₃): δ = 7.31–7.25 (m,

2H, ArH), 7.24–7.18 (m, 3H, ArH), 6.75–6.66 (br, 1H, N-H),

6.54 (d, J = 5.8 Hz, 1H, 2-H), 6.45 (dd, J = 5.8, 1.6 Hz, 1H, 1-H),

4.95 (dd, J = 4.8, 1.6 Hz, 1H, 6-H), 4.75 (d, J = 10.6 Hz, 1H, 7-

H), 4.51 (d, J = 10.6 Hz, 1H, 7-H), 4.37 (dd, J = 14.6, 5.8 Hz,

1H, 11-H), 4.30 (dd, J = 14.6, 5.8 Hz, 1H, 11-H), 2.41 (dd, J =

12.0, 4.9 Hz, 1H, 5-H), 2.32 (d, J = 14.8 Hz, 1H, 9-H), 2.23 (d, J

= 14.8 Hz, 1H, 9-H), 1.25 (d, J = 12.0 Hz, 1H, 5-H); ¹³C NMR

(100 MHz, CDCl₃): δ = 179.2 (8), 168.6 (10), 137.8 (1), 137.4

(ArC), 131.4 (2), 128.8 (ArCH), 127.8 (ArCH), 94.5 (3), 78.5

(6), 69.6 (7), 52.8 (4), 43.9 (11), 42.5 (9), 36.6 (5); HRMS (ESI)

m/z calculated for C₁₇H₁₇NNaO₄322.1050 (M+Na)⁺, found

322.1049, 0.3 ppm error.

General procedure for the hydrogenation of secondary

amides to imides. Secondary amide (1.9 mmol, 1.0 eq.) was

dissolved in MeOH (1 mL) under nitrogen, the addition of

EtOAc (1 mL) may be necessary in some cases to obtain a

solution. In a separate flask Pd/C (10 wt%) was suspended in

MeOH (1 mL) under nitrogen. H₂was bubbled through the

suspension of Pd/C in MeOH for 10 minutes. The solution of

secondary amide was then added to the catalyst suspension. The

reaction mixture was allowed to stir at room temperature for 16

hours under H₂(1 bar). The reaction mixture was then passed

through a pad of celite® and concentrated in vacuo.

N-(4-Methoxybenzyl)-2-[(1S*,5S*,7S*))-4-oxo-3,10

dioxatricyclo[5.2.1.0^1,5]dec-8-en-5-yl]acetamide (20e): Acid 12

(0.50 g, 2.4 mmol, 1.0 eq.) and 4-methoxybenzylamine (0.31 mL,

2.4 mmol, 1.0 eq.) were coupled according to the general

procedure for the synthesis of secondary amides. The crude

material was then purified by flash column chromatography

(80:20, EtOAc:PE) to give the target compound 20e as a

crystalline solid (0.24 g, 31%). R_F= 0.25 (80:20, EtOAc:PE);

m.p. 150.3–150.9 °C; IR (Neat) υ_max3259 (m), 3088 (w), 2954

(1S*,2S*,4R*)-1’-Butyl-1-(hydroxymethyl)spiro[7-

oxabicyclo[2.2.1]heptane-2,3’-pyrrolidine]-2’,5’-dione (21a):

Butyl amide 20a (0.10 g) was hydrogenated according to the

general procedure for the hydrogenation of secondary amides.

The crude material was purified using flash column

chromatography (80:20–90:10, EtOAc:PE) to give the target

compound 21a as a clear oil (0.77 g, 76%). R_F= 0.33 (90:10,

EtOAc:PE); IR (Neat) υ_max3460 (w), 2958 (w), 2874 (w), 1771

(w) and 1685 (s) cm^-1; ¹H NMR (400 MHz, CDCl₃): δ = 4.72 (t, J

= 5.3 Hz, 1H, 6-H), 3.93 (d, J = 5.3 Hz, 2H, 7-H), 3.49 (t, J = 7.3

Hz, 2H, 11-H), 2.97 (d, J = 17.9 Hz, 1H, 9-H), 2.71 (d, J = 17.9

Hz, 1H, 9-H), 2.52 (ddd, J = 12.1, 5.5, 2.6 Hz, 1H, 5-H), 2.02–

1.91 (m, 2H, 1-H), 1.78–1.62 (m, 3H, 2-H, OH), 1.59–1.49 (m,

3H, 12-H/5-H), 1.37–1.23 (m, 2H, 13-H), 0.91 (t, J = 7.3 Hz, 3H,

14-H); ¹³C NMR (100 MHz, CDCl₃): δ = 180.5 (8), 175.7 (10),

90.4 (3), 77.4 (6), 62.7 (7), 52.6 (4), 45.3 (5), 39.2 (9), 38.8 (11),

31.3 (2), 29.7 (12/1), 20.1 (13), 13.7 (14); HRMS (ESI) m/z

calculated for C₁₄H₂₁NNaO₄290.1363 (M+Na)⁺, found 290.1368,

-2.5 ppm error.

1

(w), 1758 (s), 1635 (m), 1561 (m) and 1508 (m) cm^-1; H NMR

(400 MHz, DMSO-d₆): δ = 8.41 (t, J = 5.8 Hz, 1H, N-H), 7.20–

7.15 (m, 2H, ArH), 6.90–6.85 (m, 2H, ArH), 6.55 (dd, J = 5.8,

1.7 Hz, 1H, 1-H), 6.52 (d, J = 5.8 Hz, 1H, 2-H), 5.00 (dd, J = 4.6,

1.7 Hz, 1H, 6-H), 4.74 (d, J = 10.6 Hz, 1H, 7-H), 4.47 (d, J =

10.6 Hz, 1H, 7-H), 4.15 (d, J = 5.8 Hz, 2H, 11-H), 3.72 (s, 3H,

12-H), 2.21 (s, 2H, 9-H), 2.17 (dd, J = 11.9, 4.7 Hz, 1H, 5-H),

1.51 (d, J = 11.9 Hz, 1H, 5-H); ¹³C NMR (100 MHz, DMSO-d₆):

δ = 177.7 (8), 168.0 (10), 158.3 (ArC), 137.5 (1), 131.0 (2), 128.7

(ArCH), 113.68 (ArCH), 94.0 (9), 78.0 (6), 68.2 (7), 55.1 (12),

52.2 (4), 41.7 (11), 40.6 (9), 36.1 (5); HRMS (ESI) m/z

calculated for C₁₈H₂₀NO₅330.1338 (M+H)⁺, found 330.1341, -

0.9 ppm error.

(1S*,2S*,4R*)-1’-Butyl-1-(hydroxymethyl)spiro[7-

(1S*,2S*,4R*)-1’-Hexyl-1-(hydroxymethyl)spiro[7-

oxabicyclo[2.2.1]

hept-2-ene-6,3’-pyrrolidine]-2’,5’-dione

oxabicyclo[2.2.1] heptane-2,3’-pyrrolidine]-2’,5’-dione (21b):

Hexyl amide 20b (400 mg) was hydrogenated according to the

general procedure for the hydrogenation of secondary amides.

The crude material was purified by flash column chromatography

(70:30, EtOAc:PE) to give the target compound 21b as a clear oil

(378 mg, 94%). R_F= 0.32 (80:20, EtOAc:PE); IR (Neat) υ_max

(22): Acid 12 (2.0 g, 9.5 mmol, 1.0 eq.) was suspended in

anhydrous CH₂Cl₂(5 mL) under an atmosphere of nitrogen. The

suspension was cooled to 0 °C and oxalyl chloride 2.0 M solution

in CH₂Cl₂(12 mL, 24 mmol, 2.5 eq.) was added dropwise over

10 minutes, followed by DMF (4 drops). The suspension was

stirred at ambient temperature until a solution was obtained, after

which it was concentrated in vacuo, to yield the crude acid

chloride as a light brown solid. The crude material was

redissolved in fresh anhydrous CH₂Cl₂(10 mL) and cooled to 0

°C and a solution of butylamine (4.7 mL, 48 mmol, 5.0 eq.) in

CH₂Cl₂(10 mL) was added dropwise. The solution was allowed

to stir at ambient temperature overnight before diluting with

CH₂Cl₂(30 mL) and H₂O (50 mL). The organic layer was

separated and further washed with 1M HCl_(aq)(50 mL), H₂O (50

mL) and brine (50 mL). The organic layer was then dried

(MgSO₄), filtered and concentrated in vacuo. The crude material

was purified by flash column chromatography (80:20,

EtOAc:PE) to give the target spirocyclic imide 22 as a white

crystalline solid (839 mg, 33%). Crystals suitable for X-ray

analysis were grown by slow evaporation of an acetone solution.

R_F= 0.25 (80:20, EtOAc:PE); m.p. 69.2–70.0 °C; IR (Neat) υ_max

3460 (w), 2931 (m), 2859 (w), 1771 (m) and 1686 (s) cm^-1; H

1

NMR (400 MHz, CDCl₃): δ = 4.68 (t, J = 5.2 Hz, 1H, 6-H), 3.89

(s, 2H, 7-H), 3.44 (t, J = 7.5 Hz, 2H, 11-H), 2.96 (d, J = 18.4 Hz,

1H, 9-H), 2.68 (d, J = 18.4 Hz, 1H, 9-H), 2.48 (ddd, J = 11.9, 5.2,

2.5 Hz, 1H, 5-H), 2.34–2.18 (br, 1H, OH), 2.02–1.82 (m, 2H, 1-

H/2-H), 1.75–1.60 (m, 2H, 1-H/2-H), 1.58–1.47 (m, 3H, 5-H/12-

H), 1.30–1.19 (m, 6H, 15-H/14-H/13-H), 0.84 (t J = 7.2 Hz, 3H,

16-H); ¹³C NMR (100 MHz, CDCl₃): δ = 180.5 (8), 175.8 (10),

90.4 (3), 77.1 (6), 62.6 (7), 52.6 (4), 45.2 (5), 39.2 (9), 39.0 (11),

31.4 (12), 31.2 (1), 29.6 (2), 27.5 (13), 26.5 (14), 22.6 (15), 14.1

(16); HRMS (ESI) m/z calculated for C₁₆H₂₅NNaO₄318.1676

(M+Na)⁺, found 318.1676, 0.1 ppm error.

(1S*,2S*,4R*)-1-(Hydroxymethyl)-1’-octyl-spiro[7-

oxabicyclo[2.2.1] heptane-2,3’-pyrrolidine]-2’,5’-dione (21c):

Octyl amide 20c (0.10 g) was hydrogenated according to the

10

Tetrahedron

general procedure for the hydrogenation of secondary amides.

catalyst suspension. The reaction mixture was allowed to stir at

room temperature for 16 hours under H₂(1 bar). The reaction

mixture was then passed through a pad of celite® and

concentrated in vacuo to give the target compound 18 as a white

powder (0.93 g, 93%). m.p. 184.6–185.2 °C; IR (Neat) υ_max2991

The crude material was purified using flash column

chromatography (70:30, EtOAc:PE) to give the target compound

21c as a clear oil (0.098 g, 98%). R_F= 0.26 (70:30, EtOAc:PE);

IR (Neat) υ_max3452 (w), 2926 (m), 2856 (m), 1771 (w) and 1688

(s) cm^-1; ¹H NMR (400 MHz, CDCl₃): δ = 4.69 (t, J = 4.9 Hz, 1H,

6-H), 3.90 (s, 2H, 7-H), 3.45 (t, J = 7.3 Hz, 2H, 11-H), 2.97 (d, J

= 18.3 Hz, 2H, 9-H), 2.69 (d, J = 18.3 Hz, 1H, 9-H), 2.52–2.45

(m, 1H, 5-H), 2.13–2.04 (br, 1H, OH), 2.01–1.88 (m, 2H, 1-H),

1.77–1.61 (m, 2H, 2-H), 1.58–1.47 (m, 3H, 5-H/12-H), 1.30–1.18

(m, 10H, 13-H/14-H/15-H/16-H/17-H), 0.85 (t, J = 6.3 Hz, 3H,

18-H); ¹³C NMR (100 MHz, CDCl₃): δ = 180.5 (8), 175.8 (10),

90.4 (3), 77.4 (6), 62.7 (7), 52.6 (4), 45.3 (5), 39.2 (9), 39.1 (11),

31.9 (31.3), 29.6 (1), 29.3 (2), 29.2 (14), 27.6 (15), 26.9 (16),

22.7 (17), 14.2 (18); HRMS (ESI) m/z calculated for C₁₈H_29-

NNaO₄346.1989 (M+Na)⁺, found 346.1992, -0.5 ppm error.

(m), 1713 (s) cm^-1; H NMR (400 MHz, CD₃OD): δ = 4.59 (d, J

1

= 10.6 Hz, 1H, 7-H), 4.53 (t, J = 5.4 Hz, 1H, 6-H), 4.48 (d, J =

10.6 Hz, 1H, 7-H), 2.88 (d, J = 15.4 Hz, 1H, 9-H), 2.58 (d, J =

15.4 Hz, 1H, 9-H), 2.27 (ddd, J = 12.5, 5.1, 2.3 Hz, 1H, 5-H),

2.04–1.89 (m, 2H, 1-H/2-H), 1.80 (d, J = 12.5 Hz, 1H, 5-H),

1.77–1.67 (m, 2H, 1-H/2-H); ¹³C NMR (100 MHz, CD₃OD): δ =

181.9 (8), 173.3 (10), 93.4 (3), 77.7 (6), 70.6 (7), 54.5 (4), 45.9

(5), 40.1 (9), 29.7 (1), 25.2 (2); HRMS (ESI) m/z calculated for

C₁₀H₁₃O₅213.0769 (M+H)⁺, found 213.0763, 2.8 ppm error.

General procedure for the synthesis of hydrogenated

secondary amides. Hydrogenated acid 18 (0.5 g, 2.4 mmol, 1.0

eq.) was suspended in anhydrous CH₂Cl₂(5 mL) under an

atmosphere of nitrogen. The suspension was cooled to 0 °C and

oxalyl chloride 2.0 M solution in CH₂Cl₂(3.0 mL, 5.9 mmol, 2.5

eq.) was added dropwise over 10 minutes, followed by DMF (2

drops). The suspension was stirred at ambient temperature until a

solution was obtained, after which it was concentrated in vacuo,

to yield the crude acid chloride as a light brown solid. The crude

material was redissolved in fresh anhydrous CH₂Cl₂(5 mL) and

cooled to 0 °C. Pyridine (0.19 mL, 2.4 mmol, 1.0 eq.) was then

added dropwise, followed by the primary amine (2.4 mmol, 1.0

eq.) in CH₂Cl₂(5 mL). The solution was allowed to stir at

ambient temperature overnight before diluting with CH₂Cl₂(30

mL) and H₂O (50 mL). The organic layer was separated and

further washed with 1M HCl_(aq)(50 mL), H₂O (50 mL) and brine

(50 mL). The organic layer was dried (MgSO₄), filtered and

concentrated in vacuo.

(1S*,2S*,4R*)-1’-Benzyl-1-(hydroxymethyl)spiro[7-

oxabicyclo[2.2.1] heptane-2,3’-pyrrolidine]-2’,5’-dione (21d):

Benzyl amide 20d (400 mg) was hydrogenated according to the

general procedure for the hydrogenation of secondary amides.

The crude material was purified using flash column

chromatography (80:20–90:10, EtOAc:PE) to give the target

compound 21d as a clear oil (381 mg, 95%). Crystals suitable for

X-ray analysis were grown by slow evaporation of an acetone

solution. R_F= 0.36 (90:10, EtOAc:PE); IR (Neat) υ_max3490 (m),

2978 (w), 1772 (m) and 1692 (s) cm^-1; H NMR (400 MHz,

1

CDCl₃): δ = 7.40–7.35 (m, 2H, Ar-H), 7.31–7.23 (m, 3H, Ar-H),

4.67 (t, J = 5.7 Hz, 1H, 6-H), 4.65 (d, J = 14.1 Hz, 1H, 11-H),

4.60 (d, J = 14.1 Hz, 1H, 11-H), 3.80–3.67 (m, 2H, 7-H), 2.99 (d,

J = 18.4 Hz. 1H, 9-H), 2.70 (d, J = 18.4. Hz, 1H, 9-H), 2.49 (ddd,

J = 12.1 Hz, 5.7, 2.4 Hz, 1H, 5-H), 2.03–1.86 (m, 2H, 2-H/1-H),

1.77–1.57 (m, 3H, 2-H/3-H/OH), 1.53 (d, J = 12.1 Hz, 1H, 5-H);

¹³C NMR (100 MHz, CDCl₃): δ = 180.2 (8), 175.3 (10), 136.0

(ArC), 129.0 (ArCH), 128.7 (ArCH), 128.1 (ArCH), 90.3 (3),

77.2 (6), 62.9 (7), 52.9 (4), 44.9 (5), 42.6 (11), 39.3 (9), 31.3 (2),

29.9 (1); HRMS (ESI) m/z calculated for C₁₇H₁₉NNaO₄324.1206

(M+Na)⁺, found 324.1206, 0.2 ppm error.

N-Butyl-2-[(1S*,5S*,7R*)-4-oxo-3,10-dioxatricyclo

[5.2.1.0^1,5]decan-5-yl]acetamide (19a): Hydrogenated acid 18

(0.50 g, 2.4 mmol, 1.0 eq.) and butylamine (0.24 mL, 2.4 mmol,

1.0 eq.) were coupled according to the general procedure for the

synthesis of hydrogenated secondary amides. The crude material

was then purified by flash column chromatography (80:20,

EtOAc:PE) to give the target compound 19a as a white solid

(0.21 g, 33%). R_F= 0.27 (80:20, EtOAc:PE); m.p. 58.1–58.5 °C;

IR (Neat) υ_max3319 (w), 2958 (w), 2868 (w), 1765 (s), 1635 (s),

1553 (m) cm^-1; ¹H NMR (400 MHz, CDCl₃): δ = 6.26 (br. s, 1H,

N-H), 4.55 (t, J = 5.5 Hz, 1H, 6-H), 4.50 (s, 2H, 7-H), 3.25–3.12

(m, 2H, 11-H), 2.65 (d, J = 15.0 Hz, 1H, 9-H), 2.41 (d, J = 15.0

Hz, 1H, 9-H), 2.42–2.37 (m, 1H, 5-H), 2.24 (ddd, J = 12.2, 8.8,

3.2 Hz, 2-H), 2.02–1.92 (m, 1H, 1-H), 1.75 (t, J = 12.2, 5.5 Hz,

1H, 2-H), 1.61 (d, J = 12.5 Hz, 1H, 5-H), 1.61–1.55 (m, 1H, 1-

H), 1.50–1.40 (m, 2H, 12-H), 1.36–1.27 (m, 2H, 13-H), 0.89 (t, J

= 7.4 Hz, 3H, 14-H); ¹³C NMR (100 MHz, CDCl₃): δ = 180.6 (8),

168.5 (10), 92.3 (3), 76.3 (6), 69.8 (7), 54.2 (4), 44.6 (5), 41.4

(9), 39.6 (11), 31.4 (12), 29.0 (1), 24.6 (2), 20.1 (13), 13.8 (14);

HRMS (ESI) m/z calculated for C₁₄H₂₂NO₄268.1555 (M+H)⁺,

found 268.1549, 2.2 ppm error.

(1S*,2S*,4R*)-1’-(4-Methoxybenzyl)-1-

(hydroxymethyl)spiro[7-oxabicyclo[2.2.1]heptane-2,3’-

pyrrolidine]-2’,5’-dione (21e): 4-Methoxybenzyl amide 20e

(0.10 g) was hydrogenated according to the general procedure for

the hydrogenation of secondary amides. The crude material was

purified using flash column chromatography (80:20, EtOAc:PE)

to give the target compound 21e as a white crystalline solid

(0.098 g, 98%). R_F= 0.29 (80:20, EtOAc:Hexane); IR (Neat) υ_max

3416 (w), 3312 (w), 2943 (w), 2835 (w), 1758 (w), 1672 (s),

1609 (m), 1508 (s) cm^-1; H NMR (400 MHz, CDCl₃): δ = 7.35–

1

7.30 (m, 2H, Ar-H), 6.83–6.78 (m, 2H, Ar-H), 4.67 (t, J = 5.3 Hz,

1H, 6-H), 4.59 (d, J = 14.0 Hz, 1H, 11-H), 4.54 (d, J = 14.0 Hz,

1H, 11-H), 3.79–3.68 (m, 2H, 7-H), 3.75 (s, 3H, 12-H), 2.97 (d, J

= 18.2 Hz, 1H, 9-H), 2.68 (d, J = 18.2 Hz, 1H, 9-H), 2.48 (ddd, J

= 12.0, 5.5, 2.5 Hz, 1H, 5-H), 2.02–1.93 (m, 1H, 1-H), 1.93–1.86

(m, 1H, 2-H), 1.71–1.58 (m, 1H, 1-H/2-H), 1.52 (d, J = 12.0 Hz,

1H, 5-H); ¹³C NMR (100 MHz, CDCl₃): δ = 180.2 (8), 175.4

(10), 159.3 (ArC), 130.4 (ArCH), 128.2 (ArC), 113.9 (ArCH),

90.3 (3), 77.2 (6), 62.8 (7), 55.3 (12), 52.9 (4), 44.8 (5), 42.0

(11), 39.2 (9), 31.2 (2), 29.9 (1); HRMS (ESI) m/z calculated for

C₁₈H₂₂NO₅332.1504 (M+H)⁺, found 332.1498, 1.8 ppm error.

2-[(1S*,5S*,7R*)-4-Oxo-3,10-dioxatricyclo[5.2.1.0^1,5]decan-

5-yl]acetic acid (18): Acid 12 (1.0 g, 4.8 mmol, 1.0 eq.) was

dissolved in MeOH (5 mL) under nitrogen. In a separate flask

Pd/C (10 wt%) was suspended in MeOH (1 mL) under nitrogen.

H₂was bubbled through the suspension of Pd/C in MeOH for 10

minutes. The solution of secondary amide was then added to the

N-Hexyl-2-[(1S*,5S*,7R*)-4-oxo-3,10-dioxatricyclo

[5.2.1.0^1,5]decan-5-yl]acetamide (19b): Hydrogenated acid 18

(0.50 g, 2.4 mmol, 1.0 eq.) and hexylamine (0.31 mL, 2.4 mmol,

1.0 eq.) were coupled according to the general procedure for the

synthesis of hydrogenated secondary amides. The crude material

was then purified by flash column chromatography (70:30,

EtOAc:PE) to give the target compound 19b as a yellow oil (0.43

g, 61%). R_F= 0.27 (70:30, EtOAc:PE); IR (Neat) υ_max3312 (w),

2950 (m), 2924 (m), 1762 (s), 1643 (s), 1538 (s) cm^-1; H NMR

1

(400 MHz, CDCl₃): δ = 6.28 (br. s, 1H, N-H), 4.54 (t, J = 5.3 Hz,

1H, 6-H), 4.49 (s, 2H, 7-H), 3.24–3.11 (m, 2H, 11-H), 2.65 (d, J

11

= 14.6 Hz, 1H, 9-H), 2.41 (d, J = 14.6 Hz, 1H, 9-H), 2.41–2.37

(m, 1H, 5-H), 2.23 (ddd, J = 12.5, 8.9, 3.1 Hz, 1H, 2-H), 2.01–

1.91 (m, 1H, 1-H), 1.79–1.70 (m, 1H, 2-H), 1.60 (d, J = 12.7 Hz,

1H, 5-H), 1.60–1.55 (m, 1H, 1-H), 1.49–1.40 (m, 2H, 12-H),

1.32–1.22 (m, 6H, 13-H/14-H/15-H), 0.85 (t, J = 6.7 Hz, 3H, 16-

H); ¹³C NMR (100 MHz, CDCl₃): δ = 180.6 (8), 168.5 (10), 92.3

(3), 76.2 (6), 69.7 (7), 54.2 (4), 44.6 (5), 41.3 (9), 39.9 (11), 31.5

(13), 29.3 (12), 29.0 (1), 26.6 (14), 24.55 (2), 22.6 (15), 14.1

(16); HRMS (ESI) m/z calculated for C₁₆H₂₆NO4 296.1870

(M+H)⁺, found 296.1862, 2.7 ppm error.

(m) cm^-1; ¹H NMR (400 MHz, CDCl₃): δ = 8.49 (t, J = 5.7 Hz,

1H, N-H), 7.21–7.14 (m, 2H, ArH), 6.91–6.84 (m, 2H, ArH),

4.48 (t, J = 5.4 Hz, 1H, 6-H), 4.37 (d, J = 10.4 Hz, 1H, 7-H), 4.33

(d, J = 10.4 Hz, 1H, 7-H), 4.22–4.12 (m, 2H, 11-H), 3.73 (s, 3H,

12-H), 2.66 (d, J = 14.4 Hz, 1H, 9-H), 2.46 (d, J = 14.4 Hz, 1H,

9-H), 2.10–2.03 (m, 1H, 5-H), 2.02–1.94 (m, 1H, 1-H), 1.85–1.76

(m, 1H, 2-H), 1.72 (d, J = 12.1 Hz, 1H, 5-H), 1.64–1.54 (m, 2H,

1-H/2-H); ¹³C NMR (100 MHz, CDCl₃): δ = 179.4 (8), 168.4

(10), 158.3 (ArC), 131.0 (ArC), 128.7 (ArCH), 113.7 (ArCH),

91.8 (3), 75.5 (6), 68.5 (7), 55.1 (12), 53.2 (4), 44.4 (5), 41.7

(11), 39.9 (9), 28.5 (2), 24.2 (1); HRMS (ESI) m/z calculated for

C₁₈H₂₂NO₅332.1498 (M+H)⁺, found 332.1498, 2.7 ppm error.

N-Octyl-2-[(1S*,5S*,7R*)-4-oxo-3,10-dioxatricyclo

[5.2.1.0^1,5]decan-5-yl]acetamide (19c): Hydrogenated acid 18

(0.50 g, 2.4 mmol, 1.0 eq.) and octylamine (0.39 mL, 2.4 mmol,

1.0 eq.) were coupled according to the general procedure for the

synthesis of hydrogenated secondary amides. The crude material

was then purified by flash column chromatography (70:30,

EtOAc:PE) to give the target compound 19c as a yellow waxy

solid (0.51 g, 67%). R_F= 0.3 (70:30, EtOAc:PE); m.p. 53.4–54.0

°C; IR (Neat) υ_max3394 (w), 2917 (m), 2850 (m), 1754 (s), 1665

(s) and 1523 (m) cm^-1; ¹H NMR (400 MHz, CDCl3): δ = 6.30 (br.

s, 1H, N-H), 4.54 (t, J = 5.2 Hz, 1H, 6-H), 4.49 (s, 2H, 7-H),

3.21–3.11 (m, 2H, 11-H), 2.65 (d, J = 14.5 Hz, 1H, 9-H), 2.40 (d,

J = 14.5 Hz, 1H, 9-H), 2.40–2.35 (m, 1H, 5-H), 2.22 (ddd, J =

12.3, 8.6, 3.1 Hz, 1H, 2-H), 2.01–1.90 (m, 1H, 1-H), 1.79–1.69

(m, 1H, 2-H), 1.60 (d, J = 12.5 Hz, 1H, 5-H), 1.60–1.54 (m, 1H,

1-H), 1.49–1.39 (m, 2H, 12-H), 1.31–1.17 (m, 10H, 13-H/14-

H/15-H/16-H/17-H), 0.84 (t, J = 6.6 Hz, 3H, 18-H); ¹³C NMR

(100 MHz, CDCl₃): δ = 180.6 (8), 168.5 (10), 92.25 (3), 76.2 (6),

69.7 (7), 54.2 (4), 44.6 (5), 41.4 (9), 39.9 (11), 31.9 (13), 29.4

(4), 29.3 (12/15) 29.0 (1), 26.9 (16), 24.6 (2), 22.7 (17), 14.2

(18); HRMS (ESI) m/z calculated for C₁₈H₃₀NO₄324.2184

(M+H)⁺, found 324.2175, 2.8 ppm error.

4.3. Cell culture and stock solutions

A549 human lung carcinoma cells were obtained from the

American Type Culture Collection (ATCC; Middlesex UK) and

used in a passage window of 25. HCT 116 colorectal carcinoma

cells, OVCAR-3 human ovarian adenocarcinoma cells, MDA-

MB-231 human breast adenocarcinoma cells, MCF7 human

breast adenocarcinoma cells and PC-3 human prostate

adenocarcinoma cells were obtained from AMS Biotechnology

(Europe) Limited (Abingdon, UK) and used in a passage window

of 10. Cells were cultured in F-12K Medium (A549, PC-3),

McCoy's 5A Medium (HCT 116), RPMI-1640 Medium (MDA-

MB-231, OVCAR-3) or Minimum Essential Medium (MCF7)

(Sigma-Aldrich) supplemented with 10% (v/v) FBS (Sigma-

Aldrich) and at 37 °C with 5% CO₂. Stock solutions of

compounds were prepared as 40 mM solutions in DMSO and

stored at 4 °C.

4.4. Cytocompatibility evaluation in cancer cell panel

The cytotoxicity of the compounds was assessed using the

PrestoBlue™ Cell Viability assay at two fixed concentrations of

100 µM and 200 µM at three time points of 24, 48 and 72 h.

Cells were seeded into 96 well plates at a density of 3 x 10³cells

per well and allowed to adhere in growth media overnight at 37

°C and 5% CO₂. Cell media were discarded and replaced with

fresh media (100 μL) containing the appropriate compound and

concentration. Triton X-100 applied at 0.5% (v/v) in culture

medium was used as a cell death (positive) control and culture

medium containing 0.5% DMSO was used as a negative control.

After 24, 48 or 72 h exposure, the medium was removed and

replaced with 100 µL fresh medium containing 10% (v/v)

PrestoBlue reagent for 60 minutes. Cell viability was then

assessed by measuring resultant fluorescence readings at 544/620

nm (λ_ex/λ_em). Relative metabolic activity was calculated by

setting values from the negative control as 100% and positive

control values as 0% metabolic activity.

N-Benzyl-2-[(1S*,5S*,7R*)-4-oxo-3,10-dioxatricyclo

[5.2.1.0^1,5]decan-5-yl]acetamide (19d): Hydrogenated acid 18

(0.50 g, 2.4 mmol, 1.0 eq.) and benzylamine (0.26 mL, 2.4 mmol,

1.0 eq.) were coupled according to the general procedure for the

synthesis of hydrogenated secondary amides. The crude material

was then purified by flash column chromatography (75:25,

EtOAc:PE) to give the target compound 19d as a white

crystalline solid (0.55 g, 77%). R_F= 0.28 (75:25, EtOAc:PE);

m.p. 122.2–122.7 °C; IR (Neat) υ_max3326 (w), 2961 (w), 1765

(s), 1635 (s) and 1538 (s) cm^-1; ¹H NMR (400 MHz, CDCl₃): δ =

7.27–7.15 (m, 5H, ArH), 6.75–6.69 (br. s, 1H, N-H), 4.45 (t, J =

5.3 Hz, 1H, 6-H), 4.38 (s, 2H, 7-H), 4.31 (dd, J = 14.9, 6.1 Hz,

1H, 11-H), 4.26 (dd, J = 14.9, 6.1 Hz, 1H, 11-H), 2.62 (d, J =

14.0 Hz, 1H, 9-H), 2.34 (d, J = 14.0 Hz, 1H, 9-H), 2.27 (ddd, J =

12.5, 5.2, 2.4 Hz, 1H, 5-H), 2.10 (ddd, J = 12.4, 8.7, 3.2 Hz, 1H,

1-H), 1.93–1.82 (m, 1H, 2-H), 1.70–1.60 (m, 1H, 1-H), 1.53 (d, J

= 12.5 Hz, 1H, 5-H), 1.53–1.47 (m, 1H, 2-H); ¹³C NMR (100

MHz, CDCl₃₎: δ = 180.5 (8), 168.6 (10), 137.8 (ArC), 128.7

(ArCH), 127.8 (ArCH), 127.6 (ArCH), 92.2 (3), 76.2 (6), 69.6

(7), 54.0 (4), 44.6 (5), 43.8 (11), 41.1 (9), 29.0 (2), 24.5 (1);

HRMS (ESI) m/z calculated for C₁₇H₂₀NO₄302.1399 (M+H)⁺,

found 302.1392, 2.3 ppm error.

Compound 21c underwent further dose-response analysis in

all cell lines to calculate the EC₅₀value. Cells were seeded into

96 well plates at a density of 3 x 10³cells per well and allowed to

adhere in growth media overnight at 37 °C and 5% CO₂. Cell

media were discarded and replaced with fresh media (100 μL)

containing 17c (5–250 µM). Triton X-100 applied at 0.5% (v/v)

in culture medium was used as a cell death (positive) control and

culture medium containing 0.5% DMSO was used as a negative

control. After 48 h exposure, the medium was removed and

replaced with 100 µL fresh medium containing 10% (v/v)

PrestoBlue reagent for 60 minutes. Cell viability was then

assessed by measuring resultant fluorescence readings at 544/620

nm (λ_ex/λ_em). Relative metabolic activity was calculated by

setting values from the negative control as 100% and positive

control values as 0% metabolic activity. EC₅₀values were

calculated using the dose-response fitting function in OriginPro

software.

N-(4-Methoxybenzyl)-2-[(1S*,5S*,7R*)-4-oxo-3,10-

dioxatricyclo[5.2.1.0^1,5]decan-5-yl]acetamide

(19e):

Hydrogenated acid 18 (0.50 g, 2.4 mmol, 1.0 eq.) and 4-

methoxybenzylamine (0.31 mL, 2.4 mmol, 1.0 eq.) were coupled

according to the general procedure for the synthesis of

hydrogenated secondary amides. The crude material was then

purified by flash column chromatography (80:20, EtOAc:PE) to

give the target compound 19e as a white powder (0.45 g, 58%).

R_F= 0.29 (80:20, EtOAc:PE); m.p. 160.7–161.4 °C; IR (Neat)

υ_max3259 (w), 2954 (w), 1762 (s), 1635 (m), 1568 (m) and 1508

12

Tetrahedron

18. Grogan, C. H.; Rice, L. M. Bicyclic Imides and Isoindolines 1. J.

Med. Chem. 1963, 6 (6), 802–805.

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14

Tetrahedron

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships

that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered

as potential competing interests:

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