An approach to "escape from flatland": Chemo-enzymatic synthesis and biological profiling of a library of bridged bicyclic compounds

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An Approach to “Escape from Flatland”: Chemo-enzymatic

Synthesis and Biological Profiling of a Library of Bridged Bicyclic

Compounds

Received 00th January 20xx,

Accepted 00th January 20xx

N. V. Suryanarayana Birudukota^a, Raimo Franke^aand Bernd Hofer^a*

DOI: 10.1039/x0xx00000x

www.rsc.org/

A major reason for the low success rate in current drug development through chemical synthesis has been ascribed to the

large fraction of quasi planar candidate molecules. Therefore, an “escape from flatland” strategy has been recommended

for the generation of bioactive chemical entities. In a first attempt to test this recommendation, we synthesized a small

collection of bridged bicyclic compounds possessing a rigid spherical core structure by combining a group of cyclic dienes

with a collection of dienophiles. We started from planar biphenyl analogues and, by enzymatic dioxygenation, transformed

them into hydroxylated diene structures. Using a small library of newly synthesized dienophiles, the dienes were

converted into bridged bicycles via the Diels-Alder reaction. The resulting collection of 78 structures was first tested for

bioactivity in a generic assay based on interference with the proliferation of mammalian cells. A more mechanism-targeted

bioactivity profiling method, exploiting cellular impedance monitoring, was subsequently used to obtain suggestions for

the mode of action exerted by those compounds that were the most active in the proliferation assay. Proteasome

inhibition could be confirmed for 8 of a series of 9 respective candidates. Whilst 7 of these molecules showed relatively

weak interference with proteasome activity, one candidate exerted a moderate but distinct inhibition. This result appears

remarkable in view of the small size of the compound library, which was synthesized following a few basic considerations.

It encourages the application of diverse synthetic approaches to further investigate the role of spherical shape for the

success of compound libraries.

by Reymond and co-workers⁷, considering molecular shape as

one of the important characteristics of bioactive molecules,

revealed that the vast majority of synthesized molecules were

INTRODUCTION

Within the last 50 years, the rate of drug discovery is almost

unchanged and clearly below the required level.¹Synthetic

compound libraries as source for new drug candidates typically

suffer from low success in clinical development.^2,3-5An obvious

reason for this failure may be the chemistry-driven synthetic

strategies by which compounds are produced in view of easy

accessibility rather than structural complexity. Strategies for

the chemical synthesis of compound libraries should

increasingly take into account structural features that

distinguish drugs and bioactive compounds from compounds

in general.

distributed in space regions characteristic for flat molecules

(rod or disc shapes), while very few compounds were found in

the space harboring three-dimensional structures. Proteins as

the main drug targets, however, typically use three-

dimensional sub-structures to bind small molecules. Thus, the

synthesis of small molecules with intrinsic three-

dimensionality appears of considerable interest in order to

populate unexplored regions of the chemical space and to find

candidates complementary to protein cavities. The presence of

a rigid scaffold⁶and few rotatable bonds is frequently

observed in bioactive molecules.⁶A respective survey indeed

showed that molecules with a significant three-dimensional

portion showed higher success rates in drug development.⁸

The rationale for this success may be their rigid spherical

structure, as the rigid nature of a small molecule can play a

crucial role in binding to macromolecules, due to lower

entropic losses in comparison to a flexible ligand with the

same interaction pattern.⁹Moreover, the rigidity of a ligand

typically contributes to increase the selectivity.¹⁰This may be

achieved directly or, in cases of a flexible target and a rigid

decoy, by inducing a fit only in the desired target.¹⁰For these

reasons, an “escape from flatland” has been suggested as a

A

comparison between marketed drugs, compounds

originating from combinatorial synthesis and natural products

indicated that the structural diversity of combinatorial

compound collections is substantially lower than that of either

drugs or natural products and that the former are restricted to

only a confined region of the chemical space.⁶A recent study

^a.Department of Chemical Biology, Helmholtz Centre for Infection Research, D-

38124 Braunschweig, Germany. Phone: (+49-531) 61813409; Fax: (+49-531)

61813499; E-mail: bernd.hofer@helmholtz-hzi.de

Electronic Supplementary Information (ESI) available: ¹H NMR, ¹³C NMR spectra of

all the compounds and the table of numerical values obtained in the impedance

measurement. See DOI: 10.1039/x0xx00000x

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strategy to increase the success rate in the synthesis of the dioxygenation product derived from bromobenzene, was

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DOI: 10.1039/C5OB02539G

bioactive compounds and potential drug candidates.^7,8

also used in the synthesis of BBs.

The above-mentioned features of spherical structures

encouraged us to throw focus on the synthesis of rigid sp³-rich

compounds. Out of the many strategies to obtain such

molecules, we decided on a brief two-step synthetic pathway

that permits the combination of building blocks containing

sub-structures that are related to “privileged” scaffolds. As one

of the starting components we used aromatic compounds,

more precisely, hetero-aromatic analogues of biphenyl, which

is regarded as a privileged structure. We decided for N-

substituted analogues in order to introduce additional

hydrogen bond donors and acceptors into the final products.

Aromatic molecules are chemically highly stable. However,

they may be enzymatically functionalized in a mild and

selective way, for example, by introducing simultaneously two

hydroxy groups and a conjugated diene structure (Fig. 1). This

reaction is catalyzed by bacterial aryl-hydroxylating

dioxygenases (ARHDOs). The resulting versatile dienediol (DD)

synthons may, for example, be used in [4+2] cycloaddition

reactions that introduce a bridged bicyclic structure (Fig. 1). If

required, protection and deprotection steps for the two

hydroxy groups may additionally be carried out. A wide range

of dienophiles (DPs) was used as partners in the cycloaddition

reactions, among them molecules that contain privileged sub-

structures.

Fig. 2 Structures of hetero-aryl dienediols prepared for the synthesis of bridged

bicycles.

Synthesis of dienophiles (DPs)

An overview on the DPs used in this project is given in Fig. 3

.

Five of the molecules (D01 D03 D23 D25 and D26) were

,

obtained from commercial sources. The others were

synthesized, following two routes (Scheme 1). Chemically, the

DPs may be subdivided into groups i - vii, as indicated in the

legend of Fig. 3. Route A was used for the synthesis of DPs

belonging to groups ii, iii, v and vi. Route B was applied to the

synthesis of DPs belonging to group iv. All of the dienophiles

synthesized were N-substituted maleimides, as the

electrophilic maleimide moiety is expected to show good

reactivity towards dienes in Diels-Alder reactions.¹¹

Fig. 1 Devised chemo-enzymatic route to synthesize bridged bicycles as a compound

type that possesses a rigid three-dimensional structure. ARHDO: aryl-hydroxylating

dioxygenase. Only one of the possible stereoisomers is shown.

As our approach was a structure-oriented, not a target-

oriented synthetic strategy, we first used a generic poly-target

biological screen for a broad assessment of bioactivity in the

obtained compound collection. In order to elucidate the mode

of action of the most active candidates, we applied a profiling

method that exploits cellular impedance monitoring. Finally,

we attempted to verify positive profiling results by appropriate

biochemical assays.

Results & discussion

Scheme 1 Reagents and conditions: (i) EtOAC, RT, 4 h; (ii) NaOAc, Ac₂O, RT- 80 C, 2 h;

(iii) H⁺, MeOH/EtOH, reflux, overnight; (iv) maleicanhydride, EtOAc, RT, 4 h; (v) ArCHO,

aq NaOH, 0 C-RT, overnight; (vi) NaOAc, Ac₂O, RT- 80 C, 2 h.

Transformation of aromatic compounds into cyclic cis-dienediols

In order to obtain the diene components for the synthesis of

bridged bicycles (BBs), five partly hetero-aromatic biphenyl

analogues, 2-, 3-, and 4-phenylpyridine, 4-phenylpyrimidine

and 1-phenyl-1H-imidazole, were converted into cis-dienediols

through dioxygenation by recombinant bacterial cells that

efficiently synthesize an ARHDO (H. Overwin et al., in

The maleimides of route A (Scheme 1) were readily

obtained by reacting the appropriate substituted amine (

with maleic anhydride ( ) in ethyl acetate, leading to the

corresponding N-aryl or -alkylmaleamic acid ), which

2)

1

(

3

precipitates in ethyl acetate. Without any further purification,

this open intermediate was subsequently cyclized in acetic

anhydride in the presence of sodium acetate to the desired

preparation). The resulting DD structures are shown in Fig. 2

.

In addition to these compounds, a commercially available DD,

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maleimide (

chromone were included as precursors for the synthesis of N-

substitued maleimides. The 4 -cinnamate-based maleimides

D09 D11) were obtained by reacting the (E)-4-aminocinnamic

4). Privileged structures like cinnamic acid and

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ʹ

(

-

acid with maleic anhydride and a subsequent cyclization, in

two instances followed by esterification. For the preparation of

D22, 7-amino-2-methylchromone was used as building block.

Scheme

3

Isomers formed in cycloadditions between dienediol 9P and various

dienophiles. Syn and anti are assigned with respect to the isopropylidene ring.

Table

1 Bridged bicyclic compounds obtained from cycloaddition reactions

between dienediols 11U and/or 11P and collection of dienophiles.^a

Dieno

phile

D01

D02

D03

D04

D05

D06

D07

Bridged

bicycle

Dieno

phile

D07

D08

D09

D10

D11

D12

D13

D14

D15

D16

D17

Bridged

bicycle

Dieno

phile

D18

D19

D20

D21

D22

D23

D24

D25

D26

Bridged

bicycle

BrDUD01S

BrDPD01A

BrDUD02S

BrDPD02A

BrDPD02S

BrDUD03S

BrDPD03S

BrDPD03A

BrDUD04S

BrDPD04S

BrDPD04A

BrDUD05S

BrDUD05A

BrDPD05S

BrDPD05A

BrDUD06S

BrDUD06A

BrDPD06S

BrDPD06A

BrDUD07S

BrDUD07A

BrDPD07S

BrDPD07A

BrDUD08S

BrDUD08A

BrDPD09S

BrDUD10S

BrDUD10A

BrDPD10S

BrDPD10A

BrDPD11A

BrDUD12S

BrDUD13S

BrDPD13S

BrDPD13A

BrDUD14S

BrDPD15S

BrDPD15A

BrDUD16S

BrDUD17S

BrDPD17S

BrDPD17A

BrDUD18S

BrDPD18A

BrDUD19S

BrDUD20S

BrDPD20S

BrDPD20A

BrDUD21S

BrDPD21A

BrDUD22S

BrDUD23S

BrDPD23S

BrDPD23A

BrDUD24S

BrDPD24S

BrDPD24A

BrDUD25S

BrDUD26S

Fig. 3 Overview of structures of dienophiles used for the synthesis of bridged bicycles.

They may chemically be subdivided into the following groups: (i) triazoline derivatives

(D01), (ii) aryl maleimides (D02-D08), (iii) cinnamate maleimides (D09-D11), (iv)

chalcone maleimides (D12-D21), (v) chromone maleimides (D22), (vi) alkyl maleimides

(D23-D24) and (vii) quinones (D25-D26).

The maleimides of route B (D12

Claisen-Schmidt

-

D21) were obtained by

Therefore, 4-

) was reacted with maleic anhydride to

). The intermediate

condensation.¹²

aminoacetophenone (

5

yield the N-substituted maleamic acid (

6

^aCompounds are ordered by the dienophile number (for structures see Fig.

). The compound codes shown contain the following information. BrD

was further reacted with appropriate arylaldehydes under

basic conditions to afford the intermediate, maleamic acid

3

designates that the dienediol derived from bromobenzene was used. ‘P’ or

‘U’ stands for protected or unprotected, respectively; this is followed by the

dienophile number; the final letter indicates the stereochemistry of the

structure, either endo–syn (S) or endo–anti (A).

with an α,β-unsaturated carbonyl skeleton (

cyclized to yield the desired maleimide-chalcone conjugates

). Of these compounds, the chalcone-based maleimides D13

D20 D21 and the chromone-based maleimide D22 were not

7), which was

(8

,

Table 2 Bridged bicyclic compounds obtained from cycloaddition reactions between

previously described in the literature.

dienediols of heterocyclic biphenyl analogues and nine selected dienophiles.^a

Synthesis of bridged bicycles

Dienop

hile

Bridged

bicycle

Dienop

hile

Bridged

bicycle

Dienop

hile

Bridged

bicycle

A first round of BB syntheses was carried out with the

commercial DD, (1S-cis)-3-bromo-3,5-cyclohexadiene-1,2-diol

D01

D06

D10

Py2DUD01S

Py3DUD01S

Py4DUD01S

PmDUD01S

Py3DUD06S

PmDUD06S

Py3DUD10S

D10

D13

D17

PmDUD10S

ImDUD10S

Py3DUD13S

PmDUD13S

Py3DUD17S

PmDUD17S

D19

D20

D21

Py3DUD19S

PmDUD19S

Py3DUD20S

PmDUD20S

Py3DUD21S

PmDUD21S

(9U

, Scheme 2). As synthetic applications involving cis-diol

metabolites often require protection of the hydroxy groups,¹³

they were derivatized to an acetonide (9P, Scheme 2), as

previously reported.¹⁴

^aCompounds are ordered according to the dienophile number (for

structures see Fig. 3). In the compound codes, Py2D Py3D Py4D PmD and

,

ImD designate dienediols 12 to 16

(Fig. 2). Further details of compound

codes are given in the legend of Table 1

.

Scheme 2 Protection of the vicinal dienediol hydroxy groups with 2,2-dimethoxy

propane.

The acetonide was reacted with all dienophiles shown in Fig. 3

,

except for D08 D12 D14 D16 D25 and D26, which were

,

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synthesized later, to afford bridged bicyclic structures in a with protected and unprotected DDs, higher reactivity was

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simple one-step reaction. The rates of these reactions, when observed with protected DDs under otherwise identical

performed at RT, were sluggish, thus requiring long reaction conditions.¹⁶However, with DDs 9U and 9P the reactivity

times (4-7 days). Refluxing the solution of the protected DD observed towards several dienophiles was the same. All DPs

(

9P) and a dienophile in chloroform resulted in good yields. yielded about 90% of the endo-syn isomer, probably due to the

Except D01, all of the dienophiles yielded a mixture of two directing effect of bond formation with the diol hydrogens.¹⁷

A

products. NMR characterization showed that they were the list of all cycloaddition products obtained from DDs 9U and 9P

endo isomers of the expected product. These structures were is shown in Table 1. The compound codes given there,

identified by two-dimensional NMR spectroscopy with NOE together with the structures shown in Fig. 2, Fig. 3 and

data. A through-space interaction between one of the methyl Scheme 3, allow to deduce the sterical structures of all listed

groups of the isopropylidene ring and the methine protons of compounds. Additionally, all structures are shown in the

the maleimide moiety identified the endo-syn isomer. syn Supplementary Information.

refers to the isopropylidenedioxy ring face of the diene

Nine dienophiles were selected for the synthesis of

(Scheme 3). For the endo-anti isomer, a similar proximity was cycloadducts with hetero-aryl DDs. This was based on

observed between the methine protons of the maleimide ring reactivity and yields with DDs 9U and 9P and on the initial

and the protons on the carbons bonded to the two oxygens of results of the biological activity screening (see below) of the

the isopropylidene ring. With four exceptions, the syn product first collection of bridged bicycles derived from them. Six of

was the major isomer. With D10

,

D11 and D23, the anti isomer the selected dienophiles (D13

, D17, D18, D19, D20 and D21,

had been formed in moderate excess (60-65%). In case of D01

,

see Fig. 3), were chalcone-based maleimides. The others were

the anti isomer was exclusively obtained, as previously N-(3,4,5-trimethoxy phenyl)maleimide (D06) and 4-phenyl-

reported.¹⁵The typically observed preference for the syn 1,2,4-triazoline-3,5-dione (D01). Because of the low yields with

isomer is in accordance with previous findings,¹⁶which had the acetonide deprotection of the bridged bicyclic products

shown that, while most DPs yield anti isomers in high excess, from the DD 9P, all cycloadditions were carried out with

maleimides have a significant tendency to increase formation unprotected hetero-aryl DDs. The first cycloaddition reactions

of the syn isomer. A strong syn selection is generally noticed with the selected dienophiles were performed with DD 14

(Fig.

when the diol groups of the dienes are unprotected (see ) and dienophiles D06 D10 and D17 under the same

2

,

results below).^17,18This is ascribed to the formation of conditions used for DDs 9U and 9P. However, they resulted in

hydrogen bonds between the diol and the dienophile, which only about 10% yields of the desired products, accompanied

direct the latter to the syn face.¹⁷For maleimides, we propose by several by-products. Therefore, different reaction

a similar effect also for protected diols. The two oxygens are conditions were explored. As a result, preparative reactions

still available as hydrogen bond acceptors. Hydrogens at were repeated in a sealed tube with chloroform as solvent

double- or triple-bonded carbons possess a low acidity. In the under elevated temperature and pressure for 3 days. This

case of maleimides, this is enhanced not only by the vicinal increased yields with DD 14 to about 16%. However, reactions

oxygens, but additionally by the vicinal nitrogen. Therefore, performed with DDs 11 and 13 (Fig. 2) under these conditions

the formations of weak bonds between these hydrogens and afforded products in significantly increased yields of 30-45%

the acetonide oxygens may exert a weak directing effect and 40-60%, respectively, with all of the selected dienophiles.

towards the syn face. The only striking exception was D01

,

DDs 10 and 12 were reacted with dienophile D01. They

which exclusively yielded the anti isomer, in agreement with showed similar reactivity to that of DD 11. Altogether, 20

literature data.^15,17,19This has been ascribed to electrostatic bridged bicyclic compounds were synthesized with the five

repulsion between the lone electron pairs of the acetonide hetero-aryl DDs (Table 2 and Supplementary Information). The

oxygens and of the triazolidinone nitrogens upon approach of predominant products formed were the endo-syn isomers with

the dienophile at the syn face.^16,17We note that the absence of an approximately 9-fold excess over the endo-anti isomers.

any hydrogens at the reacting bond of this DP may also The lower reactivity of the hetero-aryl-substituted DDs in the

disfavour syn formation. In case of unprotected DDs, when cycloaddition reactions, compared to the bromo-substituted

hydrogen bond formation between the reaction partners is DD 9U, may be due to increased electron-withdrawal from the

possible, a significant increase of the syn isomer was DD ring, caused by the aryl nitrogens, in conjunction with the

observed.¹⁷

extended conjugated system of the molecules. Reduced

To deprotect cycloadducts, compounds were refluxed electron densities of the diene carbons are expected to make

under acidic conditions, as previously reported.²⁰This resulted them more stable towards attack by the DP.²¹All bridged

in poor yields (less than 30%). Therefore, Diels-Alder reactions bicyclic compounds synthesized were not previously reported.

were attempted without prior protection of the hydroxy

groups. Cycloadditions were performed with the DD 9U and

Assessment of bioactivity

the entire collection of dienophiles (Fig. 3). Cycloaddition

As there is no assay for a general assessment of any kind of

products from all dienophiles were obtained successfully in

bioactivity, we determined the influence of our compounds on the

moderate to good yields in the range of 40-70%, except with

quinone-based dienophiles (D25 and D26), where yields were

the best available option. The half maximal inhibitory concentration

lower (25-30%). In cycloaddition reactions reported previously

proliferation of a mammalian cell line ²², which appeared to us to be

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values for proliferation (IC₅₀) of the bridged bicycles derived from

bromobenzene and the hetero-aromatic biphenyl analogues are

shown in Table 3. As can be seen, BBs synthesized with cinnamate-

and chalcone-based dienophiles typically showed higher

bioactivities than the other compounds.

BrDUD06S, carrying a 3,4,5-trimethoxyphenyl moiety, showed

an IC₅₀value of 44.1 µM, but its analogue, carrying a 3,4,5-

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trimethoxychalcone moiety, compound BrDUD19S, exerted

the same effect at an almost 10-fold lower concentration (4.3

µM). Protection of the diol hydroxy groups resulted in no clear

tendency. Both, increases and decreases in activity were

observed. When hydroxy groups were protected, the anti-

isomer always showed higher bioactivity than the syn-isomer.

This may be seen, for example, in the case of compounds

BrDPD17A and BrDPD17S. Amongst the cycloadducts with a

chalcone moiety, different substitutions of the terminal phenyl

ring result in different IC₅₀values. The 3,4,5-trimethoxy and 4-

Table 3 Bioactivity values of bridged bicyclic compounds as determined in a cell viability

assay.^a

IC₅₀

(µM)

IC₅₀

(µM)

Compound

Group (i)

BrDUD01S

BrDPD01A

Py2DUD01S

Py3DUD01S

Py4DUD01S

PmDUD01S

Group (ii)

BrDUD02S

BrDPD02A

BrDPD02S

BrDUD03S

BrDPD03S

BrDPD03A

BrDUD04S

BrDPD04S

BrDPD04A

BrDUD05S

BrDUD05A

BrDPD05S

BrDPD05A

BrDUD06S

BrDUD06A

BrDPD06S

BrDPD06A

Py3DUD06S

PmDUD06S

BrDUD07S

BrDUD07A

BrDPD07S

BrDPD07A

BrDUD08S

BrDUD08A

Group (iii)

BrDPD09S

BrDUD10S

BrDUD10A

BrDPD10S

BrDPD10A

Py3DUD10S

PmDUD10S

ImDUD10S

BrDPD11A

Group (iv)

BrDUD12S

BrDUD13S

BrDPD13S

BrDPD13A

Py3DUD13S

PmDUD13S

BrDUD14S

BrDPD15S

BrDPD15A

BrDUD16S

BrDUD17S

BrDPD17S

BrDPD17A

Py3DUD17S

PmDUD17S

BrDUD18S

BrDPD18A

BrDUD19S

Py3DUD19S

PmDUD19S

BrDUD20S

BrDPD20S

BrDPD20A

Py3DUD20S

PmDUD20S

BrDUD21S

BrDPD21A

Py3DUD21S

PmDUD21S

Group (v)

41.2±2.8

59.2±2.8

82.4±3.5

68.7±4.2

82.4±1.4

96±4.9

38.5±1.4

47±2.8

methyl substitutions

(BrDUD19S and BrDUD16S) yielded

21.8±1.06

9.1±0.35

39.2±0.7

29.3±2.1

34.1±0.7

19.6±0.7

19.6±1.4

6.9±0.14

13.4±0.35

35.5±2.1

8.8±0.21

38.3±1.4

67±4.9

particularly low IC₅₀values of 4.3 or 6.9 µM, respectively.

When comparing the cycloaddition products derived from

hetero-aromatic biphenyl analogues with their counterparts

derived from the bromobenzene (Table 3), the former in most

cases showed higher IC₅₀values than the latter. It is also

noteworthy that, among the former compounds, again a

compound which possesses the 3,4,5-trimethoxy substitution

at the aromatic ring (Py3DUD19S) exhibited the lowest IC₅₀

value (12 µM).

NC*

NC

44.1±1.4

NC

Bioactivity profiling

In order to obtain suggestions about the mode of action with

which the most active of these molecules interfere, a label-

free bioactivity profiling method, measurement of cellular

impedance, was applied.²³The 16 most active of the

bromobenzene-derived molecules and the 14 most active of

the biphenyl-analog-derived molecules (Table 3) were selected

for this analysis.

18.1±2.8

16.9±1.06

4.3±0.14

12±0.7

30.9±0.35

22.3±2.1

29.4±1.06

24.2±1.06

37.2±2.8

37.1±0.35

31.6±0.7

32.9±1.4

33.5±5.3

40.1±1.4

DMSO

Saframycin

Etoposid

Camptothecin

Cytochalasin D

Latrunculin B

64±2.83

NC

Rhizopodin

A

Py3DUD10S

Py3DUD20S

Py3DUD21S

ImDUD10S

Py3DUD01S

Velcade

PmDUD13S

PmDUD20S

Py3DUD17S

PmDUD21S

Py3DUD13S

PmDUD19S

Py4DUD01S

PmDUD06S

MG132

39.2±1.06

NC

BrDUD22S

Group (vi)

BrDUD23S

BrDPD23S

BrDPD23A

BrDUD24S

BrDPD24S

BrDPD24A

Group (vii)

BrDUD25S

BrDUD26S

NC

SB203580

SB202190

Cruentaren A

Oligomycin

Neopeltolid

Myxothiazol A

D_10

Simvastatin

Mevastatin

Cycloheximid

Gephyronic Acid

Anisomycin

Py3DUD19S

Taxol

Epothilon B

Tubulysin B

Griseofulvin

Nocodazol

Scriptaid

NC

25.4±2.1

NC

37±0.7

12.3±1.06

49.3±3.53

80.5±1.43

64.4±5.6

NC

Oxamflatin

Fig. 4 Heatmap deduced from the cellular impedance measurement of 14 BBs

derived from biphenyl analogues, clustered with 26 reference compounds. On

the x-axis, basis spline coeficients derived from the cubic smoothing splines

fitting of the TCRPs (corresponding to time-segments of the TCRPs, for details

see Materials and Methods) are shown for the measurement time after

compound addition of 66.5 h. The values of the basis spline coeficients were the

Z-transpormed, indicates how many standard deviations the descriptors deviate

from the mean value 0 of the descriptors. The profiles were clustered leading to

a dendrogram representation of TCRP similarity on the y-axis. Compound names

23.5±1.4

40.2±0.7

^aStandard deviations are indicated. Compound groups are defined in the legend of Fig.

3. Compound codes are explained in Tables 1 and 2. *NC-Not considered (IC₅₀> 100

µM).

More specifically, such differences were also observed in the

comparisons of the IC₅₀values of pairs of compounds that are

identical except for the absence or presence, respectively, of

the α,β-unsaturated keto moiety. For example, compound

or codes (Tables 1 and 2) are indicated at the right margin.

This journal is © The Royal Society of Chemistry 20xx

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Organic & Biomolecular Chemistry

Page 6 of 18

ARTICLE

It had been observed that cellular impedance is sensitive to

perturbations, as brought about, for example, by the bromobenzene-derived compounds yielded no clear

interaction of cellular components with small molecules.²⁴suggestions, the data from the 14 selected bridged bicyclic

Moreover, it has been shown that the change of this compounds based on the biphenyl analogues indicated curve

parameter over time can be characteristic for the type of similarities with the known proteasome inhibitors MG-132 and

perturbation.²³Thus, the similarity of a recorded impedance velcade (Fig. 4).

Journal Name

While the impedance measurements w_Vit_ieh_{w Ar}t_tih_cle_{e Onl}1_in6_e

DOI: 10.1039/C5OB02539G

curve, a so-called time-dependent cellular response profile

(TCRP) with the curves generated by compounds with known Verification of profiling suggestions

biological activities, yields suggestions about the cellular

The proteasome, a multi-catalytic complex, is responsible for

process the molecule in question may interfere with.

most of the intracellular protein degradation, including

Dimensionality reduction of the TCRPs can be achieved by a

proteins that regulate cell cycle and apoptosis. Malignant cells

spline-fitting method.²⁵The basis spline coefficients can be

have a defective cell cycle with high proliferation rate.

used as descriptors to calculate distance values (Euclidean

Therefore, these cells largely depend on the proteasome

distance) between test and reference compounds. The

degradation for further cell cycle progression. Proteasome

descriptor profiles of the TRCPs can be visualized in the form

inhibition thus leads to preferential death of proliferating

malignant cells.²⁶

of heat maps, where an associated dendrogram constructed

by hierarchical clustering of the descriptor profiles of the

Nine compounds were selected for a specific assay

TCRPs indicates curve similarities.

monitoring proteasome inhibition. The assay performed

measures the proteolytic activity associated with the

proteasome via bioluminescence. A proteasome substrate

peptide that was conjugated with a luminogenic reporter

group, aminoluciferin, was added to KB 3‒1 cells. The cellular

proteasome cleaves the peptide to release the aminoluciferin.

The luminescence signal is proportional to proteasome

activity. A known proteasome inhibitor, MG-132, was used as

positive control. Eight out of the nine compounds showed

weak inhibition, whereas one compound, PmDUD21S, showed

stronger inhibition. (Fig. 5a) The assay was repeated with

different amounts of this compound to determine the

concentration-dependency of the effect (Fig. 5b). This clearly

confirmed the inhibitory effect at concentrations of 25 µg/mL

or above. The compound showed similar proteasome

inhibition activity to the known proteasome inhibitor MG-132

at an approximately eightfold higher concentration.

Conclusions

The described results show that in a small collection of 78

compounds, which was synthesized based just on a few

general considerations, it was possible to identify a molecule

with a moderate, but distinctive biological activity. The

principal consideration was to synthesize molecules that

possess a rigid spherical structure. Another consideration was

to include compounds with privileged (sub-) structures into the

building blocks used.

One type of such molecules are biphenyl derivatives. Such

aromatic compounds are chemically rather inert. However,

enzyme-catalyzed reactions may be used for their

functionalization. For the current work, we successfully used

dioxygenation that transforms aromatic rings into synthetically

versatile dienediols, which may readily be converted into rigid

spherical structures. One should be cautious to draw too far-

reaching conclusions from the limited survey of the current

work. This initial study, however, encouraged us to extend our

research on spherical molecules. This is particularly true, as

many of the physico-chemical parameters regarded important

Fig. 5. Bioluminescent proteasome inhibiton assay (a) with the compounds selected

from the cellular impedance measurement profiling and (b) with the most active

compound at different concentrations. Proteolysis is proportional to the relative

luminescence units determined. In part (a), each compound was used at

a

concentration of 50 µg/mL (92 µM). The leftmost columns show the solvent control

(DMSO and methanol in 1:1 ratio (v/v)) and the positive control (4 uM MG-132). In part

(b), column 1 shows the solvent control, DMSO and MeOH in a 1:1 ratio (v/v); column 2

shows MG-132 as positive control (4 µM); columns 3-7 show the effects of compound

PmDUD21S at 20, 25, 30, 35 and 40 µg/mL, corresponding to 37, 46, 56, 65 and 74 µM.

Standard deviations are indicated.

6 | J. Name., 2012, 00, 1-3

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Organic & Biomolecular Chemistry

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Journal Name

ARTICLE

for bioactivity have not yet been optimized in this initial (2.3 mmol in 2 mL) was added dropwise wit_Vh_iewst_Ai_rr_tir_ci_len_Og_n._linA_e

approach. Thus, there is considerable space for improvement precooled mixture of oxoaminobutenoic acid (

^{DOI: 10.10}7³⁹,^/C1⁵.3^OBm⁰²m⁵³o⁹l^G),

along these lines. One of the next steps envisaged is the and sodium hydroxide (2.6 mmol) in water (2 mL) was added

diversification of compound libraries in terms of sizes and dropwise to the stirring solution of the aryl aldehyde over a

structure types of the three-dimensional scaffolds. With a period of 15 min with stirring. The reaction mixture was stirred

simultaneous improvement of (and also the adoption of at RT for 14 h, acidified with 2 N hydrochloric acid and stirred

additional) bio-profiling methods, it appears promising to for another 1h. The resultant precipitate was filtered, washed

further explore the influence of spherical shape on the success with water and vacuum-dried. The completely dried solid

rate of compound collections.

intermediate (9) was added to the mixture of NaOAc (0.7

mmol) and acetic anhydride (7.7 mmol), which was preheated

to 90

at 90



C for 20 min. The resultant reaction mixture was stirred

Experimental section



C. After 1 h, the reaction was allowed to cool to RT and

quenched with ice-cold water (5 mL). The aqueous layer was

extracted with ethyl acetate (2 x 15 mL), and the combined

organic layers were dried over Na₂SO₄and concentrated under

reduced pressure. The crude material was purified by column

chromatography using dichloromethane as eluent to afford

the desired compounds in 40-60% yields.

Materials and Methods

NMR data (¹H and ¹³C) was recorded with instruments Bruker

DPX-Bruker AV II-300 (300.1 MHz for 1H NMR, ¹³C NMR with

75.5 MHz, T = 296 K), AV III-400 (400.1 MHz for 1H NMR, ¹³C

NMR with 100.6 MHz, T = 296 K), Bruker DRX-400 (400.1 MHz

for 1H NMR, ¹³C NMR with 100.6 MHz, T = 300 K), and Bruker

AV II-600 (600.1 MHz for 1H NMR; ¹³C NMR with 150.3 MHz).

(E)-1-(4-(3-(4-Fluorophenyl)acryloyl)phenyl)-1H-pyrrole-2,5-dione

(D13)

The chemical shifts

 are given in ppm and referenced to the

¹H NMR (500 MHz, CDCl₃): δ 6.90 (s, 2H), 7.09-7.14 (m, 2H),

7.43 (d, J = 15.72 Hz, 1H), 7.54-7.58 (m, 2H), 7.61-7.66 (m, 2H),

7.78 (d, J = 15.56 Hz, 1H), 8.11 (d, J = 8.85 Hz, 2H); ¹³C NMR

(125 MHz, CDCl₃): δ 116.3, 121.5, 125.5, 129.4, 130.5, 131.1,

134.5, 135.3, 137.0, 144.0, 163.2, 165.2, 169.0, 189.3; MS

(NSI): m/z 321 [M+H]⁺.

internal solvent standard. Multiplicities of NMR signals are

indicated as follows: s (singlet), d (doublet), t (triplet), m

(multiplet), dd (doublet of doublets), dt (doublet of triplets)

and br. s (broad singlet). TLC was performed on pre-coated

aluminum sheets, silica gel 60 F₂₅₄with concentrating zone 20

x 2.5 cm (Merck). Silica gel (J. T. Baker) with pore size of 60 Å

and HPLC grade solvents were used for the column

chromatography purification. High-resolution mass spectra

were recorded with nano-spray ionization (HRMS-NSI) in the

positive mode, using a Thermo Fischer Scientific LTQ Orbitrap

Velos Pro instrument. All non-aqueous reactions were

conducted in dried glassware under nitrogen atmosphere. All

yields refer to isolated compounds after the final purification

process, unless otherwise stated.

(E)-1-(4-(3-(4-(Methylthio)phenyl)acryloyl)phenyl)-1H-pyrrole-2,5-

dione (D20)

¹H NMR (500 MHz, CDCl₃): δ 2.51 (s, 3H), 6.89 (s, 2H), 7.22-7.28

(m, 2H), 7.46 (d, J = 15.72 Hz, 1H), 7.56 (d, J = 8.54 Hz, 4H),

7.78 (d, J = 15.56 Hz, 1H), 8.07-8.12 (m, 2H); ¹³C NMR (125

MHz, CDCl₃): δ 15.2, 120.7, 125.5, 126.0, 128.9, 129.5, 131.3,

134.5, 135.1, 137.2, 142.7, 144.9, 169.0, 189.4; MS (NSI): m/z

350 [M+H]⁺.

General procedure for the synthesis of Dienophiles

Route A:²⁷Appropriate aryl or alkyl amine (

added to the solution of maleic anhydride (

2

, 5.8 mmol) was

, 5.1 mmol) in

(E)-1-(4-(3-(3-Nitrophenyl)acryloyl)phenyl)-1H-pyrrole-2,5-dione

(D21)

1

¹H NMR (500 MHz, DMSO-d₆): δ 7.24 (s, 2H), 7.60 (d, J = 8.70

Hz, 2H), 7.76 (t, J = 8.01 Hz, 1H), 7.89 (d, J = 15.72 Hz, 1H), 8.20

(d, J = 15.72 Hz, 1H), 8.28 (ddd, J = 8.24, 2.29, 0.92 Hz, 1H),

8.32 (d, J = 8.70 Hz, 2H), 8.34-8.37 (m, 1H), 8.80 (t, J = 1.91 Hz,

1H); ¹³C NMR (125 MHz, DMSO-d₆): δ 123.1, 124.8, 126.3,

129.4, 130.4, 134.9, 135.2, 135.9, 136.0, 136.6, 141.7, 148.5,

169.5, 188.3; MS (NSI): m/z 349 [M+H]⁺.

ethyl acetate (15 mL) and stirred at RT for 1 h. The resultant

solid precipitate was filtered and dried under vacuum to get

the intermediate, N-substituted aminobutenoic acid (

mixture of NaOAc (2.55 mmol) and acetic anhydride (25.5

mmol) was heated to 80 C in an oil bath until all NaOAc had

dissolved. The intermediate ( ) was added to this solution and

stirred at 80 C. After 1 h, the reaction was cooled to RT and

diluted with ice-cold water (15 mL). The aqueous layer was

3). A



3



extracted with ethyl acetate (2 x 15 mL). The organic layers 1-(2-Methyl-4-oxo-4H-chromen-7-yl)-1H-pyrrole-2,5-dione (D22)

¹H NMR (300 MHz, CDCl₃): δ 2.39 (d, J=0.57 Hz, 4H), 6.18 (d, J =

were combined and dried over Na₂SO₄. The organic layer was

evaporated at 30 C under reduced pressure. The obtained

crude compounds were purified by column chromatography

using petroleum ether and ethyl acetate as eluents to obtain

compounds in 40-65% yield.

0.75 Hz, 1H), 6.92 (s, 2H), 7.49 (dd, J = 8.67, 1.88 Hz, 1H), 7.57

(d, J = 1.70 Hz, 1H), 8.25 (d, J = 8.67 Hz, 1H); ¹³C NMR (75 MHz,

CDCl₃): δ 20.61, 110.9, 114.3, 121.7, 122.4, 126.6, 134.5, 135.8,

156.5, 166.6, 168.7, 177.4; MS (NSI): m/z 256 [M+H]⁺.

Route B:¹²A solution of the appropriate aromatic aldehyde (8,

1.5 mmol) in ethanol (1 mL) was cooled in an ice bath to 0 C,

Procedure for the protection of (1S-cis)-3-bromo-3,5-

cyclohexadiene-1,2-diol (9U)¹⁴

after which a precooled solution of sodium hydroxide in water

This journal is © The Royal Society of Chemistry 20xx

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ARTICLE

Journal Name

1

2,2-Dimethoxypropane (1.1 mmol) and a catalytic amount of BrDUD02S (endo-syn): H NMR (300 MHz, DMSO-d₆): δ 3.07-

View Article Online

p-toluenesulfonic acid were added to a pre-cooled solution of 3.14 (m, 1H), 3.49-3.55 (m, 1H), 3.61-3^D.6^O7^I:(¹m^0.1,⁰1³H^9/)^C,⁵3^O.^B6⁰8²-⁵3³.⁹7^G8

the DD (9U, 0.52 mmol) in dichloromethane (4 mL). The (m, 2H), 5.46-5.63 (m, 2H), 6.23 (dd, J = 8.67, 6.40 Hz, 1H),

reaction mixture was allowed to warm to RT and stirred until 6.33-6.40 (m, 1H), 7.19-7.29 (m, 2H), 7.31-7.42 (m, 2H); ¹³C

the reaction was complete (monitored by TLC). The reaction NMR (75 MHz, DMSO-d₆): δ 44.4, 64.2, 70.5, 115.7, 116.0,

mixture was neutralized with triethylamine (0.2 mmol), and 128.4, 129.0, 131.2, 135.9, 159.8, 163.0, 175.0, 177.1; HRMS

dichloromethane was evaporated under reduced pressure at (NSI): Calcd for C₁₆H₁₄BrFNO₄[M+H]⁺382.009, found 382.0085.

25 C. The crude compound was purified by flash column

chromatography using ethylacetate and petroleum ether as

eluents to obtain the pure compound in 90% yield.

4-Bromo-6-(4-fluorophenyl)-2,2-dimethyl-3a,4,4a,7a,8,8a-hexa-

hydro-5H-4,8-eth-eno[1,3]dioxolo[4,5-f]isoindole-5,7(6H)-dione

BrDPD02S (endo-syn): ¹H NMR (600 MHz, CDCl₃): δ 1.42 (s,

3H), 1.58 (s, 3H), 3.52-3.56 (m, 1H), 3.64 (dd, J = 8.44, 2.93 Hz,

1H), 3.74 (d, J = 8.44 Hz, 1H), 4.31 (dd, J = 8.25, 3.85 Hz, 1H),

4.40 (d, J = 8.44 Hz, 1H), 6.18 (dd, J = 8.44, 6.24 Hz, 1H), 6.40

(d, J = 8.44 Hz, 1H), 7.10-7.15 (m, 2H), 7.18-7.22 (m, 2H); ¹³C

NMR (150 MHz, CDCl₃): δ 24.4, 26.3, 36.6, 40.2, 44.2, 57.5,

75.1, 82.0, 113.4, 116.0, 116.3, 127.5, 128.2, 128.3, 131.0,

137.1, 161.1, 163.5, 174.4, 176.5; HRMS (NSI): Calcd for

C₁₉H₁₈BrFNO₄[M+H]⁺422.0403, found 422.0398. BrDPD02A

General procedure for [4+2] cycloadditions with protected (1S-cis)-

3-bromo-3,5-cyclohexadiene-1,2-diol (9P)

A mixture of the protected DD (9P, 0.07 mmol) and a

dienophile (0.08 mmol) in chloroform (4 mL) was refluxed

under a drying tube for 2-4 days.²⁸Reaction progress was

monitored by TLC. After the reaction was completed, the

solvent was removed under reduced pressure at 25 C and the

crude compound was purified by column chromatography

using ethyl acetate and petroleum ether as eluents to afford

cycloadducts in 40-70% yield.

1

(endo-anti): H NMR (600 MHz, CDCl₃): δ 1.35 (s, 3H), 1.40 (s,

3H), 3.05 (dd, J = 8.44, 2.93 Hz, 1H), 3.15 (d, J = 8.44 Hz, 1H),

3.56-3.59 (m, 1H), 4.41-4.47 (m, 2H), 6.07 (dd, J = 8.25, 6.42

Hz, 1H), 6.29 (d, J = 8.80 Hz, 1H), 7.10-7.15 (m, 2H), 7.16-7.21

(m, 2H); ¹³C NMR (150 MHz, CDCl₃): δ 25.1, 25.4, 35.9, 42.2,

46.7, 57.6, 78.0, 83.7, 110.7, 116.1, 116.4, 127.3, 128.2, 128.3,

129.7, 134.5, 160.7, 164.0, 172.4, 174.6; HRMS (NSI): Calcd for

C₁₉H₁₈BrFNO₄[M+H]⁺422.0403, found 422.0398.

General procedure for [4+2] cycloadditions with unprotected (1S-

cis)-3-bromo-3,5-cyclohexadiene-1,2-diol (9P)²⁸

A mixture of DD (9U, 0.07 mmol) and a dienophile (0.08 mmol)

in toluene (5 mL) was refluxed with a drying tube for 2-4 days.

Reaction progress was monitored by TLC. After the reaction

was completed, the solvent was removed under reduced

pressure at 25 C and the crude compound was purified by

column chromatography using ethyl acetate and petroleum

ether as eluents to afford cycloadducts in 40-70% yield.

4-Bromo-2-(4-bromophenyl)-8,9-dihydroxy-3a,4,7,7a-tetrahydro-

1H-4,7-ethanoisoindole-1,3(2H)-dione

BrDUD03S (endo-syn): ¹H NMR (300 MHz, Acetone-d₆): δ 3.25

(dtd, J = 6.31, 3.06, 3.06, 1.32 Hz, 1H), 3.60-3.66 (m, 1H), 3.72-

3.77 (m, 1H), 3.84-3.93 (m, 2H), 4.83 (d, J = 3.39 Hz, 1H), 5.17

(d, J = 3.58 Hz, 1H), 6.24 (dd, J = 8.67, 6.40 Hz, 1H), 6.35-6.40

(m, 1H), 7.20 (d, J = 8.85 Hz, 2H), 7.65 (d, J = 8.67 Hz, 2H); ¹³C

NMR (75 MHz, Acetone-d₆): δ 40.2, 40.9, 45.3, 63.9, 65.5, 71.8,

122.2, 129.6, 132.1, 132.7, 137.4, 175.3, 177.3; HRMS (NSI):

Calcd for C₁₆H₁₄Br₂NO₄[M+H]⁺441.9289, found 441.9284.

5-Bromo-10,11-dihydroxy-2-phenyl-5,8-dihydro-1H-5,8-ethano-

[1,2,4]triazolo[1,2-a]pyridazine-1,3(2H)-dione

1

BrDUD01S (syn): H NMR (500 MHz, Acetone-d₆): δ 4.03-4.14

(m, 2H,) 4.92-4.96 (m, 1H), 5.07 (d, J= 2.75 Hz, 1H), 5.42 (br. s.,

1H), 6.61-6.65 (m, 1H), 6.67-6.72 (m, 1H), 7.38-7.51 (m, 5H);

¹³C NMR (125 MHz, Acetone-d₆): δ 56.1, 63.9, 70.0, 71.2,

126.3, 128.1, 128.8, 130.3, 132.2, 136.8, 153.9, 154.3; HRMS

(NSI): Calcd for For C₁₄H₁₃BrN₃O₄[M+H]⁺366.0089, found

366.0084.

4-Bromo-6-(4-bromophenyl)-2,2-dimethyl-3a,4,4a,7a,8,8a-hexa-

hydro-5H-4,8-eth-eno[1,3]dioxolo[4,5-f]isoindole-5,7(6H)-dione

BrDPD03S (endo-syn): ¹H NMR (300 MHz, CDCl₃): δ 1.42 (s, 3H),

1.58 (s, 3H), 3.51-3.57 (m, 1H), 3.60-3.66 (m, 1H), 3.71-3.76 (m,

1H), 4.27-4.34 (m, 1H), 4.37-4.42 (m, 1H), 6.17 (dd, J = 8.48,

6.59 Hz, 1H), 6.40 (d, J = 8.48 Hz, 1H), 7.09-7.15 (m, 2H), 7.53-

7.60 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 24.4, 26.3, 36.6, 40.2,

44.3, 47.1, 57.4, 66.3, 75.0, 82.0, 113.4, 122.6, 124.8, 127.5,

127.9, 131.0, 131.8, 132.3, 137.1, 174.1, 176.3; HRMS (NSI):

Calcd for C₁₉H₁₈Br₂NO₄[M+H]⁺481.9602, found 481.9597.

4-Bromo-2,2-dimethyl-7-phenyl-3a,4,10,10a-tetrahydro-6H-4,10-

etheno[1,3]dio-xolo[4,5-d][1,2,4]triazolo[1,2-a]pyridazine-6,8(7H)-

dione

BrDPD01A (anti): ¹H NMR (300 MHz, CDCl₃): δ 1.41 (d, J = 5.65

Hz, 6H), 4.62-4.82 (m, 2H), 5.20 (ddd, J = 5.56, 4.05, 1.32 Hz,

1H), 6.30 (ddd, J = 8.43, 5.79, 0.85 Hz, 1H), 6.59 (dt, J = 8.48,

1.32 Hz, 1H), 7.32-7.52 (m, 5H); ¹³C NMR (75 MHz, CDCl₃): δ

25.5, 25.6, 51.9, 68.0, 74.9, 81.3, 112.5, 125.8, 128.3, 128.7,

129.2, 130.9, 134.8, 154.3, 154.8; HRMS (NSI): Calcd for

C₁₇H₁₇BrN₃O₄[M+H]⁺406.0402, found 406.0397.

1

BrDPD03A (endo-anti): H NMR (300 MHz, CDCl₃): δ 1.36 (s,

3H), 1.41 (s, 3H), 3.03-3.08 (m, 1H), 3.13-3.18 (m, 1H), 3.55-

3.63 (m, 1H), 4.41-4.50 (m, 2H), 6.07 (dd, J = 8.57, 6.31 Hz, 1H),

6.26-6.32 (m, 1H), 7.07-7.15 (m, 2H), 7.54-7.62 (m, 2H); ¹³C

NMR (75 MHz, CDCl₃): δ 25.1, 25.4, 35.9, 42.3, 46.7, 57.6, 78.0,

83.7, 110.7, 122.8, 127.9, 129.7, 130.4, 132.4, 134.5, 172.1,

4-Bromo-2-(4-fluorophenyl)-8,9-dihydroxy-3a,4,7,7a-tetrahydro-

1H-4,7-ethano-isoindole-1,3(2H)-dione

8 | J. Name., 2012, 00, 1-3

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174.4; HRMS (NSI): Calcd for C₁₉H₁₈Br₂NO₄[M+H]⁺481.9602, 8.14 Hz, 1H), 3.79-3.86 (m, 9H), 4.32 (dd, J = 8.14,_V4_ie._w07_ArtH_iclz_e,_O1_nH_lin)_e,

found 481.9597.

4.38-4.43 (m, 1H), 6.20 (dd, J = 8.39, 6^D.3^O6^{I: 1}H⁰z^.1,⁰1³H^9/)^C,⁵6^O.^B3⁰9²-⁵6³.⁹4^G5

(m, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 24.4, 26.3, 36.6, 40.1,

44.2, 56.3, 57.5, 60.9, 75.1, 82.1, 104.1, 113.4, 127.1, 131.0,

137.1, 138.4, 153.5, 174.5, 176.7; HRMS (NSI): Calcd for

C₂₂H₂₅BrNO₇[M+H]⁺494.0814, found 494.0809. BrDPD06A

(endo-anti):¹H NMR (300 MHz, CDCl₃): δ 1.33-1.39 (m, 3H),

1.42 (s, 3H), 3.01-3.09 (m, 1H), 3.12-3.19 (m, 1H), 3.59 (dt, J =

5.75, 2.78 Hz, 1H), 3.80-3.88 (m, 9H), 4.39-4.51 (m, 2H), 6.10

(dd, J = 8.67, 6.22 Hz, 1H), 6.32 (d, J = 8.67 Hz, 1H), 6.40 (s, 2H);

¹³C NMR (75 MHz, CDCl₃): δ 25.1, 25.4, 35.9, 42.2, 46.6, 56.3,

57.6, 60.9, 78.1, 83.7, 104.1, 110.7, 126.9, 129.7, 134.6, 138.4,

153.5, 172.5, 174.7; HRMS (NSI): Calcd for C₂₂H₂₅BrNO₇[M+H]⁺

494.0814, found 494.0809.

2-(4-Acetylphenyl)-4-bromo-8,9-dihydroxy-3a,4,7,7a-tetrahydro-

1H-4,7-ethano-isoindole-1,3(2H)-dione

BrDUD04S (endo-syn): ¹H NMR (400 MHz, DMSO-d₆): δ 2.64 (s,

3H), 3.08-3.15 (m, 1H), 3.55 (dd, J = 8.39, 2.80 Hz, 1H), 3.67 (d,

J = 8.14 Hz, 1H), 3.69-3.75 (m, 2H), 5.50 (d, J = 4.07 Hz, 1H),

5.60 (d, J = 4.58 Hz, 1H), 6.24 (dd, J = 8.65, 6.61 Hz, 1H), 6.38

(d, J = 8.65 Hz, 1H), 7.37 (d, J = 8.65 Hz, 2H), 8.09 (d, J = 8.65

Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 26.8, 44.5, 64.2, 70.5,

127.0, 128.8, 131.3, 136.0, 136.4, 174.8, 176.8, 197.2; HRMS

(NSI): Calcd for C₁₈H₁₇BrNO₅[M+H]⁺406.029, found406.024 .

6-(4-Acetylphenyl)-4-bromo-2,2-dimethyl-3a,4,4a,7a,8,8a-hexahy-

dro-5H-4,8-eth-eno[1,3]dioxolo[4,5-f]isoindole-5,7(6H)-dione

4-Bromo-6-(4-methoxyphenyl)-2,2-dimethyl-3a,4,4a,7a,8,8a-hexa-

hydro-5H-4,8-etheno[1,3]dioxolo[4,5-f]isoindole-5,7(6H)-dione

BrDPD04S (endo-syn): ¹H NMR (300 MHz, CDCl₃): δ 1.43 (s,

3H), 1.58 (s, 3H), 2.61 (s, 3H), 3.52 - 3.59 (m, 1H), 3.63-3.69 (m,

1H), 3.73-3.79 (m, 1H), 4.28-4.35 (m, 1H), 4.38-4.43 (m, 1H),

6.19 (dd, J = 8.57, 6.50 Hz, 1H), 6.42 (d, J = 8.48 Hz, 1H), 7.33-

7.41 (m, 2H), 7.99-8.06 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ

24.4, 26.3, 26.7, 36.6, 40.3, 44.3, 57.4, 75.0, 82.0, 113.4, 126.4,

129.1, 131.0, 135.7, 136.9, 137.1, 174.0, 176.2, 196.9; HRMS

(NSI): Calcd for C₂₁H₂₁BrNO₅[M+H]⁺446.0603, found 446.0598.

BrDPD05S (endo-syn): ¹H NMR (300 MHz, CDCl₃): δ 1.42 (s,

3H), 1.58 (s, 3H), 3.51-3.57 (m, 1H), 3.59-3.65 (m, 1H), 3.70-

3.75 (m, 1H), 3.81 (s, 3H), 4.28-4.34 (m, 1H), 4.37-4.43 (m, 1H),

6.17 (dd, J = 8.48, 6.40 Hz, 1H), 6.40 (d, J = 8.48 Hz, 1H), 6.94

(d, J = 9.04 Hz, 2H), 7.07-7.16 (m, 2H); ¹³C NMR (75 MHz,

CDCl₃): δ 24.4, 26.3, 36.6, 40.1, 44.2, 55.5, 57.6, 75.1, 82.1,

113.3, 114.4, 124.3, 127.6, 131.0, 137.0, 159.6, 174.7, 176.9;

HRMS (NSI): Calcd for C₂₀H₂₁BrNO₅[M+H]⁺434.0603, found

434.0598. BrDPD05A (endo-anti): ¹H NMR (300 MHz, CDCl₃): δ

1.36 (s, 3H), 1.41 (s, 3H), 3.01-3.07 (m, 1H), 3.11-3.17 (m, 1H),

3.55-3.62 (m, 1H), 3.82 (s, 3H), 4.41-4.49 (m, 2H), 6.07 (dd, J =

8.48, 6.22 Hz, 1H), 6.29 (d, J = 8.48 Hz, 1H), 6.92-6.98 (m, 2H),

7.08-7.15 (m, 2H); ¹³C NMR (75 MHz CDCl₃): δ 25.1, 25.4, 35.9,

42.2, 46.6, 55.5, 57.8, 78.1, 83.7, 110.6, 114.5, 124.1, 127.6,

129.7, 134.5, 159.7, 172.7, 175.0; HRMS (NSI): Calcd for

C₂₀H₂₁BrNO₅[M+H]⁺434.0603, found 434.0598.

1

BrDPD04A (endo-anti): H NMR (300 MHz, CDCl₃): δ 1.37 (s,

3H), 1.42 (s, 3H), 2.61 (s, 3H), 3.06-3.12 (m, 1H), 3.16-3.22 (m,

1H), 3.60 (dt, J = 5.89, 2.80 Hz, 1H), 4.41-4.51 (m, 2H), 6.09 (dd,

J = 8.57, 6.31 Hz, 1H), 6.31 (d, J = 8.67 Hz, 1H), 7.33-7.40 (m,

2H), 8.00-8.07 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 25.1, 25.4,

26.7, 35.9, 42.3, 46.8, 57.6, 78.0, 83.7, 110.7, 126.4, 129.1,

129.8, 134.5, 135.4, 137.0, 172.0, 174.3, 196.9; HRMS (NSI):

Calcd for C₂₁H₂₁BrNO₅[M+H]⁺446.0603, found 446.0598.

4-Bromo-8,9-dihydroxy-2-(4-methoxyphenyl)-3a,4,7,7a-tetrahy-

dro-1H-4,7-etha-noisoindole-1,3(2H)-dione

4-Bromo-8,9-dihydroxy-2-(3,4,5-trimethoxyphenyl)-3a,4,7,7a-

tetrahydro-1H-4,7-ethanoisoindole-1,3(2H)-dione

1

BrDUD05S (endo-syn): H NMR (500 MHz, Acetone-d₆): δ 3.28

BrDUD06S (endo-syn): ¹H NMR (400 MHz, Acetone-d₆): δ 3.21-

3.26 (m, 1H), 3.59 (dd, J = 8.39, 2.80 Hz, 1H), 3.71 (d, J = 8.14

Hz, 1H), 3.74 (s, 3H), 3.79 (s, 6H), 3.84-3.93 (m, 2H), 4.82 (d, J =

4.07 Hz, 1H), 5.16 (d, J = 4.07 Hz, 1H), 6.24 (dd, J = 8.90, 6.36

Hz, 1H), 6.37 (d, J = 8.65 Hz, 1H), 6.49 (s, 2H); ¹³C NMR (100

MHz, Acetone-d₆): δ 40.2, 40.7, 45.2, 56.6, 60.6, 64.0, 65.5,

71.8, 105.9, 129.2, 132.0, 137.4, 139.3, 154.3, 175.4, 177.5;

HRMS (NSI): Calcd for C₁₉H₂₁BrNO₇[M+H]⁺454.0501, found

454.0496. BrDUD06A (endo-anti): ¹H NMR (300 MHz, Acetone-

d₆): δ 3.29-3.36 (m, 2H), 3.36-3.43 (m, 1H), 3.72-3.80 (m, 9H),

4.04-4.14 (m, 1H), 4.22-4.31 (m, 1H), 4.59 (d, J = 4.52 Hz, 1H)

4.78 (d, J = 6.22 Hz, 1H), 6.11-6.20 (m, 1H), 6.24-6.30 (m, 1H),

6.48 (s, 2H); ¹³C NMR (75 MHz, Acetone-d₆): δ 39.6, 43.4, 48.6,

56.6, 60.5, 64.8, 70.1, 75.2, 105.9, 127.0, 129.1, 131.3, 135.5,

154.3, 173.8, 176.1; HRMS (NSI): Calcd for C₁₉H₂₁BrNO₇[M+H]⁺

454.0501, found 454.0496.

(br. s., 1H), 3.54-3.57 (m, 1H), 3.63-3.67 (m, 1H), 3.73-3.76 (m,

3H), 3.85 (d, J = 3.20 Hz, 2H), 6.12 (s, 1H), 6.29-6.33 (m, 1H),

6.84-6.89 (m, 2H), 7.02-7.06 (m, 2H); ¹³C NMR (125 MHz,

Acetone-d₆): δ 39.2, 39.9, 44.1, 55.3, 62.7, 64.5, 70.7, 114.2,

124.8, 127.9, 131.0, 136.7, 159.5, 162.4, 175.2, 177.2; HRMS

(NSI): Calcd for C₁₇H₁₇BrNO₅[M+H]⁺394.029, found 394.0285.

BrDUD05A (endo-anti): ¹H NMR (300 MHz, Acetone-d₆): δ 3.30

(s, 2H), 3.36-3.43 (m, 1H), 3.82 (s, 3H), 4.08 (d, J = 7.35 Hz, 1H),

4.22-4.31 (m, 1H), 4.59 (d, J = 4.33 Hz, 1H), 4.77 (br. s., 1H),

6.14 (dd, J = 8.67, 6.03 Hz, 1H), 6.22-6.31 (m, 1H), 6.93-7.02

(m, 2H), 7.05-7.15 (m, 2H); ¹³C NMR (125 MHz, Acetone-d₆): δ

39.6, 43.4, 48.6, 55.8, 64.8, 70.1, 75.2, 114.8, 126.2, 128.9,

131.3, 135.5, 160.4, 174.1, 176.3; HRMS (NSI): Calcd for

C₁₇H₁₇BrNO₅[M+H]⁺394.029, found 394.0285.

4-Bromo-2,2-dimethyl-6-(3,4,5-trimethoxyphenyl)-3a,4,4a,7a,8,8a-

hexahydro-5H-4,8-etheno[1,3]dioxolo[4,5-f]isoindole-5,7(6H)-

dione

BrDPD06S (endo-syn): ¹H NMR (400 MHz, CDCl₃): δ 1.42 (s, 3H),

1.58 (s, 3H), 3.52-3.57 (m, 1H), 3.60-3.65 (m, 1H), 3.74 (d, J =

3-(4-Bromo-8,9-dihydroxy-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-

4,7-ethanoiso-indol-2-yl)phenyl acetate

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Organic & Biomolecular Chemistry

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Journal Name

1

BrDUD07S (endo-syn): H NMR (300 MHz, Acetone-d₆): δ 2.26 131.0, 131.7, 135.1, 147.5, 173.0, 175.3; HRMS (NSI): Calcd for

View Article Online

DOI: 10.1039/C5OB02539G

+

(s, 3H), 3.25 (dtd, J = 6.29, 3.03, 3.03, 1.41 Hz, 1H), 3.61-3.66 C₁₆H₁₄BrN₂O₆[M+H] 409.0035, found 409.0029.

(m, 1H), 3.72-3.78 (m, 1H), 3.83-3.96 (m, 2H), 4.85 (d, J = 3.96

(E)-3-(4-(4-Bromo-2,2-dimethyl-5,7-dioxo-3a,4,4a,5,7,7a,8,8a-

octahydro-6H-4,8-etheno[1,3]dioxolo[4,5-f]isoindol-6-yl)phenyl)-

acrylic acid

Hz, 1H), 5.17 (d, J = 4.52 Hz, 1H), 6.24 (dd, J = 8.67, 6.40 Hz,

1H), 6.38 (d, J = 8.67 Hz, 1H), 7.03 (t, J = 2.17 Hz, 1H), 7.15

(dddd, J = 12.24, 8.10, 2.07, 0.94 Hz, 2H), 7.44-7.53 (m, 1H); ¹³

C

BrDPD09S (endo-syn): ¹H NMR (400 MHz, Acetone-d₆): δ 1.41

(s, 3H), 1.55 (s, 3H), 3.41-3.47 (m, 1H), 3.70 (dd, J = 8.65, 3.05

Hz, 1H), 3.76-3.83 (m, 1H), 4.39-4.48 (m, 2H), 6.31 (dd, J = 8.39,

6.36 Hz, 1H), 6.42 (d, J = 8.65 Hz, 1H), 6.57 (d, J = 15.77 Hz, 1H),

7.27-7.33 (m, 2H), 7.70 (d, J = 15.77 Hz, 1H), 7.78 (d, J = 8.65

Hz, 2H); ¹³C NMR (100 MHz, Acetone-d₆): δ 24.6, 26.3, 37.6,

41.4, 45.6, 59.7, 76.0, 83.0, 113.6, 120.4, 128.0, 129.3, 132.4,

135.0, 135.4, 137.4, 144.2, 167.5, 175.0, 177.2; HRMS (NSI):

Calcd for C₂₂H₂₁BrNO₆[M+H 474.0552, found 474.0547.

NMR (75 MHz, Acetone-d₆): δ 20.9, 40.2, 40.9, 45.3, 47.4, 65.5,

71.8, 121.2, 122.6, 124.8, 130.1, 132.1, 134.4, 137.4, 151.9,

169.4, 175.2, 177.3; HRMS (NSI): Calcd for C₁₈H₁₇BrNO₆[M+H]⁺

422.0239, found 422.0234. BrDUD07A (endo-anti): ¹H NMR

(300 MHz, Acetone-d₆): δ 2.26 (s, 4H), 3.28-3.36 (m, 3H), 3.40-

3.45 (m, 1H), 4.05-4.13 (m, 1H), 4.27 (d, J = 4.52 Hz, 1H), 4.60

(d, J = 4.90 Hz, 1H), 4.75-4.81 (m, 1H), 6.11-6.20 (m, 1H), 6.25-

6.31 (m, 1H), 7.02 (t, J = 2.07 Hz, 1H), 7.09-7.21 (m, 2H), 7.44-

7.54 (m, 1H); ¹³C NMR (75 MHz, Acetone-d₆): δ 20.9, 39.6,

43.5, 48.7, 70.1, 75.2, 121.2, 122.7, 124.8, 130.1, 131.4, 134.3,

135.5, 151.9, 169.4, 173.7, 175.9; HRMS (NSI): Calcd for

C₁₈H₁₇BrNO₆[M+H]⁺422.0239, found 422.0234.

Methyl-(E)-3-(4-(4-bromo-2,2-dimethyl-5,7-dioxo-3a,4,4a,5,7,7a,-

8,8a-octahydro-6H-4,8-etheno[1,3]dioxolo[4,5-f]isoindol-6-

yl)phenyl)acrylate

3-(4-Bromo-2,2-dimethyl-5,7-dioxo-3a,4,4a,5,7,7a,8,8a-octahydro-

6H-4,8-etheno-[1,3]dioxolo[4,5-f]isoindol-6-yl)phenyl acetate

BrDUD10S (endo-syn): ¹H NMR (400 MHz, Acetone-d₆): δ 3.26

(dtd, J = 6.36, 2.92, 2.92, 1.53 Hz, 1H), 3.64 (dd, J = 8.14, 3.05

Hz, 1H), 3.72-3.79 (m, 4H), 3.85-3.96 (m, 2H), 4.83 (d, J = 4.07

Hz, 1H), 5.18 (d, J = 4.07 Hz, 1H), 6.25 (dd, J = 8.65, 6.61 Hz,

1H), 6.39 (d, J = 8.65 Hz, 1H), 6.59 (d, J = 16.28 Hz, 1H), 7.31 (d,

J = 8.65 Hz, 2H), 7.69 (d, J = 16.28 Hz, 1H), 7.78 (d, J = 8.65 Hz,

2H); ¹³C NMR (100 MHz, Acetone-d₆): δ 40.4, 41.1, 45.5, 47.6,

51.9, 64.1, 65.6, 71.9, 119.9, 128.2, 129.4, 135.3, 137.5, 144.3,

167.4, 175.5, 177.5; HRMS (NSI): Calcd for C₂₀H₁₉BrNO₆[M+H]⁺

448.0395, found 448.0390. BrDUD10A (endo-anti): ¹H NMR

(400 MHz, Acetone-d₆): δ 3.32-3.37 (m, 2H), 3.40-3.47 (m, 1H),

3.76 (s, 3H), 4.11 (t, J = 6.10 Hz, 1H), 4.28 (d, J = 4.58 Hz, 1H),

4.60 (d, J = 5.09 Hz, 1H), 4.79 (d, J = 6.10 Hz, 1H), 6.13-6.20 (m,

1H), 6.28 (d, J = 8.14 Hz, 1H), 6.59 (d, J = 16.28 Hz, 1H), 7.30 (d,

J = 8.65 Hz, 2H), 7.69 (d, J = 16.28 Hz, 1H), 7.78 (d, J = 8.65 Hz,

2H); ¹³C NMR (100 MHz, Acetone-d₆): δ 39.6, 43.6, 48.7, 51.8,

64.7, 70.1, 75.2, 119.8, 128.1, 129.3, 131.4, 135.0, 135.3,

135.5, 144.1, 167.3, 173.7, 176.0; HRMS (NSI): Calcd for

C₂₀H₁₉BrNO₆[M+H]⁺448.0395, found 448.0390.

BrDP07S (endo-syn): ¹H NMR (600 MHz, CDCl₃): δ 1.42 (s, 3H),

1.58 (s, 3H), 2.28 (s, 3H), 3.52-3.55 (m, 1H), 3.63 (dd, J = 8.44,

2.93 Hz, 1H), 3.73 (d, J = 8.44 Hz, 1H), 4.30 (dd, J = 8.25, 3.85

Hz, 1H), 4.39 (d, J = 8.07 Hz, 1H), 6.17 (dd, J = 8.44, 6.60 Hz,

1H), 6.39 (d, J = 8.80 Hz, 1H), 7.07 (t, J = 2.02 Hz, 1H), 7.14

(ddd, J = 7.89, 5.50, 2.02 Hz, 2H), 7.43 (t, J = 8.07 Hz, 1H); ¹³C

NMR (150 MHz, CDCl₃): δ 21.1, 24.4, 26.3, 36.6, 40.2, 44.2,

57.5, 75.0, 77.2, 82.0, 113.3, 119.6, 121.9, 123.4, 129.6, 131.0,

137.1, 150.8, 168.9, 174.1, 176.2; HRMS (NSI): Calcd for

C₂₁H₂₁BrNO₆[M+H]⁺462.0548, found 462.0547. BrDP07A

1

(endo-anti): H NMR (600 MHz, CDCl₃): δ 1.36 (s, 3H), 1.41 (s,

3H), 2.29 (s, 3H), 3.04 (dd, J = 8.44, 2.93 Hz, 1H), 3.14 (d, J =

8.80 Hz, 1H), 3.58 (dt, J = 5.96, 2.71 Hz, 1H), 4.41-4.47 (m, 2H),

6.04-6.09 (m, 1H), 6.29 (d, J = 8.44 Hz, 1H), 7.06 (t, J = 2.02 Hz,

1H), 7.12-7.16 (m, 2H), 7.44 (t, J = 8.25 Hz, 1H); ¹³C NMR (150

MHz, CDCl₃): δ 21.1, 25.1, 25.4, 35.9, 42.2, 46.6, 57.7, 77.2,

78.0, 83.7, 110.7, 119.7, 122.1, 123.4, 129.7, 132.2, 134.5,

150.8, 168.9, 172.1, 174.3; HRMS (NSI): Calcd for C₂₁H₂₁BrNO₆

[M+H]⁺462.0548, found 462.0547.

Methyl-(E)-3-(4-(4-bromo-2,2-dimethyl-5,7-dioxo-3a,4,4a,5,7,-

7a,8,8a-octahydro-6H-4,8-etheno[1,3]dioxolo[4,5-f]isoindol-6-

yl)phenyl)acrylate

4-Bromo-8,9-dihydroxy-2-(4-nitrophenyl)-3a,4,7,7a-tetrahydro-1H-

4,7-ethanoiso-indole-1,3(2H)-dione

BrDPD10S (endo-syn): ¹H NMR (400 MHz, CDCl₃): δ 1.42 (s, 3H),

1.58 (s, 3H), 3.55 (dt, J = 6.36, 3.43 Hz, 1H), 3.63-3.67 (m, 1H),

3.73-3.77 (m, 1H), 3.81 (s, 3H), 4.31 (dd, J = 8.14, 4.07 Hz, 1H),

4.38-4.42 (m, 1H), 6.18 (dd, J = 8.39, 6.36 Hz, 1H), 6.38-6.47

(m, 2H), 7.28 (d, J = 8.14 Hz, 2H), 7.59 (d, J = 8.65 Hz, 2H), 7.68

(d, J = 15.77 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 24.4, 26.3,

36.6, 40.3, 44.3, 51.8, 57.5, 75.0, 82.0, 113.4, 119.2, 126.7,

128.6, 131.0, 133.1, 134.8, 137.1, 143.5, 167.1, 174.2, 176.4;

HRMS (NSI): Calcd for C₂₃H₂₃BrNO₆[M+H]⁺488.0709, found

488.0703. BrDPD10A (endo-anti): ¹H NMR (400 MHz, CDCl₃): δ

1.37 (s, 3H), 1.41 (s, 3H), 3.07 (dd, J = 8.39, 2.80 Hz, 1H), 3.15-

3.20 (m, 1H), 3.57-3.62 (m, 1H), 3.81 (s, 3H), 4.42-4.49 (m, 2H),

6.08 (dd, J = 8.65, 6.10 Hz, 1H), 6.30 (d, J = 8.65 Hz, 1H), 6.45

(d, J = 16.28 Hz, 1H), 7.28 (s, 2H), 7.57-7.62 (m, 2H), 7.68 (d, J =

BrDUD08S (endo-syn): ¹H NMR (300 MHz, Acetone-d₆): δ 3.25-

3.34 (m, 1H), 3.65-3.73 (m, 1H), 3.77-3.84 (m, 1H), 3.85-3.97

(m, 2H), 4.85 (d, J = 3.96 Hz, 1H), 5.16-5.22 (m, 1H), 6.27 (dd, J

= 8.67, 6.40 Hz, 1H), 6.36-6.44 (m, 1H), 7.55-7.63 (m, 2H), 8.30-

8.40 (m, 2H); ¹³C NMR (75 MHz, Acetone-d₆): δ 40.2, 41.1,

45.5, 63.8, 65.4, 71.7, 124.8, 128.5, 132.2, 137.4, 139.1, 147.9,

175.1, 177.2; HRMS (NSI): Calcd for C₁₆H₁₄BrN₂O₆[M+H]⁺

409.0035, found 409.0029. BrDUD08A (endo-anti): ¹H NMR

(300 MHz, Acetone-d₆); δ 3.33-3.44 (m, 2H), 3.47-3.52 (m, 1H),

4.09-4.16 (m, 1H), 4.26-4.33 (m, 1H), 4.61 (d, J = 4.90 Hz, 1H),

4.78-4.84 (m, 1H), 6.14-6.21 (m, 1H), 6.25-6.33 (m, 1H), 7.58

(d, J = 9.23 Hz, 2) 8.36 (d, J = 9.23 Hz, 2H); ¹³C NMR (75 MHz,

Acetone-d₆): δ 39.1, 40.6, 43.2, 48.4, 69.5, 74.7, 124.4, 128.0,

10 | J. Name., 2012, 00, 1-3

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15.77 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 25.1, 25.4, 35.9, (endo-anti): ¹H NMR (300 MHz, CDCl₃): δ 1.36 (s,_V3_iewH)_A,_rti1_cl._e4_O1_nl(_ins_e,

42.3, 46.7, 51.9, 57.6, 78.0, 83.7, 110.7, 119.3, 126.7, 128.7, 3H), 3.06-3.13 (m, 1H), 3.16-3.23 (m, 1^DH^O),^I:3¹⁰.5^.17⁰-³3⁹.^/6^C5^5O(^Bm⁰,²⁵1³H^9G),

129.7, 132.8, 134.5, 134.9, 143.4, 167.1, 172.2, 174.5; HRMS 4.41-4.51 (m, 2H), 6.06-6.14 (m, 1H), 6.32 (d, J = 8.67 Hz, 1H),

(NSI): Calcd for C₂₃H₂₃BrNO₆[M+H]⁺488.0709, found 488.0703. 7.11 (t, J = 8.57 Hz, 2H), 7.35-7.44 (m, 3H), 7.63 (dd, J = 8.76,

5.37 Hz, 2H), 7.77 (d, J = 15.82 Hz, 1H), 8.07 (d, J = 8.48 Hz, 2H);

¹³C NMR (75 MHz, CDCl₃): δ 25.2, 25.5, 36.0, 42.5, 46.9, 57.7,

Ethyl-(E)-3-(4-(4-bromo-2,2-dimethyl-5,7-dioxo-

3a,4,4a,5,7,7a,8,8a-octahydro-6H-4,8-etheno[1,3]dioxolo[4,5-

f]isoindol-6-yl)phenyl)acrylate

78.1, 83.8, 110.9, 116.3, 116.5, 121.6, 121.6, 126.5, 129.4,

129.9, 130.6, 130.6, 131.1, 134.7, 135.3, 138.3, 144.4, 163.1,

165.6, 172.2, 174.4, 189.4; HRMS (NSI): Calcd for C₂₈H₂₄BrFNO₅

[M+H]⁺552.0822, found 552.0816.

BrDPD11A (endo-anti): ¹H NMR (300 MHz, CDCl₃): δ 1.28-1.47

(m, 9H), 3.04-3.12 (m, 1H), 3.14-3.21 (m, 1H), 3.56-3.63 (m,

1H), 4.27 (q, J = 7.03 Hz, 2H), 4.41-4.51 (m, 2H), 6.08 (dd, J =

8.57, 6.31 Hz, 1H), 6.30 (d, J = 8.67 Hz, 1H), 6.44 (d, J = 16.01

Hz, 1H), 7.23-7.31 (m, 2H), 7.59 (d, J = 8.48 Hz, 2H), 7.63-7.72

(m, 1H); ¹³C NMR (75 MHz, CDCl₃): δ 14.3, 25.1, 25.4, 35.9,

42.3, 46.7, 57.5, 60.7, 78.0, 83.7, 110.7, 119.8, 126.7, 128.6,

129.7, 132.7, 134.5, 135.0, 143.1, 166.7, 172.2, 174.5; HRMS

(NSI): Calcd for C₂₄H₂₂NO₆Br [M+H]⁺502.0865, found 502.0854.

(E)-4-Bromo-2-(4-(3-(4-chlorophenyl)acryloyl)phenyl)-8,9-dihydro-

xy-3a,4,7,7a-tetrahydro-1H-4,7-ethanoisoindole-1,3(2H)-dione

BrDUD14S (endo-syn): ¹H NMR (500 MHz, Acetone-d₆): δ 3.32

(dtd, J = 6.33, 3.09, 3.09, 1.22 Hz, 1H), 3.72 (dd, J = 8.54, 3.05

Hz, 1H), 3.83 (d, J = 8.39 Hz, 1H), 3.90-3.99 (m, 2H), 5.00 (d, J =

4.27 Hz, 1H), 5.31 (t, J = 5.11 Hz, 1H), 6.31 (dd, J = 8.70, 6.41

Hz, 1H), 6.45 (d, J = 8.70 Hz, 1H), 7.49 (d, J = 8.70 Hz, 2H), 7.55

(d, J = 8.54 Hz, 2H), 7.81-7.85 (m, 1H), 7.91-7.98 (m, 3H), 8.27

(d, J = 8.70 Hz, 2H); ¹³C NMR (125 MHz, Acetone-d₆): δ 40.2,

41.0, 45.4, 63.9, 65.3, 71.7, 123.5, 127.8, 129.8, 129.9, 131.1,

132.1, 134.8, 136.6, 137.4, 137.5, 138.4, 143.6, 175.3, 177.4,

189.1; HRMS (NSI): Calcd for C₂₅H₂₀BrClNO₅[M+H]⁺528.0213,

found 528.0208.

4-Bromo-2-(4-cinnamoylphenyl)-8,9-dihydroxy-3a,4,7,7a-tetra-

hydro-1H-4,7-eth-anoisoindole-1,3(2H)-dione

BrDUD12S (endo-syn): ¹H NMR (500 MHz, CDCl₃): δ 3.48 (dddd,

J = 5.46, 4.39, 2.29, 1.14 Hz, 1H), 3.68-3.73 (m, 2H), 3.91-4.01

(m, 2H), 6.21 (dd, J = 8.62, 6.49 Hz, 1H), 6.42 (dd, J = 8.70, 1.22

Hz, 1H), 7.39-7.45 (m, 5H), 7.48 (d, J = 15.72 Hz, 1H), 7.61-7.67

(m, 2H), 7.81 (d, J = 15.72 Hz, 1H), 8.05-8.11 (m, 2H); ¹³C NMR

(125 MHz, CDCl₃): δ 38.7, 40.0, 44.0, 62.7, 64.8, 71.1, 121.8,

126.4, 128.6, 128.8, 129.1, 129.3, 130.8, 131.2, 134.7, 135.4,

136.3, 138.1, 145.6, 174.3, 176.3, 189.6; HRMS (NSI): Calcd for

C₂₅H₂₁NO₅Br [M+H]⁺494.0603, found 494.0598.

(E)-4-Bromo-6-(4-(3-(4-bromophenyl)acryloyl)phenyl)-2,2-dimeth-

yl-3a,4,4a,7a,8,-8a-hexahydro-5H-4,8-etheno[1,3]-dioxolo[4,5-

f]isoindole-5,7(6H)-dione

BrDPD15S (endo-syn): ¹H NMR (400 MHz, CDCl₃): δ 1.43 (s,

3H), 1.59 (s, 3H), 3.57 (dt, J = 6.48, 3.12 Hz, 1H), 3.68 (dd, J =

8.65, 3.05 Hz, 1H), 3.76-3.80 (m, 1H), 4.32 (dd, J = 8.14, 4.07

Hz, 1H), 4.41 (d, J = 8.14 Hz, 1H), 6.21 (dd, J = 8.39, 6.36 Hz,

1H), 6.43 (d, J = 8.65 Hz, 1H), 7.39-7.60 (m, 7H), 7.74 (d, J =

15.77 Hz, 1H), 8.07 (d, J = 8.65 Hz, 2H); ¹³C NMR (100 MHz,

CDCl₃): δ 24.3, 26.3, 36.6, 40.3, 44.3, 57.4, 75.0, 81.9, 113.4,

122.2, 125.1, 126.4, 129.2, 129.8, 131.0, 132.3, 133.6, 135.4,

137.1, 137.8, 144.1, 174.0, 176.2,189.2; HRMS (NSI): Calcd for

C₂₈H₂₄Br₂NO₅[M+H]⁺612.0021, found 612.0016. BrDPD15A

(endo-anti): ¹H NMR (400 MHz, CDCl₃): δ 1.37 (s, 3H), 1.42 (s,

3H), 3.10 (dd, J = 8.39, 2.80 Hz, 1H), 3.17-3.23 (m, 1H), 3.59-

3.65 (m, 1H), 4.44-4.50 (m, 2H), 6.07-6.14 (m, 1H), 6.32 (d, J =

8.65 Hz, 1H), 7.39-7.60 (m, 7H), 7.74 (d, J = 15.77 Hz, 1H), 8.08

(d, J = 8.14 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 25.0, 25.3,

(E)-4-Bromo-2-(4-(3-(4-fluorophenyl)acryloyl)phenyl)-8,9-dihydr-

oxy-3a,4,7,7a-tetrahydro-1H-4,7-ethanoisoindole-1,3(2H)-dione

BrDUD13S (endo-syn): ¹H NMR (500 MHz, CDCl₃): δ 3.48 (ddd, J

= 6.41, 3.28, 1.60 Hz, 1H), 3.71 (d, J = 1.53 Hz, 2H) 3.92-3.96

(m, 1H), 3.97-4.01 (m, 1H), 6.21 (dd, J = 8.70, 6.41 Hz, 1H), 6.42

(dd, J = 8.62, 1.14 Hz, 1H), 7.12 (t, J = 8.62 Hz, 2H), 7.38-7.43

(m, 3H), 7.64 (dd, J = 8.54, 5.34 Hz, 2H), 7.78 (d, J = 15.72 Hz,

1H), 8.07 (d, J = 8.70 Hz, 2H); ¹³C NMR (125 MHz, CDCl₃): δ

38.7, 40.0, 43.9, 62.6, 64.7, 71.0, 116.1, 116.3, 121.5, 126.4,

128.6, 129.2, 130.5, 131.2, 135.4, 136.3, 137.9, 144.2, 163.2,

165.2, 174.2, 176.3, 189.3; HRMS (NSI): Calcd for C₂₅H₂₀BrFNO₅

[M+H]⁺512.0509, found 512.0503.

(E)-4-Bromo-6-(4-(3-(4-fluorophenyl)acryloyl)phenyl)-2,2-dimethyl- 35.9, 42.3, 46.7, 57.6, 78.0, 83.6, 110.7, 122.2, 125.1, 126.4,

129.3, 129.9, 132.3, 133.6, 134.5, 135.2, 137.9, 144.1, 172.0,

174.3, 189.2; HRMS (NSI): Calcd for C₂₈H₂₄Br₂NO₅[M+H]⁺

612.0021, found 612.0016.

3a,4,4a,7a,8,-8a-hexahydro-5H-4,8-etheno[1,3]dioxolo[4,5-

f]isoindole-5,7(6H)-dione

BrDPD13S (endo-syn): ¹H NMR (300 MHz, CDCl₃): δ 1.43 (s, 3H),

1.59 (s, 3H), 3.53-3.60 (m, 1H), 3.65-3.71 (m, 1H), 3.76-3.81 (m,

1H), 4.30-4.35 (m, 1H), 4.40-4.44 (m, 1H), 6.21 (dd, J = 8.48,

6.59 Hz, 1H), 6.43 (d, J = 8.48 Hz, 1H), 7.08-7.16 (m, 2H), 7.37-

7.45 (m, 3H), 7.60-7.67 (m, 2H), 7.78 (d, J = 15.82 Hz, 1H), 8.07

(d, J = 8.67 Hz, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 24.4, 26.3,

36.7, 40.3, 44.4, 57.5, 75.0, 82.0, 113.4, 116.4, 121.5, 126.4,

129.3, 129.6, 130.6, 131.1, 131.8, 135.4, 137.1, 138.0, 139.5,

144.2, 162.6, 165.9, 174.1, 176.3, 189.3; HRMS (NSI): Calcd for

C₂₈H₂₄BrFNO₅[M+H]⁺552.0822, found 552.0816. BrDPD13A

(E)-4-Bromo-8,9-dihydroxy-2-(4-(3-(p-tolyl)acryloyl)phenyl)-

3a,4,7,7a-tetrahydro-1H-4,7-ethanoisoindole-1,3(2H)-dione

1

BrDUD16S (endo-syn): H NMR (500 MHz, CDCl₃): δ 2.40 (s,

3H), 3.46-3.50 (m, 1H), 3.68-3.73 (m, 2H), 3.92-4.00 (m, 2H),

6.19-6.24 (m, 1H), 6.42 (dd, J = 8.62, 1.30 Hz, 1H), 7.24 (d, J =

7.93 Hz, 2H), 7.39-7.46 (m, 3H), 7.54 (d, J = 8.24 Hz, 2H), 7.79

(d, J = 15.72 Hz, 1H), 8.05-8.09 (m, 2H); ¹³C NMR (125 MHz,

CDCl₃): δ 21.6, 38.7, 40.0, 44.0, 62.6, 64.8, 71.1, 120.8, 126.4,

This journal is © The Royal Society of Chemistry 20xx

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ARTICLE

Journal Name

128.6, 129.3, 129.8, 131.2, 132.0, 135.2, 136.3, 138.2, 141.4, 138.4, 145.9, 149.3, 151.7, 174.3, 176.4, 189.9;_VH_iewRM_ArtS_icle(_ON_nS_liI_n)_e:

145.7, 174.3, 176.3, 189.8; HRMS (NSI): Calcd for C₂₆H₂₃NO₅Br Calcd for C₂₇H₂₅BrNO₇[M+H]⁺554.0814, ^Dfo^Ou^I:n¹d^0.15⁰5³4⁹.^/0^C8^5O09^B.^02539G

[M+H]⁺508.0759, found, 508.0754.

(E)-4-Bromo-6-(4-(3-(3,4-dimethoxyphenyl)acryloyl)phenyl)-2,2-

(E)-4-Bromo-8,9-dihydroxy-2-(4-(3-(4-methoxyphenyl)acrylo-

yl)phenyl)-3a,4,7,7a-tetrahydro-1H-4,7-ethanoisoindole-1,3(2H)-

dione

dimethyl-3a,4,-4a,7a,8,8a-hexahydro-5H-4,8-etheno-

[1,3]dioxolo[4,5-f]isoindole-5,7(6H)-dione

1

BrDPD18A (endo-anti): H NMR (300 MHz, CDCl₃): δ 1.37 (s,

BrDUD17S (endo-syn): ¹H NMR (300 MHz, CDCl₃): δ 3.27 (dd, J 3H), 1.42 (s, 3H), 3.10 (dd, J = 8.48, 2.83 Hz, 1H), 3.17-3.23 (m,

= 18.08, 3.20 Hz, 2H), 3.48 (ddd, J = 4.76, 3.16, 1.51 Hz, 1H), 1H), 3.58-3.65 (m, 1H), 3.95 (d, J = 4.33 Hz, 6H), 4.46 (s, 2H),

3.71 (d, J = 1.51 Hz, 2H), 3.86 (s, 3H), 3.92-4.02 (m, 2H), 6.21 6.06-6.14 (m, 1H), 6.32 (d, J = 8.67 Hz, 1H), 6.91 (d, J = 8.29 Hz,

(dd, J = 8.67, 6.40 Hz, 1H), 6.42 (dd, J = 8.67, 1.13 Hz, 1H), 6.90- 1H), 7.15 (d, J = 1.70 Hz, 1H), 7.20-7.25 (m, 1H), 7.28-7.36 (m,

6.99 (m, 2H), 7.31-7.44 (m, 3H), 7.55-7.64 (m, 2H), 7.79 (d, J = 1H), 7.40 (d, J = 8.48 Hz, 2H), 7.75 (d, J = 15.64 Hz, 1H), 8.07 (d,

15.64 Hz, 1H), 8.04-8.10 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): δ J = 8.48 Hz, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 25.1, 25.4, 35.9,

38.7, 40.0, 44.0, 55.5, 62.7, 64.8, 71.1, 114.5, 119.6, 126.4, 42.3, 46.8, 56.0, 57.6, 78.0, 83.7, 110.2, 110.8, 111.2, 119.9,

127.5, 129.2, 129.9, 130.4, 131.2, 135.2, 136.4, 138.4, 145.5, 123.4, 126.4, 127.7, 129.3, 129.8, 134.6, 134.9, 138.6, 145.9,

161.9, 174.3, 176.3, 189.7; HRMS (NSI): Calcd for C₂₆H₂₃BrNO₆149.4, 151.7, 172.1, 174.4, 189.9; HRMS (NSI): Calcd for

[M+H]⁺524.0709, found 524.0703.

C₃₀H₂₉BrNO₇[M+H]⁺594.1127, 594.1122.

(E)-4-Bromo-6-(4-(3-(4-methoxyphenyl)acryloyl)phenyl)-2,2-

dimethyl-3a,4,4a,-7a,8,8a-hexahydro-5H-4,8-etheno[1,3]diox-

olo[4,5-f]isoindole-5,7(6H)-dione

(E)-4-Bromo-8,9-dihydroxy-2-(4-(3-(3,4,5-trimethoxyphenyl)-

acryloyl)phenyl)-3a,4,7,7a-tetrahydro-1H-4,7-ethanoisoindole-

1,3(2H)-dione

BrDPD17S (endo-syn): ¹H NMR (600 MHz, CDCl₃): δ 1.43 (s, 3H), BrDUD19S (endo-syn): ¹H NMR (300 MHz, CDCl₃): δ 3.48 (td, J =

1.59 (s, 3H), 3.55-3.59 (m, 1H), 3.67 (dd, J = 8.44, 2.93 Hz, 1H), 3.16, 1.22 Hz, 1H), 3.71 (d, J = 1.32 Hz, 3H), 3.88-3.99 (m, 11H),

3.76-3.80 (m, 1H), 3.86 (s, 3H), 4.32 (dd, J = 8.07, 3.67 Hz, 1H), 6.20 (dd, J = 8.67, 6.59 Hz, 1H), 6.42 (dd, J = 8.67, 1.13 Hz, 1H),

4.41 (d, J = 8.07 Hz, 1H), 6.20 (dd, J = 8.44, 6.60 Hz, 1H), 6.43 6.86 (s, 2H), 7.33 (d, J = 15.64 Hz, 1H), 7.41 (d, J = 8.48 Hz, 2H),

(d, J = 8.44 Hz, 1H), 6.93-6.96 (m, 2H), 7.36 (d, J = 15.41 Hz, 7.70 (d, J = 15.64 Hz, 1H), 8.06 (d, J = 8.48 Hz, 2H); ¹³C NMR (75

1H), 7.39-7.42 (m, 2H), 7.58-7.62 (m, 2H), 7.78 (d, J = 15.77 Hz, MHz, CDCl₃): δ 38.7, 40.0, 44.0, 56.3, 61.1, 62.6, 64.8, 71.0,

1H), 8.07 (d, J = 8.80 Hz, 2H); ¹³C NMR (150 MHz, CDCl₃): δ 105.8, 121.5, 126.4, 129.3, 130.2, 131.2, 135.3, 136.4, 138.2,

24.4, 26.3, 36.6, 40.3, 44.3, 55.5, 57.5, 75.0, 82.0, 113.4, 114.5, 140.7, 145.9, 153.6, 174.3, 176.4, 190.0; HRMS (NSI): Calcd for

119.6, 126.3, 127.5, 129.2, 130.4, 131.0, 135.1, 137.1, 138.4, C₂₈H₂₇NO₈Br [M+H]⁺584.092 found, 584.0915.

145.4, 161.9, 174.0, 176.3, 189.6; HRMS (NSI): Calcd for

C₂₉H₂₇BrNO₆[M+H]⁺564.1022, found 564.1016. BrDPD17A

(E)-4-Bromo-8,9-dihydroxy-2-(4-(3-(4-(methylthio)phenyl)acr-

yloyl)phenyl)-3a,4,-7,7a-tetrahydro-1H-4,7-ethanoisoindole-

(endo-anti): ¹H NMR (500 MHz, CDCl₃): δ 1.37 (s, 3H), 1.42 (s,

3H), 3.10 (dd, J = 8.54, 2.75 Hz, 1H), 3.20 (d, J = 8.39 Hz, 1H),

3.60-3.63 (m, 1H), 3.86 (s, 3H), 4.44-4.50 (m, 2H), 6.11 (dd, J =

8.62, 6.33 Hz, 1H), 6.32 (dd, J = 8.70, 0.76 Hz, 1H), 6.93-6.96

(m, 2H), 7.36 (d, J = 15.56 Hz, 1H), 7.38-7.41 (m, 2H), 7.58-7.62

1,3(2H)-dione

1

BrDUD20S (endo-syn): H NMR (500 MHz, CDCl₃): δ 2.52 (s,

3H), 3.44-3.49 (m, 1H), 3.67-3.73 (m, 2H), 3.92-4.00 (m, 2H),

6.20 (dd, J = 8.70, 6.56 Hz, 1H), 6.42 (dd, J = 8.70, 1.07 Hz, 1H),

7.24-7.28 (m, 2H), 7.38-7.46 (m, 3H), 7.55 (d, J = 8.39 Hz, 2H),

7.76 (d, J = 15.72 Hz, 1H), 8.07 (d, J = 8.85 Hz, 2H); ¹³C NMR

(125 MHz, CDCl₃): δ 38.7, 40.0, 44.0, 62.6, 64.8, 71.0, 120.7,

126.0, 126.4, 128.9, 129.2, 131.2, 135.3, 136.4, 138.2, 142.8,

145.2, 174.3, 176.4, 189.6; HRMS (NSI): Calcd for C₂₆H₂₃BrNO₅S

[M+H]⁺540.048, found 540.0475.

(m, 2H), 7.79 (d, J = 15.56 Hz, 1H), 8.07 (d, J = 8.70 Hz, 2H); ¹³

C

NMR (125 MHz, CDCl₃): δ 25.1, 25.4, 35.9, 42.3, 46.7, 55.5,

57.6, 78.0, 83.7, 110.7, 114.5, 119.5, 126.3, 127.4, 129.2,

129.8, 130.4, 134.5, 134.8, 138.6, 145.5, 161.9, 172.1, 174.4,

189.6; HRMS (NSI): Calcd for C₂₉H₂₇BrNO₆[M+H]⁺564.1022,

found 564.1016.

(E)-4-Bromo-2-(4-(3-(3,4-dimethoxyphenyl)acryloyl)phenyl)-8,9-

dihydroxy-3a,4,-7,7a-tetrahydro-1H-4,7-ethanoisoindole-1,3(2H)-

dione

(E)-4-Bromo-2,2-dimethyl-6-(4-(3-(4-(methylthio)phenyl)-acryl-

oyl)phenyl)-3a,4,4a,7a,8,8a-hexahydro-5H-4,8-etheno[1,3]dio-

xolo[4,5-f]isoindole-5,7(6H)-dione

1

BrDPD20S (endo-syn): ¹H NMR (400 MHz, CDCl₃): δ 1.43 (s, 3H),

1.59 (s, 3H), 2.52 (s, 3H), 3.57 (dt, J = 6.61, 3.31 Hz, 1H), 3.65-

3.70 (m, 1H), 3.76-3.80 (m, 1H), 4.32 (dd, J = 8.14, 3.56 Hz, 1H),

4.40-4.43 (m, 1H), 6.21 (dd, J = 8.39, 6.36 Hz, 1H), 6.43 (d, J =

8.14 Hz, 1H), 7.24-7.29 (m, 2H), 7.39-7.46 (m, 3H), 7.55 (d, J =

8.65 Hz, 2H), 7.77 (d, J = 15.77 Hz, 1H), 8.07 (d, J = 8.65 Hz, 2H);

¹³C NMR (100 MHz, CDCl₃): δ 15.1, 24.4, 26.3, 36.7, 40.3, 44.3,

57.5, 75.0, 77.2, 82.0, 113.4, 120.8, 126.0, 126.4, 128.9, 129.2,

131.0, 131.2, 135.3, 137.1, 138.2, 142.8, 145.0, 174.1, 176.3,

BrDUD18S (endo-syn): H NMR (300 MHz, CDCl₃): δ 3.34-3.52

(m, 3H), 3.60-3.73 (m, 3H), 3.75-3.88 (m, 1H), 3.95 (d, J = 4.71

Hz, 6H), 6.20 (dd, J = 8.67, 6.40 Hz, 1H), 6.42 (dd, J = 8.67, 1.13

Hz, 1H), 6.91 (d, J = 8.48 Hz, 1H), 7.15 (d, J = 1.88 Hz, 1H), 7.21-

7.25 (m, 1H), 7.32 (d, J = 15.64 Hz, 1H), 7.38-7.43 (m, 2H), 7.75

(d, J = 15.64 Hz, 1H), 8.06 (d, J = 8.67 Hz, 2H); ¹³C NMR (75

MHz, CDCl₃): δ 38.7, 40.0, 44.0, 56.0, 62.6, 64.8, 71.0, 110.2,

111.2, 120.0, 123.4, 126.4, 127.7, 129.2, 131.2, 135.2, 136.4,

12 | J. Name., 2012, 00, 1-3

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Organic & Biomolecular Chemistry

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ARTICLE

189.5; HRMS (NSI): Calcd for C₂₉H₂₇BrNO₅S [M+H]⁺580.0793,

found 580.0788. BrDPD20A (endo-anti): H NMR (400 MHz,

4-Bromo-2-ethyl-8,9-dihydroxy-3a,4,7,7a-tetrahydro-1H-4,7-

View Article Online

1

DOI: 10.1039/C5OB02539G

ethanoisoindole-1,3(2H)-dione

BrDUD23S (endo-syn): ¹H NMR (400 MHz, Acetone-d₆): δ 1.02

(t, J = 7.12 Hz, 3H), 3.10-3.21 (m, 1H), 3.34-3.48 (m, 3H), 3.51-

3.59 (m, 1H), 3.76-3.89 (m, 2H), 4.75 (br. s., 1H), 5.09 (br. s.,

1H), 6.08 (dd, J = 8.65, 6.10 Hz, 1H), 6.23 (d, J = 8.65 Hz, 1H);

¹³C NMR (100 MHz, Acetone-d₆): δ 13.1, 33.9, 39.9, 40.5, 44.9,

64.0, 65.4, 71.7, 131.8, 137.1, 176.0, 178.0; HRMS (NSI): Calcd

for C₁₅H₁₉BrNO₄[M+H]⁺356.0497, found 356.0491.

CDCl₃): δ 1.36 (s, 3H), 1.41 (s, 3H), 2.51 (s, 3H), 3.09 (dd, J =

8.39, 2.80 Hz, 1H), 3.16-3.21 (m, 1H), 3.58-3.63 (m, 1H), 4.43-

4.49 (m, 2H), 6.07-6.12 (m, 1H), 6.31 (d, J = 8.65 Hz, 1H), 7.24-

7.28 (m, 2H), 7.37-7.45 (m, 3H), 7.54 (d, J = 8.14 Hz, 2H), 7.76

(d, J = 15.77 Hz, 1H), 8.06 (d, J = 8.65 Hz, 2H); ¹³C NMR (100

MHz, CDCl₃): δ 15.2, 25.1, 25.4, 35.9, 42.3, 46.8, 57.6, 78.0,

83.7, 110.8, 120.7, 126.0, 126.4, 128.9, 129.3, 129.8, 131.2,

134.6, 135.0, 138.4, 142.8, 145.1, 172.1, 174.3, 189.5; HRMS

(NSI): Calcd for C₂₉H₂₇BrNO₅S [M+H]⁺580.0793 found,

580.0788.

4-Bromo-6-ethyl-2,2-dimethyl-3a,4,4a,7a,8,8a-hexahydro-5H-4,8-

etheno[1,3]-dioxolo[4,5-f]isoindole-5,7(6H)-dione

BrDPD23S (endo-syn): ¹H NMR (300 MHz, CDCl₃): δ 1.10 (t, J =

7.25 Hz, 3H), 1.40 (s, 3H), 1.54 (s, 3H), 3.40-3.58 (m, 5H), 4.23-

4.28 (m, 1H), 4.33-4.37 (m, 1H), 6.01-6.08 (m, 1H), 6.28 (d, J =

9.04 Hz, 1H); ¹³C NMR (75 MHz, CDCl₃): δ 13.0, 24.4, 26.2, 34.0,

36.3, 40.1, 44.1, 57.7, 75.1, 82.1, 113.2, 130.7, 136.8, 175.3,

177.3; HRMS (NSI): Calcd for C₁₅H₁₉BrNO₄[M+H]⁺356.0497,

(E)-4-Bromo-8,9-dihydroxy-2-(4-(3-(3-nitrophenyl)acryloyl)phenyl)-

3a,4,7,7a-tetrahydro-1H-4,7-ethanoisoindole-1,3(2H)-dione

BrDUD21S (endo-syn): ¹H NMR (500 MHz, CDCl₃): δ 3.49 (ddq, J

= 6.30, 3.29, 1.53, 1.53, 1.53 Hz, 1H), 3.72 (d, J = 1.68 Hz, 2H),

3.93-3.99 (m, 2H), 6.22 (dd, J = 8.62, 6.49 Hz, 1H), 6.43 (dd, J =

8.62, 1.30 Hz, 1H), 7.45 (d, J = 8.70 Hz, 2H), 7.58-7.66 (m, 2H),

7.84 (d, J = 15.72 Hz, 1H), 7.93 (d, J = 7.78 Hz, 1H), 8.11 (d, J =

8.85 Hz, 2H), 8.27 (ddd, J = 8.20, 2.25, 0.99 Hz, 1H), 8.51 (t, J =

1.91 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 38.7, 40.0, 44.0,

62.6, 64.8, 71.1, 122.5, 124.3, 124.9, 126.6, 129.4, 130.2,

131.2, 134.4, 135.8, 136.4, 136.5, 137.4, 142.4, 148.8, 174.2,

176.3, 188.7; HRMS (NSI): Calcd for C₂₅H₂₀N₂O₇Br [M+H]⁺

539.0454, found 539.0448.

1

found 356.0492. BrDPD23A (endo-anti): H NMR (300 MHz,

CDCl₃): δ 1.11 (t, J = 7.16 Hz, 3H), 1.34 (s, 3H), 1.39 (s, 3H), 2.86

(dd, J = 8.29, 2.64 Hz, 1H), 2.95-3.01 (m, 1H), 3.46-3.56 (m, 3H),

4.35-4.43 (m, 2H), 5.95 (dd, J = 8.57, 6.31 Hz, 1H), 6.18 (dd, J =

8.67, 0.94 Hz, 1H); ¹³C NMR (75 MHz, CDCl₃): δ 12.9, 25.0, 25.4,

34.2, 35.6, 42.2, 46.6, 57.8, 78.1, 83.7, 110.6, 129.4, 134.2,

173.3, 175.4; HRMS (NSI): Calcd for C₁₅H₁₉BrNO₄[M+H]⁺

356.0497, found 356.0492.

(E)-4-Bromo-2,2-dimethyl-6-(4-(3-(3-nitrophenyl)acryloyl)phenyl)-

3a,4,4a,7a,8,-8a-hexahydro-5H-4,8-etheno[1,3]dioxolo[4,5-

f]isoindole-5,7(6H)-dione

4-Bromo-8,9-dihydroxy-2-(2-morpholinoethyl)-3a,4,7,7a-tetra-

hydro-1H-4,7-ethanoisoindole-1,3(2H)-dione

1

BrDUD24S (endo-syn): H NMR (700 MHz, DMSO-d₆): δ 2.30

1

BrDPD21A (endo-anti): H NMR (300 MHz, CDCl₃): δ 1.37 (s,

(dt, J = 12.53, 6.10 Hz, 6H), 2.99 (dtd, J = 6.35, 3.07, 3.07, 1.29

Hz, 1H), 3.29-3.33 (m, 1H), 3.39-3.45 (m, 3H), 3.51 (t, J = 4.41

Hz, 4H), 3.60-3.68 (m, 2H), 5.38-5.41 (m, 1H), 5.45-5.48 (m,

1H), 6.03 (dd, J = 8.82, 6.45 Hz, 1H), 6.18 (d, J = 8.61 Hz, 1H);

¹³C NMR (175 MHz, DMSO-d₆): δ 35.2, 38.7, 43.9, 52.9, 54.7,

64.2, 66.2, 70.5, 130.9, 135.7, 175.6, 177.7; HRMS (NSI): Calcd

for C₁₆H₂₂BrN₂O₅[M+H]⁺401.0711, found 401.0707.

3H), 1.42 (s, 3H), 3.07-3.24 (m, 2H), 3.59-3.65 (m, 1H), 4.41-

4.51 (m, 2H), 6.11 (dd, J = 8.67, 6.22 Hz, 1H), 6.30-6.36 (m, 1H),

7.41-7.48 (m, 2H), 7.56-7.67 (m, 2H), 7.80-7.88 (m, 1H), 7.92

(d, J = 7.91 Hz, 1H), 8.09-8.15 (m, 2H), 8.28 (dd, J = 9.32, 1.22

Hz, 1H), 8.51 (t, J = 1.88 Hz, 1H); ¹³C NMR (75 MHz, CDCl₃): δ

25.1, 25.4, 35.9, 42.4, 46.8, 57.6, 77.2, 78.0, 83.7, 110.8, 122.5,

123.7, 124.3, 124.9, 126.6, 129.4, 129.8, 130.1, 134.4, 134.6,

135.6, 136.5, 137.5, 138.2, 142.4, 148.8, 172.1, 174.3, 188.7;

4-Bromo-2,2-dimethyl-6-(2-morpholinoethyl)-3a,4,4a,7a,8,8a-

HRMS (NSI): Calcd for C₂₈H₂₄BrN₂O₇[M+H]⁺579.0767, found hexahydro-5H-4,8-etheno[1,3]dioxolo[4,5-f]isoindole-5,7(6H)-

579.0761.

dione

BrDPD24S (endo-syn): ¹H NMR (300 MHz, CDCl₃): δ 1.40 (s, 3H),

1.55 (s, 3H), 2.37-2.54 (m, 6H), 3.38-3.51 (m, 2H), 3.53-3.69 (m,

7H), 4.23-4.31 (m, 1H), 4.32-4.39 (m, 1H), 6.04 (dd, J = 8.29,

6.78 Hz, 1H), 6.27 (d, J = 8.48 Hz, 1H); ¹³C NMR (75 MHz,

CDCl₃): δ 24.5, 26.4, 35.9, 36.4, 40.3, 44.2, 55.4, 57.8, 67.2,

75.2, 82.2, 113.4, 130.8, 136.9, 175.5, 177.6; HRMS (NSI): Calcd

for C₁₉H₂₆BrN₂O₅[M+H]⁺441.1025, found 441.1020.

4-Bromo-8,9-dihydroxy-2-(2-methyl-4-oxo-4H-chromen-7-yl)-

3a,4,7,7a-tetra-hydro-1H-4,7-ethanoisoindole-1,3(2H)-dione

BrDUD22S (endo-syn): ¹H NMR (300 MHz, Acetone-d₆): δ 2.45

(d, J = 0.57 Hz, 3H), 3.32 (dtd, J = 6.22, 3.01, 3.01, 1.32 Hz, 1H),

3.68-3.75 (m, 1H), 3.80-3.86 (m, 1H), 3.88-4.01 (m, 2H), 4.89

(d, J = 4.14 Hz, 1H), 5.19-5.27 (m, 1H), 6.21 (d, J = 0.75 Hz, 1H),

6.32 (dd, J = 8.67, 6.40 Hz, 1H), 6.41-6.49 (m, 1H), 7.37 (dd, J =

8.48, 1.88 Hz, 1H), 7.47 (d, J = 1.70 Hz, 1H), 8.16 (d, J = 8.48 Hz,

1H); ¹³C NMR (75 MHz, Acetone-d₆): δ 20.3, 40.3, 40.9, 45.5,

63.9, 65.5, 71.8, 111.1, 117.0, 123.8, 124.1, 126.4, 132.2,

137.4, 137.8, 157.0, 167.7, 175.1, 176.9, 177.2; HRMS (NSI):

Calcd for C₂₀H₁₇BrNO₆[M+H]⁺446.0239, found 446.0235.

1

BrDPD24A (endo-anti): H NMR (400 MHz, CDCl₃): δ 1.34 (s,

3H), 1.39 (s, 3H), 2.38-2.51 (m, 6H), 2.89 (dd, J = 8.14, 2.54 Hz,

1H), 3.00 (d, J = 8.14 Hz, 1H), 3.48 (dt, J = 5.85, 2.67 Hz, 1H),

3.54-3.67 (m, 6H), 4.35-4.43 (m, 2H), 5.95 (dd, J = 8.65, 6.10

Hz, 1H), 6.17 (d, J = 8.65 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ

25.1, 25.4, 35.6, 36.0, 42.2, 46.6, 53.4, 55.2, 57.8, 67.0, 78.1,

83.8, 110.6, 129.4, 134.2, 173.4, 175.6; HRMS (NSI): Calcd for

C₁₉H₂₆BrN₂O₅[M+H]⁺441.1025, found 441.1020.

J. Name., 2013, 00, 1-3 | 13

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Organic & Biomolecular Chemistry

Page 14 of 18

ARTICLE

Journal Name

Py3DUD06S (endo-syn): ¹H NMR (500 MHz, CDCl₃)_V:_iδ_ew3_A._r2_tic8_le(_Od_nd_li,_neJ

= 8.39, 2.59 Hz, 1H), 3.81 (d, J = 5.95 Hz,^D1^O1^I:H¹)⁰,^.13⁰.³9⁹7^/C(⁵d^O, ^BJ⁰=²⁵6³.⁹4^G1

Hz, 2H), 5.06-5.16 (m, 2H), 6.32 (s, 2H), 6.61 (dd, J = 8.70, 6.26

Hz, 1H), 6.75 (d, J = 8.70 Hz, 1H), 7.52-7.57 (m, 1H), 8.04 (d, J =

7.93 Hz, 1H), 8.64 (dd, J = 5.04, 1.37 Hz, 1H), 9.16 (br. s., 1H);

¹³C NMR (125 MHz, CDCl₃): δ 31.6, 32.1, 36.5, 44.4, 47.6, 53.5,

56.5, 61.0, 104.2, 122.4, 126.4, 126.9, 129.9, 131.1, 132.5,

138.7, 139.5, 145.9, 153.3, 153.7, 160.7, 174.5, 179.4; HRMS

(NSI): Calcd for C₂₄H₂₅N₂O₇[M+H]⁺453.1662, found 453.1656.

1-Bromo-9,10-dihydroxy-1,4,4a,8a-tetrahydro-1,4-ethano-naphth-

alene-5,8-dione

BrDUD25S (endo-syn): ¹H NMR (300 MHz, Acetone-d₆): δ 3.22

(dtd, J = 6.38, 3.07, 3.07, 1.41 Hz, 1H), 3.59-3.68 (m, 2H) 3.79-

3.85 (m, 2H), 4.75 (br. s., 1H), 5.13 (br. s., 1H), 6.15 (dd, J =

8.67, 6.59 Hz, 1H), 6.33 (dd, J = 8.57, 1.22 Hz, 1H), 6.64-6.69

(m, 1H) 6.74-6.79 (m, 1H); ¹³C NMR (75 MHz, Acetone-d₆): δ

43.3, 45.7, 49.0, 65.0, 70.8, 72.4, 132.7, 138.6, 142.6, 143.7,

196.1, 198.9; HRMS (NSI): Calcd for C₁₂H₁₂BrO₄[M+H]⁺

298.9919, found 298.9913.

Methyl-(E)-3-(4-(8,9-dihydroxy-1,3-dioxo-4-(pyridin-3-yl)-1,3,3a,4,-

7,7a-hexahyd-ro-2H-4,7-ethanoisoindol-2-yl)phenyl)acrylate

1-Bromo-11,12-dihydroxy-1,4,4a,9a-tetrahydro-1,4-ethano-anthr-

acene-9,10-dione

Py3DUD10S (endo-syn): ¹H NMR (500 MHz, CDCl₃): δ 3.37 (dt, J

= 3.24, 1.66 Hz, 1H), 3.51 (d, J = 8.39 Hz, 1H), 3.73 (dd, J = 8.32,

2.98 Hz, 1H), 3.77 (s, 3H), 3.86 (dd, J = 8.54, 3.51 Hz, 1H), 4.12

(d, J = 8.39 Hz, 1H), 6.39 (d, J = 16.02 Hz, 1H), 6.42-6.51 (m,

2H), 7.15 (d, J = 8.54 Hz, 2H), 7.52 (d, J = 8.39 Hz, 2H), 7.61 (d, J

= 16.02 Hz, 1H) 7.76 (dd, J = 8.24, 5.34 Hz, 1H), 8.43 (d, J = 8.24

Hz, 1H), 8.59 (d, J = 5.19 Hz, 1H), 8.96 (s, 1H); ¹³C NMR (125

MHz, CDCl₃): δ 38.7, 39.3, 40.3, 50.3, 51.8, 63.6, 70.4, 119.1,

125.1, 126.6, 128.6, 129.0, 129.1, 132.5, 132.9, 133.4, 134.7,

140.7, 141.2, 142.9, 143.5, 143.5, 167.2, 176.8, 177.6; HRMS

(NSI): Calcd for C₂₅H₂₃N₂O₆[M+H]⁺447.1556, found 447.1551.

1

BrDUD26S (endo-syn): H NMR (300 MHz, Acetone-d₆): δ 3.30-

3.37 (m, 1H), 3.75-3.90 (m, 4H), 4.81 (d, J = 3.77 Hz, 1H), 5.18

(d, J = 5.27 Hz, 1H), 5.95-6.10 (m, 2H), 7.72-7.89 (m, 4H); ¹³C

NMR (75 MHz, Acetone-d₆): δ 43.8, 46.8, 49.8, 65.0, 66.6, 72.3,

126.4, 127.1, 132.7, 134.4, 134.9, 138.6, 195.5, 197.6; HRMS

(NSI): Calcd for C₁₆H₁₄BrO₄[M+H]⁺349.0048, found 349.0070.

General procedure for [4+2] cycloadditions with hetero-aryl DDs

The appropriate dienophile (0.04 mmol) was added to a

solution of a DD (0.03 mmol) in methanol (0.2 mL) and

chloroform (3 mL). This mixture was heated to 100-110 °C in a

sealed vessel and stirred for 3-4 days until the DDs had reacted

completely (monitored by TLC). Subsequently, the solvent was

(E)-2-(4-(3-(4-Fluorophenyl)acryloyl)phenyl)-8,9-dihydroxy-4-

(pyridin-3-yl)-3a,4,-7,7a-tetrahydro-1H-4,7-ethanoisoindole-

1,3(2H)-dione

evaporated under reduced pressure at 25 C and the crude

1

Py3DUD13S (endo-syn): H NMR (500 MHz, CDCl₃): δ 3.03 (d, J

compound was purified by flash column column

chromatography using methanol and dichloromethane as

eluents to obtain pure compounds in 22-45% yields.

= 7.32 Hz, 1H), 3.42 (d, J = 8.39 Hz, 2H), 3.80 (dd, J = 8.47, 3.43

Hz, 1H), 4.11 (d, J = 8.39 Hz, 1H), 6.41-6.44 (m, 2H), 7.02 (t, J =

8.54 Hz, 2H), 7.23 (d, J = 8.70 Hz, 2H), 7.31 (d, J = 15.72 Hz, 1H),

7.54 (dd, J = 8.70, 5.34 Hz, 2H), 7.65 (d, J = 15.87 Hz, 1H), 7.70

(d, J = 5.49 Hz, 1H), 7.94 (d, J = 8.70 Hz, 2H), 8.34-8.40 (m, 1H),

8.53 (br. s., 1H), 8.90 (br. s., 1H); ¹³C NMR (125 MHz, CDCl₃): δ

38.6, 39.3, 40.3, 46.0, 63.5, 70.4, 116.0, 121.2, 125.0, 126.3,

128.7, 129.1, 130.5, 133.0, 135.3, 137.7, 139.6, 141.8, 142.4,

143.9, 144.5, 163.1, 165.1, 176.7, 177.6, 189.8; HRMS (NSI):

Calcd for C₃₀H₂₄N₂O₅F [M+H]⁺511.1669, found 511.1664.

10,11-Dihydroxy-2-phenyl-5-(pyridin-2-yl)-5,8-dihydro-1H-5,8-

ethano[1,2,4]-triazolo[1,2-a]pyridazine-1,3(2H)-dione

Py2DUD01S (endo-syn): ¹H NMR (500 MHz, CDCl₃): δ 4.10-4.15

(m, 1H), 4.45 (d, J = 8.39 Hz, 1H), 5.13 (ddd, J = 6.22, 2.71, 1.45

Hz, 1H), 6.66-6.69 (m, 1H), 6.73-6.77 (m, 1H), 7.27-7.31 (m,

1H), 7.34-7.44 (m, 5H), 7.74 (dt, J = 7.97, 0.97 Hz, 1H), 7.89 (td,

J = 7.82, 1.75 Hz, 1H),8.58-8.61 (m, 1H); ¹³C NMR (125 MHz,

CDCl₃): δ 55.8, 64.0, 65.6, 66.2, 124.8, 125.4, 125.6, 125.8,

128.2, 129.0, 129.1, 130.9, 131.3, 133.6, 139.6, 146.3, 154.5;

HRMS (NSI): Calcd for C₁₉H₁₇N₄O₄[M+H]⁺365.125, found

365.1244.

(E)-8,9-Dihydroxy-2-(4-(3-(4-methoxyphenyl)acryloyl)phenyl)-4-

(pyridin-3-yl)-3a,4,7,7a-tetrahydro-1H-4,7-ethanoisoindole-

1,3(2H)-dione

Py3DUD17S (endo-syn): ¹H NMR (700 MHz, CDCl₃): δ 3.39 (d, J

= 8.39 Hz, 2H), 3.63-3.66 (m, 1H), 3.73 (d, J = 1.29 Hz, 3H), 3.78

(dd, J = 8.39, 3.23 Hz, 1H), 4.08 (d, J = 8.17 Hz, 1H), 6.38-6.43

(m, 2H), 6.82 (d, J = 7.53 Hz, 2H), 7.18-7.21 (m, 2H), 7.23 (d, J =

15.70 Hz, 1H), 7.48 (d, J = 7.53 Hz, 2H), 7.62 (d, J = 15.49 Hz,

1H), 7.64-7.68 (m, 1H), 7.88-7.92 (m, 2H), 8.31 (d, J = 8.17 Hz,

1H), 8.50 (d, J = 4.95 Hz, 1H), 8.86 (br. s., 1H); ¹³C NMR (175

MHz, CDCl₃): δ 38.5, 39.2, 40.2, 46.0, 55.1, 63.4, 70.3, 114.2,

119.0, 124.8, 126.2, 127.0, 128.9, 130.2, 132.7, 132.8, 134.9,

138.0, 139.9, 142.1, 144.2, 145.8, 161.8, 176.6, 177.6, 190.1;

HRMS (NSI): Calcd for C₃₁H₂₇N₂O₆[M+H]⁺523.1869, found

523.1864.

10,11-Dihydroxy-2-phenyl-5-(pyridin-3-yl)-5,8-dihydro-1H-5,8-

ethano-[1,2,4]triazo-lo-[1,2-a]pyridazine-1,3(2H)-dione

Py3DUD01S (endo-syn):¹H NMR (500 MHz, CD₃OD): δ 4.03-

4.10 (m, 2H), 5.01-5.04 (m, 1H), 6.73-6.75 (m, 2H), 7.34-7.41

(m, 3H), 7.43-7.51 (m, 3H), 8.14-8.22 (m, 1H), 8.48-8.56 (m,

1H), 8.91 (d, J = 1.83 Hz, 1H); ¹³C NMR (125 MHz, CD₃OD): δ

58.6, 64.3, 68.2, 68.6, 124.6, 127.6, 129.8, 130.3, 131.3, 132.9,

134.5, 136.0, 138.1, 149.4, 150.0, 156.4, 156.9; HRMS (NSI):

Calcd for C₁₉H₁₇N₄O₄[M+H]⁺365.125, found 365.1244.

8,9-Dihydroxy-4-(pyridin-3-yl)-2-(3,4,5-trimethoxyphenyl)-

3a,4,7,7a-tetrahydro-1H-4,7-ethanoisoindole-1,3(2H)-dione

14 | J. Name., 2012, 00, 1-3

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Page 15 of 18

Organic & Biomolecular Chemistry

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Journal Name

ARTICLE

(E)-8,9-Dihydroxy-4-(pyridin-3-yl)-2-(4-(3-(3,4,5-trimethoxy-

phenyl)acryloyl)phen-yl)-3a,4,7,7a-tetrahydro-1H-4,7-ethano-

isoindole-1,3(2H)-dione

8,9-Dihydroxy-4-(pyrimidin-4-yl)-2-(3,4,5-trimethoxyphenyl)-

View Article Online

DOI: 10.1039/C5OB02539G

3a,4,7,7a-tetrahydro-1H-4,7-ethanoisoindole-1,3(2H)-dione

PmDUD06S (endo-syn): ¹H NMR (500 MHz, CDCl₃): δ 3.54-3.58

(m, 1H), 3.70 (dd, J = 8.54, 2.90 Hz, 1H), 3.79-3.83 (m, 9H),

3.88-3.92 (m, 3H), 6.32 (s, 2H), 6.52 (dd, J = 8.62, 6.48 Hz, 1H),

6.61 (d, J = 8.54 Hz, 1H), 7.61 (dd, J = 5.42, 1.30 Hz, 1H), 8.80

(d, J = 5.34 Hz, 1H), 9.24 (d, J = 1.22 Hz, 1H); ¹³C NMR (125

MHz, CDCl₃): δ 38.2, 40.6, 43.1, 49.7, 56.4, 61.0, 65.1, 67.8,

104.2, 121.0, 127.2, 130.5, 133.1, 138.5, 153.7, 157.2, 157.6,

169.3, 176.8, 177.7; HRMS (NSI): Calcd for C₂₃H₂₄N₃O₇[M+H]⁺

454.1614, found 454.1609.

1

Py3DUD19S (endo-syn): H NMR (500 MHz, CDCl₃): δ 3.58-3.66

(m, 2H), 3.88-3.92 (m, 9H), 3.98 (dd, J = 8.32, 3.28 Hz, 1H), 4.24

(d, J = 8.39 Hz, 1H), 6.45-6.58 (m, 2H), 6.84 (s, 2H), 7.27-7.35

(m, 3H), 7.66 (d, J = 15.56 Hz, 1H), 7.74-7.82 (m, 1H), 8.00 (d, J

= 8.54 Hz, 2H), 8.41 (d, J = 8.24 Hz, 1H), 8.52 (d, J = 4.58 Hz,

1H), 9.14 (br. s., 1H); ¹³C NMR (125 MHz, CDCl₃): δ 38.9, 39.2,

40.2, 50.4, 56.3, 61.0, 63.9, 71.0, 105.8, 105.8, 121.3, 125.5,

126.4, 129.2, 130.1, 132.5, 133.7, 135.1, 138.3, 140.7, 142.6,

144.0, 144.4, 145.8, 153.5, 174.4, 176.5, 177.0, 189.8; HRMS

(NSI): Calcd for C₃₃H₃₁N₂O₈[M+H]⁺583.208, found 583.2075.

Methyl-(E)-3-(4-(8,9-dihydroxy-1,3-dioxo-4-(pyrimidin-4-yl)-

1,3,3a,4,7,7a-hexa-hydro-2H-4,7-ethanoisoindol-2-yl)phenyl)-

acrylate

(E)-8,9-Dihydroxy-2-(4-(3-(4-(methylthio)phenyl)acryloyl)phenyl)-

4-(pyridin-3-yl)-3a,4,7,7a-tetrahydro-1H-4,7-ethano-isoindole-

1,3(2H)-dione

PmDUD10S (endo-syn): ¹H NMR (500 MHz, CDCl₃): δ 3.53-3.58

(m, 1H) 3.72 (dd, J = 8.47, 2.82 Hz, 1H) 3.80 (s, 3H) 3.88-3.93

(m, 3H) 6.42 (d, J = 16.02 Hz, 1H) 6.49 (dd, J = 8.62, 6.49 Hz,

1H) 6.59 (d, J = 8.54 Hz, 1H) 7.20 (d, J = 8.54 Hz, 2H) 7.55 (d, J =

8.54 Hz, 2H) 7.57-7.60 (m, 1H) 7.65 (d, J = 16.02 Hz, 1H) 8.78

(d, J = 5.34 Hz, 1H) 9.22 (s, 1H); ¹³C NMR (125 MHz, CDCl₃): δ

38.2, 40.5, 42.9, 49.7, 51.9, 64.9, 67.7, 119.2, 120.9, 126.6,

128.6, 130.4, 133.0, 134.7, 143.4, 157.3, 157.6, 167.1, 168.8,

176.3, 177.3; HRMS (NSI): Calcd for C₂₄H₂₂N₃O₆[M+H]⁺

448.1508, found 448.1503.

1

Py3DUD20S (endo-syn): H NMR (500 MHz, CDCl₃): δ 2.48 (s,

3H), 3.50-3.55 (m, 2H), 3.76-3.79 (m, 1H), 3.86-3.90 (m, 1H),

4.14 (d, J = 8.39 Hz, 1H), 6.49-6.55 (m, 2H), 7.22 (d, J = 8.54 Hz,

2H), 7.27 (d, J = 8.85 Hz, 2H), 7.37 (d, J = 15.56 Hz, 1H), 7.50 (d,

J = 8.39 Hz, 2H), 7.70 (d, J = 15.72 Hz, 1H), 7.94 (dd, J = 8.24,

5.95 Hz, 2H), 7.99 (d, J = 8.70 Hz, 2H), 8.65 (s, 1H), 9.06 (d, J =

1.98 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 15.0, 38.7, 39.4,

40.3, 50.4, 63.5, 70.3, 120.5, 123.5, 124.2, 125.9, 126.3, 128.9,

129.2, 131.0, 131.7, 133.9, 138.2, 138.6, 141.3, 142.2, 142.9,

143.7, 145.3, 145.6, 176.7, 177.3, 189.8; HRMS (NSI): Calcd for

C₃₁H₂₇N₂O₅S [M+H]⁺539.1640, found 539.1635.

(E)-2-(4-(3-(4-Fluorophenyl)acryloyl)phenyl)-8,9-dihydroxy-4-

(pyrimidin-4-yl)-3a,4,7,7a-tetrahydro-1H-4,7-ethanoisoindole-

1,3(2H)-dione

PmDUD13S (endo-syn): ¹H NMR (500 MHz, CDCl₃): δ 3.56-3.60

(m, 1H), 3.75 (dd, J = 8.54, 2.90 Hz, 1H), 3.89-3.95 (m, 3 H),

6.50-6.54 (m, 1H), 6.61 (d, J = 8.54 Hz, 1H), 7.12 (t, J = 8.62 Hz,

2H), 7.33-7.36 (m, 2H), 7.38 (d, J = 15.87 Hz, 1H), 7.58-7.65 (m,

3H), 7.76 (d, J = 15.72 Hz, 1H), 8.03-8.06 (m, 2H), 8.82 (d, J =

5.34 Hz, 1H), 9.25 (d, J = 1.22 Hz, 1H); ¹³C NMR (125 MHz,

CDCl₃): δ 38.3, 40.6, 43.2, 49.6, 65.0, 67.6, 116.2, 116.3, 120.8,

121.5, 126.4, 129.3, 130.3, 130.5, 131.0, 133.1, 135.3, 138.0,

144.3, 157.5, 157.7, 163.2, 165.2, 168.7, 176.2, 177.1, 189.3;

HRMS (NSI): Calcd for C₂₉H₂₃N₃O₅F [M+H]⁺512.1621, found

512.1616.

(E)-8,9-Dihydroxy-2-(4-(3-(3-nitrophenyl)acryloyl)phenyl)-4-

(pyridin-3-yl)-3a,4,-7,7a-tetrahydro-1H-4,7-ethanoisoindole-

1,3(2H)-dione

Py3DUD21S (endo-syn): ¹H NMR (500 MHz, CDCl₃): δ 3.58-3.61

(m, 1H), 3.63 (d, J = 8.54 Hz, 1H), 3.80 (dd, J = 8.39, 2.90 Hz,

1H), 3.97 (dd, J = 8.39, 3.36 Hz, 1H), 4.26 (d, J = 8.39 Hz, 1H),

6.49-6.52 (m, 2H), 7.38 (d, J = 8.70 Hz, 2H), 7.57 (d, J = 15.72

Hz, 1H), 7.62 (t, J = 7.93 Hz, 1H), 7.81 (d, J = 15.72 Hz, 1H) 7.90

(d, J = 7.63 Hz, 1H), 7.94-8.00 (m, 2H), 8.05 (d, J = 8.85 Hz, 2H),

8.13 (dd, J = 4.96, 1.30 Hz, 1H), 8.24-8.28 (m, 1H), 8.49 (t, J =

1.91 Hz, 1H), 8.76 (d, J = 2.14 Hz, 1H); ¹³C NMR (150 MHz,

CDCl₃): δ 38.8, 39.4, 40.3, 50.7, 63.8, 70.6, 122.5, 124.2, 124.9,

126.5, 129.6, 130.1, 132.4, 133.6, 134.4, 135.0, 135.6, 138.2,

142.4, 143.5, 148.7, 176.7, 177.3, 188.8, 192.0; HRMS (NSI):

Calcd for C₃₀H₂₄N₃O₇[M+H]⁺538.1614, found 538.1609.

(E)-8,9-Dihydroxy-2-(4-(3-(4-methoxyphenyl)acryloyl)phenyl)-4-

(pyrimidin-4-yl)-3a,4,7,7a-tetrahydro-1H-4,7-ethanoisoindole-

1,3(2H)-dione

PmDUD17S (endo-syn): ¹H NMR (500 MHz, CDCl₃): δ 3.54-3.59

(m, 1H), 3.73 (dd, J = 8.54, 2.90 Hz, 1H), 3.85 (s, 3H), 3.89-3.94

(m, 3H), 6.51 (dd, J = 8.62, 6.49 Hz, 1H), 6.60 (d, J = 8.24 Hz,

10,11-Dihydroxy-2-phenyl-5-(pyridin-4-yl)-5,8-dihydro-1H-5,8-

ethano[1,2,4]-triazolo-[1,2-a]pyridazine-1,3(2H)-dione

Py4DUD01A (endo-syn): ¹H NMR (500 MHz, CD₃OD): δ 3.77 (dd, 1H), 6.93 (d, J = 8.85 Hz, 2H), 7.32 (dd, J = 12.13, 3.43 Hz, 3H),

J = 2.82, 1.91 Hz, 1H), 4.04 (s, 1H), 4.97 (ddd, J = 6.10, 2.82, 7.58 (d, J = 8.85 Hz, 3H), 7.76 (d, J = 15.56 Hz, 1H), 8.03 (d, J =

0.99 Hz, 1H), 6.77 (dd, J = 8.39, 6.10 Hz, 1H), 6.98 (dt, J = 8.39, 8.70 Hz, 2H), 8.80 (d, J = 5.34 Hz, 1H), 9.23 (d, J = 1.22 Hz, 1H);

1.14 Hz, 1H), 7.29-7.34 (m, 2H), 7.41-7.50 (m, 4H), 7.77-7.81 ¹³C NMR (125 MHz, CDCl₃): δ 38.3, 40.6, 43.1, 49.6, 55.5, 65.0,

(m, 2H), 8.59 (d, J = 5.95 Hz, 2H); ¹³C NMR (125 MHz, CD₃OD): δ 67.7, 114.5, 119.5, 120.8, 126.3, 127.4, 129.2, 130.4, 133.1,

59.8, 68.2, 76.1, 78.9, 124.9, 127.3, 127.5, 127.8, 129.3, 129.7, 135.1, 138.4, 145.5, 157.4, 157.7, 161.9, 168.8, 176.2, 177.1,

130.0, 130.2, 130.3, 132.1, 132.6, 147.4, 149.8, 156.4, 158.1; 189.6; HRMS (NSI): Calcd for C₃₀H₂₆N₃O₆[M+H]⁺524.1821,

HRMS (NSI): Calcd for C₁₉H₁₇N₄O₄[M+H]⁺365.125, found found 524.1816.

365.1244.

J. Name., 2013, 00, 1-3 | 15

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Organic & Biomolecular Chemistry

Page 16 of 18

ARTICLE

Journal Name

(E)-8,9-Dihydroxy-4-(pyrimidin-4-yl)-2-(4-(3-(3,4,5-

trimethoxyphenyl)acryloyl)-phenyl)-3a,4,7,7a-tetrahydro-1H-4,7-

ethanoisoindole-1,3(2H)-dione

132.3, 133.4, 135.0, 135.2, 137.6, 144.1, 167.3, 175.7, 177.7;

HRMS (NSI): Calcd for C₂₃H₂₂N₃O₆[M+H] 436.1508, found

436.1503.

View Article Online

DOI: 10.1039/C5OB02539G

+

PmDUD19S (endo-syn): ¹H NMR (500 MHz, CDCl₃): δ 3.57 (dd, J

= 3.81, 2.59 Hz, 1H), 3.75 (s, 2H), 3.89 - 3.92 (m, 11H), 6.52 (dd,

J = 8.62, 6.48 Hz, 1H), 6.62 (d, J = 8.54 Hz, 1H), 6.84 (s, 2H),

7.30 (d, J = 15.72 Hz, 1H), 7.34 (d, J = 8.70 Hz, 2H), 7.59 (dd, J =

5.42, 1.30 Hz, 1H), 7.68 (d, J = 15.72 Hz, 1H), 8.02 (d, J = 8.70

Hz, 2H), 8.81 (d, J = 5.34 Hz, 1H), 9.24 (d, J = 1.22 Hz, 1H); ¹³C

NMR (125 MHz, CDCl₃): δ 38.4, 40.7, 43.1, 49.8, 56.4, 61.2,

65.0, 67.8, 105.9, 120.9, 121.5, 126.4, 129.4, 130.2, 130.5,

133.1, 135.3, 138.3, 140.8, 145.9, 153.6, 157.5, 157.8, 168.9,

176.3, 177.3, 189.9; HRMS (NSI): Calcd for C₃₂H₃₀N₃O₈[M+H]⁺

584.2033, found 584.2027.

Cell proliferation assay (MTT assay)

The procedure of Mosmann²²was modified as follows. A

suspension (120 µl) of L929 cells (mouse fibroblast) was added

into each well of a 96-well micro-titre plate (MTP). The test

compounds, dissolved in either DMSO or DMSO/Methanol

(1/1, v/v), were serially diluted and the final compound

dilutions and the solvent controls (60 µl) were transferred to

the cells in two replicates to reach final concentrations in the

range 37 µg/mL to 0.2 ng/mL. After 5 days of incubation at 37



C, the metabolic activity of the cells was determined by using

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT). Twenty µl of MTT (0.5 mg/mL) in PBS was added to the

cells. After 2h of incubation the micro-titre plates were

centrifuged (1744 x g) and the medium was removed by

tapping. The precipitate was washed with 100 µl of PBS and

the formazan crystals were dissolved by adding 100 µl of acidic

isopropanol (0.4% hydrochloric acid). The absorbance of the

colored solution was measured in a plate reader at 595 nm.

(E)-8,9-Dihydroxy-2-(4-(3-(4-(methylthio)phenyl)acryloyl)phenyl)-

4-(pyrimidin-4-yl)-3a,4,7,7a-tetrahydro-1H-4,7-ethano-isoindole-

1,3(2H)-dione

1

PmDUD20S (endo-syn): H NMR (500 MHz, CDCl₃): δ 2.52 (s,

3H), 3.56-3.60 (m, 1H), 3.74 (dd, J = 8.54, 2.90 Hz, 1H), 3.90-

3.95 (m, 3H), 6.52 (dd, J = 8.62, 6.48 Hz, 1H), 6.61 (d, J = 8.39

Hz, 1H), 7.23-7.28 (m, 2H), 7.32-7.36 (m, 2H), 7.40 (d, J = 15.72

Hz, 1H), 7.52-7.56 (m, 2H), 7.59 (dd, J = 5.49, 1.37 Hz, 1H), 7.75

(d, J = 15.72 Hz, 1H), 8.02-8.05 (m, 2H), 8.81 (d, J = 5.34 Hz,

1H), 9.24 (d, J = 1.37 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ

15.1, 38.3, 40.6, 43.1, 49.6, 65.0, 67.6, 120.7, 120.8, 126.0,

126.3, 128.9, 129.2, 130.3, 131.1, 133.1, 135.2, 138.2, 142.8,

145.1, 157.4, 157.7, 168.7, 176.2, 177.1, 189.5; HRMS (NSI):

Calcd for C₃₀H₂₆N₃O₅S [M+H]⁺540.1593, found 540.1588.

Cellular impedance measurement

24

A. Experimental procedure

:

For time-dependent cell

response profiling, 60 µl of medium was added to each well of

a 96 well plate equipped with gold electrodes (E-96 plate,

Roche/Acea Biosciences). A background measurement was

recorded prior to the the addition of 120 µL of a suspension of

L-929 cells (100000 cells/mL). After each step, the E-96 Plate

was incubated for 30 min at RT and then placed on the reader

in the incubator for continuous recording of impedance as

reflected by the cell index. After 24 h of incubation the cells

were treated with the test compounds dissolved in

DMSO/MeOH (1/1, v/v). The same mixture of solvent was also

used as solvent control in the E-96 plate. Compound quantities

corresponding to their IC₉₀values in the MTT assay were

added to the cells in triplicates in volumes of 1-3 µl. The

impedance measurement was run for 5 days.²⁴

(E)-8,9-Dihydroxy-2-(4-(3-(3-nitrophenyl)acryloyl)phenyl)-4-

(pyrimidin-4-yl)-3a,4,7,7a-tetrahydro-1H-4,7-ethanoisoindole-

1,3(2H)-dione

PmDUD21S (endo-syn): ¹H NMR (500 MHz, CDCl₃): δ 3.56-3.60

(m, 1H), 3.76 (dd, J = 8.39, 2.90 Hz, 1H), 3.91-3.95 (m, 3H), 6.53

(dd, J = 8.70, 6.41 Hz, 1H), 6.62 (d, J = 8.54 Hz, 1H), 7.38 (d, J =

8.70 Hz, 2H), 7.55-7.66 (m, 3H), 7.82 (d, J = 15.72 Hz, 1H), 7.91

(d, J = 7.78 Hz, 1H), 8.06-8.10 (m, 2H), 8.27 (ddd, J = 8.20, 2.25,

0.99 Hz, 1H), 8.50 (t, J = 1.91 Hz, 1H), 8.82 (d, J = 5.49 Hz, 1H),

9.25 (d, J = 1.37 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 38.3,

40.6, 43.2, 49.6, 65.0, 67.6, 120.8, 122.5, 124.2, 124.9, 126.5,

129.4, 130.1, 130.3, 133.1, 134.4, 135.8, 136.5, 137.3, 142.4,

148.8, 157.5, 157.7, 168.7, 176.1, 177.1, 188.6; HRMS (NSI):

Calcd for C₂₉H₂₃N₄O₇[M+H]⁺539.1566, found 539.1561.

B. Data analysis: The time-dependent cellular response

profiles (TCRP) were recorded by the Roche RTCA software.

The statistical programming language R, Version 2.12.2 (R

Development Core Team, 2011) was used for the execution of

data mining and processing. The raw cell index (CI) data

provided by RTCA software was normalized by dividing the cell

indices for each time point after the compound addition, by

the cell index at the reference point as suggested by Abassi

and co-workers.²³The last measurement recorded before the

addition of compounds was used as the reference point. Only

the readings after compound addition were considered for

subsequent analysis. The outliers out of triplicates were

detected and removed by the median polish method. Cubic

smoothing splines fitting was used to reduce the

dimensionality of the data set and to approximate the TCRPs.²⁵

Methyl-(E)-3-(4-(8,9-dihydroxy-4-(1H-imidazol-1-yl)-1,3-dioxo-

1,3,3a,4,7,7a-hexahydro-2H-4,7-ethanoisoindol-2-

yl)phenyl)acrylate

ImDUD10S (endo-syn): ¹H NMR (500 MHz, Acetone-d₆): δ 3.36

(dtd, J = 6.37, 3.07, 3.07, 1.22 Hz, 1H), 3.75 (s, 5H), 3.96 (dd, J =

8.62, 3.13 Hz, 1H), 4.24 (d, J = 8.39 Hz, 1H), 6.49 (dd, J = 8.77,

6.48 Hz, 1H), 6.57 (d, J = 16.02 Hz, 1H), 6.72 (d, J = 8.85 Hz, 1H),

6.90-6.97 (m, 1H), 7.25-7.29 (m, 2H), 7.41 (t, J = 1.30 Hz, 1H),

7.67 (d, J = 16.02 Hz, 1H), 7.72-7.77 (m, 2H), 7.98 (t, J = 1.07

Hz, 1H); ¹³C NMR (125 MHz, Acetone-d₆): δ 30.7, 39.6, 41.3,

41.8, 51.8, 65.6, 68.0, 118.9, 119.8, 126.0, 128.0, 129.0, 129.3,

16 | J. Name., 2012, 00, 1-3

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Page 17 of 18

Organic & Biomolecular Chemistry

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Journal Name

ARTICLE

R. P. Hertzberg and A. J. Pope, Curr. Opin. Chem. Biol., 2000,

4

5

6

7

8

9

Spline basis coefficients were used as descriptors to construct

the distance matrix which was used for hierarchical cluster

analysis to construct dendrograms. The distance matrix

provided numerical values (Euclidean distance) to assess

similarities between reference compounds with known

biological activity and test compounds. These numerical values

were used to select the compounds for further biological

activity assays. A heatmap was generated which displays the Z-

transformed values of the 22 descriptors which correspond to

the basis spline coefficients. Hierarchical cluster analysis was

done for the reference compounds together with the test

compounds.

View Article Online

4

, 445.

A. M. Davis, D. J. Keeling, J. Steel, N. P. Tomkinson and A. C.

Tinker, Curr. Top. Med. Chem., 2005, , 421.

DOI: 10.1039/C5OB02539G

5

M. Feher and J. M. Schmidt, J. Chem. Inf. Comput. Sci., 2003,

43, 218.

L. Ruddigkeit, L. C. Blum and J. L. Reymond, J. Chem. Inf.

Model. 2012, 52, 2864.

F. Lovering, J. Bikker and C. Humblet, J. Med. Chem., 2009,

52, 6752.

G. Klebe and H. J. Boehm, J. Recept. Signal Transduct. Res.,

1997, 17, 459.

10 D. J. Huggins, W. Sherman and B. Tidor. J. Med. Chem., 2012,

55, 1424.

11 S. A. Khattab, M. A. Khalifa and S. A. Mahgoub, J. Prakt.

Chemie, 1997, 3, 432.

12 A. Jha, C. Mukherjee, A. J. Rolle, E. De Clercq, J. Balzarini and

J. P. Stables, Bioorg. Med. Chem. Lett., 2007, 17, 4545.

13 T. Hudlicky, E. E. Boros, H. F. Olivo and J. S. Merola, J. Org.

Chem., 1992, 57, 1026.

14 S. V. Ley, A. J. Redgrave, S. C. Taylor, S. Ahmed and D. W.

Ribbons, Synlett, 1991, 741.

15 V. M. Kobal, D. T. Gibson, R. E. Davis and A. Garza, J. Am.

Chem. Soc., 1973, 95, 4420.

16 J. R. Gillard and J. D. Burnell, J. Chem. Soc. Chem. Commun.,

1989, 1439.

17 G.N. Jenkins, D.W. Ribbons, D.A. Widdowson, A.M.Z. Slawin

and D.J. Williams. J. Chem. Soc. Perkin Trans. I, 1995, 2647.

18 M. F. Mahon, K. Molloy, C. A. Pittol, R. J. Pryce, S. M. Roberts,

G. Ryback, V. Sik, J. O. Williams and J. A. Winders, J. Chem.

Soc. Perkin Trans. I, 1991, 1255.

Proteasome inhibition assay

All contents supplied in the commercial kit (Promega) were

used in the assay according to the instructions provided.²⁹All

solid substrates or reagents supplied were dissolved in the

appropriate buffer solution. Each reaction including solvent

control was done in duplicate. KB 3

into a 96 well white walled MTP with 15,000 cells/well. Cells

were incubated for 2 h at 37 C. Test compounds (1-2 µl),

dissolved in DMSO/MeOH (1/1, v/v) were added to the cells,

and the cells were incubated for another 2h at 37 C. The MTP

was allowed to equilibrate to RT. The proteasome reagent (50

µl) was added to the cells and the contents of the wells were

mixed on a plate shaker for 2 minutes, followed by the

incubation for 20 minutes at RT. Subsequently, the

luminescence of each well was measured with a plate reader.

‒1 cells (50 µl) were seeded



19 C. A. Pittol, R. J. Pryce, S. M. Roberts, G. Ryback, V. Sik and J.

O. Williams, J. Chem. Soc. Perkin Trans. I, 1989, 1160.

20 (a) I. C. Cotterill, S. M. Roberts and J. O. Williams, J. Chem.

Soc. Chem. Commun., 1988, 1628; (b) T. Hudlicky and H. F.

Olivo, J. Am. Chem. Soc., 1992, 114, 9694.

21 A. F. Carey and R. J. Sundberg, Advanced Organic Chemistry:

Part B: Reactions and Synthesis (5^thed.) New York: Springer.

Acknowledgements

ISBN 0387448993 (part A), ISBN 0387683542 (part B), 2007

22 T. Mosmann, J. lmmunol. Methods, 1983, 65, 55.

.

The authors are grateful to Heike Overwin and Enno Michaelis

from the Department of Chemical Biology, and to Myriam

González, Valentina Méndez and Michael Seeger, Univ. Técnica

Federico Santa María, Valparaíso, Chile, for providing us with

the hetero-aromatic dienediols. We thank Christel Kakoschke

for NMR measurements and Andrea Abrahamik and Anja

Meier for HRMS analyses. We are indepted to Ronald Frank for

a critical reading of the manuscript.

23 Y. A. Abassi, B. Xi, W. Zhang, P. Ye, S. L. Kirstein, M. R.

Gaylord, S. C. Feinstein, X. Wang, and X. Xu, Chem. Biol.,

2009, 16, 712.

24 K. Solly, X.Wang, X. Xu, B. Strulovici and W. Zheng, ASSAY

Drug Dev. Technol., 2004, 2, 363.

25 T. Schneider, Y. Muthukumar, B. Hinkelmann, R. Franke, M.

Döring, C. Jacob and F. Sasse, Med. Chem. Commun., 2012, 3,

784.

26 (a) D. Chauhan, T. Hideshima and K. C. Anderson, Annu. Rev.

Pharmacol. Toxicol., 2005, 45, 465; (b) A. F. Alexei F. Kisselev,

W. A. van der Linden and H. S. Overkleeft, Chem. Biol., 2012,

19, 99.

This work was supported by the HZI Graduate School and

the Department of Chemical Biology, Helmholtz Centre for

Infection Research, Braunschweig, Germany.

27 B. Budke, J. H. Kalin, M. Pawlowski, A. S. Zelivianskaia, M.

Wu, A. P. Kozikowski and P. P. Connell, J. Med. Chem., 2013,

56, 254.

28 F. Fringuelli, The Diels-Alder reaction: Selected practical

methods. New York: Wiley. ISBN: 047180343X, 2002.

29 R. A. Moravec, M. A. O'Brien, W. J. Daily, M. A. Scurria, L.

Bernad and T. L. Riss, Anal. Biochem., 2009, 387, 294.

References

1

2

B. Munos, Nat. Rev., 2009,

D. J. Payne, M. N. Gwynn, D. J. Holmes and D. L. Pompliano,

Nat. Rev. Drug Discov., 2007, , 29.

T. Mander, Drug Discov. Today, 2000,

8, 959.

6

3

5, 223.

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Organic & Biomolecular Chemistry

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DOI: 10.1039/C5OB02539G

An Approach to “Escape from Flatland”: Chemo-enzymatic Synthesis and

Biological Profiling of a Library of Bridged Bicyclic Compounds

N. V. Suryanarayana Birudukota, Raimo Franke and Bernd Hofer

A small library of 78 bridged bicyclic compounds were synthesized via a chemo-enzymatic

pathway. Biological evaluation suggested that rigid spherical scaffolds are useful to enhance

the success rate of compound libraries for drug development.

Article Doi

An approach to "escape from flatland": Chemo-enzymatic synthesis and biological profiling of a library of bridged bicyclic compounds

DOI: 10.1039/c5ob02539g

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Article abstract of DOI:10.1039/c5ob02539g

Full text of DOI:10.1039/c5ob02539g

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