Photochromic Dopamine Receptor Ligands Based on Dithienylethenes and Fulgides

A Journal of

Accepted Article

Title: Photochromic Dopamine Receptor Ligands Based on

Dithienylethenes and Fulgides

Authors: Daniel Lachmann, Carolin Studte, Barbara Männel, Harald

Huebner, Peter Gmeiner, and Burkhard König

This manuscript has been accepted after peer review and appears as an

Accepted Article online prior to editing, proofing, and formal publication

of the final Version of Record (VoR). This work is currently citable by

using the Digital Object Identifier (DOI) given below. The VoR will be

published online in Early View as soon as possible and may be different

to this Accepted Article as a result of editing. Readers should obtain

the VoR from the journal website shown below when it is published

to ensure accuracy of information. The authors are responsible for the

content of this Accepted Article.

To be cited as: Chem. Eur. J. 10.1002/chem.201702147

Link to VoR: http://dx.doi.org/10.1002/chem.201702147

Supported by

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

Photochromic Dopamine Receptor Ligands Based on

Dithienylethenes and Fulgides

Daniel Lachmann^[a][‡], Carolin Studte^{[a] [‡]}, Barbara Männel^[b], Harald Hübner^[b], Peter Gmeiner^[b]and

Burkhard König*^[a]

aromatic piperazines (1,4-DAPs), hydroxybenzoxazinone

substituted piperazines as well as conformationally restricted

dopamine analogs involving aminoindanes. The synthesis of D₂,

D₃and D₄receptor agonists and antagonists with individual

subtype selectivity or functional selectivity (biased signaling) has

received considerable attention, in recent years.^[3a-f]Although a

better understanding of the mechanism of GPCR-promoted drug

action was achieved, there is still a need to develop selective

molecular tools to obtain more insight into the dynamics or

receptors function.

Abstract: We describe the incorporation of the well-investigated

class of photochromic dithienylethenes (DTEs) and fulgides into

known dopamine receptor ligands like 1,4-disubstituted aromatic and

hydroxybenzoxazinone piperazines as well as aminoindanes.

Subtype and functional selective photochromic ligands were

obtained and characterized by NMR and UV-VIS spectroscopic

measurements. The photophysical properties of the DTE based

dopamine ligands revealed

a high fatigue resistance for the

diarylmaleimides, but the ringclosure could not be accomplished in

polar solvents due to a known twisted intramolecular charge transfer

(TICT). Several cyclopentene-DTEs showed high PSS, but a fast

degradation by forming an irreversible byproduct. Focusing on the

fulgides, high photostationary states and switching in polar solvents

were possible. The compounds 43, 45 and 46 containing the

isopropyl group showed only isomerization between the open E-form

Photopharmacology can address this issue in a non-invasive

way with high spatial and temporal precision. The importance of

this concept is revealed by the increasing number of

photochromic enzyme inhibitors,^[4a-f]photochromic ligands for

receptors and ion channels,^[5a-e]photoswitchable antibiotics,^[6a-b]

photo-switches, which are applicable in vivo using visible-

light,^[7a-c]and recently even photochromic ligands, which change

and the closed C-form. At

a concentration of 1 nM, the

cyclopentene-DTE 29-open showed a more than 11-fold higher

activation of D_2S, a pharmacologically important G protein-coupled

receptor, than its photochromic congener 29-closed. Interestingly,

the fulgimide-based pair 52-(E)-open/52-closed could be discovered

as an alternative photoswitch with inverse activation properties

exhibiting four-fold higher activity in the closed state.

their intrinsic activity.^[8]Recently, photochromic azobenzol based

[5c]

opioids

and dopamine receptor ligands, embedded in a

Introduction

phenethyl-propyl-hydroxytetraline (PPHT) structure, were

developed by Trauner et. al.^[9]

G-protein coupled receptors (GPCRs) are a vibrant field of

research because their dysfunction is linked to numerous

diseases like central nervous system (CNS) disorders, cancer or

inflammatory diseases.^[1a-c]Therefore, more than 30% of the

approved drugs target the GPCR family. Focusing on the

dopamine receptors several CNS disorders like schizophrenia,

drug addiction, Parkinson’s and Huntington’s disease are linked

to their dysfunction.^[2]Privileged structures targeting the

dopamine D₂-like receptors are derivatives of 1,4-disubstituted

Azobenzenes as well as dithienylethenes (DTEs) and fulgides

convert light-induced between two isomers, which differ for the

azobenzenes in geometry and dipole moment and for the DTEs

in conformational flexibility and electronic properties.^[10]Both

classes of switches show a high fatigue resistance, but only the

open and closed form of the DTEs and fulgides are thermally

stable, which makes them interesting candidates for

photopharmaceuticals.^[11a-b]Especially fulgides are very

interesting for biological applications, because they show high

fatigue resistance and are mostly water soluble and switchable

in aqueous buffer solutions (Scheme 1).^[12a-b]

[a]

[b]

[‡]

D. Lachmann, Dr. C. Studte, Prof. Dr. B. König

University of Regensburg Faculty of Chemistry and Pharmacy

Institute of Organic Chemistry, Universitätsstrasse 31

93053 Regensburg (Germany)

a)

E-mail: Burkhard.Koenig@ur.de

Dr. B. Männel, Dr. H. Hübner, Prof. Dr. P. Gmeiner

Friedrich-Alexander University, Emil Fischer Center

Department of Chemistry and Pharmacy, Schuhstrasse 19

91052 Erlangen (Germany)

Email: peter.gmeiner@fau.de

These authors contributed equally to this work.

Supporting information for this article is given via a link at the end of

the document.

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

b)

switches and 2) the diarylmaleic anhydrides. Both types of DTEs

have been successfully applied in photopharmacology.^[21]

Furthermore,

the

isomerization

wavelength

for

the

chromophores can be bathochromically shifted, reducing cell

damage otherwise caused by high energy light.^[11a]The

photoswitches differ in their attachment mode to the

pharmacological headgroups. Introducing two different sterically

demanding photochromic groups to the pharmacological

headgroups allows us to investigate the importance of the size

of the heterocyclic appendage for the affinity and subtype

selectivity.

Scheme 1. a) Switching principle of dithienylethene based switches. b)

Photochromic fulgides/fulgimides, depicted in three forms after irradiation

with UV light.

Two major strategies are commonly applied to introduce a

Privileged structures for aminergic GPCR ligands are 1,4-DAPs

3, 5, 7 and 9, aminoindanes 11 and 13 and the benzoxazinon

piperazine 17. A coupling reaction between the pharmacophoric

head groups and the photoswitches gave the target structures.

The 2-methoxy- and 2-methylthiophenylpiperazine building

blocks were synthesized as described in literature.^[17]

Commercially available 1-(2-methoxyphenyl)- and 1-(2-

(methylthio)phenyl)piperazine were alkylated with N-(4-

photoswitchable moiety into

a

bioactive compound: the

chromophore can either be embedded into the structure of a

pharmacophore or attached to one or two pharmacophores via a

suitable linker.^{[10, 13]}In each case, it is desirable that the light

induced switching between the two isomers results in a distinct

difference in affinity or intrinsic activity. This would allow the

photocontrol of biological functions and offer the possibility to

investigate drug targets like GPCRs very precisely.

bromobutyl)-

or

N-(2-bromoethyl)phthalimide

to

give

We envisioned that a formal exchange of a structural appendage

of known dopamine ligands by a photoswitchable unit would

lead to potent photochromic dopaminergic ligands (Figure 1).

Herein, we discuss the synthesis, photophysical characterization

and biological evaluation of photochromic dopamine receptor

ligands based on dithienylethene- and fulgide-type scaffolds.

intermediates 2, 4, 6 and 8 in good yields. Hydrazinolyses of the

respective phthalimides yielded building blocks 3, 5, 7, and 9.

The same protocol was used to obtain hydroxybenzoxazinone

substituted piperazine 17. The synthesis of precursor 14 is

described in the supporting information (SI1). Alkylation of 14

with N-(2-bromoethyl)phthalimide gave compound 15.

Subsequent hydrazinolysis and acid mediated cleavage of the

benzyl protecting groups yielded compound 17. Building blocks

11 and 13 were readily accessible following a two-step literature

procedure.^[18]

2-Propylaminoindane

oxalate

was

reacted

with

4-

bromobutyronitrile, respectively 2-bromoacetonitrile to afford

compounds 10 and 12. Reduction of the nitrile group by LiAlH₄

yielded compounds 11 and 13 (Scheme 2).

Headgroups A:

Y

N

Y

N

Y

N

a

b

O

N

NH₂

Figure 1. Structures of known dopamine ligands: 1,4-DAP FAUC 346^[14]

aminoindane FAUC 185^[15]and benzoxazinone derivative^[16]1, where the blue

,

NH

n

O

Y = O, S

2 (Y = O, n = 2)

4 (Y = O, n = 4)

6 (Y = S, n = 2)

8 (Y = S, n = 4)

3 (Y = O, n = 2)

5 (Y = O, n = 4)

7 (Y = S, n = 2)

9 (Y = S, n = 4)

coloured moieties are replaced by

dithienylethene or fulgide.

a

photochromic diarylmaleimide,

Headgroups B:

NH₂

N

n

NH

N

d

e

n

N

Results and Discussion

*oxalate

10 (n = 1)

12 (n = 3)

11 (n = 1)

13 (n = 3)

Due to their thermal stability, we investigated DTEs first. Within

this classes two kind of photoswitches were used to synthesize

photoresponsive dopamine ligands: 1) the cyclopentene based

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

Headgroup C:

O

A stock solution for the biological testing was prepared with

dimethyl sulfoxide as solvent. Dilutions thereof were used to

study the photophysical properties. Even though it is reported

that diarylmaleimides cannot be reversibly toggled between their

HN

BnN

’RN

HO

O

N

BnO

O

N

a

b

’RO

O

N

O

N

NH

N

NH₂

O

14

15

16 (R’ = Bn)

17 (R’ = H)

c

two photoisomers in polar solvents due to

a

twisted

Scheme 2. Synthesis of pharmacophore building blocks, headgroups A, B and

C. Reagents and conditions: a) N-(4-bromobutyl)- or N-(2-bromo-

ethyl)phthalimid, K₂CO₃, KI, MeCN, 75 °C, 5–16h (75–90%); b) Hydrazine

hydrate, EtOH, 75 °C, 2h, then 2 M HCl, EtOH, 75 °C, 2h (60–85%); c) MsOH,

toluene, 100°C, 2h (89%); d) 4-bromobutyronitrile or 2-bromoacetonitrile,

K₂CO₃, KI, MeCN, 75 °C, 16h (88%); e) LiAlH₄, Et₂O, 0 °C  r.t., 1h (71–90%).

intramolecular charge transfer (TICT), we observed a reversible

photoisomerization in dimethyl sulfoxide for all photochromic

ligands based on diarylmaleimides.^[21a-c]

The photophysical properties of the photochromic dopamine

receptor ligands 21-25 and 26-32 were investigated by

absorption spectroscopy. Therefore, the dissolved compounds

were irradiated with UV-light (312 or 400 nm) which resulted in a

color change of the solution accompanied with new absorption

maxima, characteristic for each of the photochromic ligands. The

resulting isosbestic points indicate a clean two-component

switching. In addition, the PSS was determined by HPLC

measurements. All photophysical properties of the compounds

21-25 and 26-32 are summarized in Table 1.

The DTE and diarylmaleic based pharmacophores, containing

the 1,4-DAP, were used with two different spacer lengths.

Depending on the DTEs, two different strategies were applied to

combine photoswitch 18 with headgroups 3, 5, 11, 13, 17 and

cyclopentene-DTE 20 with the pharmacophores 3, 5, 7, 9, 11, 13

and 17. In case of the diarylmaleic anhydride 18, the coupling

was performed with the core of the DTE, while the cyclopentene

based DTE 20 was coupled with one of the thiophene moieties.

Diarylmaleimide 18^[19]was easily accessible according to

literature as well as the precursor 19, which was then

carboxylated on only one thiophene moiety to obtain 20.^[20a-b]

The syntheses of the diarylmaleic anhydride based

photochromic ligands 21-25 were performed under slightly basic

conditions using potassium carbonate as base and are outlined

in Scheme 3.

Table 1. UV-Vis data of the open and closed form of the synthesized

ligands 21-25 and 26-32 (50 or 100 µM in DMSO) after irradiation with λ =

312 or 410 nm.

Entry

Compound

λ_maxopen

λ_maxclosed

Isosbestic

PSS^[a]

[nm]

points [nm]

1

21

22

23

24

25

26

27

28

29

30

31

32

260, 378

261, 390

264, 360

263, 391

261, 385

259

355, 493

356, 509

355, 500

354, 504

354, 500

491

300, 376,

413

305, 377,

413

301, 379,

413

300, 376,

411

46%

38%

49%

52%

72%

83%

73%

72%

77%

71%

76%

61%

2

3

4

5

300, 412,

380

295

6

7

270

490

293

292

291

292

291

300

8

262

491

Scheme 3. Syntheses of the diarylmaleimide based photochromic ligands 21 -

25.

9

259

489

10

11

12

262

494

Standard peptide coupling conditions using HBTU and N,N-

diisopropylethylamine were applied to synthesize the

photochromic ligands 26 - 32 (Scheme 4).

264

490

263

503

[a] Determined by HPLC measurements.

Entries 1-5 represent the photophysical characterization of the

diarylmaleimide based photochromic ligands 21, 22, 23, 24 and

25, whereas entries 6-12 summarize the cyclopentene based

photochromic ligands 26-32. A slightly bathochromic shift is

observed for the new arising absorption maxima corresponding

to the closed photoisomers of the diarylmaleimides 21-25, which

is characteristic for this class of DTEs.^[11a]To our surprise, the

Scheme 4. Syntheses of the cyclopentene-DTE based photochromic ligands

26 - 32.

Following these two synthetic strategies, we were able to readily

obtain various photochromic ligands in just one step.

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

photoconversion of the diarylmaleimides 21, 22, 23 and 24

(excluded 25) is not as efficient as for the cyclopentene ligands

26-32. Compared to a recently reported PSS of 94% for a

diarylmaleimide photoswitch, with a free maleimide core and a

phenyl substitution pattern on the thiophene moieties,^[4a]the

substitution on the nitrogen atom of the maleimide core with the

pharmacophores dramatically decreases the efficacy of the

photoisomerization. In contrast, the cyclopentenes 26-32 exhibit

sufficiently high photoconversions shown by PSS ranging from

61-83%. Surprisingly, the cycle performance exhibits a better

fatigue resistance for the maleimide-based switches 21-25.

Figure 2 exemplary shows the UV-Vis absorption spectra and

the cycle performance for the diarylmaleimide based ligand 23

and for the cyclopentene based ligand 30.

formation, which is related to the substitution pattern of the

DTEs is not yet fully understood.^[22a-c]

Scheme 5. Formation of the irreversible by-product 33 of the ligand 30

(R = 11) upon continuous UV irradiation.

The photoisomerization can be monitored by UV-Vis absorption

spectroscopy and is depicted in Figure SI2a-c. By irradiating the

dissolved compound 30 with λ = 312 nm (Herolab, 6 W) one new

maximum is arising at 494 nm, which is decreasing upon further

UV irradiation.

A new absorption maximum is observed,

hypsochromically shifted to 360 nm, which is attributed to the

irreversible occurring by-product 33. The HPLC traces in Figure

SI2d show that upon reaching the PSS, approximately 55% of

the by-product has already been formed and its formation is

completed after 192 sec. Similar to the results by Hecht et. al.

[22a]

we could confirm the structure of 33 with 2D-NMR

spectroscopy experiments (see SI3, Scheme SI3-1 and SI3-2).

In a second series of dopamine receptor ligands, fulgides were

synthesized, promising reversible switching in aqueous

solution^[12a-b]and good fatigue resistance. The synthesis of the

benzothiophene fulgide 37 was performed according to the

experimental protocol of Stobbe et. al. and Mattay et. al.^[23a-b]

The challenging step, the Stobbe condensation, was

accomplished by enolization of isopropylidenesuccinate and

subsequent reaction with 34. This reaction results in a mixture of

syn/anti lactones 36 whereby the syn-isomer 36 is the favoured

one due to the isopropylgroup of 34. The diacids were obtained

after an elimination reaction and subsequent saponification by

aqueous potassium hydroxide solution. For the anhydride

formation, the crude diacids were treated with acetyl chloride to

form the fulgide 37E (Scheme 6).

Figure 2. Comparison of the photochromic properties of diarylmaleimide

based ligand 23 (a, c) (100 µM in DMSO) with the cyclopentene based

ligand 30 (b, d) (100 µM in DMSO); a) and b) represent the UV-Vis

absorption spectra upon continuous irradiation with light of λ = 312 nm

(Herolab, 6 W), respectively; c) and d) represent the cycle performance

using λ = 312 nm (Herolab, 6 W) for the ring closing and λ = 530 nm

(green LED, 2.5 W) for the ring opening reaction, respectively:

absorption spectra of ligand 23 ) UV-Vis absorption spectra of ligand

30; arrows indicate the characteristic changes in the absorption spectra;

) repetitive switching cycles at 500 nm of ligand 23 ) repetitive

switching cycles at 494 nm of ligand 30

a) UV-Vis

;

b

c

; d

.

The arrows indicate the characteristic changes in the spectra

upon continuous irradiation with light of λ = 312 nm (Herolab,

6 W), accompanied by a characteristic color change of the

samples. The cycle performance showed that ligand 23 is stable

over at least six switching cycles, whereas a fast degradation of

cyclopentene 30 was observed.

Further investigations by continuous irradiation with UV light

(λ = 312 nm) revealed the formation of an irreversible by-product

33 (Scheme 5). The phenomenon of this annulated isomer has

been reported previously, but the mechanism of the by-product

Scheme 6. Benzothiophene fulgide 37 synthesis according Stobbe et. al.;

Reagents and conditions: a) Isopropylidenesuccinate, LDA, THF, -78 °C  r.t.,

48h (16%); b) Step 1, KOH, EtOH, H₂O, 70 °C, 24h; Step 2, AcCl, CH₂Cl₂,

40 °C, 20h (87%).

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

The indolyl-isopropyl fulgide 38, 5-methylthiophene-isopropyl

fulgide 39, benzothiophenemethyl fulgide 40 and indolylmethyl

fulgide 41 were synthesized according to literature.^[24-26]Only the

E-isomers were isolated and used for fulgimide formation.

The target fulgimides were prepared in a two-step reaction. First,

the succinamic acids 42, 44, 47, 49 and 51 were obtained by

heating the amines 7, 11 and 17 with the fulgides 37 - 41 in

chloroform or DMF. Second the imides were formed by an active

ester method with DCC, HOBt and DIPEA or MSNT and Me-

imidazole to obtain the desired fulgimides 43, 45, 46, 48, 50 and

52 (Scheme 7).

Table 2. Photochemical properties of fulgimides 43-52.

Entry

Com-

pound

λ_max

open

[nm]

λ_max

closed

[nm]

Isosbestic

points

[nm]

PSS^[a]

Z:E:C

PSS^[b]

Z:E

1

2

3

4

5

6

7

8

9

43^[c]

45^[c]

45^[d]

46^[c]

48^[c]

50^[c]

50^[d]

52^[c]

52^[d]

307

360

356

272

328

370

363

473

543

525

532

486

564

290, 342,

347

310, 344,

420

305, 337,

417

360

0:30:70

0:20:80

0:19:81

0:16:84

19:36:45

6:16:78

4:18:78

4:12:84

4:10:86

-

315, 357

306, 436

475

19:81

6:94

4:96

325,

546

563

302, 325,

430

300, 320,

409

279,

366

300,

545

[a] Closing of the open E-isomers with UV light, PSS determined by HPLC

measurements. [b] Opening of the closed mixture with visible light, PSS

determined by HPLC measurements. [c] Solvent: DMSO. [d] Solvent: Tris-

buffer + 10% DMSO.

Scheme 7. Fulgimide synthesis with fulgide 37 (Ar = benzothiophene, R³

isopropyl), 38 (Ar = indole, R³= isopropyl), 39 (Ar = 5-methyl-thiophene, R³

isopropyl), 40 (Ar = benzothiophene, R³= methyl), 41 (Ar = indole, R³

=

The UV/Vis absorption spectra changes are exemplarily shown

for compound 45 in Figure 3 upon irradiation with 410 nm light

in periods of 2 s. New absorption bands arise at 543 nm and 330

nm, the band at 360 nm decreases and belongs to the open

isomer. The opening of the closed photoisomers was

accomplished by irradiation with light of 505 or 530 nm (1-2 min)

for 43, 45, 46, 48, 50 and 52.

methyl) and amines 7, 11, 17. Reaction conditions: a) amine A, B or C, CHCl₃

or DMF, 60 °C, 4-48h; b) DCC, HOBt, DIPEA or MSNT, Me-imidazole, CHCl₃

or DMF, 25 °C, 24-48h.

The fulgimide 46 was formed after heating in CHCl₃for 24 h

without adding coupling reagents. No fulgimide could be

obtained with amine 9 and 13.

Applications of photoswitches in biological assays require their

solubility in aqueous buffer. Hence, the photoisomerization of

the fulgimides 45, 50 and 52 were carried out in polar solvents to

obtain UV/Vis absorption spectra. Table 2 summarizes the

photostationary states (PSS), isosbestic points as well as the

absorption maxima of 43 and 45, determined in DMSO. The

method for the PSS determination by HPLC is exemplarily

shown for compound 48 in the SI4. The indolyl fulgimides 45,

50 and 52 were also measured in a 10% DMSO – tris-buffer

solution (pH = 7.4) (Table 2, Entry 3,7 and 9), whereby a

hypsochromic shift of 18 nm of the absorption maximum in the

visible light range was observed.

Figure 3. Left) the photochromic properties of a solution of 45 (50 µM in

DMSO). The slightly yellow open form turned purple after 12 s irradiation with

410 nm. The absorption spectra showed a new absorption band at 543 nm.

Right) Repetitive switching of 45 is depicted on the right side.

The cycle performances of compounds 43, 45, 46, 48, 50 and 52

were studied by alternating irradiation at the appropriate

wavelengths (43  312_closenm, 505_opennm; 45  400_closenm,

530_opennm; 46, 48  365_closenm, 530_opennm; 50, 52  400_close

nm, 530_opennm). The compounds showed good fatigue

resistance and only little degradation was observed. Exemplarily,

the cycle performance of compound 45 is depicted in Figure 3.

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

The biological investigation of all test compounds was targeted

towards their ability to activate the dopamine D_2Sreceptor. Our

studies were directed to the identification of compound pairs

exhibiting different activation profiles for the open and the closed

photostationary state. Therefore, we applied screening assays

for G-protein mediated signaling and arrestin recruitment at a

fixed concentration (10 µM). The G-protein pathway was

investigated utilizing the IP-One^®assay (Cisbio) with HEK 293T

cells transiently co-expressing D_2Sand the hybrid G-protein

G_qi5HAwhile -arrestin-2 recruitment was determined applying

the PathHunther^®assay (DiscoverX). All activation data are

summarized in Table 3, SI5 and SI6 in comparison to the

reference ligand quinpirole. In the arrestin assay, the

cyclopentene DTEs 26-30, 32, the diarylmaleimides 21-23, 25

and the fulgimides 43, 45, 46, 48, 50 and 52 showed E_maxvalues

less than 15%. Only DTE 31 and the maleimide 24 both bearing

the indanylamine moiety and a 4-atom spacer between the

pharmacophoric headgroup and the photochromic entity (13)

showed E_maxvalues of 70% (31-open), 76% (31-closed), 33%

(24-open), and 37% (24-closed), respectively. Additional

measurement of arrestin recruitment for those compounds at

100 nM revealed E_maxvalues less than 10% and no differences

in the activation pattern of the open and closed conformation.

Whereas weak arrestin recruitment was observed, the

determination of the G-protein mediated signaling revealed

agonist properties with efficacies in the range of 40 to 95% for all

test compounds (Figure SI5, Table SI5-1, SI5-2). More detailed

measurements at 100 nM and 1 nM showed a dose-dependent

range of efficacies allowing a precise evaluation of the open and

closed conformations of each photochromic ligand. Within the

series of maleimides, the indanylamine 24 showed the highest

E_maxvalues of 95% for both conformers, which is similar to the

rank order of agonist effects derived from the arrestin assay, but

without any difference between the open and the closed form.

This observation can be confirmed by the results for the

cylopentene DTEs, where 31 (indanylamine with headgroup 13)

showed an E_maxvalue of 95% for the open and the closed

conformation, respectively. Clear differences in E_maxbetween

both photochromic states could be observed for the methyl ether

substituted phenylpiperazines 27 and 29 both carrying a 4-

carbon spacer and only differing in the heteroatom of the ether

group (O for 27, S for 29). For 29, E_maxvalues of 77% (10 µM),

80% (100 nM) and 32% (1 nM) have been determined for the

open state, while efficacies of 70% (10 µM), 47% (100 nM) and

3% (1 nM) could be measured for the closed form (Table 3,

Figure 4). This data indicate a 11-fold more efficient receptor

activation by the open conformer compared to the closed

derivative at low concentration (1 nM). In contrast, the

methoxyphenylpiperazine 27 showed stronger receptor

activation profile in the closed state compared to the open

derivative, when a 3-fold increase of efficacy was determined for

27-closed at 100 nM (closed-open: 49%-16% at 100 nM, 15%-

11% at 1 nM). A similar selectivity profile could be detected for

the

indolyl

fulgimides

45

and

52.

The

methylthiophenylpiperazine 45-closed showed a 2-fold increase

of efficacy at 100 nM compared to the open state, while the

benzoxazinone 52-closed revealed a 4-fold improve of activity at

1 nM (for 45: closed-open: 45%-25% at 100 nM, 8%-5% at 1 nM,

for 52: closed-open: 47%-48% at 100 nM, 40%-10% at 1 nM).

To learn more about the mechanistic relations of these selective

photoswitchable ligands, we investigated the binding affinities of

the most promising compounds 27-open, 27-closed, 29-open,

29-closed, 45-open, 45-closed, and 52-(E)-open, 52-closed at

the dopamine D_2Sreceptor in a competition binding experiment.

As all compounds displayed K_ivalues between 9 and 17 nM

(Table SI6), the state-selective intrinsic activity appears to be

controlled by the individual ability to preferentially stabilize the

active state of the receptor rather than by different binding

affinities.

Table 3. Functional screening of the most promising photoactive ligands 27,

29, 45 and 52 for G-protein mediated activation of the dopamine D_2Sreceptor

applying an IP accumulation assay^[a]

.

E

_max[% ± SEM]^[b]

100 nM

photo-

comp.

active state

10 µM

1 nM

27

29

open

closed

open

85 ± 2.6

67 ± 7.4

77 ± 3.8

70 ± 6.8

68 ± 2.8

62 ± 2.4

58 ± 6.9

69 ± 6.6

16 ± 9.6

49 ± 2.0

80 ± 2.8

47 ± 5.2

25 ± 5.3

45 ± 7.8

48 ± 8.5

47 ± 4.2

11 ± 9.8

15 ± 0.9

32 ± 4.6

2.9 ± 3.2

5.6 ± 3.2

7.7 ± 2.6

10 ± 3.8

40 ± 4.6

closed

open

45

closed

open

52^[c]

closed

[a] IP accumulation determined by applying the IP-One^®assay (from Cisbio)

with HEK 239T cells co-transfected with the cDNA of the dopamine D_2S

receptor and that of the hybrid G-protein Gqi5HA. [b] Emax value ± S.E.M.

derived from 4 to 8 individual experiments each done in quadruplicate relative

to the maximum effect of quinpirole. [c] The open isomer describes only the E-

isomer.

An overall analysis of the data confers that the photoswitches 29

and 52 are most promising biochemical tools for the regulation

of the pharmacologically important dopamine D_2Sreceptor. The

potency of the more active isomers is comparable to the

activation power of the reference dopaminergic agent quinpirole.

The compounds require a very low dose of 1nM to exhibit a

degree of receptor activation that strongly depends on the

photostationary state of the ligand. Hence, the presence of the

cyclopentene-DTE based photochromic ligands 29 in the open

state induces an 11-fold higher G protein-promoted signaling

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

than the existence of 29-open in the same concentration. As a

complementary pair of photochromic ligands, the fulgimide 52

shows four-fold higher D_2Sreceptor activation in the closed state

compared to 52-(E)-open (Figure 4).

reversible, light-induced control of photochromic ligands when

bound to the receptor are in progress.

Experimental Section

Starting materials were purchased from commercial suppliers and used

without any further purification. Solvents were used in p.a. quality and

dried according to common procedures, if necessary. Dry nitrogen was

used as inert gas atmosphere. Thin-layer chromatography (TLC) for

reaction monitoring was performed with alumina plates coated with

Merck silica gel 60 F₂₅₄(layer thickness: 0.2 mm) and analyzed under

UV-light (254 nm). Flash column chromatography was performed with

Sigma Aldrich MN silica gel 60M (0.040-0.063 mm, 230-400 mesh) as

stationary phase or a reversed phase column (KP-C18-HS) on a Biotage

Isolera One automated flash purification system with UV-Vis detector.

NMR spectra were recorded using a Bruker Avance 300 (¹H: 300 MHz,

¹³C: 75 MHz, T = 295 K), Bruker Avance 360 (¹H: 360 MHz, ¹³C: 91 MHz,

T = 295 K), Bruker Avance 400 (¹H 400.1: MHz, ¹³C: 100.6 MHz, T =

300K) or a Bruker Avance 600 (¹H: 600 MHz, ¹³C: 151 MHz, T = 295 K)

instrument. The spectra are referenced against the NMR solvent and are

reported as follows: ¹H: chemical shift δ (ppm), multiplicity, integration,

coupling constant (J in Hz). ¹³C: chemical shift δ (ppm), abbreviations: (+)

Figure 4. Activation of G-protein mediated receptor signaling determined by

an IP accumulation assay using D₂s and the G-protein hybrid Gqi5HA.

Accumulation of IP for the most promising photoswitchable ligands 27 and 52

in comparison to the reference quinpirole all determined at 1 nM. The DTE 29

shows an 11-fold improved activity for the open state (dark blue) than the

closed state (light blue). In contrary, fulgimide 52 is 4-fold less active in the

open conformation than for the closed one. For comparison, receptor

activation induced by 1 nM of quinpirole (striped) reveals an efficacy of 28 %

(derived from the dose-response curve, Figure SI4). Bars represent average

efficacy [%±S.E.M.] derived from 4 to 8 individual experiments each done in

quadruplicates.

=

primary/tertiary, (−) = secondary, (q) = quaternary carbon. The

assignment resulted from DEPT, COSY, HMBC and HSQC experiments.

Mass spectra were measured with a Finnigan MAT 95, Finnigan MAT

SSQ 710 A, ThermoQuest Finnigan TSQ 7000 or an Agilent Q-TOF 6540

UHD instrument. Standard hand-held lamps (Herolab, 312 nm, 6 W) and

LEDs with 365 nm (3.2 W) 400 nm (2.5 W) were used for visualizing TLC

and to perform the ring closure reactions. The ring opening reactions

were performed using LEDs with 505 nm (2.5 W) and 530 nm (2.5 W)

emission maximum. Absorption spectra were recorded on a Varian Cary

50 Bio UV/Vis spectrophotometer. Preparative HPLC was performed on

a Knauer HPLC (column: Luna 10 250 x 21 mm; flow: 3 mL/min, solvent

A: H₂O [0.1 Vol% TFA], solvent B: MeCN  prep-HPLC-1) and on an

Agilent1100 Series (Column: Phenomenex Luna 10, C18, 100A, 250 x

21.2 mm, flow 20 mL/min, solvent A: H₂O [0.05 Vol% TFA], solvent B:

MeCN  prep-HPLC-2). Photostationary states of the final compounds

were measured on an Agilent 1290 Series HPLC (Column: Phenomenex

Luna C18, 3 µm, 150 x 2.00 mm, 100 Å; flow: 0.3 mL/min) and Agilent

1220 Infinity LC System (column: Phenomenex Luna, 3µ C18(2) 100A,

150 x 2.0 mm, 100 Å, 40 °C). All biological investigations were performed

in the group of Prof. Dr. P. Gmeiner by Dr. H. Hübner (University of

Erlangen-Nürnberg).

Conclusions

We succeeded in synthesizing DTE and fulgide based

photochromic dopamine receptor ligands by modifying the

structure of known pharmacologically active compounds with

photochromic motifs. The maleimides could not be switched in

aqueous solution and the cyclopentene-DTEs had a low fatigue

resistance, whereas the fulgimides showed excellent

photophysical properties in aqueous solution. At a concentration

of 1 nM, the cyclopentene-DTE 29-open showed a more than

11-fold higher activation of D_2S, a pharmacologically important G

protein-coupled receptor, than its photochromic congener 29-

closed. Interestingly, the fulgimide-based pair 52-(E)-open/52-

closed could be discovered as an alternative photoswitch with

inverse activation properties exhibiting four-fold higher activity in

the closed state. Further studies on the optimization of GPCR-

regulating photoswitches and biological investigations including

NMR-spectra are available in the supporting informations (SI7).

4-(2-Methoxyphenyl)-1-[2-(N-phthalimido)ethyl]piperazine (2)

1-(2-Methoxyphenyl)piperazine (673 mg, 3.5 mmol, 1.0 eq) and N-(2-

bromoethyl)phthalimide (1.07 g, 4.2 mmol, 1.2 eq) were dissolved in

MeCN (12 mL). K₂CO₃(725 mg, 5.2 mmol, 1.5 eq) and a catalytic

amount of KI were added and the reaction mixture was stirred under

reflux for 5 h. After cooling down to room temperature, the precipitate

was filtered off and washed with acetonitrile. The solvent was evaporated

and the crude product was purified via column chromatography

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

(CH₂Cl₂/MeOH 100:1) to yield 2 (914 mg, 71%) as a colorless solid.

Analytical data as described in literature.^[16]

chromatography (CH₂Cl₂/MeOH 40:1  12:1) to yield 3 (178 mg, 73%)

as a colorless solid. Analytical data as described in literature. ^[16]

4-(2-Methoxyphenyl)-1-[4-(N-phthalimido)butyl]piperazine (4)

Compound 4 was prepared as described for 2, using a solution of 1-(2-

methoxyphenyl)piperazine (600 mg, 3.1 mmol, 1.0 eq), N-(4-

bromobutyl)phthalimide (967 mg, 3.4 mmol, 1.1 eq), K₂CO₃(646 mg, 4.7

mmol, 1.5 eq) and a catalytic amount of KI in MeCN (10 mL), allowing a

reaction time of 16h. Purification of the crude product by column

chromatography yielded 4 (1.1 g, 88%) as yellow oil. Analytical data as

described in literature. ^[16]

1-(4-Aminobutyl)-4-(2-methoxyphenyl)piperazine (5)

Compound 5 was prepared as described for 3, using a solution of 4-(2-

methoxyphenyl)-1-[4-(N-phthalimido)butyl]piperazine

(4)

(260 mg,

0.66 mmol, 1.0 eq), 80% hydrazine hydrate solution (62 µL, 0.99 mmol,

1.5 eq) in EtOH (1.3 mL) and HCl (2 M, 0.7 mL). Purification of the crude

product by flash chromatography yielded 5 (137 mg, 79%) as a light

yellow oil. Analytical data as described in literature. ^[16]

1-(2-Aminoethyl)-4-(2-methylthiophenyl)piperazine (7)

4-(2-Methylthiophenyl)-1-[2-(N-phthalimido)ethyl]piperazine (6)

Compound 7 was prepared as described for 3, using a solution of 4-(2-

Compound 6 was prepared as described for 2, using a solution of 1-(2-

(methylthio)-phenyl)piperazine (650 mg, 3.1 mmol, 1.0 eq), N-(2-

bromoethyl)phthalimide (871 mg, 3.4 mmol, 1.1 eq), K₂CO₃(646 mg,

4.7 mmol, 1.5 eq) and a catalytic amount of KI in MeCN (10 mL), allowing

a reaction time of 16h. Purification of the crude product by column

chromatography yielded 6 (890 mg, 75%) as a yellow solid. ¹H NMR (360

MHz, CDCl₃): δ = 2.40 (s, 3H), 2.59 – 2.85 (m, 6H), 2.86 – 3.05 (m, 4H),

3.86 (t, J = 6.6 Hz, 2H), 6.98 – 7.04 (m, 1H), 7.05 – 7.15 (m, 3H), 7.71

(dd, J = 5.4, 3.1 Hz, 2H), 7.85 (dd, J = 5.5, 3.0 Hz, 2H); ¹³C NMR (91

MHz , CDCl₃): δ = 14.57 (+), 35.53 (−), 51.76 (−), 53.63 (−), 55.96 (−),

119.8 (+), 123.3 (+), 124.4 (+), 125.0 (+), 132.4 (q), 134.0 (+), 135.0 (q),

149.7 (q), 168.5 (q); MS (ESI, [M+H]⁺): m/z = 381.9.

methylthiophenyl)-1-[2-(N-phthalimido)ethyl]piperazine

(6)

(400 mg,

1.0 mmol, 1.0 eq), 80% hydrazine hydrate solution (0.1 mL, 1.6 mmol,

1.6 eq) in EtOH (2.1 mL) and HCl (2 M, 1.2 mL). Purification of the crude

product by flash chromatography yielded 7 (233 mg, 82%) as a yellow oil.

¹H NMR (360 MHz, CDCl₃): δ = 2.41 (s, 3H), 2.51 (t, J = 6.2 Hz, 2H),

2.65 (m, 4H), 2.84 (t, J = 6.2 Hz, 2H), 3.08 – 2.97 (m, 4H), 7.16 – 7.02 (m,

4H); ¹³C NMR (151 MHz, CDCl₃): δ = 14.56 (+), 39.05 (−), 51.83 (−),

53.82 (−), 61.36 (−), 119.7 (+), 124.4 (+), 124.5 (+), 125.0 (+), 135.1 (q),

149.7 (q); MS (ESI, [M+H]⁺): m/z = 251.9.

1-(4-Aminobutyl)-4-(2-methylthiophenyl)piperazine (9)

Compound 9 was prepared as described for 3, using a solution of 4-(2-

methoxyphenyl)-1-[4-(N-phthalimido)butyl]piperazine

(8)

(400 mg,

4-(2-Methoxyphenyl)-1-[4-(N-phthalimido)butyl]piperazine (8)

0.98 mmol, 1.0 eq), 80% hydrazine hydrate solution (0.1 mL, 1.5 mmol,

1.5 eq) in EtOH (2 mL). Purification of the crude product by flash

chromatography yielded 9 (156 mg, 57%) as a yellow oil. ¹H NMR (360

MHz, CDCl₃): δ = 1.42 – 1.69 (m, 4H), 2.41 (s, 3H), 2.40 – 2.47 (m, 2H),

2.58 – 2.69 (s, 4H), 2.69 – 2.89 (m, 2H), 2.93 – 3.15 (m, 4H), 7.02 – 7.18

(m, 4H); ¹³C NMR (151 MHz, CDCl₃): δ = 14.56 (+), 24.55 (−), 31.92 (−),

42.22 (−), 51.77 (−), 53.77 (−), 58.72 (−), 119.8 (+), 124.3 (+), 124.5 (+),

125.1 (+), 135.1 (q), 149.7 (q); MS (ESI, [M+H]⁺): m/z = 280.17.

Compound 8 was prepared as described for 4, using a solution of 1-(2-

(methylthio)phenyl)piperazine (650 mg, 3.1 mmol, 1.0 eq), N-(4-

bromobutyl)phthalimide (967 mg, 3.4 mmol, 1.1 eq), K₂CO₃(646 mg,

4.7 mmol, 1.5) and a catalytic amount of KI in MeCN (10 mL), allowing, a

reaction time of 16h. Purification of the crude product by column

1

chromatography yielded 8 (1.15 g, 90%) as a yellow solid. H NMR (360

MHz, CDCl₃): δ = 1.53 – 1.68 (m, 2H), 1.68 – 1.90 (m, 2H), 2.40 (s, 3H),

2.44 – 2.57 (m, 2H), 2.62 – 2.80 (m, 4H), 2.91 – 3.15 (m, 4H), 3.73 (t, J =

7.0 Hz, 2H), 7.01 – 7.07 (m, 1H), 7.07 – 7.14 (m, 3H), 7.64 – 7.78 (m,

2H), 7.78 – 7.93 (m, 2H); ¹³C NMR (91 MHz, CDCl₃): δ = 14.55 (+), 24.25

(−), 26.77 (−), 37.98 (−), 51.57 (−), 53.71 (−), 58.18 (−), 119.8 (+), 123.3

(+),

4-[4-Benzyl-5-(benzyloxy)-2H-benzo[b][1,4]oxazin-3(4H)-one-8-yl]-1-[2-

(N-phthalimido)ethyl] piperazine (15)

Compound 15 was prepared as described for 2, using a solution of 4-[4-

benzyl-5-(benzyloxy)-2H-benzo[b][1,4]oxazin-3(4H)-one-8-yl]piperazine

(14) (339 mg, 0.79 mmol, 1.0 eq) and N-(2-bromoethyl) phthalimide

(220 mg, 0.89 mmol, 1.1 eq) , K₂CO₃(163 g, 1.18 mmol, 1.5 eq) and a

catalytic amount of KI in MeCN (8 mL). Purification by flash

chromatography (CH₂Cl₂/MeOH 50:1) to yielded 15 (318 mg, 67 %) as a

colorless solid. ¹H NMR (600 MHz, CDCl₃): δ = 2.58 – 2.80 (m, 6H), 2.84

– 3.02 (m, 4H), 3.85 (t, J = 6.3 Hz, 2H), 4.56 (s, 2H), 4.90 (s, 2H), 5.38 (s,

2H), 6.50 (d, J = 9.0 Hz, 1H), 6.56 (d, J = 9.0 Hz, 1H), 7.08 – 7.17 (m,

3H), 7.28 – 7.32 (m, 2H), 7.32 – 7.39 (m, 3H), 7.69 – 7.73 (m, 2H), 7.82 –

7.89 (m, 2H). ¹³C NMR (91 MHz , CDCl₃): δ = 35.39 (−), 46.84 (−), 51.17

(−), 53.27 (−), 55.87 (−), 68.91 (−), 71.61 (−), 107.8 (+), 114.2 (+), 120.1

(q), 123.3 (+), 123.5 (q), 126.9 (+), 127.0 (+), 127.7 (+), 128.3 (+), 128.3

(+), 128.8 (+), 132.4 (q), 134.0 (+), 134.1 (q), 136.5 (q), 137.4 (q), 142.7

(q), 145.1 (q), 166.7 (q), 168.5 (q); MS (ESI, [M+H]⁺): m/z = 603.2.

124.4 (+), 124.6 (+), 125.1 (+), 132.3 (q), 134.0 (+), 135.1 (q), 149.6 (q),

168.6 (q); MS (ESI, [M+H]⁺): m/z = 410.3.

1-(2-Aminoethyl)-4-(2-methoxyphenyl)piperazine (3)

4-(2-Methoxyphenyl)-1-[2-(N-phthalimido)ethyl]piperazine (2) (350 mg,

0.96 mmol, 1.0 eq) was dissolved in EtOH (2.0 mL) and 80% hydrazine

hydrate solution (90 µL, 1.4 mmol, 1.5 eq) was added. The reaction

mixture was stirred under reflux for 2 h, then HCl (2 M, 1.1 mL) was

added and stirring was continued under reflux for two hours. After cooling

down to room temperature, the precipitate was filtered off. The solvent

was evaporated and the residue was dissolved in H₂O. The pH was

adjusted to 8 with NaOH (1 M) and the product was extracted with

CH₂Cl₂. The combined organic layers were dried over Na₂SO₄and the

solvent was evaporated. The crude product was purified by flash

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

1-(2-Aminoethyl)-4-[4-benzyl-5-(benzyloxy)-2H-benzo[b][1,4]oxazin-

3(4H)-one-8-yl]pipera-zine (16)

2.1 mmol, 5.5 eq), KI (63 mg, 0.38 mmol, 1.0 eq) and 4-

bromobutyronitrile (90 µL, 0.91 mmol, 2.4 eq) in MeCN (2.5 mL).

Purification of the crude product by flash chromatography (PE/EtOAc 9:1)

yielded 12 (81 mg, 88%) as a brownish oil. Analytical data as described

in literature.²

Compound 16 was prepared as described for 3, using a solution of 4-[4-

benzyl-5-(benzyloxy)-2H-benzo[b][1,4]oxazin-3(4H)-one-8-yl]-1-[2-(N-

phthalimido)ethyl] piperazine (15) (310 mg, 0.51 mmol, 1.0 eq) and

80% hydrazine hydrate solution (50 μL, 0.77 mmol, 1.5 eq) in EtOH

(5 mL). Purification by flash chromatography (CH₂Cl₂/MeOH 20:1, 0.1%

2-[(N-Indan-2-yl)(propyl)amino]ethane-1-amine (11)

1

NH₃) yielded 16 (206 mg, 85%) as a colorless solid. H NMR (600 MHz,

2-[(N-Indan-2-yl)(propyl)amino]acetonitrile (10) (149 mg, 0.69 mmol, 1.0

eq) was dissolved in Et₂O (7 mL) and cooled to 0°C. LiAlH₄(0.35 mL,

1.4 mmol, 2.0 eq) was added dropwise and the mixture was stirred at

room temperature for 1 h. Saturated NaHCO₃was added at 0°C. The

precipitate was filtered off and the product was extracted with CH₂Cl₂.

The organic layers were dried over Mg₂SO₄and evaporated. Purification

of the crude product by flash chromatography (CH₂Cl₂/MeOH 15:1, 0.1%

NH₃) yielded 11 (107 mg, 71%) as a colorless oil. ¹H NMR (600 MHz,

CDCl₃): δ = 0.89 (t, J = 7.4 Hz, 3H), 1.46 – 1.56 (m, 2H), 2.47 – 2.56 (m,

2H), 2.60 (t, J = 6.1 Hz, 2H), 2.80 (m, 2H), 2.90 (dd, J = 15.5, 8.3 Hz, 2H),

3.03 (dd, J = 15.5, 7.9 Hz, 2H), 3.74 (p, J = 8.1 Hz, 1H), 7.10 – 7.15 (m,

2H), 7.17 (dt, J = 7.2, 3.6 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃): δ = 12.04

(+), 20.79 (−), 36.36 (−), 40.15 (−), 53.92 (−), 53.96 (−), 62.94 (+), 124.6

(+), 126.4 (+), 142.0 (q); MS (ESI, [M+H]⁺): m/z = 219.0.

DMSO-d): δ = 2.51 – 2.60 (m, 6H), 2.81 – 2.95 (m, 6H), 4.60 (s, 2H),

5.02 (s, 2H), 5.29 (s, 2H), 6.63 (d, J = 9.1 Hz, 1H), 6.71 (d, J = 9.1 Hz,

1H), 7.00 (d, J = 7.2 Hz, 2H), 7.15 (t, J = 7.3 Hz, 1H), 7.21 (t, J = 7.4 Hz,

2H), 7.44 – 7.31 (m, 5H); ¹³C NMR (91 MHz, DMSO-d): δ = 35.84 (−),

45.63 (−), 50.32 (−), 52.60 (−), 54.68 (−), 68.22 (−), 70.65 (−), 107.9 (+),

113.9 (+), 119.5 (q), 126.4 (+), 126.8 (+), 127.7 (+), 127.9 (+), 128.2 (+),

128.5 (+), 135.5 (q), 136.5 (q), 137.4 (q), 142.1 (q), 144.2 (q), 166.3 (q);

MS (ESI, [M+H]⁺): m/z = 473.3.

1-(2-Aminoethyl)-4-(5-Hydroxy-2H-benzo[b][1,4]oxazin-3(4H)-one-8-

yl)piperazine (17)

1-(2-Aminoethyl)-4-[4-benzyl-5-(benzyloxy)-2H-benzo[b][1,4]oxazin-

3(4H)-one-8-yl]piperaz-ine (16) (120 mg, 0.25 mmol, 1.0 eq) was

suspended in toluene (3.6 mL) and methansulfonic acid (0.2 mL,

2.5 mmol, 10 eq) was added. The reaction mixture was stirred under

reflux for 3 h. After cooling down to room temperature, the solvent was

removed. Purification of the crude product by preparative flash

chromatography (CH₂Cl₂/MeOH 12:1, 0.5% NH₃) yielded 17 (65 mg,

4-[(N-Indan-2-yl)(propyl)amino]butane-1-amine (13)

Compound 13 was prepared as described for 11, using a solution of 4-

[(N-indan-2-yl)(propyl) amino]butyronitrile (30 mg, 0.12 mmol, 1.0 eq) and

LiAlH₄(75 µL, 0.30 mmol, 2.5 eq) in Et₂O (0.85 mL). Purification of the

crude product by flash chromatography (CH₂Cl₂/MeOH 15:1, 0.1% NH₃)

yielded 13 (18 mg, 57%) as a colorless oil. Analytical data as described

in literature. ^[18]

1

89%) as a colorless solid. H NMR (600 MHz, DMSO-d): δ = 2.55 – 2.71

(m, 4H), 2.81 – 2.93 (m, 4H), 2.93 – 3.06 (m, 2H), 3.10 – 3.24 (m, 2H),

4.48 (s, 2H), 6.44 (s, 2H), 9.78 (s, 1H), 9.48 (s, 1H); ¹³C NMR (101 MHz,

DMSO-d): δ = 33.5 (−), 47.9 (−), 51.9 (−), 52.6 (−), 66.9 (−), 108.6 (+),

112.8 (+), 116.7 (q), 131.1 (q), 137.6 (q), 141.7 (q), 164.4 (q); MS (ESI,

[M+H]⁺): m/z = 293.1.

General preparation of diarylmaleimide based switches 21-24:

To a suspension of 18 (61 mg, 0.17 mmol, 1.0 eq) and K₂CO₃(23 mg,

0.17 mmol, 1.0 eq) in CH₂Cl₂(1.5 mL) was added an amine of A or B

(0.17 mmol, 1.0 eq). After stirring for 0.25 - 3 h at r.t., water (1 mL) was

added and the aqueous phase was extracted with CH₂Cl₂(2 x 4 mL). The

combined organic phases were dried over MgSO₄, filtered and the

solvent was removed under reduced pressure. The crude product was

first purified by automated flash column chromatography and second by

preparative HPLC.

2-[(N-Indan-2-yl)(propyl)amino]acetonitrile (10)

2-Propylaminoindane oxalate (210 mg, 0.82 mmol, 1.0 eq), K₂CO₃

(634 mg, 4.6 mmol, 5.6 eq) and KI (136 mg, 0.82 mmol, 1.0 eq) were

suspended in MeCN (5.5 mL) and stirred for 5 min. Chloroacetonitrile

(0.13 mL, 2.0 mmol, 2.4 eq) was added dropwise and the mixture was

stirred under reflux for 2 h. After cooling down to room temperature, the

solvent was removed under reduced pressure. Water was added to the

residue and the pH was adjusted to 7 with NaOH (1 M). The product was

extracted with DCM. The organic layers were dried over Na₂SO₄and

evaporated. Purification of the crude product by flash chromatography

(PE/EtOAc 10:1) yielded 10 (153 mg, 89%) as a yellow oil. ¹H NMR (600

MHz, CDCl₃): δ = 0.95 (t, J = 7.4 Hz, 3H), 1.53 (dq, J = 14.7, 7.3 Hz, 2H),

2.56 – 2.67 (m, 2H), 2.88 (dd, J = 14.8, 9.2 Hz, 2H), 3.14 (dd, J = 14.7,

7.4 Hz, 2H), 3.47 – 3.59 (m, 1H), 3.67 (s, 2H), 7.11 – 7.17 (m, 2H), 7.17

– 7.22 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ = 11.83 (+), 20.57 (−),

37.89 (C1, C3), 40.24 (−), 53.57 (−), 64.30 (+), 115.5 (q), 124.6 (+),

126.8 (+), 141.0 (q); MS (ESI, [M+H]⁺): m/z = 215.0.

3,4-Bis(5-chloro-2-methylthiophen-3-yl)-1-(2-(4-(2-methoxyphenyl)-

piperazin-1-yl)ethyl)-1H-pyrrole-2,5-dione (21)

Yellow solid: 68% yield; reaction time: 2 h; flash chromatography:

CH₂Cl₂/MeOH: 0-5% MeOH; preparative HPLC (prep-HPLC-1, gradient

A/B: 0-25 min: 80/20, 25-35 min: 5/95), t_R= 19.6 min; R_f(CH₂Cl₂/MeOH:

10/1): 0.10; ¹H NMR (600 MHz, CDCl₃) δ = 1.98 (s, 6H), 3.08-3.20 (m,

2H), 3.30-3.40 (m, 2H), 3.43 (t, 2H, J = 5.7 Hz), 3.46-3.57 (m, 2H), 3.87

(s, 3H), 3.92-4.02 (m, 2H), 4.05 (t, 2H, J = 5.7 Hz), 6.88- 6.90 (m, 3H),

6.92- 6.98 (m, 2H), 7.07-7.10 (m, 1H); ¹³C NMR (151 MHz, CDCl₃)

δ = 14.8 (+), 32.5 (−), 47.4 (−), 52.3 (−), 55.0 (−), 55.5 (+), 111.4 (+),

119.1 (+), 121.3 (+), 124.9 (+), 125.7 (q), 127.0 (q), 127.3 (+), 133.1 (q),

138.2 (q), 140.9 (q), 152.0 (q), 169.8 (q). HR-MS (ESI, [M+H]⁺): calcd. for

C₂₇H₂₈Cl₂N₃O₃S₂: 576.0944, found 576.0945; UV/Vis (50 µM in DMSO):

4-[(N-Indan-2-yl)(propyl)amino]butyronitrile (12)

Compound 12 was prepared as described for 10, using a solution of 2-

propylaminoindane oxalate (80 mg, 0.38 mmol, 1.0 eq), K₂CO₃(294 mg,

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

open isomer: λ_max= 261 nm, 390 nm; closed isomer: λ_max= 356 nm,

509 nm.

Dithienylmaleimide 18 (50.0 mg, 0.14 mmol, 1 eq) was dissolved in DMF

(5 mL). Than K₂CO₃(19.1 mg, 0.14 mmol, 1eq) and amine 17 (40.5 mg,

0.14 mmol, 1 eq) were added and the mixture was stirred for 24 h at

room temperature. Water (5 mL) was added and the aqueous layer was

extracted with CH₂Cl₂(3x 50 mL). The combined organic layers were

dried over Na₂SO₄and the solvent was reduced in vacuo. The crude

product was first purified by automated flash chromatography

(CH₂Cl₂/MeOH: 0-10% MeOH) and second purified by preparative HPLC

(prep-HPLC-2, gradient A/B: 0-15 min: 30/70, 15-20 min: 2/98, t_R= 8.2

min) to obtain 25 (6.0 mg, 9.5 µmol, 9%) as a light yellow solid. R_f

(CH₂Cl₂/MeOH, 20:1): 0.55; ¹H-NMR (400 MHz, DMSO-d): δ = 1.95 (s,

6H), 2.82-2.96 (m, 2H), 3.22-3.35 (m, 2H), 3.35-3.48 (m, 4H), 3.67-3.73

(m, 2H), 3.40-3.96 (m, 2H), 4.51 (s, 2H), 6.44 (d, J = 8.7 Hz, 1H), 6.50 (d,

J = 8.7 Hz, 1H), 6.98 (s, 2H); ¹³C-NMR (151 MHz, DMSO-d6): δ = 14.7

(+), 32.7 (−), 47.6 (−), 51.4 (−), 53.2 (−), 66.8 (−), 108.4 (+), 112.5 (+),

116.8 (q), 125.0 (q), 126.2 (q), 127.6 (+), 131.6 (q), 132.7 (q), 137.5 (q),

140.3 (q), 141.4 (q), 164.3 (q), 169.5 (q); (ESI, [M+H]⁺): calcd. for

C₂₈H₂₆Cl₂N₄O₅S₂: 633.0794, found 633.08; UV/Vis (50µM in DMSO):

open isomer: λ_max= 370 nm, closed isomer: λ_max= 564 nm.

3,4-Bis(5-chloro-2-methylthiophen-3-yl)-1-(4-(4-(2-methoxyphenyl)-

piperazin-1-yl)butyl)-1H-pyrrole-2,5-dione (22)

Orange solid: 80% yield; reaction time: 0.25 h; flash chromatography:

CH₂Cl₂/MeOH: 0-10% MeOH; preparative HPLC (prep-HPLC-1, gradient

A/B: 0-25 min: 80/20, 25-35 min 5/95), t_R= 19.2 min; R_f(CH₂Cl₂/MeOH:

1

10/1): 0.63; H NMR (600 MHz, CDCl₃) δ = 1.72-1.81 (m, 2H), 1.82-1.91

(m, 2H), 1.94 (s, 6H), 3.09- 3.17 (m, 4H), 3.27 (t, 2H, J = 12.3 Hz), 3.49-

3.55 (m, 2H), 3.62- 3.71 (m, 4H), 3.87 (s, 3H), 6.86- 6.89 (m, 1H), 6.90 (s,

2H), 6.92- 6.97 (m, 2H), 7.08 (ddd, J = 8.1, 6.2, 2.8 Hz, 1H); ¹³C NMR

(151 MHz, CDCl₃) δ = 14.9 (+), 20.8 (−), 25.8 (−), 37.2 (−), 47.6 (−),

52.2(−), 55.5 (+), 56.5 (−), 111.4 (+), 119.0 (+), 121.3 (+), 124.7 (+),

125.8 (q), 127.1 (+), 127.2 (q), 132.5 (q), 138.6 (q), 140.5 (q), 152.1 (q),

170.1 (q); (ESI, [M+H]⁺): calcd. for C₂₉H₃₂Cl₂N₃O₃S₂: 604.1257, found

604.1253; UV/Vis (100 µM in DMSO): open isomer: λ_max= 260 nm,

378 nm; closed isomer: λ_max= 355 nm, 493 nm.

3,4-Bis(5-chloro-2-methylthiophen-3-yl)-1-(2-((2,3-dihydro-1H-inden-2-

yl)(propyl)amino)ethyl)-1H-pyrrole-2,5-dione (23)

General preparation of cyclopentene-DTE based switches 26-31:

Orange solid: 15% yield; reaction time: 3 h; no flash chromatography,

preparative HPLC (prep-HPLC-1, solvent A: H₂O [0.05 Vol% TFA],

solvent B: MeCN, gradient A/B: 0-30 min: 60/40, 30-40 min 5/95),

To a solution of 20 (44 mg, 0.13 mmol, 1.0 eq) and HBTU (46 mg,

0.12 mmol, 0.95 eq) in CH₂Cl₂(1 mL) at 0 °C were added DIPEA (45 µL,

0.26 mmol, 2.0 eq) and a solution of amines A or B (0.13 mmol, 1.0 eq)

in CH₂Cl₂(0.2 mL). The reaction was allowed to come to RT overnight.

Water (2 mL) was added and the aqueous phase was extracted with

CH₂Cl₂(3 x 3 mL). The combined organic phases were dried over

MgSO₄, filtered and the solvent was removed under reduced pressure.

The crude product at 26 and 27 was first purified by automated flash

column chromatography (CH₂Cl₂/MeOH: 0-10% MeOH). In general, the

cyclopentene-DTEs 26-31 were then purified by preparative HPLC.

t

_R= 21.6 min; ¹H NMR (300 MHz, DMSO-d₆+ D₂O) δ = 0.97 (t, 3H,

J = 7.3 Hz), 1.68-1.81 (m, 2H), 1.91 (s, 6H), 3.18-3.26 (m, 4H), 3.30-3.37

(m, 4H), 3.13-3.21 (m, 4H), 3.29- 3.40 (m, 4H), 3.88-4.00 (m, 2H), 4.31-

4.44 (m, 1H), 6.99 (s, 2H), 7.19-7.23 (m, 2H), 7.25-7.29 (m, 2H); ¹³C

NMR (151 MHz, DMSO-d₆) δ = 10.9 (+), 14.3 (+), 16.3 (−), 32.6 (−), 34.3

(−), 47.1 (−), 51.9 (−), 62.9 (+), 124.4 (+), 125.0 (q), 126.2 (q), 127.2 (+),

127.6 (+), 132.7 (q), 138.9 (q), 140.2 (q), 169.4 (q); HR-MS (ESI,

[M+H]⁺): calcd. for C₂₈H₂₉Cl₂N₂O₂S₂: 559.1042; found 559.1048; UV/Vis

(100 µM in DMSO): open isomer: λ_max= 264 nm, 360 nm; closed isomer:

λ_max= 355 nm, 500 nm.

4-(2-(5-Chloro-2-methylthiophen-3-yl)cyclopent-1-en-1-yl)-N-(4-(4-(2-

methoxyphenyl)piperazin-1-yl)butyl)-5-methylthiophene-2-carboxamide

(26)

3,4-Bis(5-chloro-2-methylthiophen-3-yl)-1-(4-((2,3-dihydro-1H-inden-2-

yl)(propyl)amino)butyl)-1H-pyrrole-2,5-dione (24)

Light beige solid: 38% yield; after stirring over night + 2 d at r.t.;

preparative HPLC (prep-HPLC-1, gradient A/B: 0-25 min: 60/40, 25-

40 min 5/95), t_R= 14.5 min; R_f(CH₂Cl₂/MeOH: 100/1): 0.42; ¹H NMR

(600 MHz, DMSO-d): δ = 1.49-1.58 (m, 2H), 1.64-1.73 (m, 2H), 1.81 (s,

3H), 1.89 (s, 3H), 1.95-2.04 (m, 2H), 2.68-2.78 (m, 4H), 2.86-2.93 (m,

2H), 3.13- 3.20 (m, 4H), 3.21-3.26 (m, 2H), 3.49-3.56 (m, 4H), 3.78 (s,

3H), 6.81 (s, 1H), 6.88-6.92 (m, 1H), 6.93-6.95 (m, 1H), 6.97-6.99 (m,

1H), 7.00-7.04 (m, 1H), 7.50 (s, 1H), 8.38 (t, 1H, J = 5.7 Hz), 9.37 (bs,

1H); ¹³C NMR (151 MHz, DMSO-d): δ = 13.8 (+), 14.1 (+), 20.9 (−), 22.3

(−), 26.3 (−), 37.8 (−), 38.1 (−), 38.4 (−), 47.1 (−), 51.3 (−), 55.3 (−), 55.4

(+), 111.9 (+), 118.3 (+), 120.8 (+), 123.5 (q), 123.6 (+), 127.3 (+), 128.6

(+), 133.0 (q), 133.9 (q), 134.3 (q), 135.0 (q), 135.8 (q), 136.0 (q),138.9

(q), 139.3 (q), 151.8 (q), 160.9 (q); HR-MS (ESI, [M+H]⁺): calcd. for

C₃₁H₃₉ClN₃O₂S₂: 584.2167; found 584.2169; UV/Vis (50 µM in DMSO):

open isomer: λ_max= 270 nm; closed isomer: λ_max= 490 nm.

Orange solid: 86% yield; reaction time: 3 h; flash chromatography:

CH₂Cl₂/MeOH: 0-10% MeOH; preparative HPLC (prep-HPLC-1, gradient

A/B: 0-30 min: 60/40, 30-40 min 2/98), t_R= 16.7 min, R_f(CH₂Cl₂/MeOH:

10/1): 0.14; ¹H NMR (400 MHz, DMSO-d) δ = 0.92 (t, 3H, J = 7.3 Hz),

1.59-1.83 (m, 6H), 1.91 (s, 6H), 3.03- 3.10 (m, 2H), 3.12-3.19 (m, 2H),

3.24-3.36 (m, 4H), 3.55 (t, 2H, J = 6.7 Hz), 4.13-4.29 (m, 1H), 6.99 (s,

2H), 7.18-7.21 (m, 2H), 7.22-7.26 (m, 2H), 10.32 (bs, 1H); ¹³C NMR

(101 MHz, DMSO-d): δ = 10.9 (+), 14.2 (+), 16.5 (−), 20.3 (−), 25.2 (−),

34.3 (−), 37.1 (−), 49.9 (−), 51.9 (−), 62.7 (+), 124.2 (q), 124.3 (q), 124.7

(+), 126.4 (q), 127.1 (+), 127.7 (+), 132.4 (q), 139.1 (q), 139.9 (q), 169.8

(q); HR-MS (ESI, [M+H]⁺): calcd. for C₃₀H₃₃Cl₂N₂O₂S₂: 587.1355, found

587.1359; UV/Vis (50 µM in DMSO): open isomer: λ_max= 263 nm,

391 nm; closed isomer: λ_max= 354 nm, 504 nm.

3,4-bis(5-chloro-2-methylthiophen-3-yl)-1-(2-(4-(5-hydroxy-3-oxo-3,4-

dihydro-2H-benzo[b][1,4]oxazin-8-yl)piperazin-1-yl)ethyl)-1H-pyrrole-2,5-

dione (25)

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

4-(2-(5-Chloro-2-methylthiophen-3-yl)cyclopent-1-en-1-yl)-N-(2-(4-(2-

methoxyphenyl)piperazin-1-yl)ethyl)-5-methylthiophene-2-carboxamide

(27)

4-(2-(5-Chloro-2-methylthiophen-3-yl)cyclopent-1-en-1-yl)-N-(2-((2,3-

dihydro-1H-inden-2-yl)(propyl)amino)ethyl)-5-methylthiophene-2-

carboxamide (30)

Light beige solid: 19% yield; preparative HPLC (prep-HPLC-1, gradient

A/B: 0-25 min: 60/40, 25-40 min 5/95), t_R= 17.5 min; R_f(CH₂Cl₂/MeOH:

Light beige solid: 26% yield; preparative HPLC (prep-HPLC-1, gradient

A/B: 0-30 min: 60/40, 30-40 min 5/95), t_R= 17.1 min; R_f(CH₂Cl₂/MeOH:

100/1): 0.42. mp 178 °C. ¹H NMR (600 MHz, DMSO-d): δ = 0.92 (t, 3H,

J = 7.3 Hz), 1.65-1.76 (m, 2H), 1.79 (s, 3H), 1.90 (s, 3H), 1.95-2.04 (m,

2H), 2.69-2.77 (m, 4H), 3.13-3.21 (m, 4H), 3.29- 3.36 (m, 4H), 3.56-3.64

(m, 2H), 4.27-4.36 (m, 1H), 6.81 (s, 1H), 7.19-7.22 (m, 2H), 7.24-7.26 (m,

2H), 7.50 (s, 1H), 8.62-8.69 (m, 1H), 9.89 (bs, 1H); ¹³C NMR (151 MHz,

DMSO-d): δ = 11.3 (+), 14.3 (+), 14.7 (+), 16.9 (−), 22.8 (−), 34.7 (−),

34.8 (−), 34.9 (−), 38.3 (−), 38.6 (−), 49.6 (−), 52.7 (−), 63.5 (+), 124.2 (q),

124.9 (+), 127.1 (q), 127.6 (+), 127.7 (+), 129.8 (+), 133.5 (q), 134.6 (q),

135.2 (q), 135.4 (q), 136.6 (q), 139.4 (q),140.2 (q), 141.1 (q), 162.0 (q);

HR-MS (ESI, [M+H]⁺): calcd. for C₃₀H₃₆ClN₂OS₂: 542.1955; found

542.1957; UV/Vis (100 µM in DMSO): open isomer: λ_max= 262 nm;

closed isomer: λ_max= 494 nm.

1

10/1): 0.29; H NMR (600 MHz, DMSO-d): δ = 1.82 (s, 3H), 1.91 (s, 3H),

1.97-2.03 (m, 2H), 2.71-2.77 (m, 4H), 2.89-2.95 (m, 2H), 3.21- 3.28 (m,

2H), 3.31-3.36 (m, 2H), 3.50-3.54 (m, 2H), 3.56-3.61 (m, 2H), 3.63-3.68

(m, 2H), 3.79 (s, 3H), 6.81 (s, 1H), 6.89-6.92 (m, 1H), 6.94-6.96 (m, 1H),

6.97-6.99 (m, 1H), 7.00-7.04 (m, 1H), 7.51 (s, 1H), 8.62 (t, 1H,

J = 5.7 Hz), 9.47 (bs, 1H); ¹³C NMR (151 MHz, DMSO-d): δ = 13.8 (+),

14.2 (+), 22.3 (−), 34.0 (−), 37.8 (−), 38.1 (−), 47.0 (−), 51.5 (−), 54.8 (−),

55.4 (+), 111.9 (+), 118.3 (+), 120.8 (+), 123.5 (q), 123.6 (+), 127.3 (+),

129.3 (+), 133.0 (q), 134.0 (q), 134.1 (q), 134.9 (q), 134.9 (q), 136.1

(q),139.2 (q), 139.6 (q), 151.8 (q), 161.6 (q); HR-MS (ESI, [M+H]⁺): calcd.

for C₂₉H₃₅ClN₃O₂S₂: 556.1854; found 556.1857; UV/Vis (50 µM in

DMSO): open isomer: λ_max= 259 nm; closed isomer: λ_max= 323 nm,

491 nm.

4-(2-(5-Chloro-2-methylthiophen-3-yl)cyclopent-1-en-1-yl)-N-(4-((2,3-

dihydro-1H-inden-2-yl)(propyl)amino)butyl)-5-methylthiophene-2-

carboxamide (31)

4-(2-(5-Chloro-2-methylthiophen-3-yl)cyclopent-1-en-1-yl)-5-methyl-N-(2-

(4-(2-(methylthio)phenyl)piperazin-1-yl)ethyl)thiophene-2-carboxamide

(28)

Light beige solid: 70% yield; preparative HPLC (prep-HPLC-1, gradient

A/B: 0-30 min: 60/40, 30-40 min 5/95, t_R= 20.2 min); ¹H NMR (600 MHz,

DMSO-d): δ = 0.91 (t, 3H, J = 7.3 Hz), 1.52-1.59 (m, 2H), 1.63-1.68 (m,

2H), 1.63-1.73 (m, 2H), 1.80 (s, 3H), 1.89 (s, 3H), 1.94-2.07 (m, 2H),

2.69-2.76 (m, 4H), 3.04- 3.11 (m, 2H), 3.11-3.18 (m, 4H), 3.22-3.27 (m,

2H), 3.28-3.31 (m, 2H), 4.13-4.25 (m, 1H), 6.81 (s, 1H), 7.19-7.21 (m,

2H), 7.22-7.26 (m, 2H), 7.50 (s, 1H), 8.36 (t, 1H, J = 5.7 Hz), 9.38 (bs,

1H); ¹³C NMR (151 MHz, DMSO-d): δ = 11.4 (+), 14.3 (+), 14.6 (+), 17.3

(−), 21.1 (−), 22.8 (−), 26.7 (−), 34.8 (−), 34.9 (−), 38.3 (−), 38.5 (−), 38.6

(−), 50.9 (−), 52.6 (−), 63.3 (+), 124.1 (q), 124.8 (+), 124.9 (+), 127.7 (+),

127.8 (+), 129.1 (+), 133.5 (q), 134.4 (q), 134.8 (q), 135.4 (q), 136.2 (q),

136.4 (q), 139.4 (q), 139.5 (q), 139.5 (q), 161.5 (q); HR-MS (ESI,

[M+H]⁺): calcd. for C₃₂H₄₀ClN₂OS₂: 567.2265; found 567.2279; UV/Vis

(50 µM in DMSO): open isomer: λ_max= 264 nm; closed isomer:

λ_max= 490 nm.

Light beige solid: 29% yield; preparative HPLC (prep-HPLC-1, gradient

A/B: 0-35 min: 60/40, 35-40 min 5/95), t_R= 17.8 min; ¹H NMR (600 MHz,

DMSO-d): δ = 1.83 (s, 3H), 1.92 (s, 3H), 1.99-2.03 (m, 2H), 2.40 (s, 3H),

2.72-2.78 (m, 4H), 2.97-3.05 (m, 2H), 3.16-3.29 (m, 4H), 3.35-3.43 (m,

2H), 3.55-3.62 (m, 2H), 3.65-3.75 (m, 2H), 6.82 (s, 1H), 7.12-7.17 (m,

3H), 7.18-7.20 (m, 1H), 7.53 (s, 1H), 8.63 (bs, 1H), 9.65 (bs, 1H); ¹³C

NMR (151 MHz, DMSO-d): δ = 13.6 (+), 13.8 (+), 14.2 (+), 22.3 (−), 34.1

(−), 37.9 (−), 38.1 (−), 48.1 (−), 51.9 (−), 55.0 (−), 119.7 (+), 123.7 (q),

124.6 (+), 125.0 (+), 127.3 (+), 129.3 (+), 133.0 (q), 134.1 (q), 134.2 (q),

134. 4 (q), 134.9 (q), 136.1 (q), 139.7 (q), 147.1 (q), 157.8 (q), 158.0 (q);

HR-MS (ESI, [M+Na]⁺): calcd. for C₂₉H₃₄ClN₃NaOS₃: 594.1445; found

594.1446; UV/Vis (50 µM in DMSO): open isomer: λ_max= 262 nm; closed

isomer: λ_max= 491 nm.

4-(2-(5-Chloro-2-methylthiophen-3-yl)cyclopent-1-en-1-yl)-5-methyl-N-(4-

(4-(2-(methylthio)phenyl)piperazin-1-yl)butyl)thiophene-2-carboxamide

(29)

4-(2-(5-chloro-2-methylthiophen-3-yl)cyclopent-1-en-1-yl)-N-(2-(4-(5-

hydroxy-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-8-yl)piperazin-1-

yl)ethyl)-5-methylthiophene-2-carboxamide (32)

Light beige solid: 26% yield; preparative HPLC (prep-HPLC-1, gradient

A/B: 0-25 min: 60/40, 30-35 min 5/95), t_R= 16.1 min; ¹H NMR (600 MHz,

DMSO-d): δ = 1.52-1.60 (m, 2H), 1.67-1.76 (m, 2H), 1.83 (s, 3H), 1.91 (s,

3H), 1.98-2.06 (m, 2H), 2.41 (s, 3H), 2.73-2.79 (m, 4H), 2.96-3.05 (m,

2H), 3.10- 3.18 (m, 2H), 3.20-3.29 (m, 4H), 3.30-3.35 (m, 2H), 3.56-3.62

(m, 2H), 6.84 (s, 1H), 7.13-7.15 (m, 1H), 7.17-7.19 (m, 2H), 7.19-7.22 (m,

1H), 7.53 (s, 1H), 8.41 (t, 1H, J = 5.7 Hz), 9.59 (bs, 1H); ¹³C NMR

(151 MHz, DMSO-d): δ = 14.0 (+), 14.3 (+), 14.6 (+), 21.4 (−), 22.8 (−),

26.8 (−), 38.3 (−), 38.6 (−), 38.8 (−), 48.6 (−), 52.1 (−), 55.8 (−), 120.2 (+),

124.1 (q), 125.1 (+), 125.5 (+), 125.7 (+), 127.8 (+), 129.1 (+), 133.5 (q),

134.4 (q), 134.8 (q), 134.9 (q), 135.5 (q), 136.3 (q), 136.4 (q),139.4 (q),

147.8 (q), 161.4 (q); HR-MS (ESI, [M+H]⁺): calcd. for C₃₁H₃₉ClN₃OS₃:

603.1939; found 603.1939; UV/Vis (50 µM in DMSO): open isomer:

λ_max= 259 nm; closed isomer: λ_max= 489 nm.

Compound 20 (20.0 mg, 0.059 mmol, 1.0 eq) and HBTU (21.3 mg, 0.06

mmol, 1.0 eq) were dissolved in DMF (2 mL). DIPEA (0.02 mL, 0.12

mmol, 2 eq) and a solution of amine 17 (17.3 mg, 0.06 mmol, 1.0 eq) in

DMF (0.5 mL) was added. The mixture was stirred at room temperature

for 4h. The solvent was evaporated and the crude mixture was first

purified by automated flash column chromatography (CH₂Cl₂/MeOH: 0-

20% MeOH) and second purified by preparative HPLC (prep-HPLC-2,

gradient A/B: 0-15 min: 70/30, 15-20 min: 5/95, t_R= 14.1 min) to afford 32

(2.00 mg, 0.003 mmol, 6%) as a light yellow solid. R_f(CH₂Cl₂/MeOH,

20:1): 0.78; ¹H-NMR (400 MHz, DMSO-d): δ = 1.83 (s, 3H), 1.92 (s, 3H),

2.01 (qi, J = 7.3 Hz, 2H), 2.75 (q, J = 6.9 Hz, 4H), 2.90 (t, J = 11.4 Hz, 2H),

3.17-3.27 (m, 2H), 3.35-3.41 (m, 4H), 3.54-3.68 (m, 4H), 4.51 (s, 2H),

6.45 (d, J= 8.7 Hz, 1H), 6.50 (d, J= 8.8 Hz, 1H), 6.82 (s, 1H), 7.52 (s, 1H),

8.62 (bs, 1H), 9.62 (bs, 1H), 9.88 (bs, 1H); ¹³C-NMR (151 MHz, DMSO-

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

d): δ = 13.8 (+), 14.2 (+), 22.3 (−), 34.0 (−), 37.9 (−), 38.2 (−), 47.7 (−),

51.5 (−), 54.9 (−), 66.8 (−), 108.4 (+), 112.5 (+), 116.8 (q), 123.7 (q),

127.3 (+), 129.3 (+), 131.6 (q), 133.0 (q), 134.1 (q), 134.2 (q), 134.9 (q),

136.1 (q), 137.5 (q), 139.6 (q), 141.4 (q), 161.6 (q), 164.3 (q); HR-MS

(ESI, [M+H]⁺): calcd. for C₃₀H₃₃ClN₄O₄S₄: m/z = 613.1705, found

613.1708; UV/VIS:(50µM in DMSO): open isomer: λ_max= 263 nm, closed

isomer: λ_max= 503 nm.

387.1627; syn-36: R_f(CH₂Cl₂/MeCN, 100:1): 0.77; ¹H-NMR (600 MHz,

CDCl₃): δ = 0.58 (t, J = 7.1 Hz, 3H), 0.78 (d, J = 6.7 Hz, 3H), 0.97 (d, J =

6.8 Hz, 3H), 1.97 (s, 3H), 2.34 (s, 3H), 2.71 (s, 3H), 2.77 (sep, J = 6.8 Hz,

1H), 3.43-3.54 (m, 2H) 4.42 (s, 1H), 7.20-7.24 (m, 1H), 7.27-730 (m, 1H),

7.67 (t, J = 9.1 Hz, 2H); ¹³C-NMR (151 MHz, CDCl₃): δ = 13.3 (+), 16.2

(+), 17.3 (+), 17.5 (+), 20.6 (+), 24.2 (+), 38.3 (+), 56.1 (+), 61.0 (−), 89.4

(q), 121.5 (q), 122.0 (+), 123.3 (+), 123.4 (+), 123.5 (+), 128.6 (q), 137.3

(q), 138.1 (q), 138.6 (q), 152.0 (q), 168.3 (q), 170.0.(q); HR-MS (ESI,

[M+H]⁺): calcd. for C₂₂H₂₆O₄S: m/z = 387.1625, found 387.1631

2-Methyl-1-(2-methylbenzo[b]thiophen-3-yl)propan-1-one (34)

A mixture of 2-methylbenzothiophene (1.1 g, 7.4 mmol, 1.0 eq) and

isobutyryl chloride (0.9 mL, 8.2 mmol, 1.1 eq) in dry toluene (40 mL) was

cooled to 5 °C under stirring and nitrogen atmosphere. SnCl₄was

dissolved in toluene (5 mL) and added dropwise to the reaction mixture.

The solution was stirred 2 h at room temperature before it was quenched

by adding HCl-solution (2 M, 20 mL). After separation of the organic layer,

it was dried over Na₂SO₄and the solvent was evaporated. The crude

product 34 was recrystallized from hexane to obtain a yellow solid (1.1 g,

(E)-3-(2-methyl-1-(2-methylbenzo[b]thiophen-3-yl)propylidene)-4-

(propan-2-ylidene)dihydrofuran-2,5-dione (E-37)

Compound 36 (622 mg, 1.61 mmol, 1.0 eq) was dissolved in ethanol (40

mL) and after addition of KOH (4.5 g, 80.5 mmol, 50 eq) and H₂O (10

mL), the mixture was stirred for 20 h at 70 °C. The reaction mixture was

poured onto ice and quenched with an aqueous HCl-solution (2 M, 50

mL). The aqueous layer was extracted with ethyl acetate (3 x 60 mL) and

the combined organic layers were washed with brine (40 mL) and dried

over Na₂SO₄. The organic solvent was removed in vacuo to get the crude

diacid as a yellow solid (258 mg, 0.72 mmol). The diacid was suspended

in dichloromethane (30 mL) and after adding acetyl chloride (380 mg,

4.83 mmol, 3.0 eq) the solution was stirred for 1 day at 40 °C. The

mixture was neutralized with saturated NaHCO₃solution and the

aqueous layer was extracted with dichloromethane (2 x 30 mL). The

combined organic layers were dried over Na₂SO₄and the solvent was

removed in vacuo. Then, the crude product was purified by automated

flash column chromatography (cyclohexane/EtOAc, gradient 1-5%

EtOAc) to obtain a slightly yellow solid E-37 (213 mg, 0.65 mmol, 87%).

1

5.0 mmol, 70%). R_f(PE/EtOAc, 20:1): 0.46; H-NMR (400 MHz, CDCl₃):

δ = 1.23 (d, J = 6.8 Hz, 6H), 2.67 (s, 3H), 3.36 (sept, J = 6.8 Hz, 1H),

7.29-7.41 (m, 2H), 7.76 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H); ¹³C-

NMR (101 MHz, CDCl₃): δ = 15.9 (+), 18.5 (+), 40.6 (+), 121.9 (+), 122.9

(+), 124.3 (+), 125.0 (+), 133.5 (q), 137.9 (q), 138.5 (q), 144.9 (q), 205.0

(q); HR-MS (ESI, [M+H]⁺): calcd. for C₁₃H₁₄OS: m/z = 219.0838, found

219.0839.

(anti/syn)-Ethyl 2-isopropyl-2-(2-methylbenzo[b]thiophen-3-yl)-5-oxo-4-

(propan-2-ylidene)tetrahydrofuran-3-carboxylate (36)

Diisopropylamine (1.17 g, 16.0 mmol, 1.6 eq) was dissolved in

tetrahydrofuran and cooled to −78 °C under nitrogen atmosphere. After

addition of n-butyllithium (0.96 g, 15.0 mmol, 1.5 eq) the mixture was

stirred at this temperature for 30 min. Diisopropylidenesuccinate 15 (3.22

g, 15.0 mmol, 1.2 eq) was added and the mixture was stirred at −78 °C

for 1h. Then compound 34 (2.20 g, 10.1 mmol, 1.0 eq) was added and

the mixture was allowed to warm to room temperature overnight. The

reaction mixture was stirred for 24 h at room temperature and then

quenched with HCl-solution (2 M, 60 mL). The aqueous layer was

extracted with ethyl acetate (3 x 100 mL) and the combined organic

layers were dried over Na₂SO₄. The solvent was removed in vacuo and

the crude product was purified by automated flash column

chromatography (CH₂Cl₂/MeCN, gradient 0 - 3% MeCN then 3 - 20%) to

obtain the product 36 as a colourless oil of syn/anti isomers (622 mg,

16.1 mmol, 16%). To determine the syn/anti isomers, the lactone mixture

was separated by preparative HPLC. (analytical HPLC: t_R(anti-36) =

21.76 min , t_R(syn-36) = 19.89 min , flow rate: 0.3 mL/min, A = H₂O (0.05

Vol% TFA), B = MeCN, gradient: 30-95% B); anti-36: R_f(CH₂Cl₂/MeCN,

100:1): 0.73; ¹H-NMR (600 MHz, CDCl₃): δ = 0.73 (d, J = 7.0 Hz, 3H),

1.01 (d, J = 6.4 Hz, 3H), 1.38 (t, J = 7.2 Hz, 3H), 1.66 (s, 3H), 2.28 (s,

3H), 2.66 (s, 3H), 2.94 (sep, J = 6.7 Hz, 1H), 4.23-4.32 (m, 1H), 4.34-4.40

(m, 1H), 4.53 (s, 1H), 7.29 (t, J = 7.3 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H)

7.72 (d, J = 7.9 Hz, 1H), 8.27 (d, J = 7.3 Hz, 1H); ¹³C-NMR (151 MHz,

CDCl₃): δ = 14.2 (+), 17.0 (+), 19.7 (+), 20.6 (+), 24.8 (+), 33.7 (+), 54.5

(+), 61.9 (−), 90.3 (q), 115.1 (q), 119.9 (q), 122.3 (+), 122.7 (+), 123.7 (+),

124.3 (+), 131.6 (q), 138.2 (q), 138.6 (q), 155.1 (q), 168.9 (q), 170.6 (q);

HR-MS (ESI, [M+H]⁺): calcd. for C₂₂H₂₆O₄S: m/z = 387.1625, found

1

R_f(cyclohexane/EtOAc, 9:1): 0.71; H-NMR (400 MHz, CDCl₃): δ = 0.99

(d, J = 7.0, 3H), 1.08 (s, 3H), 1.33 (d, J = 7.0, 3H), 2.13 (s, 3H), 2.30 (s,

3H), 3.98 (sep, J = 7.0 Hz, 1H), 7.27-7.38 (m, 2H), 7.48-7.52 (d, 1H),

7.71-7.75 (m, 1H); ¹³C-NMR (101 MHz, CDCl₃): δ = 15.6 (+), 21.2 (+),

23.1 (+), 23.4 (+), 26.8 (+), 33.7 (+), 120.6 (q), 122.3 (+), 123.0 (+), 124.4

(+), 124.9 (+), 125.1 (q), 132.2 (q), 136.8 (q), 138.5 (q), 157.5 (q), 158.6

(q), 159.4 (q), 162.8 (q), 163.0 (q); HR-MS (ESI, [M+H]⁺): calcd. for

C₂₀H₂₀O₃S: m/z = 341.1133, found 341.1135; UV/Vis (100 µM in DMSO):

open isomer: λ_max= 342 nm; closed isomer: λ_max= 479 nm.

(E)-3-(2-methyl-1-(2-methylbenzo[b]thiophen-3-yl)propylidene)-1-(2-(4-(2-

(methylthio)-phenyl)piperazin-1-yl)ethyl)-4-(propan-2-ylidene)pyrrolidine-

2,5-dione (43)

The phenyl piperazine derivative 7 (57.8 mg, 0.30 mmol, 1.1 eq) and

compound E-37 (71.0 mg, 0.21 mmol, 1.0 eq) were dissolved in

chloroform (30 mL) and heated to 60 °C overnight to form amide acid 42.

After cooling to room temperature, DCC (55.9 mg, 0.27 mmol, 1.3 eq),

HOBt (36.6 mg, 0.27 mmol, 1.3 eq) and DIPEA (35.0 mg, 0.27 mmol, 1.3

eq) were added. The mixture was stirred for 3 days and quenched with

saturated NaHCO₃(10 mL) solution. The aqueous layer was separated

and extracted with ethyl acetate (3 x 20 mL). The combined organic

layers were dried over Na₂SO₄and the solvent was removed under

reduced pressure. Purification was done by automated flash column

chromatography (PE/EtOAc, gradient 0-12% EtOAc) to yield 43 (93.3 mg,

0.16 mmol, 74%) as a colorless solid. R_f(PE/EtOAc, 5:1, 0.1 % NEt₃):

1

0.38; H-NMR (400 MHz, CDCl₃): δ = 0.99 (d, J = 7.0, 3H), 1.05 (s, 3H),

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

1.32 (d, J = 7.0, 3H), 2.09 (s, 3H), 2.29 (s, 3H), 2.41 (s, 3H), 2.76 (m, 6H),

3.00 (m, 4H), 3.84 (q, J = 6.8 Hz, 2H), 4.17 (sep, J = 7.0 Hz, 1H), 6.97-

7.02 (m, 1H), 7.12-7.05 (m, 3H), 7.24-7.34 (m, 2H), 7.54 (d, J = 7.4 Hz,

1H), 7.72 (d, J = 8.2 Hz, 1H); ¹³C-NMR (101 MHz, CDCl₃): δ = 14.6 (+),

15.7 (+), 21.6 (+), 22.0 (+), 23.7 (+), 26.7 (+), 32.6 (+), 34.9 (−), 51.7 (−),

53.6 (−), 55.4 (−), 119.6 (+), 122.1 (+), 123.3 (+), 123.4 (+), 123.9 (+),

124.4 (+), 124.5 (+), 125.0 (+) 128.9 (q), 133.0 (q), 135.1 (q), 136.6 (q),

138.4 (q), 139.3 (q), 144.5 (q), 149.6 (q), 152.7 (q), 168.1 (q), 168.3 (q);

HR-MS (ESI, [M+H]⁺): calcd. for C₃₃H₃₉N₃O₂S₂: m/z = 574.2556, found

574.2566; UV/Vis (50 µM in DMSO): open isomer: λ_max= 307 nm; closed

isomer: λ_max= 473 nm.

J = 7.7 Hz, 15.7 Hz, 2H), 3.54-3.62 (m, 2H), 3.67 (qi, J = 8.0 Hz, 1H), 4.34

(sep, J = 7.0 Hz), 6.71 (s, 1H), 7.05-7.11 (m, 2H), 7.12-7.17 (m, 2H); ¹³C-

NMR (151 MHz, DMSO-d): δ = 11.7 (+), 13.6 (+), 14.8 (+), 20.2 (+), 21.2

(+), 22.9 (+), 25.4 (+), 29.3 (+), 32.5 (−), 35.6 (−), 35.8 (−), 47.3 (−), 52.7

(−), 61.7 (+), 122.8 (+), 124.3 (+), 124.7 (q), 125.5 (+), 126.2 (+), 129.0

(q), 129.6 (q), 132.4 (q), 135.3 (q), 135.8 (q), 141.5 (q), 147.0 (q), 152.2

(q), 167.5 (q); HR-MS (ESI, [M+H]⁺): calcd. for C₃₁H₄₀N₂O₂S: m/z =

505.2883, found 505.2892; UV/Vis (100 µM in DMSO): open isomer:

λ_max= 272 nm; closed isomer: λ_max= 526 nm.

Preparation of amide acids 47 and 49:

The fulgide (1.0 eq) was dissolved in CHCl₃(10 mL). Than amine 11 (1.5

eq) was added and the mixture was heated up to 70 °C for 4h. The

solvent was evaporated and the crude mixture was purified by

preparative HPLC (prep-HPLC-2, gradient A/B: 0-15 min: 30/70, 15-20

min: 2/98).

Synthesis of (3E,4E)-3-ethylidene-4-(2-methyl-1-(2-methyl-1H-indol-3-

yl)propyli-dene)-1-(2-(4-(2-(methylthio)phenyl)piperazin-1-yl)ethyl)-

pyrrolidine-2,5-dione (45)

The amide acid 44 was formed of amine X3 (20.0 mg, 0.06 mmol, 1.0 eq)

and fulgide 38 (16.3 mg, 0.07 mmol, 1.1 eq) in chloroform (20 mL) at

60 °C overnight. After cooling to room temperature DCC (15.8 mg, 0.08

mmol, 1.3 eq), HOBt (10.3 mg, 0.08 mmol, 1.3 eq) and DIPEA (10.3 mg,

0.08 mmol, 1.3 eq) were added. The mixture was stirred for 4 days at

room temperature and quenched with saturated NaHCO₃(8 mL) solution.

The aqueous layer was separated and extracted with ethyl acetate (3 x

20 mL). The combined organic layers were dried over Na₂SO₄and the

solvent was evaporated. The crude product was first purified by

automated NP flash column chromatography (PE/EtOAc, gradient 0 - 8%

EtOAc), and second with RP flash column chromatography (H₂O/MeCN,

gradient 30 - 95% MeCN) to obtain 45 (8.2 mg, 0.015 mmol, 25%) as a

slightly yellow solid. R_f(PE/EtOAc, 2:1, 0.1% NEt₃): 0.47; ¹H-NMR (600

MHz, CDCl₃): δ = 0.96 (d, J = 7.0 Hz, 3H), 1.00 (s, 3H), 1.41 (d, J = 7.1

Hz, 3H), 2.09 (s, 3H), 2.12 (s, 3H), 2.41 (s, 3H), 2.65-2.95 (m, 6H), 2.98-

3.12 (m, 4H), 3.80-3.94 (m, 2H), 4.32 (sep, J = 7.0 Hz), 7.00-7.05 (m,

1H), 7.07-7.11 (m, 4H), 7.12-7.15 (m, 1H), 7.24-7.26 (m, 1H), 7.28-7.30

(m, 1H), 7.46-7.48 (m, 1H), 8.00 (s, 1H); ¹³C-NMR (151 MHz, CDCl₃): δ =

13.2 (+), 14.5 (+), 21.1 (+), 21.9 (+), 23.8 (+), 26.9 (+), 32.3 (+), 34.8 (−),

35.0 (−), 53.5 (−), 55.3 (−), 110.2 (+), 110.4 (q), 114.2 (q), 119.5 (q),

119.7 (+), 119.8 (q), 120.4 (+), 120.7 (+), 121.2 (q), 121.6 (+), 123.9 (q),

124.4 (+), 124.7 (+), 125.1 (+), 127.5 (q), 132.0 (q), 135.1 (q), 135.5 (q),

168.2 (q), 168.7 (q); HR-MS (ESI, [M+H]⁺): calcd. for C₃₃H₁₄N₄O₂S: m/z =

557.2945, found 557.2946; UV/Vis (50 µM in DMSO): open isomer:

(E)-3-((2-((2,3-dihydro-1H-inden-2-yl)(propyl)amino)ethyl)carbamoyl)-4-

(2-methylbenzo-[b]thiophen-3-yl)-2-(propan-2-ylidene)pent-3-enoic acid

(47)

Colourless solid: 18% yield; preparative HPLC: t_R= 7.9 min; ¹H-NMR

(400 MHz, CDCl₃): δ = 0.96-1.07 (m, 3H), 1.75 (s, 6H) 1.78-1.94 (m, 2H),

2.26 (s, 3H), 2.33 (s, 3H), 2.99-3.17 (m, 2H), 3.25-3.46 (m, 6H), 3.65-

3.99 (m, 2H), 4.15-4.26 (m, 1H), 7.17-7.26 (m, 6H), 7.49-7.58 (m, 1H),

7.64-7.69 (m, 1H); ¹³C-NMR (151 MHz, CDCl₃): δ = 11.2 (+), 14.4 (+),

17.3 (−), 21.7 (+), 22.2 (+), 24.4 (+), 34.6 (−), 36.1 (−), 51.8 (−), 54.5 (−),

54.7 (−) 64.3 (+), 114.7 (q), 122.0 (+), 122.4 (+), 122.5 (q), 123.8 (+),

123.9 (+), 124.4 (+), 124.5 (+), 124.7 (+), 128.1 (+), 132.8 (q), 135.7 (q),

138.1 (q), 138.2 (q), 138.4 (q), 138.8 (q), 151.2 (q), 169.0 (q), 172.2 (q);

HR-MS (ESI, [M+H]⁺): calcd. for C₃₂H₃₉N₂O₃S: m/z = 531.2676, found

531.2676.

(E)-3-((2-((2,3-dihydro-1H-inden-2-yl)(propyl)amino)ethyl)carbamoyl)-4-

(2-methyl-1H-indol-3-yl)-2-(propan-2-ylidene)pent-3-enoic acid (49)

Yellow solid: 48% yield; preparative HPLC: t_R= 6.9 min; ¹H-NMR (400

MHz, DMSO-d): δ = 0.98 (t, J = 7.2 Hz, 3H), 1.66 (s, 3H) 1.69-1.79 (m,

2H), 1.82 (s, 3H), 2.07 (s, 3H), 2.25 (s, 3H), 3.15-3.29 (m, 6H), 3.32-3.43

(m, 2H), 3.50-3.56 (m, 2H), 4.30-4.41 (m, 1H), 6.88 (dt, J = 0.79 Hz, 8.0

Hz, 1H), 6.96 (dt, J = 1.1 Hz, 7.9 Hz, 1H) 7.18-7.25 (m, 4H), 7.26-7.31 (m,

2H); ¹³C-NMR (151 MHz, DMSO-d): δ = 10.9 (+), 12.2 (+), 16.9 (−), 21.8

(+), 22.1 (+), 23.9 (+), 34.1 (−), 34.3 (−), 34.5 (−), 49.1 (−), 52.7 (−), 63.0

(+), 110.5 (+), 113.5 (q), 115.2 (q), 118.1 (q), 118.5 (+), 118.6 (+), 120.1

(q), 124.4 (+), 126.6 (+), 127.2 (+), 132.2 (q), 135.4 (q), 139.0 (q), 139.4

(q), 139.6 (q), 168.3 (q), 169.5 (q); HR-MS (ESI, [M+H]⁺): calcd. for

C32H39N3O3: m/z = 514.3064, found 514.3068.

λ_max= 360 nm; closed isomer: λ_max= 543 nm.

(E)-3-(1-(2,5-dimethylthiophen-3-yl)-2-methylpropylidene)-4-(propan-2-

ylidene)dihydrofuran-2,5-dione (46)

Fulgide 39 (100 mg, 0.33 mmol, 1.0 eq) and N-1-(2,3-dihydro-1H-inden-

2-yl)-N¹-propylethane-1,2-diamine 11 (108 mg, 0.49 mmol, 1.5 eq) were

dissolved in chloroform (50 mL) and heated up to 60 °C for 48 h. The

solvent was evaporated and the residue was purified by automated NP

column chromatography (PE/EtOAc, gradient 0 – 50% EtOAc, 1% NEt₃)

and second with RP flash column chromatography (H₂O/MeCN, gradient

30 - 95% MeCN) to obtain 46 (16,1 mg, 0.032 mmol, 10%) as a

colourless solid. R_f(PE/EtOAc, 9:1, 0.1% NEt₃): 0.25; ¹H-NMR (400 MHz,

DMSO-d): δ = 0.69 (d, J = 7.1 Hz, 3H), 0.80 (t, J = 7.3 Hz, 3H), 1.19 (s,

3H), 1.29 (d, J = 7.1 Hz, 3H), 1.38 (sx, J = 7.3 Hz, 2H), 1.89 (s, 3H), 2.14

(s, 3H), 2.39 (s, 3H), 2.43 (t, J = 7.2 Hz, 2H), 2.61-2.73 (m, 4H), 2.93 (dd,

Preparation of fulgimides 48 and 50:

MSNT (1.2 eq) and Me-imidazole (0.5 eq) were added to a solution of

amide acid 47 or 49 (1.0 eq) in CHCl₃at room temperature. The mixture

was stirred for 24 h at 40 °C. The solvent was reduced in vacuo and the

mixture was purified by automated column chromatography and

preparative HPLC (prep-HPLC-2, gradient A/B: 0-15 min: 30/70, 15-20

min 2/98).

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

(E)-1-(2-((2,3-dihydro-1H-inden-2-yl)(propyl)amino)ethyl)-3-(1-(2-

methylbenzo[b]thiophen-3-yl)ethylidene)-4-(propan-2-ylidene)pyrrolidine-

2,5-dione (48)

118.6 (+), 119.1 (+), 120.8 (+), 120.9 (q), 123.2 (q), 125.2 (q), 134.1 (q),

135.1 (q), 137.1 (q), 141.0 (q), 164.0 (q), 167.3 (q), 167.9 (q); HR-MS

(ESI, [M+H]⁺): calcd. for C₃₂H₃₅N₅O₅: m/z = 570.2711, found 570.2720;

UV/Vis (50 µM in DMSO): open isomer: λ_max= 363 nm; closed isomer:

λ_max= 563 nm.

White solid: 9% yield; column chromatography (PE/EtOAc: 0-50%

EtOAc); preparative HPLC: t_R= 10.3 min; R_f(PE/EtOAc, 9:1, 0.1% NEt₃):

0.82; ¹H-NMR (400 MHz, CDCl₃): δ = 1.03 (s, 3H), 1.08 (t, J = 7.3 Hz,

3H), 1.49-1.61 (m, 2H), 2.17 (s, 3H), 2.26 (s, 3H), 2.69 (s, 3H), 2.79-2.93

(m, 4H), 3.08 (m, 2H), 3.72-3.81 (m, 3H), 7.09-7.21 (m, 3H), 7.27-7.39 (m,

3H), 7.48 (t, J = 7.8 Hz, 1H), 7.74 (t, J = 7.0 Hz, 1H); ¹³C-NMR (151 MHz,

CDCl₃): δ = 11.4 (+), 15.4 (+), 17.6 (−), 22.2 (+), 22.6 (+), 26.2 (+), 31.6

(−), 34.8 (−), 35.2 (−), 53.8 (−), 64.6 (+), 122.3 (+), 122.5 (+), 124.4 (q),

124.8 (+), 124.9 (+), 125.8 (q), 128.0 (+), 135.4 (q), 136.7 (q), 138.0 (q),

138.1 (q), 138.2 (q), 138.7 (q), 153.4 (q), 167.7 (q), 168.3 (q); HR-MS

(ESI, [M+H]⁺): calcd. for C₃₂H₃₇N₂O₂S: m/z = 513.2570, found 513.2573;

UV/Vis (50 µM in DMSO): open isomer: λ_max= 328 nm; closed isomer:

λ_max= 486 nm.

IP accumulation assay

The measurement of D_2Sreceptor stimulated activation of the G-protein

mediated pathway was performed applying the IP-One HTRF^®assay

(Cisbio, Codolet, France) according to the manufacturer’s protocol. In

brief, HEK-293T cells were grown to a confluence of approx. 70% and

transiently cotransfected with the cDNAs of the human D_2Sreceptor (in

pcDNA3.1) and the hybrid G-protein Gqi5-HA (Gq protein with the last

five amino acids at the C-terminus replaced by the corresponding

sequence of Gi; gift from The J. David Gladstone Institutes, San

Francisco, CA)^[27,28]applying TransIT-293 Mirus transfection reagent

(MoBiTec, Goettingen, Germany). After one day cells were detached

from the culture dish with Versene (Life Technologies GmbH, Darmstadt,

Germany), seeded into black 384-well plates (10000 cells/well) (Greiner

Bio-One, Frickenhausen, Germany) and maintained for 24 h at 37 °C.

Test compounds (final concentration of 10 µM, 100 nM and 1 nM) were

dissolved in stimulation buffer and added to the cells as quadruplicates in

a black box under illumination with monochromatic light of a wave length

of 625 nm or in the dark according to the photostability of the ligands.

Incubation was performed for 2 hrs at 37 °C and subsequently stopped

by adding the detection reagents (IP1-d2 conjugate and Anti-IP1cryptate

TB conjugate each dissolved in lysis buffer). After waiting for further 1 h

at room temperature time resolved fluorescence resonance energy

transfer (HTRF) was measured using the Clariostar plate reader (BMG,

Ortenberg, Germany) according to the requirements of the company.

FRET data were normalized to basal (= 0%) and the maximum effect of

quinpirole (= 100%). Average values including standard deviation are the

result of three to ten individual experiments each done in quadruplicate

and calculated using PRISM 6.0 (GraphPad software, San Diego, CA).

(E)-1-(2-((2,3-dihydro-1H-inden-2-yl)(propyl)amino)ethyl)-3-(1-(2-methyl-

1H-indol-3-yl)ethylidene)-4-(propan-2-ylidene)pyrrolidine-2,5-dione (50)

Yellow solid: 14% yield; column chromatography (CH₂Cl₂, MeOH: 0-10%

MeOH); preparative HPLC: t_R= 9.9 min; R_f(CH₂Cl₂/MeOH, 20:1): 0.62;

¹H-NMR (400 MHz, CDCl₃): δ = 0.98 (s, 3H), 1.06 (t, J = 7.3 Hz, 3H), 1.94

(q, J = 7.4 Hz, 2H), 2.18 (s, 6H), 2.79 (s, 3H), 3.28-3.43 (m, 4H), 3.44-

3.55 (m, 4H), 4.01-410 (m, 2H), 4.15 (qi, J = 8.5 Hz, 1H), 6.88 (m, 1H),

7.09-7.18 (m, 2H), 7.19-7.24 (m, 4H), 7.29 (d, J = 7.9 Hz, 1H), 7.38 (d, J

= 7.9 Hz, 1H); ¹³C-NMR (151 MHz, CDCl₃): δ = 11.4 (+), 13.7 (+), 17.6

(−), 22.3 (+), 22.8 (+), 26.3 (+), 31.1 (−), 35.1 (−), 36.6 (−), 46.8 (−), 53.5

(−), 64.5 (+), 110.8 (+), 117.5 (q), 119.8 (+), 120.7 (+), 122.1 (q), 122.3

(+), 123.4 (q), 124.8 (+), 126.3 (q), 127.8 (+), 133.0 (q), 135.6 (q), 137.6

(q), 138.4 (q), 145.5 (q), 150.3 (q), 168.1 (q), 168.5 (q); HR-MS (ESI,

[M+H]⁺): calcd. for C₃₂H₃₈N₃O₂: m/z = 496.2959 , found 496.2961; UV/Vis

(100 µM in DMSO): open isomer: λ_max= 370 nm; closed isomer:

λ_max= 564 nm.

(E)-1-(2-(4-(5-hydroxy-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-8-

yl)piperazin-1-yl)ethyl)-3-(1-(2-methyl-1H-indol-3-yl)ethylidene)-4-

(propan-2-ylidene)pyrrolidine-2,5-dione (52)

-Arrestin recruitment assay

D_2SR mediated -arrestin-2 recruitment was determined using the

Indolyl fulgide 41 (10.0 mg, 0.034 mmol, 1.0 eq) and the benzoxacinon

derivative 17 (8.87 mg, 0.041 mmol, 1.2 eq) were dissolved in DMF (4

mL). The mixture was stirred at 60 °C for 4 h and cooled to room

temperature. HOBt (4.58 mg, 0.034 mmol, 1.0 eq), HBTU (15.4 mg,

0.041 mmol, 1.2 eq) and DIPEA (4.38 mg, 0.034 mmol, 1.0 eq) were

added and the mixture was stirred for 4 h at room temperature. The

crude product was first purified by automated flash chromatography

(CH₂Cl₂/MeOH: 0-30% MeOH) and second purified by preparative HPLC

(prep-HPLC-2, gradient A/B: 0-15 min: 30/70, 15-20 min: 2/98, t_R= 5.2

min) to obtain 52 (2.0 mg, 3.5 µmol, 10%) as a white solid. R_f

(CH₂Cl₂/MeOH, 20:1): 0.15; ¹H-NMR (600 MHz, DMSO-d): δ = 0.87 (s,

3H), 2.13 (s, 3H) 2.17 (s, 3H), 2.75 (s, 3H), 2.84-2.95 (m, 2H), 3.12-3.22

(m, 2H), 3.41-3.51 (m, 4H), 3.68-3.80 (m, 2H), 3.86-4.01 (m, 2H), 4.51 (s,

2H) 6.43-6.47 (m, 1H), 6.50-6.55 (m, 1H), 6.96-7.03 (m, 1H), 7.04-7.09

(m, 1H), 7.29-7.36 (m, 2H); ¹³C-NMR (151 MHz, DMSO-d): δ = 12.8 (+),

21.2 (+), 22.0 (+), 25.1 (+), 31.6 (−), 47.2 (−), 51.2 (−), 52.8 (−), 66.4 (−),

103.3 (q), 108.0 (+), 110.6 (+), 112.2 (+), 113.6 (q), 115.6 (q), 116.4 (q),

PathHunter^®assay (DiscoverX, Birmingham, U.K.) as described

previously.^[29]In brief, HEK293 cells stably expressing the enzyme

acceptor (EA) tagged -arrestin-2 fusion protein were transiently

transfected with the ProLink tagged D2SR-ARMS2-PK2 construct

employing Mirus TransIT-293. One day (24 hrs) after transfection, cells

were detached from the culture dish with Versene (Life Technologies,

Darmstadt, Germany), seeded into 384-well plates (white plates with

transparent bottom, Greiner Bio-One, Frickenhausen, Germany) at a cell

number of 5000 cells/well and maintained for further 24 h at 37 °C, 5 %

CO₂. Test compounds at a final concentration of 10 µM, 100 nM and 1

nM were dissolved in PBS and added to the cells as quadruplicates in a

black box under illumination with monochromatic light of a wave length of

625 nm or in the dark according to the photostability of the ligands.

Incubation with the test compounds was continued for 5 hrs at 37 °C and

was stopped by adding the detection mix to every well. After further 1 h

at room temperature chemiluminescence was determined using

a

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

[5]

a) J. Broichhagen, A. Damijonaitis, J. Levitz, K. R. Sokol, P. Leippe, D.

Konrad, E. Y. Isacoff, D. Trauner, ACS Cent. Sci. 2015, 1, 383-393; b)

T. Fehrentz, C. A. Kuttruff, F. M. E. Huber, M. A. Kienzler, P. Mayer, D.

Trauner, ChemBioChem 2012, 13, 1746-1749, S46/1-S46/28; c) M.

Schoenberger, D. Trauner, Angew. Chem., Int. Ed. 2014, 53, 3264-

3267; d) A. Rullo, A. Reiner, A. Reiter, D. Trauner, E. Y. Isacoff, G. A.

Woolley, Chem. Commun. (Cambridge, U. K.) 2014, 50, 14613-14615

e) S. Pittolo, X. Gomez-Santacana, K. Eckelt, X. Rovira, J. Dalton, J.

Giraldo, C. Goudet, J.-P. Pin, A. Llobet, A. Llebaria, P. Gorostiza, Nat.

Chem. Biol. 2014, 10, 813-815

Clariostar plate reader (BMG, Ortenberg, Germany). Row data were

normalized to basal (= 0%) and the maximum effect of quinpirole (=

100%). Average values including standard deviation were derived from

four to ten individual experiments each done in quadruplicate and mean

values were calculated using PRISM 6.0 (GraphPad software, San Diego,

CA).

Dopamine D_2Sreceptor binding

[6]

[7]

a) W. A. Velema, J. P. van der Berg, M. J. Hansen, W. Szymanski, A. J.

M. Driessen, B. L. Feringa, Nat. Chem. 2013, 5, 924-928; b) W. A.

Velema, M. J. Hansen, M. M. Lerch, W. Szymanski, B. L. Feringa, A. J.

M. Driessen, Bioconjug Chem 2015, 26, 2592-2597

a) M. Dong, A. Babalhavaeji, S. Samanta, A. A. Beharry, G. A. Woolley,

Acc. Chem. Res. 2015, 48, 2662-2670; b) S. Samanta, A. A. Beharry,

O. Sadovski, T. M. McCormick, A. Babalhavaeji, V. Tropepe, G. A.

Woolley, J. Am. Chem. Soc. 2013, 135, 9777-9784; c) D. Bleger, S.

Hecht, Angew. Chem., Int. Ed. 2015, 54, 11338-11349

M. I. Bahamonde, J. Taura, S. Paoletta, A. A. Gakh, S. Chakraborty, J.

Hernando, V. Fernández-Dueñas, K. A. Jacobson, P. Gorostiza, F.

Ciruela, Bioconjugate Chem. 2014, 25, 1847-1854

M. Schönberger, Developing Photoswitchable Ligands for Transmem-

brane Receptors, Diss. 2014, LMU München

Receptor binding studies were carried out as described previously.^[30]In

brief, competition binding experiments were performed using

preparations of membranes from CHO cells stably expressing the human

D_2SR^[31]and the radioligand [³H]spiperone (specific activity = 81 Ci/mmol,

PerkinElmer, Rodgau, Germany) at a final concentration of 0.20 nM. The

assays were carried out with a protein concentration of 2 µg per assay

tube, a K_Dvalue of 0.050 nM and a corresponding B_maxvalue of 2000

fmol/mg. Test compounds were applied at final concentrations in the

range from 0.01 nM to 10000 nM each done as triplicates. Unspecific

binding was determined in the presence of haloperidol (10 µM), the

protein concentration was established by the method of Lowry using

bovine serum albumin as standard.^[32]The resulting competition curves of

the binding experiments were analyzed by nonlinear regression using the

algorithms in PRISM 6.0 (GraphPad Software, San Diego, CA). For each

[8]

[9]

[10] W. A. Velema, W. Szymanski, B. L. Feringa, J. Am. Chem. Soc. 2014,

136, 2178-2191

[11] a) M. Irie, Chem. Rev. 2000, 100, 1685-1716; b) C. Brieke, F. Rohrbach,

A. Gottschalk, G. Mayer, A. Heckel, Angew. Chem., Int. Ed. 2012, 51,

8446-8476

[12] a) X. Chen, N. I. Islamova, S. P. Gracia, J. A. DiGirolamo, W. J. Lees, J.

Org. Chem. 2009, 74, 6777-6783; b) X. Chen, N. I. Islamova, R. V.

Robles, W. J. Lees, Photochem. Photobiol. Sci. 2011, 10, 1023-1029

[13] W. Szymański, J. M. Beierle, H. A. V. Kistemaker, W. A. Velema, B. L.

Feringa, Chem. Rev. 2013, 113, 6114-6178

individual experiment, the data was fitted using

a monophasic

competition model to provide an IC₅₀value, which was then transformed

into a K_ivalue according to the equation of Cheng and Prusoff.^[33]Mean

K_ivalues ±SEM were derived from four to six individual experiments.

[14] L. Bettinetti, K. Schlotter, H. Hübner, P. Gmeiner, J. Med. Chem. 2002,

45, 4594-4597

[15] K. Schlotter, F. Böckler, H. Hübner, P. Gmeiner, J. Med. Chem. 2005,

48, 3696-3699

[16] D. Weichert, M. Stanek, H. Hübner, P. Gmeiner, Bioorg. Med. Chem.

2016, 24, 2641-2653

Acknowledgements

[17] N. Nebel, S. Maschauer, A. L. Bartuschat, S. K. Fehler, H. Hübner, P.

Gmeiner, T. Kuwert, M. R. Heinrich, O. Prante, C. Hocke, Bioorganic &

Medicinal Chemistry Letters 2014, 24, 5399-5403

This work was supported by the Deutsche Forschungs-

gemeinschaft (DFG, grant GRK 1910).

[18] N. Tschammer, M. Dörfler, H. Hübner, P. Gmeiner, ACS Chemical

Neuroscience 2009, 1, 25-35

[19] D. Wutz, C. Falenczyk, N. Kuzmanovic, B. Konig, RSC Adv. 2015, 5,

18075-18086

Keywords: Dopamine • Photoswitch • Fulgimide •

Dithienylethene • GPCR

[20] a) A. J. Myles, Z. Zhang, G. Liu, N. R. Branda, Org. Lett. 2000, 18,

2749-2751; b) Linda N. Lucas, Jaap J. D. d. Jong, Jan H. v. Esch,

Richard M. Kellogg, Ben L. Feringa, Eur. J. Org. Chem. 2003, 2003,

155-166

[1]

a) R. Lappano, M. Maggiolini, Nat Rev Drug Discov 2011, 10, 47-60; b)

N. Packiriswamy, N. Parameswaran, Genes Immun 2015, 16, 367-77;

c) P. Jeffrey Conn, A. Christopoulos, C. W. Lindsley, Nat Rev Drug

[21] a) T. Yamaguchi, M. Irie, Chem. Lett. 2004, 33, 1398-1399; b) T.

Yamaguchi, K. Uchida, M. Irie, J. Am. Chem. Soc. 1997, 119, 6066-

6071; c) M. Irie, K. Sayo, J. Phys. Chem. 1992, 96, 7671-7674

[22] a) M. Herder, B. M. Schmidt, L. Grubert, M. Paetzel, J. Schwarz, S.

Hecht, J. Am. Chem. Soc. 2015, 137, 2738-2747; b) J. R. Matis, J. B.

Schoenborn, P. Saalfrank, Phys. Chem. Chem. Phys. 2015, 17, 14088-

14095; c) M. Irie, T. Lifka, K. Uchida, S. Kobatake, Y. Shindo, Chem.

Commun. 1999, 747-750

Discov 2009, 8, 41-54

[2]

[3]

J.-M. Beaulieu, R. R. Gainetdinov, Pharmacol. Rev. 2011, 63, 182-217

a) S. Löber, H. Hübner, N. Tschammer, P. Gmeiner, Trends Pharmacol.

Sci. 2011, 32, 148-157; b) D. Möller, R. C. Kling, M. Skultety, K. Leuner,

H. Hübner, P. Gmeiner, J. Med. Chem. 2014, 57, 4861-4875; c) D.

Weichert, A. Banerjee, C. Hiller, R. C. Kling, H. Hübner, P. Gmeiner, J.

Med. Chem. 2015, 58, 2703-2717; d) N. Tschammer, J. Elsner, A.

Goetz, K. Ehrlich, S. Schuster, M. Ruberg, J. Kühhorn, D. Thompson, J.

Whistler, H. Hübner, P. Gmeiner, J. Med. Chem. 2011, 54, 2477-2491;

e) J. Elsner, F. Boeckler, F. W. Heinemann, H. Hübner, P. Gmeiner, J.

Med. Chem. 2005, 48, 5771-5779; f) C. Haubmann, H. Hübner, P.

Gmeiner, Bioorg. Med. Chem. Lett. 1999, 9, 3143-3146; g) P.

Rodriguez Loaiza, S. Löber, H. Hübner, P. Gmeiner, J. Comb. Chem.

2006, 8, 252-261

a) C. Falenczyk, M. Schiedel, B. Karaman, T. Rumpf, N. Kuzmanovic,

M. Grötli, W. Sippl, M. Jung, B. König, Chem. Sci. 2014, 5, 4794-4799;

b) D. Vomasta, C. Högner, N. R. Branda, B. König, Angew. Chem., Int.

Ed. 2008, 47, 7644-7647; c) D. Vomasta, A. Innocenti, B. König, C. T.

Supuran, Bioorg. Med. Chem. Lett. 2009, 19, 1283-1286; d) B.

Reisinger, N. Kuzmanovic, P. Löffler, R. Merkl, B. König, R. Sterner,

Angew. Chem., Int. Ed. 2014, 53, 595-598; e) J. Broichhagen, I.

Jurastow, K. Iwan, W. Kummer, D. Trauner, Angew. Chem., Int. Ed.

2014, 53, 7657-7660; f) R. Ferreira, J. R. Nilsson, C. Solano, J.

Andreasson, M. Groetli, Sci. Rep. 2015, 5, 9769

[23] a) H. Stobbe, Ber. Dtsch. Chem. Ges. 1905, 38, 3893-3678; b) F.

Strübe, R. Siewertsen, F. D. Sönnichsen, F. Renth, F. Temps, J. Mattay,

Eur. J. Org. Chem. 2011, 10, 1947-1955

[24] Dietrich.

S.,

Synthese

und

Photochrome

Eigenschaften

funktionalisierter Indolylfulgimide, Diss. 2006, Berlin

[25] B. Otto, K. Rück-Braun, Eur. J. Org. Chem. 2011, 10, 1947-1955

[26] S. I. Luyksaar, V. A. Migulin, B. B. Nabatov, M. M. Krayushkin, Russ.

Chem. Bull., Int. Ed 2010, 59, 446-451

[27] A.C. Kruse, A.M. Ring, A. Manglik, J. Hu, K. Hu, K. Eitel, H. Huebner, E.

Pardon, C. Valant, P.M. Sexton, A. Christopoulos, C.C. Felder, P.

Gmeiner, J. Steyaert, W.I. Weis, K.C. Garcia, J. Wess, and B.K. Kobilka,

Nature 2013, 504, 101-106

[28] B.R. Conklin, Z. Farfel, K.D. Lustig, D. Julius, H.R. Bourne, Nature

1993, 363, 274-276

[29] D. Möller, R.C. Kling, M. Skultety, K. Leuner, H. Hübner, P. Gmeiner, J.

Med. Chem. 2014, 57, 4861-4875

[4]

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

[30] H. Hübner, C. Haubmann, W. Utz, P. Gmeiner, J. Med. Chem. 2000, 43,

756-762

[31] G. Hayes, T.J. Biden, L.A. Selbie, J. Shine. Mol. Endocrinol. 1992, 6,

920-926

[32] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem.

1951, 193, 265-275

[33] Y.-C. Cheng, W.H. Prusoff, Biochem. Pharmacol. 1973, 22, 3099-3108

10.1002/chem.201702147

Chemistry - A European Journal

FULL PAPER

Entry for the Table of Contents (Please choose one layout)

Layout 1:

FULL PAPER

Text for Table of Contents

Author(s), Corresponding Author(s)*

Page No. – Page No.

Title

((Insert TOC Graphic here: max.

width: 5.5 cm; max. height: 5.0 cm))

Layout 2:

FULL PAPER

Daniel Lachmann, Dr. Carolin Studte,

Dr. Barbara Männel, Dr. Harald Hübner,

Prof. Dr. Peter Gmeiner, Prof. Dr.

Burkhard König*

Page No. – Page No.

Photochromic Dopamine Receptor

Ligands Based on Dithienylethenes

and Fulgides

State specific G-protein activation between the open and closed isomer of

dithienylethens and fulgimides has been shown. The photophysical properties of

the fulgimides exhibit high photostationary states and high fatigue resistance in

aqueous buffer solutions.

Article Doi

Photochromic Dopamine Receptor Ligands Based on Dithienylethenes and Fulgides

DOI: 10.1002/chem.201702147

Source and publish data:

Authors:

Article abstract of DOI:10.1002/chem.201702147

Full text of DOI:10.1002/chem.201702147

Products guided by the article

R&D Labs maybe for 102392-05-2

Relevant to this article

Hot Product