EPC syntheses and structure-activity relationships of hypoglycaemic semicyclic amidines

Eur. J. Med. Chem. 35 (2000) 377−392

S0223523400001380/FLA

377

Original article

EPC syntheses and structure–activity relationships

of hypoglycaemic semicyclic amidines

Susanne Hartmann^a, Susanne Ullrich^b,c, Charlotte Hupfer^b, August W. Frahm^a*

^aChair of Pharmaceutical Chemistry, University of Freiburg, Hermann-Herder-Str. 9, D-79104 Freiburg, Germany

^bDepartment of Physiology; University of Freiburg, D-79104 Freiburg, Germany

Received 1 June 1999; revised 19 August 1999; accepted 9 September 1999

Abstract – A series of homochiral sterically hindered mono- and bicyclic amidines was prepared as hypoglycaemic agents by lethargic

reaction of O-methylcaprolactim and 3-ethoxy-2-azabicyclo[2.2.2]oct-2-ene, respectively, with homochiral cis-2-substituted cyclopentane

amines provided by asymmetrical reductive amination of racemic 2-substituted cyclopentanones. All compounds, except the cyclohexylmethyl-

isoquinuclidone derivative which inhibited secretion at 100 µM, signiﬁcantly stimulated insulin secretion 2–8-fold at 10 µM and 100 µM in

INS-1 cells. The most potent activator was the 2-cyclopentyl-substituted caprolactam derivative 5e. The stimulatory effects on secretion

increased with rising steric hindrance of both the amidine α-carbon and the bicyclic amidine moiety itself. Enantiomeric discrimination was

observed for the 2-[(cis-2-bulkysubstituted cyclopentyl)imino]hexahydroazepine halides 5e and 5f and for the 3-[(cis-2-substituted

cyclopentyl)imino]-2-azabicyclo[2.2.2]octane halides 6a and 6c. The amidines depolarized INS-1 cells and generated action potentials,

accompanied by a decrease of membrane conductance. Simultaneously [Ca²⁺]_iincreased, probably due to Ca²⁺-entry through voltage-

dependent Ca²⁺-channels. At high concentrations, where inhibition of secretion was observed, [Ca²⁺]_istill rose upon application of the

amidines, indicating an additional inhibitory pathway downstream to the elevation of [Ca²⁺]_i. Even at high concentrations (100 µM), the

asymmetric reductive amination / mono- and bicyclic amidines / insulin secretion / INS-1 cells / structure–activity relationship

1. Introduction

The phenolethanolamine derivative (R, R)-CL 316,243

[7, 8], a highly selective ꢀ₃-receptor agonist, is a prom-

ising phase II candidate for the new target ꢀ₃-receptors

[9] in treatment of diabetes mellitus type IIb. (R)-(+)-

Etomoxir, withdrawn from development due to myocar-

dial hypertrophy, lowers blood glucose via irreversible

inhibition of carnitin-palmitoyl-transferase I (CPT I),

while the (S)-(–)-enantiomer does not show any activity

[10, 11]. N-Palmitoylcarnitine and its phosphate ana-

logues are reversible inhibitors of CPT I [12]. The

naturally occurring, highly potent (R)-enantiomers are by

far superior to the corresponding (S)-enantiomers.

Although achiral sulfonylureas, which improve insulin

secretion from pancreatic ꢀ-cells, are widely used for the

treatment of type 2 diabetes (NIDDM or non-insulin-

dependent diabetes mellitus), enantiomerically pure oral

antidiabetics of different mechanisms of action are gain-

ing growing interest in the treatment of type

II-hyperglycaemia. The sulfonylamidopyrimidine gliﬂu-

mide [1, 2] was one of the ﬁrst homochiral compounds

interacting with the sulfonylurea receptor [3], the (S)-

enantiomer being 20-fold more efficacious than the (R)-

enantiomer [4]. The insulinotropic non-sulfonylurea (+)-

repaglinide was recently launched and is twice as active

as its (–)-enantiomer [5, 6].

Semicyclic amidines are also known for their hypogly-

caemic effects, with racemic 2-[(cis-2-cyclohexyl-

cyclopentyl)imino]-hexahydroazepine hydrochloride as

the most efficacious representative developed as a result

of extensive structure–activity relationship studies [13].

Dose-dependent blood glucose lowering effects could be

observed in rats after oral application according to a

* Correspondence and reprints: awfrahm@ruf.uni-freiburg.de

^cPresent address: Department of Neurophysiology, University

of Köln, Robert-Koch-Str. 39, D-50931 Köln, Germany

Dedicated to Prof. Dr. Woldemar Schneider on the occasion of his

80th birthday.

378

generally accepted method [14]. Increasing steric hin-

drance of the C-1 atom neighbouring the amidino func-

tion by spatially demanding 2-substituents correlated

with an increasing hypoglycaemic effect. However, chiral

differentiating efficacy of the enantiomerically pure com-

pounds of this new class of hypoglycaemic agents has yet

to be elucidated.

Therefore, we have developed the EPC (enantiomeri-

cally pure compounds) synthesis of homochiral semicy-

clic amidines derived from ε-caprolactam and have tested

their chiral differentiating insulinotropic activity in vitro

in INS-1 cells and by means of electrophysiological

methods. Besides introduction of various spatially de-

manding substituents in position 2 of the aminocyclopen-

tane moiety (isopropyl, phenyl, cyclohexyl, benzyl, cy-

clopentyl and cyclohexylmethyl) we were especially

interested in the inﬂuence of increasing steric hindrance

of the semicyclic amidine moiety. Thus, the hexahy-

droazepine system was also replaced by the bicyclic

isoquinuclidine in a second series of semicyclic amidines.

In 1989 Palfreyman et al. [15] reported lactamimide

analogues representing a new chemical class of calcium

antagonists with diltiazem-like properties in in vitro test

systems. However, the most potent member N-(2,2-

diphenylpentyl)azacyclotridecan-2-imine hydrochloride

contains a large 13-membered ring, whereas the racemic

forms of the 7-membered hexahydroazepines 5b and 5c

are only weak competitive antagonists of the calcium-

induced contractions.

2. Chemistry

The homochiral semicyclic amidines 5a–f HX and

6a–f HX were prepared from the racemic 2-substituted

cyclopentanones 1a–f by means of asymmetric reductive

amination [16] and subsequent lethargic reaction of the

homochiral 2-substituted cyclopentaneamine halides 4a–f

HX with O-methylcaprolactim and 3-ethoxy-2-

azabicyclo[2.2.2]oct-2-ene, respectively, in a four-step

procedure (ﬁgure 1).

2-Isopropylcyclopentanone (1a) [17, 18], 2-phenyl-

cyclopentanone (1b) [19–21], 2-benzylcyclopentanone

(1d) [22, 23], and 2-cyclopentylcyclopentanone (1e) [24,

25] were synthesized according to common literature.

2-Cyclohexylcyclopentanone (1c) and 2-cyclohexyl-

methylcyclopentanone (1f) were accessible from 1b and

1d, respectively, by hydrogenation of the aromatic ring.

Hydrogenation of unprotected 1b with Rh/γ-Al₂O₃in

methanol/acetic acid [26] furnished a diastereomeric

mixture of 2-cyclohexylcyclopentanols and 1c in a 5:4

ratio, which was oxidized in situ to pure 1c with

pyridiniumchlorochromate on Al₂O₃[27]. However, hy-

Figure 1. Four-step procedure for the preparation of amidines

5a–f HX and 6a–f HX.

drogenation of 1d yielded 97% 1f with only negligible

amounts of the respective cyclopentanols.

Condensation of the racemic 2-substituted cyclopen-

tanones 1a–f with optically active (R)-(+)- and (S)-(–)-

1-phenylethylamine (PEA), respectively, led to the imi-

nes 2a–f. The latter were hydrogenated in situ with Raney

nickel at room temperature in a Parr shaker for a period

of 5–11 days to give the optically active, diastereomeri-

cally pure secondary cis-2-substituted N-(1-phenylethyl)-

cyclopentaneamines 3a–f in yields up to 73%. Their

halides 3a–f HX were hydrogenolysed with palladium-

on-charcoal (10%) at 50 °C in a Parr shaker within 24 h

to give the enantiomerically high grade pure primary

379

Table I. Reaction times and yields of the lethargic reaction to the

semicyclic amidines 5a–f HX and 6a–f HX.

Compound

Reaction time (days)

Yield (%)

5a HCl

5b HCl

5c HBr

5d HCl

5e HBr

5f HBr

6a HCl

6b HCl

6c HBr

6d HCl

6e HBr

6f HBr

30

16

42

17

35

21

84

63

105

63

90

86

79

85

92

56

91

73

86

76

90

Figure 2. Conﬁgurations of 7a–f, 8a–f and 8p.

substituted cyclopentaneamines. The CD spectra are con-

gruent in their positive Cotton effects at 315 and 255 nm

and their negative Cotton effect at 225 nm. Divergence in

intensity is caused by differing conformative arrange-

ments of the 2-substituents. The CD curves with extrema

at comparable wavelength ranges, but with actually

slightly deviating τ-values are strongly indicative of the

identical absolute conﬁguration of (1S, 2S)-7b and (1S,

2S)-7a, -7c–f.

154

42

cis-2-substituted cyclopentaneamine halides 4a–f HX in

yields of about 90%.

Lethargic reaction [13, 28] of 4a–f HX with

The enantiomeric excesses (ee%) of 4a–f and of the

auxiliary amines were determined by ¹⁹F-NMR spectros-

copy and HPLC via derivatization with (S)-(+)-α-

methoxy-α-triﬂuoro-methylphenylacetylchloride (Mosh-

er’s reagent) [32, 33] to the diastereomeric Mosher’s

amides 8a–f and 8p (ﬁgure 2). With more than 99.0% the

ee values of 8a–f exceeded those of the auxiliary amines,

97.9% for (R)-(+)-PEA and 97.0% for (S)-(–)-PEA,

which provides evidence for a recrystallization effect of

the secondary and primary amines 3a–f HX and 4a–f HX.

The ¹⁹F-NMR spectra of 8a–f and 8p show two singlets

for a couple of diastereomeric Mosher’s amides each,

which are slightly widened by lr-coupling to the methoxy

protons. The diastereotopic CF₃signals of 8a, 8b, 8d, 8f

and 8p range between 50 and 230 Hz, sufficient for

precise quantiﬁcation. However, the corresponding sig-

nals of 8c and 8e diverge by only 14 and 17 Hz,

respectively. Reliable spectroscopic determination of ee

values was impossible in these two cases, even though the

signals of the major diastereomer did not reveal any

shoulder in the expanded spectra. The HPLC separation

of the Mosher’s amides 8a–f and 8p were in accordance

with results derived from ¹⁹F-NMR spectroscopy. The

subsidiary diastereomer of 8c and 8e, respectively, was

below the detection limit (< 0.5%) each time.

O-methylcaprolactim

and

3-ethoxy-2-azabicyclo-

[2.2.2]oct-2-ene, respectively, led to 5a–f HX in yields of

about 85% in a period of 17–42 days, and to 6a–f HX in

yields of about 85% (with one exception) within 6–15

weeks (table I). The extremely long reaction times for the

lethargic reactions could not be overcome by well-known

accelerating methods of organic synthesis, such as acid

catalysis, pressure, sonication or microwave, partly be-

cause of the instability of the lactimethers used [13]. 5a–f

HX and 6a–f HX show E/Z-equilibria in solution. Their

composition has been reported elsewhere [29]. In solid

state Z-conﬁguration is observed [30]. The Z-conﬁgured

species is supposed to produce the biological effect, since

a complete shift of the equilibria towards Z-5a–f HX and

Z-6a–f HX was observed in polar solvents such as

methanol and dimethylsulfoxide.

Various 1-D and 2-D H- and ¹³C-NMR techniques,

1

including lr (longrange)-INEPT, COSY, HETCOR, GH-

SQCR and INADEQUATE, were used to elucidate the

structure and relative conﬁguration of all compounds and

to analyse the E/Z-equilibria of the semicyclic amidines

in solution.

The absolute conﬁguration of 4a–f was determined

indirectly by CD correlation spectroscopy of the corre-

sponding

cis-2-substituted

(1S, 2S)-cyclopentane-

aminesalicylidenes (1S, 2S)-7a–f (ﬁgure 2) with refer-

ence to cis-(1S, 2S)-2-phenylcyclopentaneamine-N-

salicylidene ((1S, 2S)-7b) [16] of known absolute

conﬁguration. The results relate well to ﬁndings in

literature [16], where like-induction [31] was reported for

the reductive amination step, thus (R)-(+)-PEA leading to

secondary cis-(1R, 1’R)-2-substituted cyclopentaneam-

ines and (S)-(–)PEA to corresponding cis-(1S, 1’S)-2-

3. Physiology

3.1. Effects of semicyclic amidines

on insulin secretion in INS-1 cells

The insulin secreting cell line INS-1 was used to test

the insulinotropic effect of the semicyclic amidines 5a–f

380

Figure 3. Effects of caprolactame derivatives 5a–f HX on insulin secretion of INS-1 cells. INS-1 cells were incubated and insulin

secretion measured as described in Experimental protocols. The compounds were tested at 10 and 100 µM in the presence of 0.5 mM

glucose and 5 µM forskolin. Results are expressed as fold-stimulation over forskolin alone and are means ± SEM for the indicated

#

number of observations (resulting from up to ﬁve independent experiments). 50 µM. * and §; signiﬁcant differences between the

enantiomers.

HX and 6a–f HX. Forskolin was present throughout

incubations since it potentiated the stimulatory effect of

amidines on secretion (data not shown). All compounds

increased insulin release at 10 µM (ﬁgures 3 and 4). The

effect varied between 43% and 369%. The most potent

activators of secretion were cyclohexyl-, benzyl-, and

cyclohexylmethyl substituted isoquinuclidone derivatives

6c HBr, 6d HCl and 6f HBr, respectively, and the

cyclopentyl substituted caprolactam derivate 5e HBr.

However, the caprolactam-derived cyclohexyl substituted

compound 5c HBr in contrast to in vivo data [13],

together with the isopropyl-derivates 5a HCl and 6a HCl

were the weakest stimulators of insulin secretion. Com-

paring the respective enantiomers, only (1’R, 2’R)-6c

HBr increased secretion more effectively than the (1’S,

2’S)-6c HBr distomer. All other enantiomers showed no

chiral differentiation at this concentration.

respectively, as well as cyclohexyl and cyclopentyl deri-

vates 5c HBr and 5e HBr further augmented insulin

release at the higher concentration. In contrast, cyclo-

hexyl and cyclopentyl derivates (1’R, 2’R)-6c HBr and

(1’R, 2’R)-6e HBr showed similar effects at 100 and

10 µM. Surprisingly, the cyclohexylmethyl derivate 6f

HBr inhibited secretion at 100 µM but had a stimulatory

effect at 10 µM. However, the cyclohexylmethyl derivate

5f HBr had a smaller activating effect at 100 µM than at

10 µM. The sulfonylurea tolbutamide was tested simul-

taneously and did not affect secretion at 10 µM but

increased insulin release 2.46 ± 0.13-fold (n = 20) at

50 µM and 2.33 ± 0.25-fold (n = 8) at 100 µM (cf. also

reference [34]). Thus, semicyclic amidines stimulate

insulin secretion in a similar concentration range to

tolbutamide. The semicyclic amidines 6c HBr, 6d HCl

and 6f HBr with the bicyclic lactam moiety were more

potent at 10 µM than the respective sterically less

hindered caprolactam compounds 5c HBr, 5d HCl and 5f

HBr.

The substances were also tested at 100 µM. Isopropyl,

phenyl and benzyl derivates of both series of amidines, 5a

HCl, 5b HCl, 5d HCl, 6a HCl, 6b HCl and 6d HCl,

381

Figure 4. Effects of isoquinuclidone derivatives 6a–f HX on insulin secretion of INS-1 cells. INS-1 cells were incubated and insulin

secretion measured as described in Experimental protocols. The compounds were tested at 10 and 100 µM in the presence of 0.5 mM

glucose and 5 µM forskolin. Results are expressed as fold-stimulation over forskolin alone and are means ± SEM for the indicated

number of observations (resulting from up to ﬁve independent experiments). * and §; signiﬁcant differences between the enantiomers.

3.2. Effects of semicyclic amidines

on [Ca²⁺]_iin INS-1 cells

At 10 µM, (1’R, 2’R)-6f HBr increased the fura-2 ﬂuo-

rescence ratio 340/380 nm from 1.32 ± 0.08 (n = 12) to

1.57 ± 0.15 (n = 5), at 100 µM to 1.84 ± 0.09 (n = 7).

Thus, although (1’R, 2’R)-6f HBr stimulated secretion

only at the lower concentration (ﬁgure 4), it further

increased [Ca²⁺]_imore consistently at the higher concen-

tration. In the case of 100 µM (1’R, 2’R)-6f HBr the

increase in [Ca²⁺]_iwas transient and returned to basal

level before the removal of the drug.

The observation that forskolin potentiated amidine-

induced secretion suggests that the amidines mainly act

by increasing [Ca²⁺]_i. They might activate the cells at

least in part by inducing depolarization due to closure of

K⁺_ATP-channels and by subsequent activation of voltage-

dependent Ca²⁺-channels.

Indeed, when measuring [Ca²⁺]_iin fura-2 loaded INS-1

cells, [Ca²⁺]_iincreased upon amidine addition (ﬁgures 5

and 6). The effect was comparable to tolbutamide-

induced [Ca²⁺]_i-increase (100 µM). [Ca²⁺]_ireturned to

basal levels after removal of (1’R, 2’R)-6a HCl with a

delay indicating that the wash-out of the compound was

slow. Since [Ca²⁺]_ioscillated in 68% of INS-1 cells,

which probably followed membrane voltage oscillations

observed previously [35], the effects on [Ca²⁺]_iwere not

quantiﬁed.

3.3. Effects of semicyclic amidines on membrane voltage

and membrane conductance in INS-1 cells

As the increase of [Ca²⁺]_imay be due to depolarization

of the plasma membrane, membrane voltage and conduc-

tance were measured using the standard whole cell mode

of the patch clamp method. Figure 7A shows a represen-

tative registration of membrane voltage [V_m] in INS-1

cells, depolarized with 100 µM (1’R, 2’R)-5a HCl. The

effects of (1’R, 2’R)-5a HCl on membrane voltage and

membrane conductance are summarized in ﬁgure 7B.

In another series of experiments the effects on [Ca²⁺]_i

of 10 µM and 100 µM (1’R, 2’R)-6f HBr were compared.

382

Figure 5. Effects of tolbutamide (Tol), (1’S, 2’S)-6a HCl (S6a) and (1’R, 2’R)-6a HCl (R6a) on [Ca²⁺]_iin INS-1 cells. Shown is a

representative trace of continuous [Ca²⁺]_irecording. The compounds were added as indicated, each at a concentration of 100 µM.

Membrane conductance was decreased 80% by 100 µM

(1’R, 2’R)-5a HCl, whereas the sulfonylurea tolbutamide

reduced membrane conductance by 95% at the same

concentration. The depolarizing effect of (1’R, 2’R)-5a

HCl was also smaller than that of tolbutamide, 15 mV and

45 mV, respectively. These observations indicate that

Figure 6. Effects of tolbutamide (Tol), (1’R, 2’R)-5a HCl (R5a) and (1’S, 2’S)-5a HCl (S5a) on [Ca²⁺]_iin INS-1 cells. Shown is a

representative trace of continuous [Ca²⁺]_irecording. The compounds were added as indicated, each at a concentration of 100 µM.

383

Figure 7. Effect of (1’R, 2’R)-5a HCl on membrane voltage in INS-1 cells. A: Shown is a representative trace of continuous

membrane voltage recording. 100 µM (1’R, 2’R)-5a HCl and 100 µM tolbutamide (Tol) were added as indicated. B. Mean ± SEM of

seven experiments.

semicyclic amidines at least partially stimulate insulin

release by depolarizing the INS-1 cells, which in succes-

sion leads to the opening of voltage-sensitive Ca²⁺

channels and to an increase of [Ca²⁺]_i.

spatially demanding cyclohexylmethyl substituted

amidines 5f HBr and 6f HBr stimulated insulin release

3–4-fold at 10 µM. At this concentration the sulfonylurea

tolbutamide had no signiﬁcant effect on insulin release

(ﬁgure 3). At 100 µM, the most potent secretagogue was

the compound (1’R, 2’R)-5e HBr, increasing insulin

release 8-fold. In comparison, 100 µM tolbutamide in-

creased secretion 2–3-fold.

4. Results and discussion

Sulfonylureas improve insulin secretion by a receptor

mediated mechanism. Structurally unrelated racemic

amidines have been described as hypoglycaemic agents

with an unelucidated mode of action [13]. In this study

we have prepared enantiomerically pure semicyclic

amidines in order to test their ability to stimulate insulin

secretion and to examine their mode of action.

The compounds had no toxic effects in INS-1 cells at

the highest concentration tested (100 µM), since i) depo-

larization and increase of [Ca²⁺]_iwere reversible (ﬁg-

ures 5–7), ii) insulin content and the secretory behaviour

of the cells were unchanged under conditions of cell

culturing for 24 h in the presence of amidines (not

shown), iii) the stimulatory effect of amidines was

inhibited by adrenaline, which is a physiological inhibitor

of insulin secretion (not shown). Thus, these substances

stimulate ꢀ-cell secretion via a controllable mechanism.

All semicyclic amidines increased insulin secretion of

INS-1 cells at 10 µM (ﬁgures 3 and 4). The ﬂexible,

Signiﬁcant differences between the potency of enanti-

omers to stimulate insulin release was observed for 5e

HBr, 5f HBr, 6a HCl and 6c HBr. Thus, the (1’R,

2’R)-eutomers of 5e HBr, 5f HBr, 6a HCl induced a 37%,

95% and 33% higher secretion than the respective (1’S,

2’S)-enantiomers. In contrast, the (1’R, 2’R)-enantiomer

of 6c HBr was 22% less potent than its (1’S, 2’S)-

enantiomer. These observations show the site of action to

possess only a weak stereoselectivity for these amidines.

In contrast to ﬁndings with other insulin secreting

cells, tolbutamide induced secretion in INS-1 cells only

in the presence of increased adenylyl cyclase activity

[34]. Synergistic effects of cAMP and [Ca²⁺]_ion insulin

secretion are also well described for native ꢀ-cells [36].

Thus, the potentiation of amidine induced secretion by

cAMP suggests a site of action involving increases in

[Ca²⁺]_i. Indeed, pronounced elevation of [Ca²⁺]_iis ob-

served upon amidine addition (ﬁgures 5 and 6). This

384

effect is reversible, but after removal of the drug, [Ca²⁺]_i

returns only slowly to basal levels indicating a reduced

wash-out of the substance. The measurements of mem-

brane voltage and membrane conductance revealed that

amidines depolarized the cells and reduced the membrane

conductance by 80% under control conditions (ﬁgure 7).

Since the whole cell conductance under control condi-

multiplets were determined by the corresponding ¹H-¹³C-

Hetcor spectra. H-NMR data are always listed in the

1

following order: multiplicity (s, singlet; d, doublet; t,

triplet; q, quartet; quint, quintet; m, multiplet; mc, centric

multiplet), number of nuclei, coupling constant(s) in Hz,

assignment. *^{, #, §, ◆}indicate protons/carbon atoms that

can be exchanged. GC MS spectra were recorded on a

Perkin Elmer QMASS 910 spectrometer on an Rtxt-35

column (Restek, Bellefonte, USA), crossbondt (35%

diphenyl-, 65% dimethylpolysiloxane) 0.25 µm, 35 m.

HPLC separation of the Mosher amides (enantiomeric

excesses (ee%)) was performed on a Lichrosorbt Si-60

column (5 µm, 250 × 4 mm, Bischoff, Leonberg, D) with

a Waters 600 multisolvent delivery system with a diode-

array detector Waters 990 or a Waters 486 Tunable

Absorbance Detector. Flash chromatography was carried

out on E. Merck silica gel 60 (0.040–0.068 mm). Thin

layer chromatography (TLC) was performed on silica gel

F₂₅₄plates from E. Merck, Darmstadt, D. Visualization

was done using ninhydrin reagent (Stahl) and heating.

Analyses (Analytical Laboratories, Chemical Institutes,

University of Freiburg, D) indicated by the symbols of

the elements or functions were within ± 0.4% of theo-

retical values.

tions has been shown to result mainly from K⁺

-

channel activity, the observed effects of amidines are^Ad^Tu^Pe

to inhibition of these channels [34].

Thus, the proposed mechanism of action involves the

closure of K⁺_ATP-channels which leads to a decrease of

membrane conductance, depolarization and subsequent

opening of voltage-activated Ca²⁺-channels. Whether the

site of action is also the SUR-molecule [35] as for

sulfonylureas, remains obscure and further studies are

needed for clariﬁcation.

Since

the

ﬂexible,

spatially

demanding

cyclohexylmethyl-substituted amidine 6f HBr inhibited

secretion at the highest concentration tested (100 µM)

while increasing [Ca²⁺]_i, it is suggested that accumulation

of this compound leads to the interference with further

membrane proteins resulting in an inhibition of secretion.

In conclusion, this study shows that enantiomerically

pure semicyclic amidines induce insulin secretion with

only weak stereoselectivity, at least in part through

inhibition of K⁺_ATP-channels and a subsequent rise in

[Ca²⁺]_i.

5.1.1. 2-Cyclohexylcyclopentanone (1c)

To

a

solution of 3.2 g (20 mmol) 2-phenyl-

cyclopentanone (1b) in 40 mL methanol abs were added

0.6 g of Rh/γ-Al₂O₃(5%) and 0.5 mL acetic acid [26].

The mixture was hydrogenated at an initial pressure of 5

bar and at room temperature in a Parr shaker. The

progress of the reaction was observed by GC/MS. After

10.5 h the reaction mixture was ﬁltered (Celite 535), the

catalyst was washed with ﬁve portions of 20 mL metha-

nol each and the ﬁltrate was evaporated after addition of

5 mL toluene for complete removal of the acetic acid. The

residue was oxidized in situ.

Hydrogenation mixture (3.2 g) was added dropwise to

22.6 g (17.6 mmol) pyridiniumchlorochromate-Al₂O₃[27]

suspended in 50 mL toluene, and stirred at room tempera-

ture over night. The oxidant was ﬁltered off and washed

with ﬁve portions of 30 mL diethylether each. The

organic phase was dried with sodium sulfate, ﬁltered and

evaporated in vacuo. A 2.9 g (88%) yield of a colourless

oil, containing 96.5% 1c (GC-MS) was condensed in situ

with (R)-(+)- and (S)-(–)-phenylethylamine, respectively.

MS (EI) m/e 166 ([M]⁺); ¹H-NMR (CDCl₃) δ 0.97–1.32

(m, 5H, H-2’b, H-6’b, H-3’b, H-4’b, H-5’b), 1.42 (mc,

1H, H-6’a*), 1.58–1.82 (m, 7H, H-3’a, H-4’a, H-5’a,

H-3b, H-4b, H-2’a*, H-1’), 1.89–2.06 (m, 4H, H-4a, H-2,

H-3a, H-5b), 2.19–2.31 (m, 1H, H-5a); ¹³C-NMR (CDCl₃)

δ 20.68 (C-4), 25.33 (C-3), 26.22 (C-3’*), 26.24 (C-4’),

5. Experimental protocols

5.1. Chemistry

Hydrogenation reactions were carried out in a Parr

shaker, constructed by the Pharmaceutical Institute of the

University of Freiburg using Rh/γ-Al₂O₃(5%) type G

207 R, Raney-nickel suspended under water type B 113

W and Pd/C (10%) type E 10 N/D (all Degussa, Frankfurt

a. M., D). Melting points (m.p.) were determined on a

Mel Temp II capillary melting point apparatus (Labora-

tory Devices, USA) and are uncorrected. IR spectra were

recorded on a Perkin Elmer 841 infrared spectrometer.

Optical rotation was determined on a Perkin Elmer 241

polarimeter with c = 1.0, ethanol. CD spectra were

recorded on a CD 6 Dichrograph System, ISA Instru-

1

ments Jobin Yvon, Longjumeau, F. H-, ¹³C-, ¹⁹F- 1-D

and 2-D-NMR spectra were recorded on a Varian Unity

300 spectrometer operating at 300, 75 and 282 MHz,

respectively. Chemical shift values are referenced to

CHCl₃, δ 7.24 in CDCl₃for ¹H-NMR, to CDCl₃, δ 77.00

for ¹³C-NMR and to 1,1,2-trichlorotriﬂuoroethane, δ

–68.03 for ¹⁹F-NMR. The chemical shifts of proton

385

26.44 (C-5’*), 28.74 (C-2’^#), 31.56 (C-6’^#), 37.49 (C-1’),

39.11 (C-5), 54.46 (C-2), 221.16 (C-1); IR (NaCl, cm^–1)

1 737 (C=O).

one third and the sec amine hydrobromide was precipi-

tated by adding diethylether and recrystallized from

ethanol/ether to give 3e HBr and 3f HBr, respectively.

The ice-cooled solution of an aliquot amount of sec

cis-2-cyclohexylcyclopentaneamine 3c, rectiﬁed by col-

umn chromatography, was acidiﬁed with triﬂuoroacetic

acid. 3c TFA was recrystallized from n-hexane/di-

ethylether (2:1). 3c HBr (73%) was hydrogenated in situ.

5.1.2. 2-Cyclohexylmethylcyclopentanone 1f

To

a

solution of 8.7 g (50 mmol) 2-benzyl-

cyclopentanone (1d) in 100 mL methanol abs were added

2.4 g of Rh/γ-Al₂O₃(5%) and 1.6 mL acetic acid. The

mixture was hydrogenated at an initial pressure of 5 bar

and at room temperature in a Parr shaker. The progress of

the reaction was observed by GC/MS. After 20.5 h the

reaction mixture was ﬁltered (Celite 535), the catalyst

was washed with ﬁve portions of 20 mL methanol each

and the ﬁltrate was evaporated after addition of 5 mL

toluene for complete removal of the acetic acid. An 8.8 g

(98%) yield of a colourless oil, containing 98.5% 1f

(GC-MS) was condensed in situ with (R)-(+)- and (S)-

(–)-phenylethylamine, respectively. MS (EI) m/e (M), 84

5.1.4.1. cis-2-Cyclohexyl-N-(1-phenylethyl)-

cyclopentaneamine triﬂuoroacetate 3c TFA

25

D

(1R, 2R)-3c TFA: m.p. 117–119 °C, ␣

–7.9°; (1S,

͓ ͔

25

1

2S)-3c TFA: m.p. 119–121 °C,

␣

͓ ͔

+8.2°; H-NMR

D

(CDCl₃) δ 0.86 (mc, 2H, H-2’b, H-6’b), 1.05–1.21 (m,

1H, H-3b), 1.21–1.39 (m, 2H, H-3’b, H-5’b), 1.42–1.71

(m, 9H, H-2’a*, H-4b, H-1’, H-2, H-3’a, H-5’a, H-5b,

H-3a, H-4’b), 1.75 (d, 3H, 6.8, ꢀ-CH₃), 1.71–1.92 (m,

4H, H-4’a, H-6’a*, H-5a, H-4a), 3.35 (s (broad (br)), 1H,

H-1), 4.35 (q, 1H, 6.8, α-CH), 7.37–7.47, (m, 3H, H-3″,

H-4″, H-5″), 7.50–7.55 (m, 2H, H-2″, H-6″), 7.71 (s (br),

1H, ⁺NH-b), 10.02 (s (br), 1H, ⁺NH-a); ¹³C-NMR (CDCl₃)

δ 18.96 (ꢀ-CH₃), 20.12 (C-4), 24.97 (C-3’, C-5’), 25.16

(C-4’), 25.55 (C-3), 29.95 (C-5), 31.24 (C-2’*), 32.50

(C-6’*), 34.91 (C-1’), 50.65 (C-2), 58.68 (C-1), 59.06

1

(100); H-NMR (CDCl₃) δ 0.70–0.92 (m, 2H, H-2’b,

H-6’b), 0.97–1.29 (m, 5H, H-7’b, H-3’b, H-4’b, H-5’b,

H-1’), 1.33–1.46 (m, 1H, H-3b), 1.51–1.75 (m, 7H,

H-2’a*, H-7’a, H-6’a*, H-3’a, H-4’a, H-5’a, H-4b),

1.86–2.25 (m, 5H, H-4a, H-5b, H-2, H-3a, H-5a); ¹³C-

NMR (CDCl₃) δ 20.54 (C-4), 25.99 (C-3’^#), 26.11

(C-5’^#), 26.35 (C-4’), 30.00 (C-3), 32.12 (C-2’*), 33.98

(C-6’*), 35.48 (C-1’), 37.27 (C-7’), 37.74 (C-5), 46.60

(C-2), 221.61 (C-1); IR (NaCl, cm^–1) 1 739 (C=O).

1

(α-CH), 116.17 (q, J_{C, F}= 294 Hz, CF₃), 127.34 (C-2″,

C-6″), 128.63 (C-3″, C-5″), 128.67 (C-4″), 135.89 (C-1″),

161.35 (q, ²J_{C, F}= 35 Hz, F₃C–COO^–). (1R, 2R)-3c TFA

anal. C₂₁H₃₀F₃NO₂(H, N); C: calcd., 65.43; found 64.80;

(1S, 2S)-3c TFA anal. C₂₁H₃₀F₃NO₂(C, H, N).

5.1.3. Preparation

of 2-substituted cyclopentaneimines 2a–f

The 2-substituted cyclopentaneimines 2a–f were pre-

pared according to a common procedure [16]. Toluene

was used as solvent. The optimized reaction times and

yields of imine condensation are: 25 h, 88% for 1c, 22 h,

80% for 1e and 14 h, 90% for 1f. The yields were

determined by ¹H-NMR (α-H-signal imine 4.48 and 4.43

ppm/PEA 4.1 ppm). For the complete NMR data see the

supporting information.

5.1.4.2. cis-2-Cyclopentyl-N-(1-phenylethyl)-

cyclopentaneamine hydrobromide 3e HBr

(1R, 2R)-3e HBr: m.p. 165–167 °C, ␣ ²⁵+1.4°; (1S,

͓ ͔

D

25

1

2S)-3e HBr: m.p. 164–165 °C,

␣

–1.7°; H-NMR

͓ ͔

(CDCl₃) δ 0.96–1.18 (m, 2H, H-2’b,^DH-5’b), 1.48–1.75

(m, 8H, H-4b, 3’-CH₂, 4’-CH₂, H-3b, H-2’a*, H-2), 1.80

(mc, 1H, H-5b), 1.92–2.13 (m, 3H, H-5’a*, H-4a, H-3a),

2.09 (d, 3H, 6.9, ꢀ-CH₃), 2.26–2.46 (m, 2H, H-5a, H-1’),

3.17 (s (br), 1H, H-1), 4.55 (s (br), 1H, α-CH), 7.36 (tt,

1H, 7.2, 1.3, H-4″), 7.43 (dt, 2H, 7.0, 1.8, H-3″, H-5″),

7.81 (td, 2H, 6.9, 1.5, H-2″, H-6″), 8.49 (s (br), 1H,

5.1.4. Preparation of cis-2-substituted

N-(1-phenylethyl)-cyclopentaneamine halides 3a–f HX

The cis-2-substituted N-(1-phenylethyl)-cyclopent-

aneamine halides 3a–f HX were prepared according to a

common procedure [16]. The optimized hydrogenation

conditions and yields (calculated from ketones over both

steps) are 11 days hydrogenation time, 73% (oil) for 2c,

11 days hydrogenation time, 54% for 2e and 5 days

hydrogenation time, 60% for 2f with 1.9 g Raney-nickel/

10 mmol 2-substituted cyclopentaneimines 2c, 2e and 2f,

respectively.

+

⁺NH-b), 9.27 (s (br), 1H, NH-a); ¹³C-NMR (CDCl₃) δ

20.41 (ꢀ-CH₃), 21.08 (C-4), 25.08 (C-4’^#), 25.10 (C-3’^#),

27.22 (C-3), 30.39 (C-5), 31.89 (C-5’*), 32.77 (C-3’*),

38.00 (C-1’), 49.38 (C-2), 59.98 (α-CH), 60.91 (C-1),

128.48 (C-2″, C-6″), 129.18 (C-4″), 129.25 (C-3″, C-5″),

136.34 (C-1″). Anal. C₁₈H₂₈NBr (C, H, N).

5.1.4.3. cis-2-Cyclohexylmethyl-N-(1-phenylethyl)-

cyclopentaneamine hydrobromide 3f HBr

The ice-cooled solution of the hydrogenation product

of 2c, 2e and 2f in ethanol was acidiﬁed with hydrobro-

mic acid 48%. The volume of the solution was reduced to

(1R, 2R)-3f HBr: m.p. 240–241 °C decomposition,

␣

͓ ͔

²⁵+54.9°; (1S, 2S)-3f HBr: m.p. 241–242 °C decom-

D

386

25

1

(C-5), 32.26 (C-5’*), 32.32 (C-2’*), 39.99 (C-1’), 50.14

(C-2), 56.06 (C-1). Anal. C₁₀H₂₀NBr (C, H, N).

position,

␣

͓ ͔

–55.0°; H-NMR (CDCl₃) δ 0.86–1.00

(m, 1H, H-6’b^D*), 1.06–1.32 (m, 5H, H-2’b*, H-1’, H-3’b,

H-4’b, H-5’b), 1.36–1.53 (m, 4H, H-4b, 7’-CH₂, H-3b),

1.58–1.80 (m, 6H, H-3a, H-3’a, H-4’a, H-5’a, H-2’a^#,

H-4a), 1.80–1.92 (m, 1H, H-6’a^#), 1.99–2.30 (m, 3H,

H-2, 5-CH₂), 2.03 (d, 3H, 6.9, ꢀ-CH₃), 2.99 (quint, 1H,

7.0, H-1), 4.30 (quint, 1H, 6.5, α-CH), 7.31–7.46 (m, 3H,

H-3″, H-4″, H-5″), 7.75 (dt, 2H, 7.4, 1.4, H-2″, H-6″),

9.11 (s (br), 1H, ⁺NH-b), 9.66 (s (br), 1H, ⁺NH-a);

¹³C-NMR (CDCl₃) δ 19.80 (C-4), 21.32 (ꢀ-CH₃), 26.15

(C-3’^#), 26.29 (C-5’^#), 26.50 (C-4’), 27.12 (C-5), 27.64

(C-3), 32.23 (C-6’*), 34.54 (C-2’*), 35.21 (C-7’), 35.37

(C-1’), 35.97 (C-2), 58.88 (α-C), 59.80 (C-1), 128.19

(C-2″, C-6″), 129.10 (C-4″), 129.25 (C-3″, C-5″), 136.25

(C-1″). Anal. C₂₀H₃₂NBr (C, H, N).

5.1.5.3. cis-2-Cyclohexylmethylcyclopentaneamine

hydrobromide 4f HBr

(1R, 2R)-4f HBr: m.p. 212–213 °C decomposition,

25

D

␣

͓ ͔

+6.5°; (1S, 2S)-4f HBr: m.p. 213–214 °C decom-

25

position, ␣ –7.3°; ¹H-NMR (CDCl₃) δ 0.77–1.01 (m,

͓ ͔

D

2H, H-6’b, H-2’b), 1.03–1.40 (m, 5H, H-3’b, H-4’b,

H-5’b, H-1’, H-7’b), 1.51 (mc, 1H, H-7’a), 1.55–1.73 (m,

6H, H-3’a, H-4’a, H-5’a, H-3b, H-4b, H-2’a*), 1.73–1.90

(m, 2H, H-6’a*, H-3a), 1.90–2.14 (m, 4H, H-4a, 5-CH₂,

H-2), 3.60 (mc, 1H, H-1), 7.92 (s (br), 3H, ⁺NH₃);

¹³C-NMR (CDCl₃) δ 21.60 (C-4), 26.09 (C-3’*), 26.19

(C-5’*), 26.54 (C-4’), 28.72 (C-3), 30.80 (C-5), 32.79

(C-2’^#), 33.74 (C-6’^#), 35.92 (C-1’), 37.04 (C-7’), 40.02

(C-2), 56.22 (C-1). Anal. C₁₂H₂₄NBr (C, H, N).

5.1.5. Preparation of cis-2-substituted

cyclopentaneamine halides 4a–f HX

The cis-2-substituted cyclopentaneamine halides 4a–f

were prepared according to a common procedure [16].

The optimized hydrogenolysis conditions and yields are:

0.8 g Pd/C / 10 mmol 3c HBr, 24 h, 61% 4c HBr, 1.0 g

Pd/C / 10 mmol 3e HBr, 24 h, 93% 4e HBr and 0.9 g

Pd/C / 10 mmol 3f HBr, 24 h, 81% 4f HBr.

5.1.6. Preparation of 2-[(cis-2-substituted

cyclopentyl)imino]hexahydroazepine halides 5a–f HX

and 3-[(cis-2-substituted cyclopentyl)imino]-2-

azabicyclo[2.2.2]octane halides 6a–f HX

Five mmol of the primary cis-2-substituted cyclopen-

taneamine halide 4a–f HX in 1 mL ethanol abs and

5.5 mmol of O-methylcaprolactim (0.7 g) and 3-ethoxy-

2-azabicyclo[2.2.2]oct-2-ene (0.84 g), respectively, were

carefully dispersed in an ultrasonic device. The reaction

mixture was stirred at room temperature. The progress of

the lethargic reaction [13] was observed by TLC (ethyl

acetate/ethanol/ammonia conc.: 90/10/1, ninhydrin re-

agent). When necessary, further ethanol abs was added to

keep the suspension liquid and further lactimether was

added to achieve complete turnover. Reaction times and

yields are listed in table I. The reaction mixture was then

resolved in methanol, ﬁltered and the ﬁltrate was evapo-

rated. The white residues were recrystallized from

methanol/acetone to give the semicyclic amidines 5a–e

HX and 6a–f HX. Analytically pure 5f HBr was collected

as residue after extraction of the raw 5f HBr with ethyl

acetate.

5.1.5.1. cis-2-Cyclohexylcyclopentaneamine

hydrobromide 4c HBr

(1R, 2R)-4c HBr: m.p. 275–278 °C decomposition,

␣

²⁵–16.5°; (1S, 2S)-4c HBr: m.p. 277–280 °C decom-

͓ ͔

D

25

1

position,

␣

+16.1°; H-NMR (CDCl₃) δ 0.88 (mc,

͓ ͔

D

2H, H-2’b, H-6’b), 1.02–1.30 (m, 2H, H-3’b, H-5’b),

1.35 (mc, 1H, H-1’), 1.43–1.71 (m, 6H, H-2, H-3’a,

H-5’a, H-4’b, H-4b, H-3b), 1.71–1.83 (m, 2H, H-3b,

H-6’a*), 1.83–2.05 (m, 4H, H-5b, H-3a, H-2’a*, H-4a),

2.11–2.23 (m, 1H, H-5a), 3.71 (s (br), 1H, H-1), 7.86 (s

(br), 3H, ⁺NH₃); ¹³C-NMR (CDCl₃) δ 20.70 (C-4), 25.45

(C-4’), 26.04 (C-3’^#), 26.07 (C-3), 26.14 (C-5’^#), 30.67

(C-5), 32.25 (C-2’*), 32.65 (C-6’*), 36.88 (C-1’), 50.44

(C-2), 54.53 (C-1); C-3, C-3’, C-4’, C-5’ were assigned

using lr INEPT spectroscopy. Anal. C₁₁H₂₂NBr (C, H,

N).

5.1.5.2. cis-2-Cyclopentylcyclopentaneamine

hydrobromide 4e HBr

5.1.6.1. 2-[(cis-2-Isopropylcyclopentyl)-

imino]hexahydroazepine hydrochloride 5a HCl

(1R, 2R)-4e HBr: m.p. 266–267 °C decomposition,

²⁵–21.9°; (1S, 2S)-4e HBr: m.p. 270–271 °C decom-

(1’R, 2’R)-5a HCl: m.p. 256–258 °C decomposition,

␣

͓ ͔

D

25

D

25

1

␣

͓ ͔

–122.8°; (1’S, 2’S)-5a HCl: m.p. 259–263 °C

position,

␣

+21.6°; H-NMR (CDCl₃) δ 0.95–1.23

͓ ͔

D

(m, 2H, H-5’b, H-2’b), 1.42–2.04 (m, 12H, 3’-CH₂,

4’-CH₂, H-2, H-4b, H-1’, 3-CH₂, H-2’b*, H-5b, H-4a),

2.07–2.26 (m, 2H, H-5a, H-5’b*), 3.65 (mc, 1H, H-1),

7.95 (s (br), 3H, ⁺NH₃); ¹³C-NMR (CDCl₃) δ 21.24

(C-4), 24.81 (C-4’^#), 25.13 (C-3’^#), 27.65 (C-3), 30.85

decomposition, ͓␣͔²⁵+115.9°; ¹H-NMR (CDCl₃) δ 0.81

(d, 3H (Z), 6.4, 3″-C^DH₃*), 0.85 (d, 3H (E), 6.4, 3″-CH₃*),

0.87 (d, 3H (Z), 6.4, 2″-CH₃*), 0.90 (d, 3H (E), 6.4,

2″-CH₃*), 1.40–2.05 (m, 14H, H-4’b, H-2’, 6-CH₂,

H-3’b, H-5’b, 4-CH₂, 5-CH₂, H-4’a, H-1″, H-3’a, H-5’a),

387

2.66 (mc, 2H (E), 3-CH₂), 2.91 (dd, 1H (Z), 14.0, 7.7,

H-3b), 3.19 (dd, 1H (Z), 14.0, 9.6, H-3a), 3.32–3.39 (m,

2H (E), 7-CH₂), 3.39–3.51 (m, 1H (Z), H-7b), 3.51–3.64

(m, 1H (Z), H-7a), 3.94 (mc, 1H (E), H-1’), 4.40 (mc, 1H

(Z), H-1’), 9.30 (d, 1H (Z), 9.3, NH_exo), 9.68 (d, 1H (E),

9.3, NH_exo), 9.72 (t, 1H (Z), 5.6, NH_endo), 10.43 (t, 1H

(E), 5.6, NH_endo); ¹³C-NMR (CDCl₃) δ Z-5a HCl: 22.03

(C-2″, C-3″), 22.10 (C-4’), 23.97 (C-4), 27.68 (C-3’),

27.76 (C-1″), 28.29 (C-6), 29.70 (C-5), 31.16 (C-3),

33.15 (C-5’), 43.59 (C-7), 51.61 (C-2’), 55.80 (C-1’),

168.02 (C-2); E-5a HCl: 21.03 (C-4’), 21.75 (C-3″*),

21.83 (C-2″*), 22.95 (C-4), 26.37 (C-3), 27.58 (C-3’),

27.89 (C-1″), 28.29 (C-6), 29.70 (C-5), 34.74 (C-5’),

43.74 (C-7), 52.16 (C-2’), 57.37 (C-1’), 171.46 (C-2); IR

(KBr, cm^–1) 3 434 (ν (NH)), 1 641 (ν (C=N)). Anal.

C₁₄H₂₇N₂Cl (C, H, N).

1.03–1.31 (m, 3H, H-3″b, H-4″b, H-5″b), 1.42–2.07 (m,

19H, H-6b, H-4’b, H-2″a, H-1″, H-4b, H-4″a, H-5b,

H-3″a, H-5″a, H-2’, H-5’b, H-6a, H-4a, H-6″a, H-3’b,

H-4’a, H-5a, H-3’a, H-5’a), 2.66 (mc, 2H (E), 3-CH₂),

2.90 (dd, 1H (Z), 13.8, 10.1, H-3b), 3.24 (dd, 1H (Z),

13.8, 7.9, H-3a), 3.38 (mc, 2H (E), 7-CH₂), 3.46 (mc, 1H

(Z), H-7b), 3.61 (mc, 1H (Z), H-7a), 3.99 (s (br), 1H (E),

H-1’), 4.47 (s (br), 1H (Z), H-1’), 8.64 (d, 1H (Z), 7.3,

+

⁺NH_exo), 9.09 (d, 1H (E), 7.3, NH_exo), 9.20 (s (br), 1H

(Z), NH_endo), 10.06 (s (br), 1H (E), NH_endo); ¹³C-NMR

(CDCl₃) δ Z-5c HBr: 21.89 (C-4’), 23.99 (C-4), 25.91

(C-3″*), 26.00 (C-5″*), 26.36 (C-4″), 27.25 (C-3’), 28.23

(C-6), 29.66 (C-5), 31.71 (C-3), 32.26 (C-2″^#), 32.56

(C-6″^#), 32.89 (C-5’), 37.20 (C-1″), 43.61 (C-7), 50.25

(C-2’), 55.63 (C-1’), 168.10 (C-2); E-5c HBr: 20.80

(C-4’), 23.03 (C-4), 25.69 (C-3″*), 25.73 (C-5″*), 26.23

(C-4″), 26.66 (C-3), 27.61 (C-3’), 28.23 (C-6), 29.66

(C-5), 32.13 (C-2″^#), 32.34 (C-6″^#), 34.58 (C-5’), 36.90

(C-1″), 43.83 (C-7), 50.74 (C-2’), 57.06 (C-1’), 171.44

(C-2); IR (KBr, cm^–1) 3 432 (ν (NH)), 1 643 (ν (C=N)).

Anal. C₁₇H₃₁N₂Br (C, H, N).

5.1.6.2. 2-[(cis-2-Phenylcyclopentyl)-

imino]hexahydroazepine hydrochloride 5b HCl

25

(1’R, 2’R)-5b HCl: m.p. 184–185 °C, ␣

͓ ͔

+113.4°;

–114.1°;

D

25

(1’S, 2’S)-5b HCl: m.p. 182–184 °C,

␣

͓ ͔

D

¹H-NMR (CDCl₃) δ 0.82 (mc, 1H, H-4b), 0.99 (mc, 1H,

H-6b), 1.11–1.67 (m, 5H, H-5b, H-6a, H-4a, H-5a,

H-4’b), 1.98–2.12 (m, 2H, H-4’a, H-3’b), 2.13–2.23 (m,

1H, H-3’a), 2.28 (mc, 2H (Z) + 1H (E), 5’-CH₂(Z), H-3b

(E)), 2.43 (dd, 1H (Z), 14.2, 11.2, H-3b), 2.82 (dd, 1H

(Z), 14.2, 7.8, H-3a), 3.05–3.18 (m, 1H (Z) + 2H (E),

H-7b (Z + E), H-2’ (E)), 3.18–3.25 (m, 2H (E), H-3a,

H-7a), 3.25–3.37 (m, 1H (Z), H-7a), 3.69 (ddd, 1H (Z),

J_{2’-H/3’a-H}= 11.5, J_{2’-H/3’-b}= J_{2’-H/1’-H}= 7.5, H-2’), 4.04

(mc, 1H (E), H-1’), 4.85 (mc, 1H (Z), H-1’), 7.18 (tt, 1H

(Z), 7.1, 1.2, H-4″), 7.24–7.34 (m, 2H (Z) + 5H (E), H-3″,

H-5″ (Z), H-2″, H-3″, H-4″, H-5″, H-6″ (E)), 7.59 (td, 2H

(Z), 7.1, 1.2, H-2″, H-6″), 9.29 (t, 1H (Z), 5.0, NH_endo),

9.59 (d, 1H (Z), 8.8, NH_exo), 10.06 (s (br), 1H (E),

NH_exo), 10.09 (s (br), 1H (E), NH_endo); ¹³C-NMR (CDCl₃)

δ Z-5b HCl: 22.83 (C-4), 24.33 (C-4’), 27.08 (C-6),

29.37 (C-5), 31.10 (C-3), 31.91 (C-5’), 32.29 (C-3’),

43.26 (C-7), 49.58 (C-2’), 56.07 (C-1’), 126.19 (C-4″),

127.54 (C-3″, C-5″), 129.92 (C-2″, C-6″), 139.81 (C-1″),

167.08 (C-2); E-5b HCl: 21.94 (C-4), 24.33 (C-4’), 25.83

(C-3), 27.34 (C-6), 28.62 (C-5), 31.63 (C-3’), 31.91

(C-5’), 43.66 (C-7), 51.88 (C-2’), 59.65 (C-1’), 127.10

(C-4″), 128.38 (C-3″, C-5″), 129.16 (C-2″, C-6″), 138.58

(C-1″), 171.77 (C-2); IR (KBr, cm^–1) 3 430 (ν (NH)),

1 650 (ν (C=N)). Anal. C₁₇H₂₅N₂Cl (C, H, N).

5.1.6.4. 2-[(cis-2-Benzylcyclopentyl)-

imino]hexahydroazepine hydrochloride 5d HCl

25

(1’R, 2’R)-5d HCl: m.p. 164–166 °C,

(1’S, 2’S)-5d HCl: m.p. 164–166 °C,

␣

͓ ͔

+30.4°;

–30.3°;

͓ ͔

D

25

D

¹H-NMR (CDCl₃) δ 1.33–1.69 (m, 9H, H-4’b, 6-CH₂,

4-CH₂, 5-CH₂, 3’-CH₂), 1.77 (mc, 1H, H-4’a), 2.02 (mc,

2H, 5’-CH₂), 2.35 (m, 1H (E), H-2’), 2.51 (part B, 1H,

J

_BA= 13.0, J_BX= 9.6, H-7″b), 2.63 (mc, part X, 1H (Z),

H-2’), 2.72 (mc, 2H, 3-CH₂), 2.87 (part A, 1H, J_AB

=

13.0, J_AX= 5.4, H-7″a), 3.27 (mc, 2H (E), 7-CH₂), 3.36

(s (br), 2H (Z), 7-CH₂), 3.55 (s (br), 1H (E), H-1’), 4.39

(mc, 1H (Z), H-1’), 7.02–7.09 (m, 1H, H-4″), 7.10–7.19

(m, 4H, H-2″, H-3″, H-5″, H-6″), 9.60 (s (br), 1H (Z),

NH_endo), 9.75 (d, 1H (Z + E), 6.4, NH_exo), 10.08 (s (br),

1H (E), NH_endo); ¹³C-NMR (CDCl₃) δ Z-5d HCl: 21.92

(C-4’), 23.93 (C-4), 28.16 (C-6), 29.75 (C-5), 29.85

(C-3’), 30.93 (C-5’), 31.41 (C-3), 35.45 (C-7″), 43.16

(C-2’), 43.71 (C-7), 56.44 (C-1’), 125.55 (C-4″), 128.04

(C-2″, C-6″), 128.83 (C-3″, C-5″), 141.10 (C-1″), 168.18

(C-2); E-5d HCl: 21.65 (C-4’), 22.19 (C-4), 26.15 (C-3),

27.67 (C-6), 29.42 (C-5), 29.59 (C-3’), 33.56 (C-5’),

35.45 (C-7″), 43.71 (C-7), 45.19 (C-2’), 57.36 (C-1’),

126.03 (C-4″), 128.35 (C-2″, C-6″), 128.60 (C-3″, C-5″),

140.33 (C-1″), 171.49 (C-2); IR (KBr, cm^–1) 3 436 (ν

(NH)), 1 644 (ν (C=N)). Anal. C₁₈H₂₇N₂Cl (C, H, N).

5.1.6.3. 2-[(cis-2-Cyclohexylcyclopentyl)-

imino]hexahydroazepine hydrobromide 5c HBr

5.1.6.5. 2-[(cis-2-Cyclopentylcyclopentyl)-

imino]hexahydroazepine hydrobromide 5e HBr

25

(1’R, 2’R)-5c HBr: m.p. 218–220 °C,

(1’S, 2’S)-5c HBr: m.p. 217–218 °C,

␣

͓ ͔

–75.0°;

+71.8°;

͓ ͔

D

25

(1’R, 2’R)-5e HBr: m.p. 227–229 °C decomposition,

D

25

D

¹H-NMR (CDCl₃) δ 0.88 (mc, 2H, H-2″b, H-6″b),

␣

͓ ͔

–55.4°; (1’S, 2’S)-5e HBr: m.p. 227–229 °C de-

388

25

+55.4°; ¹H-NMR (CDCl₃)

δ

5.1.6.7. 3-[(cis-2-Isopropylcyclopentyl)imino]-

2-azabicyclo[2.2.2]octane hydrochloride 6a HCl

composition,

␣

͓ ͔

1.00–1.22 (m, 2H, ^DH-2″b, H-5″b), 1.37–2.12 (m, 20H,

3″-CH₂, 4″-CH₂, H-4’b, H-4b, H-5″a, 6-CH₂, H-5b,

H-2″a, H-3’b, H-4a, H-2’, H-4’a, H-5’b, H-5a, H-3’a,

H-1″, H-5’a), 2.64 (mc, 2H (E), 3-CH₂), 2.92 (dd, 1H (Z),

14.2, 9.3, H-3b), 3.15 (dd, 1H (Z), 14.3, 7.9, H-3a), 3.37

(dd, 2H (E), 10.0, 5.4, 7-CH₂), 3.42–3.65 (m, 2H (Z),

7-CH₂), 3.92 (mc, 1H (E), H-1’), 4.44 (mc, 1H (Z), H-1’),

8.76 (d, 1H (Z), 9.3, ⁺NH_exo), 9.16 (t, 1H (Z), 5.0,

NH_endo), 9.23 (d, 1H (E), 8.3, ⁺NH_exo), 10.03 (s (br), 1H

(E), NH_endo); ¹³C-NMR (CDCl₃) δ Z-5e HBr: 22.38

(C-4’), 23.92 (C-4), 25.27 (C-4″^#), 25.40 (C-3″^#), 28.28

(C-6), 29.13 (C-3’), 29.74 (C-5), 31.85 (C-5″*), 31.83

(C-3), 32.40 (C-2″*), 32.66 (C-5’), 39.79 (C-1″), 43.72

(C-7), 49.88 (C-2’), 56.85 (C-1’), 168.01 (C-2); E-5e

HBr: 21.45 (C-4’), 23.04 (C-4), 25.11 (C-4″^#), 25.14

(C-3″^#), 26.69 (C-3), 27.71 (C-6), 28.12 (C-3’), 29.74

(C-5), 31.74 (C-5″*), 32.26 (C-2″*), 34.46 (C-5’), 39.94

(C-1″), 43.87 (C-7), 50.90 (C-2’), 58.60 (C-1’), 171.37

(C-2); IR (KBr, cm^–1) 3 433 (ν (NH)), 1 644 (ν (C=N)).

Anal. C₁₆H₂₉N₂Br (C, H, N).

(1’R, 2’R)-6a HCl: m.p. 229–231 °C decomposition,

25

D

␣

͓ ͔

–103.3°; (1’S, 2’S)-6a HCl: m.p. 226–228 °C

25

decomposition, ␣ +107.5°; ¹H-NMR (CDCl₃) δ 0.84

͓ ͔

(d, 3H (Z), 6.6, 3″-C^DH₃*), 0.86 (d, 3H (E), 6.8, 3″-CH₃*),

0.90 (d, 3H (Z), 6.4, 2″-CH₃*), 0.93 (d, 3H (E), 6.4,

2″-CH₃*), 1.48–2.10 (m, 16H, H-4’b, H-2’, H-8a^#, H-5’b,

#

5-CH₂, 6-CH₂, 7-CH₂, H-8b^#, 3’-CH₂, H-4’a, H-1″,

H-5’a), 3.14 (mc, 1H (E), H-4), 3.80 (s (br), 1H (Z) + 1H

(E), H-4 (Z), H-1 (E)), 3.99 (mc, 1H (E), H-1’), 4.00 (s

(br), 1H (Z), H-1), 4.37 (mc, 1H (Z), H-1’), 9.50 (d, 1H

(Z), 9.3, NH_exo), 9.77 (d, 1H (E), 9.0, NH_exo), 10.35 (s

(br), 1H (E), NH_endo), 10.52 (d, 1H (Z), 6.0, NH_endo);

¹³C-NMR (CDCl₃) δ Z-6a HCl: 22.01 (3″-CH₃*), 22.06

(C-4’), 22.09 (2″-CH₃*), 22.87 (C-5^#), 23.54 (C-8^#),

26.78 (C-6, C-7), 27.77 (C-3’), 28.06 (C-1″), 31.33 (C-4),

33.19 (C-5’), 45.97 (C-1), 51.78 (C-2’), 55.42 (C-1’),

167.66 (C-3), E-6a HCl: 21.02 (C-4’), 21.74 (3″-CH₃*),

21.83 (2″-CH₃*), 23.02 (C-5^#), 23.07 (C-8^#), 25.91

(C-7^§), 26.35 (C-6^§), 26.99 (C-3’), 28.06 (C-1″), 28.36

(C-4), 34.81 (C-5’), 46.32 (C-1), 52.08 (C-2’), 56.54

(C-1’), 170.52 (C-3); IR (KBr, cm^–1) 3 435 (ν (NH)),

1 665 (ν (C=N)). Anal. C₁₅H₂₇N₂Cl (C, H, N).

5.1.6.6. 2-[(cis-2-Cyclohexylmethylcyclopentyl)-

imino]hexahydroazepine hydrobromide 5f HBr

(1’R, 2’R)-5f HBr: m.p. 217–218 °C, (1’R, 2’R)-5f:

25

D

␣

͓ ͔

+27.3° (free base); (1’S, 2’S)-5f HBr: m.p.

25

D

1

216–217 °C, ␣

͓ ͔

–41.0°; 5f HCl: H-NMR (CDCl₃) δ

5.1.6.8. 3-[(cis-2-Phenylcyclopentyl)imino]-

0.66–0.90 (m, 2H, H-2″b, H-6″b), 0.98–1.28 (m, 6H,

H-3″b, H-4″b, H-5″b, H-1″, 7″-CH₂), 1.36–1.89 (m, 16H,

H-4’b, H-5b, H-3″a, H-4″a, H-5″a, H-3’b, 4-CH₂, 6-CH₂,

H-2″a, H-6″a, H-5a, H-3’a, H-4’a, H-5’b), 1.96 (mc, 1H,

H-5’a), 2.19–2.32 (m, 1H, H-2’), 2.58–2.65 (m, 2H (E),

3-CH₂), 2.73–2.86 (m, 1H (Z), H-3b), 3.04–3.17 (m, 1H

(Z), H-3a), 3.32–3.47 (m, 1H (Z) + 2H (E), H-7b (Z),

2-azabicyclo[2.2.2]octane hydrochloride 6b HCl

(1’R, 2’R)-6b HCl: m.p. 255–257 °C decomposition,

25

D

␣

+96.3°; (1’S, 2’S)-6b HCl: m.p. 256–258 °C

͓ ͔

25

1

decomposition, ␣

͓ ͔

–94.2°; H-NMR (CDCl₃) δ 0.72

(mc, 1H, H-8b^#), 0.^D92 (mc, 1H, H-7b*), 1.34 (mc, 2H,

H-8a^#, H-7a*), 1.46–1.72 (m, 5H, 5-CH₂, 6-CH₂, H-4’b),

1.98–2.16 (m, 2H, H-3’b, H-4’a), 2.16–2.41 (m, 3H,

H-3’a, 5’-CH₂), 2.55 (s (br), 1H (E), H-4), 3.16 (mc, 1H

(E), H-2’), 3.26 (s (br), 1H (Z), H-4), 3.65 (ddd, 1H (Z),

11.7, 7.5, 7.5, H-2’), 3.72 (s (br), 1H (Z + E), H-1), 4.04

(mc, 1H (E), H-1’), 4.78 (mc, 1H (Z), H-1’), 7.12–7.37

(m, 3H (Z) + 5H (E), H-3″, H-4″, H-5″ (Z), H -2″, H-3″,

H-4″, H-5″, H-6″ (E)), 7.62 (d, 2H (Z), 7.1, H-2″, H-6″),

9.84 (d, 1H (Z), 9.3, NH_exo), 9.92 (d, 1H (E), 3.8,

NH_endo), 10.05 (d, 1H (E), 10.6, NH_exo), 10.16 (d, 1H (Z),

3.8, NH_endo); ¹³C-NMR (CDCl₃) δ Z-6b HCl: 22.52

(C-8^#), 23.07 (C-5^#), 24.25 (C-4’), 26.28 (C-7*), 26.37

(C-6*), 31.19 (C-4), 32.00 (C-3’), 32.08 (C-5’), 45.51

(C-1), 49.86 (C-2’), 55.73 (C-1’), 126.24 (C-4″), 127.54

(C-3″, C-5″), 129.93 (C-2″, C-6″), 139.84 (C-1″), 166.74

(C-3), E-6b HCl: 21.91 (C-8^#), 24.25 (C-4’), 26.05

(C-7*), 26.20 (C-6*), 28.62 (C-4), 30.95 (C-3’), 32.64

(C-5’), 46.32 (C-1), 51.83 (C-2’), 59.02 (C-1’), 127.10

(C-4″), 128.44 (C-3″, C-5″), 129.14 (C-2″, C-6″), 138.74

7-CH (E)), 3.49–3.61 (m, 1H (Z), H-7a), 3.86 (mc, 1H

2

(E), H-1’), 4.31 (mc, 1H (Z), H-1’), 9.44 (d, 1H (Z), 8.6,

+

⁺NH_exo), 9.53 (d, 1H (E), 8.6, NH_exo), 9.59, (t, 1H (Z),

5.0, NH_endo), 10.15 (t, 1H (E), 5.0, NH_endo); ¹³C-NMR

(CDCl₃) δ Z-5f HBr: 22.00 (C-4’), 24.11 (C-4), 26.22

(C-3″*), 26.31 (C-5″*), 26.52 (C-4″), 28.32 (C-6), 29.69

(C-3’), 29.83 (C-5), 31.14 (C-5’), 31.41 (C-3), 32.69

(C-2″^#), 34.39 (C-6″^#), 35.97 (C-1″), 36.45 (C-7″), 39.44

(C-2’), 43.66 (C-7), 56.72 (C-1’), 167.93 (C-2); E-5f

HBr: 21.23 (C-4’), 22.93 (C-4), 26.22 (C-3″*), 26.31

(C-5″*), 26.39 (C-4″, C-3), 27.69 (C-6), 28.66 (C-3’),

30.07 (C-5), 32.85 (C-2″^#), 33.31 (C-5’), 34.03 (C-6″^#),

36.06 (C-1″), 37.01 (C-7″), 41.16 (C-2’), 43.81 (C-7),

58.81 (C-1’), 171.39 (C-2); 5f HBr: IR (KBr, cm^–1) 3 440

(ν (NH)), 1 650 (ν (C=N)); (1’R, 2’R)-5f HBr Anal.

C₁₈H₃₃N₂Br (H, N); C: calcd, 60.50; found, 59.67; (1’S,

2’S)-5f HBr Anal. C₁₈H₃₃N₂Br (H, N); C: calcd, 60.50;

found, 59.77.

389

(C-1″), 170.54 (C-3); C-5^#(E) hidden; IR (KBr, cm^–1)

3 435 (ν (NH)), 1 656 (ν (C=N)). Anal. C₁₈H₂₅N₂Cl (C,

H, N).

(C-4), 29.08 (C-3’), 33.87 (C-5’), 35.33 (C-7″), 45.17

(C-2’), 46.39 (C-1), 56.58 (C-1’), 126.06 (C-4″), 128.23

(C-2″, C-6″), 128.42 (C-3″, C-5″), 140.32 (C-1″), 170.61

(C-3); IR (KBr, cm^–1) 3 440 (ν (NH)), 1 642 (ν (C=N)).

Anal. C₁₉H₂₇N₂Cl (C, H, N).

5.1.6.9. 3-[(cis-2-Cyclohexylcyclopentyl)imino]-

2-azabicyclo[2.2.2]octane hydrobromide 6c HBr

(1’R, 2’R)-6c HBr: m.p. 227–228 °C decomposition,

5.1.6.11. 3-[(cis-2-Cyclopentylcyclopentyl)imino]-

2-azabicyclo[2.2.2]octane hydrobromide 6e HBr

25

␣

–59.6°; (1’S, 2’S)-6c HBr: m.p. 225–227 °C de-

͓ ͔

D

25

1

(1’R, 2’R)-6e HBr: m.p. 211–212 °C,

(1’S, 2’S)-6e HBr: m.p. 209–211 °C,

␣

͓ ͔

–47.6°;

+49.2°;

͓ ͔

composition,

␣

͓ ͔

+61.1°; H-NMR (CDCl₃) δ 0.87

D

(mc, 2H, H-2″b, ^DH-6″b), 1.01–1.28 (m, 3H, H-3″b,

H-4″b, H-5″b), 1.38–2.05 (m, 21H, H-1″, H-4’b, H-2″a,

H-8b, H-2’, H-3″a, H-4″a, H-5″a, 5-CH₂, 6-CH₂, 7-CH₂,

H-5’b, H-6″a, H-8a, H-3’b, H-4’a, H-3’a, H-5’a), 3.12 (s

(br), 1H (E), H-4), 3.83 (s (br), 1H (Z) + 1H (E), H-4 (Z),

H-1 (E)), 3.92 (s (br), 1H (E), H-1’), 4.00 (s (br), 1H (Z),

H-1), 4.40 (mc, 1H (Z), H-1’), 8.86 (d, 1H (Z), 9.8,

25

D

¹H-NMR (CDCl₃) δ 1.02–1.23 (m, 2H, H-2″b, H-5″b),

1.36–2.10 (m, 22H, 3″-CH₂, 4″-CH₂, H-4’b, H-5″a*,

#

H-8b^#, 5-CH₂, 6-CH₂, 7-CH₂, H-2″a*, H-5b, H-8a^#,

3’-CH₂, H-2’, H-4’a, H-1″, H-5’a), 3.13 (s (br), 1H (E),

H-4), 3.79 (s (br), 1H (Z) + 1H (E), H-4 (Z), H-1 (E)),

3.94 (mc, 1H (E), H-1’), 4.01 (s (br), 1H (Z), H-1), 4.36

(mc, 1H (Z), H-1’), 8.97 (d, 1H (Z), 9.6, ⁺NH_exo), 9.34 (d,

1H (E), 7.9, ⁺NH_exo), 9.92 (s (br), 1H (Z), NH_endo), 9.97

(s (br), 1H (E), NH_endo); ¹³C-NMR (CDCl₃) δ Z-6e HBr:

22.34 (C-4’), 22.99 (C-5^§), 23.47 (C-8^§), 25.20 (C-4″^#),

25.34 (C-3″^#), 26.76 (C-6, C-7), 29.10 (C-3’), 31.82 (C-4,

C-5″*), 32.36 (C-2″*), 32.67 (C-5’), 40.03 (C-1″), 46.01

(C-1), 50.05 (C-2’), 56.39 (C-1’), 167.56 (C-3); E-6e

HBr: 21.31 (C-4’), 23.08 (C-5, C-8), 25.02 (C-3″, C-4″),

26.06 (C-7^#), 26.37 (C-6^#), 27.90 (C-3’), 28.57 (C-4),

31.67 (C-5″*), 32.12 (C-2″*), 34.55 (C-5’), 40.03 (C-1″),

46.49 (C-1), 50.73 (C-2’), 57.73 (C-1’), 170.43 (C-3); IR

(KBr, cm^–1) 3 444 (ν (NH)), 1 663 (ν (C=N)). Anal.

C₁₇H₂₉N₂Br (C, H, N).

⁺NH_exo), 9.20 (d, 1H (E), 8.8, NH_exo), 9.94 (s (br), 1H

+

(Z), NH_endo), 10.02 (s (br), 1H (E), NH_endo); ¹³C-NMR

(CDCl₃) δ Z-6c HBr: 21.89 (C-4’), 22.76 (C-5⁽), 23.71

(C-8^◆), 25.86 (C-3″^§), 26.05 (C-5″^§), 26.33 (C-4″), 26.68

(C-6^#), 26.78 (C-7^#), 27.30 (C-3’), 31.83 (C-4), 32.24

(C-2″*), 32.61 (C-6″*), 32.79 (C-5’), 37.57 (C-1″), 46.00

(C-1), 50.40 (C-2’), 55.24 (C-1’), 167.69 (C-3); E-6c

HBr: 20.73 (C-4’), 23.04 (C-5^◆), 23.12 (C-8^◆), 25.67

(C-3″^#), 25.71 (C-5″ ^#), 26.19 (C-4″), 28.65 (C-4), 32.07

(C-2″*), 32.37 (C-6″*), 34.39 (C-5’), 37.03 (C-1″), 46.46

(C-1), 50.62 (C-2’), 56.19 (C-1’), 170.46 (C-3); C-6 (E),

C-7 (E), C-3’ (E) hidden; IR (KBr, cm^–1) 3 429 (ν (NH)),

1 657 (ν (C=N)). Anal. C₁₈H₃₁N₂Br (C, H, N).

5.1.6.10. 3-[(cis-2-Benzylcyclopentyl)imino]-

2-azabicyclo[2.2.2]octane hydrochloride 6d HCl

5.1.6.12. 3-[(cis-2-Cyclohexylmethylcyclopentyl)imino]-

2-azabicyclo[2.2.2]octane hydrobromide 6f HBr

25

(1’R, 2’R)-6d HCl: m.p. 244–246 °C decomposition,

(1’R, 2’R)-6f HBr: m.p. 233–234 °C,

(1’S, 2’S)-6f HBr: m.p. 233–234 °C,

␣

͓ ͔

+37.9°;

–37.8°;

͓ ͔

D

25

D

25

␣

͓ ͔

+5.5°; (1’S, 2’S)-6d HCl: m.p. 243–245 °C de-

composition, ␣ –5.5°; ¹H-NMR (CDCl₃) δ 1.32–1.72

¹H-NMR (CDCl₃) δ 0.78–0.89 (m, 2H, H-2″^Db, H-6″b),

1.00–1.32 (m, 6H, H-3″b, H-4″b, H-5″b, H-1″, H-7″b,

H-7″a), 1.49 (mc, 1H, H-4’b), 1.49–1.89 (m, 17H, H-3’b,

H-3″a, H-4″a, H-5″a, H-8b*, H-2″a, H-6″a, 5-CH₂*,

6-CH₂, 7-CH₂, H-8a*, H-3’a, H-4’a, H-5’b), 1.99 (mc,

1H, H-5’a), 2.22 (mc, 1H, H-2’), 3.08 (s (br), 1H (E),

H-4), 3.71 (s(br), 1H (Z), H-4), 3.80 (s (br), 1H (E), H-1),

3.87 (mc, 1H (E), H-1’), 3.99 (s (br), 1H (Z), H-1), 4.27

(mc, 1H (Z), H-1’), 9.03 (d, 1H (Z), 9.3, ⁺NH_exo), 9.26 (d,

25

͓ ͔

D

(m, 11H, H-4’b, H-7b*, 6-CH₂*, H-7a*, 3’-CH₂, 5-CH₂,

8-CH₂), 1.72–1.86 (m, 1H, H-4’a), 1.92 (mc, 1H, H-5’a),

2.03 (mc, 1H, H-5’b), 2.34 (m, 1H (E), H-2’), 2.49 (mc,

part B, 1H (Z + E) + 1H (Z), J_AB= 17.7, H-7″b (Z + E),

H-2’ (Z)), 2.80 (m, 1H (E), H-4), 2.92 (part A, 1H (Z +

E), J_AB= 17.7, H-7″a), 3.63 (s (br), 1H (Z), H-4), 3.74 (s

(br), 2H (E), H-1, H-1’), 3.90 (s (br), 1H (Z), H-1), 4.35

(mc, 1H, H-1’), 7.02–7.09 (m, 1H, H-4″), 7.09–7.21 (m,

4H, H-2″, H-3″, H-5″, H-6″), 9.86 (d, 1H (Z + E), 8.8,

NH_exo), 10.13 (s (br), 1H (E), NH_endo), 10.48 (d, 1H (Z),

4.0, NH_endo); ¹³C-NMR (CDCl₃) δ Z-6d HCl: 22.49

(C-4’), 23.12 (C-8^#), 23.42 (C-5^#), 26.66 (C-7*), 26.75

(C-6*), 29.56 (C-3’), 31.87 (C-5’), 31.49 (C-4), 35.21

(C-7″), 44.10 (C-2’), 46.02 (C-1), 56.05 (C-1’), 125.56

(C-4″), 128.04 (C-2″, C-6″), 128.71 (C-3″, C-5″), 141.08

(C-1″), 167.79 (C-3); E-6d HCl: 21.49 (C-4’), 22.20

(C-8^#), 23.12 (C-5^#), 25.80 (C-7*), 26.47 (C-6*), 28.32

+

1H (E), 9.0, NH_exo), 9.92 (d, 1H (Z + E), 3.7, NH_endo);

¹³C-NMR (CDCl₃) δ Z-6f HBr: 22.09 (C-4’), 23.04

(C-5*), 23.66 (C-8*), 26.28 (C-3″, C-5″), 26.53 (C-4″),

26.69 (C-7^#), 26.84 (C-6^#), 29.79 (C-3’), 31.27 (C-5’),

31.78 (C-4), 32.82 (C-6″ ^§), 34.28 (C-2″ ^§), 35.98 (C-1″),

36.63 (C-7″), 39.88 (C-2’), 45.98 (C-1), 56.62 (C-1’),

167.72 (C-3); E-6f HBr: 21.28 (C-4’), 23.04 (C-5*),

23.66 (C-8*), 26.28 (C-3″, C-5″), 26.40 (C-4″), 26.69

(C-7^#), 26.84 (C-6^#), 28.57 (C-4), 28.72 (C-3’), 32.94

390

(C-6″ ^§), 33.36 (C-5’), 34.02 (C-2″ ^§), 36.11 (C-1″), 37.22

(C-7″), 41.15 (C-2’), 46.52 (C-1), 58.28 (C-1’), 170.53

(C-3); IR (KBr, cm^–1) 3 440 (ν (NH)), 1 657 (ν (C=N)).

Anal. C₁₉H₃₃N₂Br (C, H, N).

H-5b), 1.81–2.00 (m, 3H, H-3a, H-4a, H-5a), 2.00–2.14

(m, 1H, H-2), 3.63 (dt, 1H, 4.9, 1.6, 1-H), 6.85 (dt, 1H,

7.4, 1.1, H-5″), 6.93 (dd, 1H, 8.2, 0.55, H-3″), 7.25 (dt,

1H, 8.2, 1.6, H-4″), 7.29 (dd, 1H, 8.6, 1.6, H-6″), 8.27 (s,

1H, H-α), 13.71 (s, 1H, OH); ¹³C-NMR (CDCl₃) δ 22.39

(C-4), 26.37 (C-3’^#), 26.39 (C-5’^#), 26.65 (C-4’), 29.86

(C-3), 33.23 (C-2’*), 34.06 (C-6’*), 34.36 (C-5), 36.17

(C-1’), 38.09 (C-7’), 42.78 (C-2), 73.04 (C-1), 116.93

(C-3″), 118.31 (C-5″), 118.86 (C-1″), 130.98 (C-4″),

131.82 (C-6″), 161.36 (C-2″), 162.23 (C-α); IR (KBr,

cm^–1) 3 447 (ν (OH)), 1 629 (ν (C=N)). Anal. C₁₉H₂₇NO

(C, H, N).

5.1.7. Preparation of cis-2-substituted

cyclopentaneaminesalicylidenes 7a–f

The cis-2-substituted cyclopentaneaminesalicylidenes

7a–f were prepared according to a common procedure

[16].

5.1.7.1. (1S, 2S)-cis-Cyclohexylcylo-

pentaneaminesalicylidene (1S, 2S)-7c

25

88%; m.p. 105–106°; ␣ +215°; ¹H-NMR (CDCl₃)

5.1.8. Correlation of absolute conﬁguration

͓ ͔

δ 0.81–0.99 (m, 2H, H-6’b,^DH-2’b), 0.99–1.26 (m, 4H,

H-3’b, H-4’b, H-5’b, H-1’), 1.48–1.79 (m, 9H, H-3b,

H-3’a, H-4’a, H-5’a, H-2, H-5b, H-6’a*, H-4b, H-2’a*),

1.82–2.01 (m, 3H, H-4a, H-3a, H-5a), 3.79 (t, 1H, 3.8,

H-1), 6.85 (dt, 1H, 7.5, 1.1, H-5″), 6.94 (d, 1H, 8.2,

H-3″), 7.25 (dt, 1H, 8.3, 1.6, H-4″), 7.29 (dd, 1H, 8.6, 1.8,

H-6″), 8.30 (s, 1H, H-α), 13.87 (s, 1H, OH); ¹³C-NMR

(CDCl₃) δ 21.82 (C-4), 25.89 (C-5’^#), 26.03 (C-3’^#),

26.54 (C-4’), 27.55 (C-3), 31.71 (C-6’*), 32.38 (C-2’*),

34.73 (C-5), 37.89 (C-1’), 52.29 (C-2), 70.71 (C-1),

116.94 (C-3″), 118.31 (C-5″), 118.90 (C-1″), 130.99

(C-4″), 131.85 (C-6″), 161.36 (C-2″), 162.20 (C-α); IR

(KBr, cm^–1) 3 431 (ν (OH)), 1 630 (ν (C=N)). Anal.

C₁₈H₂₅NO (C, H, N).

A solution of the cis-2-substituted cyclopentaneamine-

salicylidenes 7a–f in acetonitrile (c = 10^–3M) was

measured CD-spectroscopically. λ (nm) (θ). (1S, 2S)-7a:

313 (+1 725), 260 (+3 113), 228 (–2 381) [16]; (1S,

2S)-7b: 314 (+6 981), 252 (+12 929), 228 (+7 657) [16];

(1S, 2S)-7c: 312 (+7 276), 259 (+11 415), 225 (–3 763);

(1S, 2S)-7d: 311 (+3 469), 252 (+7 811), 220 (–1 649)

[16]; (1S, 2S)-7e: 311 (+2 864), 255 (+4 077), 225

(–3 664); (1S, 2S)-7f: 312 (+2 878), 256 (+3 747), 226

(–2 235).

5.1.9. Determination of enantiomeric excess

The diastereomeric Mosher amides: cis-2-substituted

cyclopentaneamine-α-methoxy-α-triﬂuoro-methylphenyl-

acetic acid amides (8a–f) and 1-phenylethyl-amine-α-

methoxy-α-triﬂuoromethyl-phenylacetic acid amides (8p)

were prepared according to a common procedure [16].

The residue was directly used for HPLC and ¹⁹F-NMR

spectroscopy.

5.1.7.2. (1S, 2S)-cis-Cyclopentylcylo-

pentaneaminesalicylidene (1S, 2S)-7e

25

D

1

87%; m.p. 84–85°; ␣

͓ ͔

+242°; H-NMR (CDCl₃) δ

1.01–1.19 (m, 2H, H-5’b*, H-2’b*), 1.33–1.48 (m, 2H,

H-3’b, H-4’b), 1.48–1.80 (m, 9H, H-3’a, H-4’a, H-1’,

H-5’a*, H-3b, H-5b, H-2, H-4b, H-2’a*), 1.83–2.00 (m,

3H, H-3a, H-4a, H-5a), 3.69 (t, 1H, 4.1, H-1), 6.85 (dt,

1H, 7.6, 1.1, H-5″), 6.93 (d, 1H, 8.2, H-3″), 7.25 (dt, 1H,

7.9, 1.6, H-4″), 7.29 (dd, 1H, 8.2, 1.6, H-6″), 8.30 (s, 1H,

H-α), 13.77 (s, 1H, OH); ¹³C-NMR (CDCl₃) δ 22.45

(C-4), 25.09 (C-4’^#), 25.21 (C-3’^#), 29.10 (C-3), 30.90

(C-5’*), 32.46 (C-2’*), 34.61 (C-5), 41.03 (C-1’), 53.01

(C-2), 72.58 (C-1), 116.95 (C-3″), 118.34 (C-5C″), 118.87

(C-1″), 130.99 (C-4″), 131.86 (C-6″), 161.38 (C-2″),

162.06 (C-α); IR (KBr, cm^–1) 3 452 (ν (OH)), 1 631 (ν

(C=N)). Anal. C₁₇H₂₃NO (C, H, N).

5.1.9.1. HPLC

Twenty microlitres of a 10–20 mg/mL solution of the

Mosher amides in ethyl acetate were injected on a

Lichrosorbt Si-60 column (5 µm). The ﬂow rate was 2

and 3 mL/min, respectively. The Mosher amides were

detected at 260 nm (λ_max): retention time (min) major

diastereomer/subsidiary diastereomer, α (ee%). (R)-8p

17.06/22.52, α = 1.32 (98.3%); (S)-8p: 21.49/18.76, α =

1.15 (95.8%); (1R, 2R)-8a: 9.73–10.26/–* (> 99.0%);

(1S, 2S)-8a: 12.87–13.40/–* (> 99.0%); (1R, 2R)-8a +

(1S, 2S)-8a^#: 9.87–10.40/13.07, α = 1.26; (1R, 2R)-8b^◆:

23.11/17.68, α = 1.31 (99.9%); (1S, 2S)-8b^◆: 16.80/–*

(> 99.0%); (1R, 2R)-8c: 2.77–3.54/–* (> 99.0%); (1S,

2S)-8c: 4.90/–* (> 99.0%); (1R, 2R)-8c + (1S, 2S)-8c^#:

3.40/4.70, α = 1.38; (1R, 2R)-8d^◆: 12.54/–* (> 99.0%);

(1S, 2S)-8d^◆: 18.19/14.59, α = 1.25 (98.8%); (1R,

2R)-8e: 4.90–6.61/–* (> 99.0%); (1S, 2S)-8e:

7.64–9.53/–* (> 99.0%); (1R, 2R)-8e + (1S, 2S)-8e^#:

5.40–6.50/8.07–8.30, α = 1.24; (1R, 2R)-8f: 5.04–7.14/–*

5.1.7.3. (1S, 2S)-cis-Cyclohexylmethylcylo-

pentaneaminesalicylidene (1S, 2S)-7f

25

D

1

87%; m.p. 66–67°; ␣

͓ ͔

+216°; H-NMR (CDCl₃) δ

0.79–0.92 (m, 2H, H-6’b*, H-2’b*), 1.02–1.29 (m, 6H,

H-7’b, H-7’a, H-3’b, H-4’b, H-5’b, H-1’), 1.43–1.79 (m,

8H, H-3b, H-3’a, H-4’a, H-5’a, H-6’a*, H-2’a*, H-4b,

391

(> 99.0%); (1S, 2S)-8f: 10.00–10.50/–* (> 99.0%); (1R,

2R)-8f + (1S, 2S)-8f^#: 5.91–7.17/9.51–10.90, α = 1.33;

*below detection limit (< 0.5%), ^#artiﬁcial mixture;

^◆3 mL/min.

and ¹²⁵I-labelled insulin (CIS Diagnostik GmbH, Dreie-

ich, Germany).

5.2.3. Patch clamp measurements

For patch clamp measurements, cells (0.2 × 10⁶cells)

were seeded onto glass coverslips coated with poly-L-

Orn (10 mg/L, Sigma, Munich, Germany) and used after

2–5 days of culture in standard culture medium. The

coverslip was mounted on the stage of an inverted

microscope (Axiovert 10, Zeiss, Germany) and perifused

continuously at 37 °C with a solution containing (in

mM): 140 NaCl, 5.6 KCl, 1.2 MgCl₂, 2.6 CaCl₂, 0.5

Glucose and 10.0 Hepes, pH 7.4. A ﬂowing KCl-electrode

served as a reference and appropriate corrections for

liquid junction voltages were made. The patch clamp

pipettes (Clark-Medical, Reading, GB) were pulled auto-

matically (DMZ Universal Puller, Zeitz, Augsburg, Ger-

many) and had an input R of 6–8 MX. They were ﬁlled

with a solution containing in (mM): 30 KCl, 95 potas-

sium gluconate, 1.0 MgCl₂, 1.2 NaH₂PO₄, 4.8 Na₂HPO₄,

5.0 Na₂ATP, 0.5 Na₂GTP, pH 7.15. After a GX seal was

achieved, the standard whole cell conﬁguration was

obtained by sucking of the membrane underneath the

pipette. Membrane voltage (V_m) was measured continu-

ously using the current-clamp-mode of the patch-clamp

ampliﬁer (Fröbe, U. and Busche, R., Institute of Physi-

ology of the University of Freiburg, Germany) and was

displayed by a pen recorder. After pipette capacitance was

compensated, the access conductance (G_a) was measured

as previously described [39]. Whole cell conductance

(G_m) was calculated from the total conductance (G_t)

measured in the voltage clamp mode using the equation

G_m= G_a(G_t/(G_a– G_t).

5.1.9.2. ¹⁹F-NMR

CDCl₃, standard: 1,1,2-trichlorotriﬂuoroethane, δ ma-

jor diastereomer/subsidiary diastereomer (ee%). 8-(R)-

PEA: –64.60/–64.42 (97.9%); 8-(S)-PEA: –64.42/–64.60

(97.0%); (1R, 2R)-8a: –64.72/–* (> 99.0%); (1S, 2S)-8a:

–64.55/–64.71 (97.4%); (1R, 2R)-8b: –65.21/–64.38

(99.0%); (1S, 2S)-8b: –64.38/–* (> 99.0%); (1R, 2R)-8c:

–64.63/–* (> 99.0%); (1S, 2S)-8c: –64.58/–* (> 99.0%);

(1R, 2R)-8d: –64.49/–* (> 99.0%); (1S, 2S)-8d: –64.26/

–64.49 (98.8%); (1R, 2R)-8e: –64.59/–* (> 99.0%); (1S,

2S)-8e: –64.53/–* (> 99.0%); (1R, 2R)-8f: –64.60/–64.40

(99.6%); (1S, 2S)-8f: –64.40/–* (> 99.5%); *below

detection limit (< 0.5%).

5.2. Physiology

5.2.1. Cell culture

The effects of semicyclic amidines on insulin secre-

tion, [Ca²⁺]_iand membrane voltage were tested in the

insulin secreting cell line INS-1 [37]. Cells were cultured

at a cell density of 250 × 10⁶cells/L in RPMI 1640

culture medium (GIBCO BRL, Eggenstein, Germany)

containing 11 mM glucose and supplemented with 50 µM

2-mercaptoethanol, 1 mM sodiumpyruvate, 10 mM

Hepes, 10 0000 IU/L penicillin, 100 mg/L streptomycin

and 100 g/L decomplemented foetal calf serum (Se-

romed, Berlin, Germany). The medium was changed

twice a week. Every week cells were trypsinized and

re-seeded at the same cell density.

5.2.4. [Ca²⁺]_i-Measurements

5.2.2. Insulin secretion

For measurements of insulin secretion cells (0.25 × 10⁶

cells per well) were cultured for 2 days in 24-well dishes.

After washing the cells with a modiﬁed Krebs-Ringer-

hydrogen carbonate (KRB) buffer containing (in mM):

136 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.0 CaCl₂, 1.2 KH₂PO₄,

5.0 NaHCO₃, 0.5 glucose, 10.0 Hepes, pH 7.4 and 5 g/L

BSA (fraction V, Sigma, Munich, Germany), they were

pre-incubated at 37 °C for 30 min in the same buffer and

incubated for another 30 min in buffer containing the

tested compounds as indicated in each experiment. Insu-

lin released into the supernatant and insulin content of the

cells after acidic ethanol (1.5% (v/v) HCl in 75% (v/v)

ethanol) extraction was measured by a radio-immunoassay

as described previously [38] using rat insulin (Novo

Nordisk, Bagsvaerd, Denmark) as standard, rabbit anti-

insulin porcine antiserum (Linco, Biotrend, Germany)

For [Ca²⁺]_imeasurements cells were incubated at room

temperature with 2 µmol/L fura-2 AM for 30 min. The

ﬂuorescence was monitored with a photomultiplier sys-

tem at excitation wavelengths of 340, 360 and 380 nm,

with a ﬁlter rotation rate of 10 per second. As a measure

of [Ca²⁺]_ithe ﬂuorescence emission ratio at 340/380 nm

excitation was calculated after subtraction of the auto-

ﬂuorescence as described previously [40].

5.2.5. Statistical analysis

Data are presented as mean ± SEM. Student’s paired

t-test (for patch clamp data) or Mann-Whitney U- Wil-

coxon Rank Sum W Test (for insulin secretion) (P < 0.05)

was used for statistical analyses.

Supporting information is available (4 pages).

392

[15] Palfreyman M.G., Dudley M.W., Cheng H.C., Mir A.K., Yamada S.,

Roeske W.R., Obata T., Yamamura H.I., Biochem. Pharm. 38 (1989)

2459–2465.

Acknowledgements

S. Hartmann would like to thank the ‘Dr. Hilmer-

Stiftung im Stifterverband für die Deutsche Wissen-

schaft’ for the award of a scholarship. The authors are

indebted to Mrs M. Wagner and Mr V. Brecht for

conscientious CD and NMR measurements. We are

gratefully indebted to the medical students Mrs S. Lehr

and Mr M. Herbst for electrophysiological and [Ca²⁺]_i

measurements. This work was supported by the grant

‘interdisziplinäre Projekte’ of the University of Freiburg

(Germany). Financial and substantial support from ‘Fonds

der Chemischen Industrie’ and Degussa, Frankfurt a. M.

and a generous gift of 3-ethoxy-2-azabicyclo[2.2.2]oct-

2-ene by Prof. Dr W. Schneider, Freiburg (Germany) is

gratefully acknowledged.

[16] Wiehl W., Frahm A.W., Chem. Ber. 119 (1986) 2668–2677.

[17] Wiehl W., Thesis, Bonn 1985.

[18] Cornubert R., Borrel C., Chim. Fr. 47 (1930) 301–322 modiﬁed

according to Wiehl.

[19] Kötz A., Blendermann K., Kárpáti E., Rosenbusch R., Liebigs Ann.

Chem. 400 (1913) 47–51.

[20] Wanzlick H.W., Gollmer G., Milz H., Chem. Ber. 88 (1955) 69–77.

[21] Arnold R.T., Buckley J.S., Dodson R.M., J. Am. Chem. Soc. 72

(1950) 3153–3155.

[22] Forward G.C., Whiting D.A., J. Chem. Soc. C, (1969) 1868–1873.

[23] Stork G., Brizzolara A., Landesman H., Szmuszkovicz J., Terrell R.,

J. Am. Chem. Soc. 85 (1963) 207–222.

[24] Hückel W., Maier M., Jordan E., Seeger W., Liebigs Ann. Chem. 616

(1958) 46–47.

[25] Mayer R., Chem. Ber. 89 (1965) 1443–1454.

[26] Stocker J.H., J. Org. Chem. 27 (1962) 2288–2289.

[27] Cheng Y.S., Liu W.L., Chen S.H., Synthesis (1980) 223–224.

[28] Pearson D.E., Keaton O.D., J. Org. Chem. 28 (1963) 1557–1558.

References

[1] Castañer J., Arrigoni-Martelli E., Drugs Future 1 (1976) 119–122.

[2] Schillinger E., Biochem. Pharmacol. 27 (1978) 651–653.

[29] Hartmann S., Brecht V., Frahm A.W., Magn. Reson. Chem. 37

(1999) 69–72.

[3] Sturgess N.C., Ashford M.L.J., Cook D.L., Hales C.N., Lancet 2

[30] Hartmann S., Weckert E., Frahm A.W., Acta Cryst. C 55 (1999)

(1985) 474–475.

806–808.

[4] Rufer C., Biere H., Ahrens H., Loge O., Schröder E., J. Med. Chem.

17 (1974) 708–721.

[31] Seebach D., Prelog V., Angew. Chem. 94 (1982) 696–702.

[32] Dale J.A., Dull D.L., Mosher H.S., J. Org. Chem. 34 (1969)

[5] Graul A., Castañer J., Drugs Future 21 (1996) 694–699.

2543–2549.

[6] Fuhlendorff J., Shymko R., Carr R.D., Kofod H., Diabetes 44

(Suppl. 1) (1995) Abstr. 848.

[33] Dale J.A., Mosher H.S., J. Am. Chem. Soc. 90 (1968) 3732–3738.

[34] Ullrich S., Abel K.B., Lehr S., Greger R., Pﬂügers Arch. 432 (1996)

630–636.

[7] Bloom J.D., Dutia M.D., Johnson B.D., J. Med. Chem. 35 (1992)

3081–3084.

[35] Aguilar-Bryan L., Nichols C.G., Wechsler S.W., Clement J.P., Boyd

[8] Bloom J.D., Claus T.H., Drugs Future 19 (1994) 23–26.

A.E., González G. et al., Science 268 (1995) 423–426.

[9] Emorine L.J., Marullo S., Briend-Sutren M., Patey G., Tate K.,

Delavier-Klutchko C., Strosberg A.D., Science 245 (1989)

1118–1121.

[36] Ullrich S., Wolheim C.B., J. Biol. Chem. 259 (1984) 4111–4115.

[37] Asfari M., Janjic D., Meda P., Li G., Halban P.A., Wollheim C.B.,

Endocrinol. 130 (1992) 167–178.

[10] Eistetter K., Wolf H.P.O., J. Med. Chem. 25 (1982) 109–113.

[11] Eistetter K., Wolf H.P.O., Drugs Future 11 (1986) 1034–1036.

[38] Herbert V., Lau K.S., Gottlieb C.W., Bleicher S.J., J. Clin. Endo-

crinol. Metab. 25 (1965) 1375–1384.

[12] Anderson R.C., Balestra M., Bell P.A., Deems R.O., Fillers W.S.,

[39] Kerst G., Fischer K.G., Normann C., Kramer A., Leipziger J.,

Foley J.E. et al., J. Med. Chem. 38 (1995) 3448–3450.

Greger R., Pﬂügers Arch. 430 (1995) 253–265.

[13] Grisar J.M., Claxton G.P., Carr A.A., Wiech N.L., J. Med. Chem. 16

[40] Nietschke R., Fröbe U., Greger R., Pﬂügers Arch. 417 (1991)

622–632.

(1973) 679–683.

[14] Gerritsen G.C., Dulin W.E., Diabetes 14 (1965) 507–515.

Article Doi

EPC syntheses and structure-activity relationships of hypoglycaemic semicyclic amidines

DOI: 10.1016/S0223-5234(00)00138-0

Source and publish data:

Authors:

Article abstract of DOI:10.1016/S0223-5234(00)00138-0

Full text of DOI:10.1016/S0223-5234(00)00138-0

Products guided by the article

R&D Labs maybe for 87376-12-3

Relevant to this article

Hot Product