Synthesis and structure-activity relationship of Huprine derivatives as human acetylcholinesterase inhibitors

DOI: 10.1016/j.bmc.2009.05.005

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Bioorganic and Medicinal Chemistry p. 4523 - 4536 (2009)

Update date:2022-08-05

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Authors:

Ronco, Cyril

Sorin, Geoffroy

Nachon, Florian

Foucault, Richard

Jean, Ludovic

Romieu, Anthony

Renard, Pierre-Yves

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Article abstract of DOI:10.1016/j.bmc.2009.05.005

New series of Huprine (12-amino-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinolines) derivatives have been synthesized and their inhibiting activities toward recombinant human acetylcholinesterase (rh-AChE) are reported. We have synthesized two series of Huprine analogues; in the first one, the benzene ring of the quinoline moiety has been replaced by different heterocycles or electron-withdrawing or electron-donating substituted phenyl group. The second one has been designed in order to evaluate the influence of modification at position 12 where different short linkers have been introduced on the Huprine X, Y skeletons. All these molecules have been prepared from ethyl- or methyl-bicyclo[3.3.1]non-6-en-3-one via Friedlaender reaction involving selected o-aminocyano aromatic compounds. The synthesis of two heterodimers based on these Huprines has been also reported. Activities from moderate to same range than the most active Huprines X and Y taken as references have been obtained, the most potent analogue being about three times less active than parent Huprines X and Y. Topologic data have been inferred from molecular dockings and variations of activity between the different linkers suggest future structural modifications for activity improvement.

Full text of DOI:10.1016/j.bmc.2009.05.005

Bioorganic & Medicinal Chemistry 17 (2009) 4523–4536

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Synthesis and structure–activity relationship of Huprine derivatives

as human acetylcholinesterase inhibitors

Cyril Ronco ^a,b,, Geoffroy Sorin ^a,b,, Florian Nachon ^c, Richard Foucault ^c, Ludovic Jean ^a,b

Anthony Romieu ^a,b, Pierre-Yves Renard ^a,b,d,

^aEquipe de Chimie Bio-Organique, COBRA—CNRS UMR 6014 & FR 3038, rue Lucien Tesnière, 76131, Mont-Saint-Aignan, France

^bUniversité de Rouen, Place Emile Blondel, 76821, Mont-Saint-Aignan, France

^cUnité d’enzymologie, Département de Toxicologie Centre de Recherches du Service de Santé des Armées, 24 Avenue des Maquis du Grésivaudan BP87, 38702 La Tronche, France

^dJunior Member of the Institut Universitaire de France, 103, Boulevard Saint-Michel, 75005, Paris, France

a r t i c l e i n f o

a b s t r a c t

Article history:

New series of Huprine (12-amino-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinolines) derivatives

have been synthesized and their inhibiting activities toward recombinant human acetylcholinesterase

(rh-AChE) are reported. We have synthesized two series of Huprine analogues; in the ﬁrst one, the ben-

zene ring of the quinoline moiety has been replaced by different heterocycles or electron-withdrawing or

electron-donating substituted phenyl group. The second one has been designed in order to evaluate the

inﬂuence of modiﬁcation at position 12 where different short linkers have been introduced on the Hup-

rine X, Y skeletons. All these molecules have been prepared from ethyl- or methyl-bicyclo[3.3.1]non-6-

en-3-one via Friedländer reaction involving selected o-aminocyano aromatic compounds. The synthesis

of two heterodimers based on these Huprines has been also reported. Activities from moderate to same

range than the most active Huprines X and Y taken as references have been obtained, the most potent

analogue being about three times less active than parent Huprines X and Y. Topologic data have been

inferred from molecular dockings and variations of activity between the different linkers suggest future

structural modiﬁcations for activity improvement.

Received 22 January 2009

Revised 24 April 2009

Accepted 4 May 2009

Available online 8 May 2009

Keywords:

Huprines

AChE inhibitors

Heterodimers

Alzheimer’s disease

Friedländer reaction

1. Introduction

sion where the synaptic connections are still intact and thus reduc-

ing the secondary neurodegenerative effects of the pathology.

The ubiquitous synaptic enzyme acetylcholinesterase (AChE)

terminates transmission at cholinergic synapses by rapidly hydro-

lyzing acetylcholine. As a consequence of its key physiological role,

AChE is not only the target of a repertoire of natural toxins [e.g., the

alkaloids Galanthamine and (ꢀ)-Huperzine A, or the three-ﬁngered

polypeptide toxin Fasciculin], or man made pest control agents and

warfare agents, but also the target of drugs designed to combat

neuromuscular disorders, such as myasthenia gravis and glau-

coma,¹and most recently to alleviate the cholinergic deﬁciency

associated with Alzheimer’s disease.²With this latter aim in mind,

a possible symptomatic treatment for this disease is to remedy the

abnormal low rate of neurotransmitter acetylcholine by controlling

the enzymatic activity of acetylcholinesterase. Indeed, AChE can be

reversibly inhibited,^3–5resulting in an increased neurotransmis-

Recent results show that a speciﬁc inhibition of AChE is a potential

therapeutic target also for prevention of the ﬁrst step of Alzheimer

disease, the assembly of b-amyloid peptide (Ab) into amyloid ﬁ-

brils.^6–9

Since the inhibition of AChE based on the ‘cholinergic hypothe-

sis’ remains the most powerful therapeutic strategy in treating Alz-

heimer’s disease, four different AChE inhibitors have already been

approved by the health authorities and are commercially available,

Tacrine (Cognex^Ò), Donepezil (or E2020, Aricept^Ò), Rivastigmine

(Exlon^Ò), and Galanthamine (Reminyl^Ò), whose structures are dis-

played in Figure 1.

Within these compounds, Tacrine has been the most studied

compound and the ﬁrst marketed product despite severe side ef-

fects. Today, Tacrine remains a reference structure even if the ad-

verse troubles induced, such as hepatotoxicity and gastrointestinal

disorders, led to its withdrawal from the market. Treatment having

to be taken life-long, with the enhanced liver toxicity of those drugs,

it is of prior importance to develop new more efﬁcient, selective, and

safe AChE inhibitors.

* Corresponding author. Address: Pr. Pierre-Yves RENARD Equipe de Chimie Bio-

Organique, COBRA-CNRS UMR 6014; FR 3038, Université de Rouen, rue Lucien

Tesnière, 76131, Mont-Saint-Aignan, France. Tel.: +33 2 35 52 24 14; fax: +33 2 35

52 29 59.

Huprines, empirically designed Tacrine–Huperzine A hybrids

have been described a few years ago as highly potent AChE

E-mail address: pierre-yves.renard@univ-rouen.fr (P.-Y. Renard).

These authors contributed equally to this work.

doi:10.1016/j.bmc.2009.05.005

4524

C. Ronco et al. / Bioorg. Med. Chem. 17 (2009) 4523–4536

inhibitors (Scheme 1).¹⁰Surprisingly, if many efforts have been

undertaken to develop new Tacrine analogues, the modiﬁcations

on the Huprines have been scarcely studied.

With the goal in mind to develop efﬁcient heterodimeric acyla-

tion site—peripheral site hAChE inhibitors, in a ﬁrst step, we have

decided to evaluate the possibility of further improvement of Hup-

rine‘s binding ability toward AChE acylation site. First, docking

experiments led on human AChE, and crystallographic data on Tor-

pedo California AChE complexed with Huprine¹¹allowed us to

identify on Huprine structure two preferred moieties (positions

1–4 of benzene ring B of quinoline moiety and position 9 on the

cyclohexene part) which could be favorably modiﬁed in order to

improve the binding of these inhibitors to the acylation site. Previ-

ous results have shown that the modiﬁcations on the positions 1–3

on the quinoline benzene have led to tight binding AChE inhibitors,

the most potent being Huprines X and Y displaying a chlorine atom

at position 3 (Scheme 2).^10,12,13

H₂N

Huprine Y

Huprine X

Scheme 2. Huprines X and Y.

R¹

11a

(

) n=1,2

R²

Moreover, investigation of structural modiﬁcations on positions

9¹⁰and 13^14,15on the bicyclo[3.3.1]nonane moiety led to disap-

pointing results. Taking into account that the nature of the B aro-

matic moiety has not been modiﬁed on the previous studies, we

decided to focus our synthetic efforts on the modiﬁcation of this

aromatic ring size and on its electronic density as displayed in

Scheme 3.

In a second step, aware of the interest of a homo- or heterodi-

meric dual inhibitor binding both AChE acylation and peripheral

binding sites,¹⁶and thus of the necessity to introduce a reactive

linker on the Huprine scaffold, another goal for us was to explore

Huprines binding ability when modiﬁed with such a linker,

Scheme 3. Sites of planned modiﬁcations on the Huprine scaffold. R¹represents a

methyl or an ethyl group. R²represents various

-donor or electron-donating

groups. Aminopyridine cycle (X = NH₂) or N-substituted aminopyridine cycle

(X = (CH₂)₂-Y, Y = OH, N₃, Cl, or -chloroacetyl) or 4-substituted pyridine cycle

(X = OH, Cl) is designed as cycle A and aromatic cycle fused to aminopyridine is

designed as cycle B.

depending on the nature of the linker. We took as reference the

previous results obtained on Huprine and Tacrine homodimers,

and the docking experiments, both of which show that aromatic

amino group on position 12 points toward the narrowest region

of the gorge. We thus decided to modify this aniline on the best

binding Huprine inhibitor obtained in the ﬁrst step. These results,

supported by both the inhibition activity measurements and by

modelization data will enable us to determine the best structure

for this part of the inhibitor following a progressive approach of

the construction of the linker.

In this article, we want to disclose our efforts toward the

synthesis and characterization of our ﬁrst targets as well as the

biological activity assays on the recombinant human acetylcholin-

esterase (IC₅₀measurements). A set of 24 new Huprine derivatives

has been synthesized, which can be divided into two groups

depending on the region modiﬁed: (1) the modulation of the aro-

matic part, (2) the interactions with the amino acid residues at

the bottom of the gorge. Finally, these results have also been com-

pared to those obtained from molecular modeling calculations on

the same enzyme in order to rationalize the activities observed.

NH₂

MeO

Donepezil

Tacrine

Me₂N

MeO

NMe

Galanthamine

Rivastigmine

2. Results and discussion

2.1. Chemistry

Figure 1. Commercially available AChE inhibitors.

2.1.1. Modulation of the aromatic part

As ﬁrst targets, the following Huprines 3–11 (Fig. 2) were syn-

thesized, with the goal in mind to screen a wide variety of chemical

structures, and to get a better insight into their binding ability to

AChE. In this way, taking into account the results reported by

Camps et al. for the halogenated Huprines,^10,12we have ﬁrst intro-

duced two electron-donor methoxy groups in order to increase the

electron density of the aromatic ring (3a,b), then varied its distri-

bution by introducing one or several heteroatoms within the aro-

matic ring. Thus, the quinoline moiety was replaced by 1,8 and

NH₂

Tacrine

(-)-Huperzine A

R¹

^5aN

R²

1,7

p-enriched naphthyridine moieties (4a,b, 5), furo[2,3-b]pyri-

11a

12a

dine (6a,b), oxazolo[5,4-b]pyridine (7a,b, 8), thiazolo[4,5-b]pyri-

dine (9a,b), and 2H-pyrazolo[3,4-b]pyridine (10, 11).

These Huprine analogues have been readily synthesized in four

steps from symmetrical bicyclic diketone 1 as shown in Scheme 4.

Indeed, it is well-known from the literature that nucleophilic addi-

tions of Grignard or organolithium reagents on this type of bicyclic

H₂N

Huprine

Scheme 1. Structure of Tacrine–Huperzine A hybrids (Huprines) and their starting

models. The atom numbering of the Huprine core has been addressed according to

Camps et al.¹⁰

C. Ronco et al. / Bioorg. Med. Chem. 17 (2009) 4523–4536

4525

OMe

H₂N

3a R = Me

3b R = Et

4a R = Me

4b R = Et

6a R = Me

6b R = Et

7a R = Me

7b R = Et

H₂N

9a R = Me

9b R = Et

Figure 2. Huprine derivatives synthesized through the modulation of the aromatic moiety.

R₁

Table 1

1) MeLi or EtMgCl, CH₂Cl_2,-10°C

Regioselectivity observed for the synthesis of Huprines 6–8 and 10–11

2) MsCl, NEt_3,CH₂Cl_2,-10°C

3) SiO_2,CH₂Cl_2,RT

Compound

anti/syn ratio^a

87/13

80/20

93/7

88/12

100/0

96/4

2a R₁=Me

2b R₁=Et

AlCl₃

ClCH₂CH₂Cl

reflux

H₂N

R₂

R₁

95/5

82/18

Determined by ¹H NMR.

R₂

H₂N

7–9, respectively, were synthesized following described proce-

dures (see Supplementary data).

Scheme 4. Synthetic pathway used for the preparation of Huprines.

The Friedländer condensation was then performed under classi-

cal conditions, using anhydrous AlCl₃in reﬂuxing 1,2-dichloroeth-

ane (DCE) overnight and afforded the desired Huprine analogues in

moderate to very good isolated yields (30–90%). Interestingly,

using such equilibration conditions (overnight reﬂux), the regiose-

lectivity of this reaction proved to be excellent in the case of six-

membered rings, yielding exclusively the thermodynamically fa-

vored anti Huprines.¹⁹In an opposite manner, the minor regio-

isomer syn could be observed in some ﬁve-membered rings. The

ratios anti/syn obtained for the Huprine derivatives including a

ﬁve-membered ring are displayed in Table 1. Apparently, the syn/

anti regioselectivity between Huprines depends on the nature of

the second aromatic ring.

These compounds correspond to original modiﬁcations of the

Huprine scaffold while only analogues bearing different substitu-

ents on a quinoline scaffold have been reported to date.^10,12All

the new Huprines were fully characterized through their spectro-

scopic (IR, MS, ¹H and ¹³C NMR spectra) and analytic data (elemen-

tal analysis), and their purity was checked by RP-HPLC.

diketones afford polycyclic structures such as the oxaadamentanol

intermediates required for the preparation of the key enones

2a,b.¹⁷Mesylation of the oxaadamentanols followed by a Grob-like

fragmentation¹⁸gives access to the enones 2a,b. The key step has

been then a remarkably regioselective Friedländer annulation of

enones 2a,b on the corresponding functionalized o-cyanoaniline

derivatives.¹⁹Considering that the diketone 1 is commercially

available but quite expensive, we have chosen to perform the syn-

thesis of this intermediate in two steps from methyl acetonedi-

carboxylate and propanedial following the classical procedure

described by Bertz et al.²⁰

Concerning the accessibility of the second Friedländer reaction

partner, some of them are commercially available (precursors of

Huprines X, Y, 3–4, 6a,b, 10–11) whereas o-aminocyanoaromatics

18–21 (Scheme 5) required for the preparation of Huprines 5 and

NH₂

2.1.2. Derivatization with reactive linkers to probe key

H₂N

interactions with the amino acid residues in the AChE gorge

Taking into account that none of the newly described Huprines

displayed better binding afﬁnities than parent Huprines X and Y

(vide infra) and with the goal in mind to evaluate the possibilities

to further graft a linker on the Huprine scaffold aimed at preparing

homo- or heterodimeric dual AChE binding sites inhibitors, the

new Huprines 12–17 (Fig. 3), based on Huprine X and Y skeleton

were synthesized. These molecules were chosen to achieve two

main objectives: ﬁrstly, the synthesis of these molecules will allow

H₂N

Scheme 5. O-aminocyanoaromatics 18–21 intermediates synthesized for the

preparation of Huprines 5, 7–9.

4526

C. Ronco et al. / Bioorg. Med. Chem. 17 (2009) 4523–4536

backs such as the formation of signiﬁcant amounts of regioisomers.

To avoid this problem, we have decided to apply conditions used

for the preparation of 9-chlorotacrine derivatives.^23,24Reaction of

enones 2a,b with 2-amino-4-chlorobenzoic acid in reﬂuxing POCl₃

afforded the pure desired 12-chloroquinoline derivatives 14a,b in

27% and 46% yield, respectively.

Thereafter, S_NAr reactions have been undertaken. Treatment

of 14a,b with ethanolamine in reﬂuxing n-pentanol gave mixture

of compounds,²²whereas the conditions described by Hu et al.

(with catalytic amounts of NaI in phenol at 150 °C) allowed to

furnish the desired products 15a,b in 77% and 95% yield, respec-

tively.²⁴With 15a,b in hand, introduction of different functional

groups on the linker was envisaged. Upon treatment of alcohols

15a,b with SOCl₂, the compounds 16a,b were readily obtained in

quantitative yields (97% and 99%, respectively). Finally, nucleo-

philic substitution of the chlorine atom of 16a and 16b by so-

dium azide gave products 17a and 17b in good yields (78%

and 69%, respectively, over two steps from the alcohols 15a

and 15b). In the other hand, we attempted selective reduction

of carboxamide function of 12 with various hydride sources to

access directly to 16a, but all tested conditions failed.

14a R = Me

14b R = Et

N₃

15a R = Me

15b R = Et

16a R = Me

16b R = Et

17a R = Me

17b R = Et

Figure 3. Huprine derivatives synthesized for the exploration of possible interac-

tions with the amino acid residues of the enzyme gorge.

2.1.3. Coupling of a peripheral site inhibitor to afford dual-site

inhibition

us to tackle the tricky problem of the alkylation of this type of frag-

ile aminoquinoline derivatives.²¹Secondly, the biological evalua-

tion of these derivatives will bring some interesting information

on the molecular environment at this part of the gorge and the pos-

sibilities of further interactions with the amino acid residues pres-

ent in the enzyme. We have chosen a two-carbon linker with a

further functional group aiming at the covalent association with

an AChE peripheral site binder.²²

The prepared Huprine analogues have various heteroatoms (N

from azido group, O, Cl) at the terminal position of either the

grafted spacer unit or the amino group of the parent Huprine. As

we have encountered serious difﬁculties to alkylate or acylate

the amino group of Huprine (details in Supplementary data), we

have chosen (except for acylated compound 12) to introduce the

linker by a S_NAr reaction on the 12-chloro-Huprine analogues

14a,b. To the best of our knowledge, the preparation of these 12-

chloro-derivatives has been only reported by Camps et al.,²¹but

the conditions described by the authors showed important draw-

As suggested above, the covalent association with an AChE

peripheral site binder has ﬁnally been undertaken. Huprines 17a

and 17b have respectively been coupled by a copper(I)-catalyzed

Huisgen reaction with the tetrahydroquinoline derivatives 22 and

23 prepared following described procedures (see Supplementary

data).³³The anti regioisomers 24 and 25 were thus obtained exclu-

sively (Fig. 4).

3. Structure–activity relationship

The AChE inhibitory efﬁciencies have been evaluated according

to the standard Ellman methodology (see Section 5).²⁵According to

the targeted activity, we have chosen to perform our analyses with

human AChE rather than with Torpedo Californica or Electrophorus

Electricus AChE since signiﬁcant differences in the structures and

conformations of these enzymes exist. Most of the inhibitory activ-

MeO

NO₂

( )₆

OMe

Figure 4. Dual-site inhibitors 24 and 25 and their respective tetrahydroquinoline derivative precursors 22 and 23.

C. Ronco et al. / Bioorg. Med. Chem. 17 (2009) 4523–4536

4527

Table 2

Recombinant human AChE IC₅₀values for the inhibitory activity of Huprines 3–11

Compound

IC₅₀(M)

Calculated pK_aof protonated

pyridine moiety^b

Huprine X

Huprine Y

1.10 0.02 ꢁ 10^ꢀ9

1.84 0.03 ꢁ 10^ꢀ9

1.17 0.01 ꢁ 10^ꢀ5

5.10 0.93 ꢁ 10^ꢀ6

8.10 0.24 ꢁ 10^ꢀ7

8.55 0.32 ꢁ 10^ꢀ7

6.64 0.78 ꢁ 10^ꢀ7

4.78 0.03 ꢁ 10^ꢀ6

6.45 0.28 ꢁ 10^ꢀ6

>5.0 ꢁ 10^ꢀ5

7.95

10.05

7.33

4a^c

6.71

6.90

5.70

>5.0 ꢁ 10^ꢀ5

2.71 0.33 ꢁ 10^ꢀ5

7.69 0.09 ꢁ 10^ꢀ6

7.27 0.83 ꢁ 10^ꢀ6

>5.0 ꢁ 10^ꢀ5

6.87

10.02

>5.0 ꢁ 10^ꢀ5

The conditions used for the tests were as follows: enzyme concentration of

M NaN_3,and

125 pM in 0.1 M phosphate buffer containing 15 mM NaCl, 150

15 M Bovine Serum Albumin (BSA) at pH 7.4, 25 °C (see Section 5).

Calculated using the SPARC online calculator (http://ibmlc2.chem.uga.edu/

sparc/).

Inhibition obtained on recombinant human BuChE: 3.42 0.12 ꢁ 10^ꢀ7M;

Selectivity BuChE/AChE: 0.4.

that Huprines X and Y ﬁt in the active site gorge of rh-AChE

(Fig. 5), in a similar manner than observed in the X-ray structure

of the complex Huprine X and Torpedo californica AChE.¹¹The cen-

tral 4-aminopyridine nucleus (cycle A) is stacked against Trp86

with the amino group H-bonded to a water molecule close to

Ser125. The amino group is also at 3.7 Å of Tyr337 hydroxyl, a dis-

tance too long for strong H-bonding. Protonation of 4-aminopyri-

dine is an important factor as the pyridinium proton is H-bonded

to the main chain carbonyl of His447, and the delocalized positive

charge leads to cation–p interactions with Trp86. The heterocyclic

fused-ring onto the central 4-aminopyridine nucleus of Huprine

(cycle B) ﬁts in an aromatic/hydrophobic pocket formed by resi-

Figure 5. Side view (A) and top view (B) of Huprine

X docked in human

acetylcholinesterase. The solvent accessibility surface is represented as dots.

Carbon atoms are represented in gray (rh-AChE) or cyan (Huprine X), oxygen in

red, and nitrogen in blue. Atoms of Huprine X and water molecules are represented

as spheres. Key hydrogen bonds are represented as yellow dashes. Key amino acid

residues are labeled and represented in sticks.

ities toward human AChE, reported in the literature have been ob-

tained from experiments performed on human erythrocytes. In or-

der to get more reliable values, and since we have developed an

efﬁcient production protocol of conformationally active recombi-

nant human AChE (rh-AChE), we have chosen to perform the inhib-

itory efﬁciency evaluation on this rh-AChE. Furthermore, only few

reference values are available for this enzyme.

As this study is devoted to a ﬁrst evaluation of the inhibitory

activities of Huprine derivatives 3–17, all the inhibitory potencies

(IC₅₀) have been determined with the racemic mixtures, the (ꢀ)

enantiomer of Huprines being reported to be about twice more ac-

tive than the corresponding racemic compound. For comparative

purposes, we also included the IC₅₀values of the most active

known Huprines X and Y toward the same enzyme rh-AChE. The

values obtained for these reference compounds were found to be

in good agreement, but slightly weaker than those given in the lit-

erature¹²(1.10 nM vs 0.75 nM and 1.84 nM vs 0.78 nM for ( Hup-

rines X and Y, respectively), probably due to different experimental

conditions used for the assays. Especially, in vitro AChE inhibitory

activities were measured at pH 7.4 whereas literature values are

reported at pH 8.0.

Figure 6. Huprine X, 6b and 15b docked in human acetylcholinesterase. Carbon

atoms are represented in gray (rh-AChE), cyan (Huprine X), yellow (6b) or magenta

(15b), oxygen in red, and nitrogen in blue. Atoms of Huprine X and water molecules

are represented as spheres. Key hydrogen bonds are represented as yellow dashes.

Key amino acid residues are labeled and represented in sticks.

In order to rationalize the interpretations of kinetic data, dock-

ing studies were performed on rh-AChE for each compound includ-

ing the references Huprines X and Y. Docking experiments show

4528

C. Ronco et al. / Bioorg. Med. Chem. 17 (2009) 4523–4536

dues Tyr337, Try341, Met443, Trp439, and Pro446. In particular,

cycle B is stacked against Tyr337 and chlorine interacts with

Trp439. The aliphatic bicycle ﬁts in an elbow of the active site

gorge with the ethyl/methyl at position 9 of Huprines X/Y close

to the catalytic serine. Overall, dockings of Huprines X/Y allow

evaluating the impact of the modiﬁcations made.

Finally, Huprines 6a and 6b showed the best result considering

a 5-membered ring size possibly due to better hydrophobic inter-

actions with residues in the pocket (Fig. 6).

Furthermore, the variation from methyl to ethyl group at posi-

tion 9 always led to the same range of activity without signiﬁcant

differences, even if ethyl derivatives proved often slightly more

active.

3.1. Modulation of the aromatic part

The inhibition of the most potent Huprine in this series on re-

combinant human butylcholinesterase (rh-BuChE) has been re-

ported. Interestingly, the IC₅₀value proved better on this enzyme.

Despite lower activities than expected, these ﬁrst analogues are

very interesting as the large variety of synthesized structures al-

lowed us to deﬁne which types of interactions were unfavorable

and will guide us in view of further structural modiﬁcations of

Huprine scaffold. As a general trend, the only signiﬁcant data we

could draw from these results is the inﬂuence of stacking interac-

tions between cycle B and Tyr337, larger rings being better, and

pK_aof 4-aminopyridine dependent on the nature of cycle B.

Compounds 3–11 were tested for their AChE inhibitory activity

and the results are summarized in Table 2. Regarding modiﬁcations

of the aromatic part, the best inhibitions were obtained for 4-ami-

no-1,7-naphthyridine 4 and 4-amino-1,8-naphthyridine 5 deriva-

tives which are however less active (two orders of magnitude)

than the reference compounds.

Structure–activity relationship analysis regarding the heterocy-

clic fused-ring cycle B, led to the following conclusions: introduc-

tion of a cyclopropyl- and a cyclobutyl-1,3-oxazole moieties

(compounds 7–8) was found to decrease strongly the inhibition

activity. It is important to note that during the course of our work,

Marco et al. have reported the synthesis of substituted 1,3-oxazole

Tacrine derivatives and reported similar negative results on the

inhibition of AChE.²⁶Docking results show that there is enough

room in the pocket to ﬁt the cyclopropyl and cyclobutyl groups.

So the decrease in activity seems to be related to other factors than

steric hindrance. There is a signiﬁcant decrease of the calculated

pK_awhich suggests that 7–8 pyridiniums are deprotonated at pH

7.4, thus possibly leading to less favorable interactions with

Trp86 and His447. Although the general orientations of the 6-atom

ring and 5-atom ring analogues are virtually identical, 5-atom ring

cycles have less surface contact with Tyr337, thus decrease aro-

matic–aromatic stacking interactions. Besides, despite being

hydrophobic, the small aliphatic cycles might not optimally inter-

act with the aromatic residues of the pocket.

The placement of a pyrazole heterocyclic ring (compounds 10–

11) decreased signiﬁcantly the inhibitory activity and led to some

very disappointing results. In addition to less favorable stacking of

the 5-atom ring with Tyr337, one hypothesis is that both nitrogens

might be too much hydrophilic for the hydrophobic pocket.

Noteworthing, the thiazole derivatives 9a and 9b proved to be

more active by at least one order of magnitude than the oxazole

or pyrazole analogues, despite the propyl chain being too long

3.2. Derivatization with reactive linkers to probe key

interactions with the amino acid residues in the gorge

Compounds 12–17 were submitted to the same in vitro assay to

determine their potency to inhibit rh-AChE. The results are sum-

marized in Table 3.

As previously mentioned, these structures have been designed:

(1) to conﬁrm former molecular modelization analyses on Torpedo

californica AChE showing interactions between the amino group of

the Huprine with a water molecule close to Ser125¹⁰and especially

to determine the direct impact of the lack of this group for human

AChE inhibition activity, (2) to study the inﬂuence of the attach-

ment of a linker at this position and to probe the potential interac-

tions of this added chemical moiety with some enzyme amino acid

residues, in order to choose the less detrimental linker with the

goal in mind to further design highly potent dual binding

inhibitors.

As mentioned for the underivatized Huprines, changing a

methyl group by an ethyl group at position 9 did not lead to signif-

icant differences in the inhibition activity.

As expected, compounds 14a and 14b bind the enzyme very

weakly, the decrease in inhibitory potency being four orders of

magnitude lower than that of Huprines X and Y. These results con-

ﬁrm the strong favorable effect induced by the amino group of cen-

tral pyridine nucleus which is thus of prior importance for H-

bonding, and protonation of the pyridine moiety.

for the pocket. This might be explained by favorable p–sulfur inter-

actions between the thiazole sulfur and Tyr337 on one side and the

thiopropyl sulfur and Trp439 on the other side.

In addition to this ﬁrst result, we also studied the replacement

of this amino group by another H-bond donor (i.e., hydroxyl group

for compound 13). However the hydroxyl is deprotonated and the

pyridine is protonated at pH 7.4 resulting in a drastic modiﬁcation

of electrons distribution of the aromatic cycle. Thus there is both a

Table 3

Recombinant human AChE IC₅₀values for the inhibitory activity of Huprines 12–17

Compound

IC₅₀(M)

Calculated pK_aof protonated

pyridine moiety^b

loss of an H-bond donor at position 12 and a loss of cation–p inter-

Huprine X

Huprine Y

14a

1.10 0.00002 ꢁ 10^ꢀ9

1.84 0.03 ꢁ 10^ꢀ9

2.51 0.97 ꢁ 10^ꢀ6

1.47 0.03 ꢁ 10^ꢀ5

2.24 0.39 ꢁ 10^ꢀ5

1.92 0.16 ꢁ 10^ꢀ5

9.66 0.35 ꢁ 10^ꢀ9

6.75 1.23 ꢁ 10^ꢀ9

4.33 0.19 ꢁ 10^ꢀ8

8.69 0.03 ꢁ 10^ꢀ9

1.37 0.07 ꢁ 10^ꢀ8

7.95

action with Trp86. This likely explains that 13 is 8000-fold less ac-

tive than Huprine X. This convinces us that the central 4-

aminopyridine moiety of Huprine derivatives is essential for inhi-

bition activity and cannot be replaced by an isosteric group.

Interestingly, modifying this key interaction through mono-

alkylation of amino group (suppressing possibility of double H-

bonding) resulted in very active compounds, slightly less than par-

ent Huprines X and Y, but inferior to 14 nM for all of them. In this

latter series, the only exception was compound 12 which exhibits a

micromolar inhibition activity (60-fold less potent than its reduced

analogue 16). This fact can be easily explained by the change of pK_a

and to a lesser degree by the presence of the carbonyl group which

is bulky enough to prevent the ability of inhibitor molecule 12 to ﬁt

within the narrowest region of the gorge. This valuable informa-

tion should be taken into consideration when choosing the syn-

4.63

11.86

4.08

14b

15a

8.58

15b^c

16a

8.50

8.64

17a

17b

The conditions used for the tests were as follows: enzyme concentration of

M NaN_3,and

125 pM in 0.1 M phosphate buffer containing 15 mM NaCl, 150

15 M Bovine Serum Albumin (BSA) at pH 7.4, 25 °C (see Section 5).

Calculated using the SPARC online calculator (http://ibmlc2.chem.uga.edu/

sparc/).

Inhibition obtained on recombinant human BuChE: 3.64 0.03 ꢁ 10^ꢀ6M;

Selectivity BuChE/AChE: 540.

C. Ronco et al. / Bioorg. Med. Chem. 17 (2009) 4523–4536

4529

thetic methodology for the linker introduction. Thus, alkylation

reactions of the 4-aminopyridine moiety with small hydrocarbyl

chains will be preferred.

and more heterodimeric inhibitors must be synthesized by rational

approach.

Finally, the most interesting feature of these heterodimers

proved to be their high selectivity toward AChE, since they are

about 10,000-fold more potent on AChE than on BuChE. In compar-

ison, the more selective Tacrine–Huprine heterodimer was 100-

fold more potent on AChE.^21a

Chemical variations at the terminal position of the linker in-

duced little changes in the inhibition activity (compounds 15–

17). For this sub-class of N-derivatized Huprines, the hydroxyl

group containing inhibitor 15b was found to be the most active,

(only sixfold less active than Huprine X and 3.6-fold less active

than Huprine Y). Moreover, the low inhibition value reported on

rh-BuChE for this compound showed its good selectivity for rh-

AchE (Table 3). According to docking experiments, the hydroxyl

of 15b is at H-bond distance to Tyr124, Ser125, and a conserved

water molecule nearby (Fig. 6).

These ﬁndings could be of interest since the retained strategy to

inhibit AChE involves dual-site binding, which requires the intro-

duction of a linker attached on the amino group of the Huprines.

Furthermore, these latter results clearly show that the linker must

be very slim (i.e., linear alkyl chain) at the bottom of the gorge, but

the nature of its terminal reactive group does not seem to be a cru-

cial factor. Thus, a wide range of heterobifunctional cross-linkers

bearing a linear carbon chain could be used in the context of homo-

or heterodimeric dual inhibitors. Finally, further exploration of

these interactions must be carried out in order to increase the

number of contact points between the inhibitor molecule and the

closest amino acid residues and so further improve its inhibitory

efﬁciency.

4. Conclusions

In this paper, we have reported the synthesis and structure–

activity relationship, based on recombinant human AChE inhibi-

tory activity and docking calculations, of 24 new Huprine deriva-

tives and 2 Huprine-based heterodimers. The selectivity toward

human BuChE has been evaluated for the most potent compounds.

All these analogues have been prepared from various o-cyanoaro-

matic compounds (synthesized or commercially available) via

Friedländer condensation with enones 2a,b. Concerning the series

where the aromatic part has been modiﬁed, the most active ana-

logues were the naphthyridine analogues 4a,b, and 5. The replace-

ment of the six-membered ring by a ﬁve-membered ring proved

generally detrimental on the inhibitory activity. Structure–activity

relationships have suggested that the inﬂuence of the pyridine pK_a

is of prior importance on the binding efﬁciency. The study on the

introduction of a short linker on the amino group of Huprines X

and Y showed that the nature of the linker at this position is impor-

tant, and that only compounds bearing a slim alkyl group on the

nitrogen atom have shown a weak loss of activity (inferior to one

order of magnitude). This ﬁnding is of particular relevance for fur-

ther dual-binding inhibitors design. The heterodimers displayed

better activities than the parent Huprines but without the strong

synergic effect expected. However their high selectivity for AChE

should be taken into consideration.

3.3. Coupling of a peripheral site inhibitor to afford dual-site

inhibition

Heterodimers 24 and 25 as well as their monomer precursors

were also tested for inhibition potency on recombinant human

AChE and BuChE. The results obtained are resumed in Table 4.

Taking these results into consideration, the following conclu-

sions could be drawn. Despite no strong beneﬁcal synergic effect

has been observed, these ﬁrst heterodimers 24 and 25 proved a lit-

tle (about two times) more efﬁcient than the Huprines precursors.

Several explanations are possible: the modest binding afﬁnity of

the tetrahydroisoquinoline derivatives perhaps limits the binding

ability, but an inadequate length of the tether or a bad position

of triazole inducing detrimental interactions with residues in AChE

gorge can be also responsible of this relative activity for the hetero-

dimers. However, similar results have been reported by Camps

et al.^21ain their exploration of Tacrine–Huprine heterodimers,

the gain of introducing a second inhibitor being limited compared

to the results of Sharpless et al. with Tacrine-based heterodimers

on other acetylcholinesterases.³³

5. Experimental

5.1. Enzyme kinetic studies

AChE inhibitory activity was evaluated spectrophotometrically

using a UV–vis Varian Cary 50 scan spectrophotometer equipped

with a microplate reader at 25 °C by the method of Ellman,²⁵using

recombinant human AChE (rh-AChE) or recombinant human BuChE

(rh-BuChE) and acetylthiocholine iodide or butyrylthiocholine

(0.53 mM) as substrate. The reaction took place in the presence of

125 pM of rh-ChE in a ﬁnal volume of 200

buffered solution (pH 7.4), containing 15 mM NaCl, 150

15 M Bovine Serum Albumin (BSA), and 333

M 5,5⁰-dithiobis(2-

lL of 0.1 M phosphate-

lM NaN₃,

Anyhow, more analyses (docking calculations) must be carried

out in the future to have a better overview of the interactions

nitrobenzoic) acid (DTNB) solution used to produce the yellow anion

of 5-thio-2-nitrobenzoic acid. The different inhibitory derivatives

were pre-incubated with the enzyme at 25 °C for at least 30 min be-

fore measurement. One sample without inhibitor was always pres-

ent to yield the 100% of ChE activity. The rate of change of

Table 4

Recombinant human AChE and BuChE IC₅₀values for the inhibitory activity of

heterodimers 24 and 25 and their precursors 17a, 22, 17b, and 23

absorbance (DA/min), reﬂecting the rate of hydrolysis of acetylthi-

ocholine was recorded at k = 414 nm for 20 min (kinetic mode).

These experiments were generally done at least in triplicate and

the values averaged. Data from concentration–inhibition experi-

ments of the inhibitors were calculated by nonlinear regression

analysis, which gave estimates of the IC₅₀(concentration of drug

producing 50% of enzyme activity inhibition). Recombinant human

acetylthiocholinesterase was puriﬁed as previously described.²⁷

DTNB and acetylthiocholine were purchased from Sigma.

Compound

IC₅₀(M)^ahuman AChE

IC₅₀(M)^ahuman BuChE

BuChE/AChE

270

17a

17b

8.69 0.03 ꢁ 10^ꢀ9

8.08 0.47 ꢁ 10^ꢀ6

3.95 0.33 ꢁ 10^ꢀ9

1.37 0.07 ꢁ 10^ꢀ8

2.92 0.03 ꢁ 10^ꢀ6

5.96 0.36 ꢁ 10^ꢀ9

2.35 0.06 ꢁ 10^ꢀ6

>10⁴

8.24 0.24 ꢁ 10^ꢀ7

>10⁴

The conditions used for the tests were as follows: enzyme concentration of

M NaN_3,and

125 pM in 0.1 M phosphate buffer containing 15 mM NaCl, 150

5.2. Molecular docking

M Bovine Serum Albumin (BSA) at pH 7.4, 25 °C (see Section 5).

Less than 1% of reversion was obtained at 100 lM.

Only 6% of reversion was obtained at 10

Only 7% of reversion was obtained at 10

Docking calculations were carried out using ^AUTODOCK, version

4.0.1, with the Lamarckian genetic algorithm (LGA²⁸). In order to

lM.

4530

C. Ronco et al. / Bioorg. Med. Chem. 17 (2009) 4523–4536

determine the protonation state of each derivative at pH 7.4, pK_a

was calculated using the SPARC online calculator (http://ibmlc2.-

chem.uga.edu/sparc/). The molecular models of each derivative

were built and minimized with the MM2 force ﬁeld of Chem3D

(CambridgeSoft.). Gasteiger total charges were calculated using

the PETRA online (http://www2.chemie.uni-erlangen.de/software/

petra/intro.phtml).²⁹The structure of human AChE was prepared

using MODELLER and CNS from the crystal structure of its complex

with fasciculin (pdb code 1b41). In order to take into account the

ﬂexibility of the enzyme upon Huprines binding, the structure of

hAChE was compared to the complex of Huprine X and Torpedo cal-

ifornica AChE (pdb code 1e66) and moving loops were rebuilt and

minimized using MODELLER.³⁰Structural water molecules were

conserved in the model in order to improve docking accuracy. Hup-

rines and hAChE were further prepared using ^AUTODOCKTools

1.5.2.³¹The 3D afﬁnity grid box was designed to include the full ac-

tive site gorge of human AChE. The number of grid points in the x-,

y-, and z-axes was 96, 115, and 115 with grid points separated by

0.2 Å. Docking calculations were set to 100 runs. At the end of the

calculation, ^AUTODOCKperformed cluster analysis. Docking solutions

with ligand all-atom root-mean-square deviation (rmsd) within

1.0 Å of each other were clustered together and ranked by the low-

est energy representative. Usually more than 85% of the runs lead

to solutions belonging to the same cluster. The lowest-energy solu-

tion was accepted as the one most representative of the Huprine–

AChE complex.

(c = 0.3 M) and the resulting reaction mixture was reﬂuxed over-

night. Thereafter, the mixture was cooled to room temperature

and diluted by sequential addition of deionized water, THF, aque-

ous 5 N NaOH and then stirred at room temperature for 30 min.

The organic phase was separated and the aqueous phase was ex-

tracted with CH₂Cl₂. The combined organic phases were washed

with brine, dried over MgSO_4,and concentrated under vacuum.

The resultant crude product was puriﬁed by ﬂash chromatography

on a silica gel column to give the desired Huprine.

5.3.1.1.

( )-12-Amino-2,3-dimethoxy-9-methyl-6,7,10,11-tetra-

hydro-7,11-methanocycloocta[b]quinoline (3a). This compound

was prepared according to the general procedure described above

using 2-amino-4,5-dimethoxybenzonitrile (1.81 g, 10.2 mmol), 2a

(1 g, 6.65 mmol). Puriﬁcation by ﬂash chromatography on a silica

gel column (AcOEt to CH₂Cl₂/MeOH, 92:8) afforded the desired Hup-

rine 3a as a off-white solid (600 mg, 30%); mp: 247 °C; ¹H NMR

(300 MHz, CDCl₃): d = 1.52 (s, 3H, H₁₄), 1.94–2.04 (m, 3H, H₁₀, H₁₃),

2.42–2.52 (m, 1H, H₁₃), 2.68–2.78 (m, 1H, H₇), 2.93 (br d,

J = 17.1 Hz, 1H, H₆,), 3.12 (dd, J = 17.1 Hz, J = 5.4 Hz, 1H, H₆), 3.16–

3.26 (m, 1H, H₁₁), 3.96 (s, 3H, OMe), 3.98 (s, 3H, OMe), 4.57 (br s,

2H, NH₂), 5.49–5.59 (m, 1H, H₈), 6.89 (s, 1H, H₁), 7.26 (s, 1H, H₄);

¹³C NMR (75 MHz, CDCl₃): d = 23.6 (C₁₄), 27.7 (C₁₁), 28.6 (C₇), 29.4

(C₁₀), 35.9 (C₁₃), 39.6 (C₆), 56.1 (OMe), 98.7 (C₁), 108.0 (C₄), 111.8

(C_12a), 114.9 (C_11a), 125.5 (C₈), 132.2 (C₉), 143.7 (C₁₂or C_4a), 144.7

(C₁₂or C_4a), 148.1 (C₂or C₃), 151.7 (C₂or C₃), 155.3 (C_5a); IR (KBr):

= 3470, 2985, 1651, 1438, 1254, 1162, 1004 cm^ꢀ1; MS (ESI+): m/z

5.3. Synthesis

(%): 311 (100) [M+H]⁺; HPLC: t_R= 21.4 min (purity >95%).

In general, column chromatography puriﬁcations were per-

formed on silica gel (40–63 lm) from SdS. Thin-layer chromatogra-

5.3.1.2. ( )-12-Amino-2,3-dimethoxy-9-ethyl-6,7,10,11-tetrahy-

dro-7,11-methanocycloocta[b]quinoline (3b). This compound

was prepared according to the general procedure described above

using 2-amino-3-cyanopyridine (454 mg, 2.54 mmol), 2b (250 mg,

1.66 mmol). Puriﬁcation by ﬂash chromatography on a silica gel

column (CH₂Cl₂/MeOH, 98:2) afforded the desired Huprine 3b as

a white-off solid (350 mg, 68%); mp: 149 °C (decomposition); ¹H

NMR (300 MHz, CDCl₃): d = 0.83 (t, 3H, H₁₅, J = 7.7 Hz), 1.76 (q,

2H, H₁₄, J = 7.7 Hz), 1.88 (m, 1H, H₁₀), 1.96 (d, J = 17.7 Hz, 1H,

H₁₃), 1.98–2.08 (m, 1H, H₁₀), 2.40–2.50 (m, 1H, H₁₃), 2.67–277

(m, 1H, H₇), 2.90 (d, J = 17.4 Hz, 1H, H₆,), 3.10 (dd, J = 17.4 Hz,

J = 5.4 Hz, 1H, H₆), 3.07–3.17 (m, 1H, H₁₁), 3.95 (s, 3H, OMe), 3.97

(s, 3H, OMe), 5.29 (br s, 2H, NH₂), 5.41–5.51 (m, 1H, H₈), 7.04 (s,

1H, H₁), 7.20 (s, 1H, H₄); ¹³C NMR (75 MHz, CDCl₃): d = 12.0 (C₁₅),

27.4 (C₁₁), 28.0 (C₇), 29.4 (C₁₀), 30.0 (C₁₄), 34.2 (C₁₃), 38.2 (C₆),

56.2 (OMe), 99.7 (C₁), 105.7 (C₄), 111.4 (C_12a), 114.0 (C_11a), 123.1

(C₈), 137.8 (C₉), 146.7 (C₁₂or C_4a), 148.1 (C₂or C₃, C₁₂or C_4a),

phy (TLC) was carried out on Merck DC Kieselgel 60 F-254

aluminum sheets. Compounds were visualized by one of the two

following methods: (1) illumination with a short wavelength UV

lamp (k = 254 nm) or (2) staining with a 3.5% (w/v) phosphomolyb-

dic acid solution in absolute ethanol. All solvents were dried fol-

lowing standard procedures (CH₂Cl₂, 1,2-dichloroethane, and

CH₃CN: distillation over P₂O₅, DMF, and DMSO: distillation over

BaO under reduced pressure, THF, toluene, and Et₂O: distillation

over Na/benzophenone). Triethylamine (TEA) and pyridine were

distilled from CaH₂and stored over BaO or KOH.

Melting points were recorded on a LEICA VMHB Koﬂer system

at atmospheric pressure and were uncorrected. Microanalyses

were carried out on Carlo-Erba 1106. Infrared spectra were re-

corded as KBr pellets using a Perkin Elmer FT-IR Paragon 500 spec-

trometer with frequencies given in reciprocal centimeters (cm^ꢀ1).

¹H and ¹³C NMR spectra were recorded on a Bruker DPX 300 spec-

trometer (Bruker, Wissembourg, France). Chemical shifts are ex-

pressed in parts per million (ppm) from CDCl₃(d_H= 7.26,

d_C= 77.16), DMSO-d₆(d_H= 2.50, d_C= 39.52) or CD₃OD (d_H= 3.31,

d_C= 49.00).³²¹J values are expressed in hertz. Mass spectra were

obtained with a Finnigan LCQ Advantage MAX (ion trap) apparatus

equipped with an electrospray source. All analyses were performed

in the positive mode. Analytical HPLC was performed on a Thermo

Electron Surveyor instrument equipped with a PDA detector under

151.7 (C₂or C₃), 155.3 (C_5a); IR (KBr):

m = 3364, 2963, 1645, 1514,

1412, 1284, 1122 cm^ꢀ1; MS (ESI+): m/z (%): 325 (100) [M+H]⁺;

HPLC: t_R= 19.4 min (purity 90%).

5.3.1.3. ( )-11-Amino-8-methyl-5,6,9,10-tetrahydro-6,10-meth-

anocyclooctabenzo[b][1,8]naphthyridine (4a). This compound

was prepared according to the general procedure described above

using 2-amino-3-cyanopyridine (454 mg, 2.54 mmol), 2a (250 mg,

1.66 mmol). Puriﬁcation by ﬂash chromatography on a silica gel

column (CH₂Cl₂/MeOH, 98:2) afforded the desired Huprine 4a as

a white-off solid (350 mg, 68%); mp: 251 °C; ¹H NMR (300 MHz,

CDCl₃): d = 1.52 (s, 3H, H₁₃), 1.95–2.05 (m, 3H, H₉, H₁₅), 2.44–

2.54 (m, 1H, H₁₅), 2.68–2.78 (m, 1H, H₆), 3.09 (br d, J = 17.9 Hz,

1H, H₄), 3.18 (dd, J = 17.9 Hz, J = 5.2 Hz, 1H, H₄), 3.15–3.25 (m,

1H, H₁₀), 4.98 (br s, 2H, NH₂), 5.49–5.59 (m, 1H, H₇), 7.28 (dd,

J = 8.3 Hz, J = 4.2 Hz, 1H, H₂), 8.14 (br d, J = 8.3 Hz, 1H, H₁), 8.93

(dd, J = 4.2 Hz, J = 1.8 Hz, 1H, H₃); ¹³C NMR (75 MHz, CDCl₃):

d = 23.5 (C₁₃), 27.7 (C₁₀), 28.3 (C₆), 29.1 (C₉), 35.5 (C_4a), 39.9 (C₄),

111.9 (C_11a), 116.0 (C_5a), 119.2 (C₂), 125.5 (C₇), 130.3 (C₁), 132.1

(C₈), 147.2 (C₁₁), 152.5 (C₃), 154.7 (C_12a), 161.0 (C₁₂); IR (KBr):

the following conditions: Thermo Hypersil GOLD C18 column (5 l,

4.6 ꢁ 150 mm) with CH₃CN and 0.1% aq TFA as eluents [90% TFA

(5 min), linear gradient from 0% to 100% of CH₃CN (40 min)] at a

ﬂow rate of 1.0 mL min^ꢀ1dual UV detection at 254 and 270 nm.

5.3.1. General procedure for the preparation of racemic

Huprines

To a suspension of anhydrous AlCl₃(1.5 equiv) and o-aminocy-

ano aromatic heterocycle (1.53 equiv) in dry 1,2-dichloroethane

(c = 0.5 M) was added dropwise a solution of ( )-7-alkylbicy-

clo[3.3.1]non-6-en-3-one (1 equiv) in dry 1,2-dichloroethane

C. Ronco et al. / Bioorg. Med. Chem. 17 (2009) 4523–4536

4531

= 3481, 2912, 1634, 1550, 1429, 1336 cm^ꢀ1; MS (ESI+): m/z (%):

above using 2-amino-4,5-dimethylfuran-3-carbonitrile (678 mg,

5.09 mmol), 2b (250 mg, 1.52 mmol). Puriﬁcation by ﬂash chroma-

tography on a silica gel column (cyclohexane/AcOEt, 3:2) afforded

the desired Huprine 6b as a white solid (223 mg, 52%, along with

20% of unseparable undesired isomer); mp: 218 °C (decomposi-

tion); ¹H NMR (300 MHz, CDCl₃): d = 0.88 (t, J = 7.4 Hz, 3H, H₁₅),

1.82–1.92 (m, 4H, H₁₀, H₁₄, H₁₃), 2.28 (s, 3H, 2-Me or 3-Me), 2.30

(s, 3H, 2-Me or 3-Me), 2.40–2.50 (m, 1H, H₁₃), 2.63–2.73 (m, 1H,

H₇), 2.78 (br d, J = 17.9 Hz, 1H, H₆), 3.02 (dd, J = 17.9 Hz,

J = 5.6 Hz, 1H, H₆), 3.01–3.11 (m, 1H, H₁₁), 4.39 (br s, 2H, NH₂),

5.46–5.56 (m, 1H, H₈); ¹³C NMR (75 MHz, CDCl₃): d= 10.5 (2-Me

or 3-Me), 11.4 (2-Me or 3-Me), 12.2 (C₁₅), 25.8 (C₁₁), 27.9 (C₇),

29.6 (C₁₀), 30.0 (C₁₄), 34.4 (C₁₃), 39.1 (C₆), 105.7 (C₂), 107.6 (C₁),

115.1 (C_11a), 123.6 (C₈), 137.3 (C₉), 145.1 (C₃), 146.2 (C_4a), 150.5

252 (100) [M+H]⁺; HPLC: t_R= 13.8 min (purity >95%).

5.3.1.4. ( )-11-Amino-8-ethyl-5,6,9,10-tetrahydro-6,10-metha-

nocyclooctabenzo[b][1,8]naphthyridine (4b). This compound

was prepared according to the general procedure described above

using 2-amino-3-cyanopyridine (333 mg, 2.79 mmol), 2b (300 mg,

1.82 mmol). Precipitation in acetone gave the desired Huprine 4b

as a white-off solid (270 mg, 48%); mp: 161–162 °C; ¹H NMR

(300 MHz, DMSO-d₆):d = 0.81 (t, J = 7.7 Hz, 3H, H₁₄), 1.75 (q,

J = 7.7 Hz, 2H, H₁₃), 1.83–1.93 (m, 3H, H₉, H₁₅), 2.35–2.45 (m, 1H,

H₁₅), 2.61–2.71 (m, 1H, H₆), 2.79 (d, J = 17.4 Hz, 1H, H₄), 3.02 (dd,

J = 17.4 Hz, J = 5.4 Hz, 1H, H₄), 3.32–3.42 (m, 1H, H₁₀), 5.42–5.52

(m, 1H, H₇), 6.71 (br s, 2H, NH₂), 8.04 (d, J = 5.7 Hz, 1H, H₂), 8.29

(d, J = 5.7 Hz, 1H, H₁), 8.94 (s, 1H, H₃); ¹³C NMR (75 MHz, DMSO-

d₆): d = 11.9 (C₁₄), 26.5 (C₁₀), 27.8 (C₆), 28.9 (C₉), 29.4 (C₁₃), 33.9

(C_4a), 39.5 (C₄), 115.0 (C₂), 116.5 (C_11a), 120.6 (C_5a), 122.9 (C₇),

137.7 (C₈), 139.9 (C₁), 141.8 (C_12a), 146.4 (C₁₁), 152.1 (C₃), 158.4

(C₁₂), 160.7 (C_5a); IR (KBr):

m = 3496, 2923, 1631, 1591, 1433,

1296, 1115 cm^ꢀ1; MS (ESI+): m/z (%): 283 (100) [M+H]⁺; HPLC:

t_R= 19.0 min (purity 93%).

(C₁₂); IR (KBr):

= 3339, 2897, 1662, 1574, 1415, 1358 cm^ꢀ1; MS

5.3.1.8. ( )-9-Amino-1-cyclopropyl-oxazolo[4,5-b]-6-methyl-3,4,

7,8-tetrahydro-6,10-methano cycloocta[e]pyridine (7a). This com-

pound was prepared according to the general procedure described

above using 5-amino-2-cyclopropyloxazole-4-carbonitrile 20

(456 mg, 3.05 mmol), 2a (300 mg, 2.00 mmol). Puriﬁcation by ﬂash

chromatography on a silica gel column (cyclohexane/AcOEt, 1:1)

afforded a yellow solid (270 mg, 48%, along with 7% of unseparable

isomer). Recrystallization from Et₂O gave the desired Huprine 7a

as a white solid; mp: 166 °C; ¹H NMR (300 MHz, CDCl₃): d = 1.06–

1.16 (m, 2H, CH₂cyclopropyl), 1.17–1.27 (m, 2H, CH₂cyclopropyl),

1.53 (s, 3H, H₁₄), 1.82–1.92 (m, 2H, H₁₀, H₁₃), 1.92–2.02 (m, 1H,

H₁₀), 2.06–2.16 (m, 1H, CH cyclopropyl), 2.37–2.47 (m, 1H, H₁₃),

2.61–2.71 (m, 1H, H₇), 2.77 (br d, 1H, H₆, J = 17.4 Hz), 3.02 (dd,

J = 17.4 Hz, J = 5.5 Hz, 1H, H₆), 3.04–3.14 (br s, 1H, H₁₁), 4.66 (br s,

2H, NH₂), 5.46–5.56 (m, 1H, H₈); ¹³C NMR (75 MHz, CDCl₃): d = 8.8

(CH₂cyclopropyl), 9.77 (CH₂cyclopropyl), 22.7 (C₁₄), 27.4 (C₁₁),

28.5 (C₇), 29.0 (C₁₀), 35.8 (C₁₃), 39.1 (C₆), 116.6 (C_11a), 118.3 (C₁),

125.5 (C₈), 132.0 (C₉), 142.1 (C_4a), 151.1 (C₁₂), 158.4 (C_5a), 164.5

(ESI+): m/z (%): 266 (100) [M+H]⁺; HPLC: t_R= 15.9 min (purity 93%).

5.3.1.5. ( )-11-Amino-8-ethyl-5,6,9,10-tetrahydro-6,10-metha-

nocyclooctabenzo[b][1,7]naphthyridine (5). This compound was

prepared according to the general procedure described above using

3-aminoisonicotinonitrile 19 (150 mg, 1.25 mmol), 2b (135 mg,

0.82 mmol). Puriﬁcation by ﬂash chromatography on a silica gel

column (CH₂Cl₂/MeOH, 98:2) afforded the desired Huprine 5 as a

white-off solid (234 mg, 70%); mp: 192 °C; ¹H NMR (300 MHz,

CDCl₃): d = 0.84 (t, J = 7.4 Hz, 3H, H₁₄), 1.78 (q, J = 7.4 Hz, 2H,

H₁₃), 1.87–1.97 (m, 1H, H₉), 1.97 (br d, J = 16.8 Hz, 1H, H₁₅),

1.99–2.09 (m, 1H, H₉), 2.43–2.53 (m, 1H, H₁₅), 2.69–2.79 (m, 1H,

H₆), 3.07 (dt, J = 17.9 Hz, J = 1.8 Hz, 1H, H₄), 3.17 (dd, J = 17.9 Hz,

J = 5.2 Hz, 1H, H₄), 3.16–3.26 (m, 1H, H₁₀), 5.00 (br s, 2H, NH₂),

5.47–5.57 (m, 1H, H₇), 7.25 (dd, J = 8.3 Hz, J = 4.2 Hz, 1H, H₂), 8.16

(dd, J = 8.3 Hz, J = 1.8 Hz, 1H, H₁), 8.92 (dd, J = 4.2 Hz, J = 1.8 Hz,

1H, H₃); ¹³C NMR (75 MHz, CDCl₃): d = 12.1 (C₁₄), 27.8 (C₁₀), 28.3

(C₆), 29.4 (C₉), 30.0 (C₁₃), 34.1 (C_4a), 40.4 (C₄), 111.9 (C_11a), 116.2

(C_5a), 119.1 (C₂), 123.6 (C₇), 129.9 (C₁), 137.4 (C₈), 146.6 (C₁₁),

(C₃); IR (KBr):

m = 3339, 2906, 1742, 1651, 1471, 1334, 1291,

1107 cm^ꢀ1MS (ESI+): m/z (%): 282 (100) [M+H]⁺; HPLC:

;

152.4 (C₃), 155.1 (C_12a), 161.5 (C₁₂); IR (KBr):

= 3330, 2892,

t_R= 17.9 min (purity >95%).

1623, 1547, 1490, 1332 cm^ꢀ1; MS (ESI+): m/z (%): 266 (100)

[M+H]⁺; HPLC: t_R= 15.1 min (purity >95%).

5.3.1.9. ( )-9-Amino-1-cyclopropyl-oxazolo[4,5-b]-6-ethyl-3,4,7,

8-tetrahydro-6,10-methano cycloocta[e]pyridine (7b). This com-

pound was prepared according to the general procedure described

above using 5-amino-2-cyclopropyloxazole-4-carbonitrile 20 (300

mg, 2.01 mmol), 2b (216 mg, 1.31 mmol). Puriﬁcation by ﬂash chro-

matography on a silica gel column (cyclohexane/AcOEt, 1:1) afforded

a yellow solid. Recrystallization from Et₂O furnished the desired

Huprine 7b as a pale orange solid (390 mg, 70%, along with 12% of

unseparable undesired isomer); mp: 134 °C; ¹H NMR (300 MHz,

CDCl₃): d = 0.80 (t, J = 7.3 Hz, 3H, H₁₅), 1.01–1.11 (m, 2H, CH₂cyclo-

5.3.1.6. ( )-9-Amino-7-methyl-2,3-dimethylfuro[2,3-b]-4,5,8,9-

tetrahydro-5,9-methanocyclo octan[e]pyridine (6a). This com-

pound was prepared according to the general procedure described

above using 2-amino-4,5-dimethylfuran-3-carbonitrile (678 mg,

5.09 mmol), 2a (500 mg, 3.33 mmol). Puriﬁcation by ﬂash chroma-

tography on a silica gel column (cyclohexane/AcOEt, 3:2) afforded

the desired Huprine 15a as a white solid (510 mg, 57%, along with

13% of inseparable isomer); mp: 251 °C; ¹H NMR (300 MHz, CDCl₃):

d = 1.53 (s, 3H, H₁₄), 1.79–1.89 (m, 1H, H₁₀), 1.88 (br d, J = 17.1 Hz,

1H, H₁₃), 1.93–2.03 (m, 1H, H₁₀), 2.28 (s, 3H, 2-Me or 3-Me), 2.30

(s, 3H, 2-Me or 3-Me), 2.33–2.43 (m, 1H, H₁₃), 2.60–2.70 (m, 1H,

H₇), 2.78 (br d, J = 17.9 Hz, 1H, H₆), 3.02 (dd, J = 17.9 Hz, J = 5.6 Hz,

1H, H₆), 3.03–3.09 (m, 1H, H₁₁), 5.46–5.56 (m, 1H, H₈); ¹³C NMR

(75 MHz, CDCl₃): d = 9.3 (2-Me or 3-Me), 10.3 (2-Me or 3-Me),

22.4 (C₁₄), 25.8 (C₁₁), 27.4 (C₇), 28.2 (C₁₀), 34.7 (C₁₃), 37.8 (C₆),

104.6 (C₂), 106.4 (C₁), 113.9 (C_11a), 124.5 (C₈), 130.8 (C₉), 143.9

propyl), 1.11–1.21 (m, 2H, CH₂cyclopropyl), 1.80–1.90 (m, 4H, H₁₀

H₁₃, H₁₄), 1.92–2.02 (m, 2H, H₁₀, H₁₃), 2.06–2.16 (m, 1H, CH cyclopro-

pyl), 2.37–2.47 (m, 1H, H₁₆), 2.61–2.71 (m, 1H, H₇), 2.73 (br d,

J = 17.4 Hz, 1H, H₆), 2.97 (dd, J = 17.4 Hz, J = 5.5 Hz, 1H, H₆,), 3.00–

3.10 (br s, 1H, H₁₁), 4.62 (br s, 2H, NH₂), 5.46–5.56 (m, 1H, H₈); ¹³C

NMR (75 MHz, CDCl₃): d = 8.8 (CH₂cyclopropyl), 9.8 (CH₂cyclopro-

pyl), 12.2 (C₁₅), 27.4 (C₁₁), 28.4 (C₇), 29.3 (C₁₀), 30.0 (C₁₄), 34.2

(C₁₃), 39.2 (C₆), 116.6 (C_11a), 118.3 (C₁), 123.5 (C₈), 137.4 (C₉),

(C₃), 145.1 (C_4a), 149.3 (C₁₂), 159.6 (C_5a); IR (KBr):

= 3489, 2919,

142.1 (C_4a), 151.2 (C₁₂), 158.4 (C_5a), 164.5 (C₃); IR (KBr): m = 3339,

1632, 1590, 1434, 1360, 1297, 1115 cm^ꢀ1; MS (ESI+): m/z (%): 269

(100) [M+H]⁺; HPLC: t_R= 17.3 min (purity >95%).

2906, 1742, 1651, 1471, 1334, 1291, 1107 cm^ꢀ1; HPLC: t_R= 17.8 min

(purity >95%).

5.3.1.7. ( )-9-Amino-7-methyl-2,3-dimethylfuro[2,3-b]-4,5,8,9-

tetrahydro-5,9-methanocyclo octan[e]pyridine (6b). This com-

pound was prepared according to the general procedure described

5.3.1.10.

( )-9-Amino-1-cyclobutyl-oxazolo[4,5-b]-6-methyl-

3,4,7,8-tetrahydro-6,10-methano cycloocta[e]pyridine (8). This

compound was prepared according to the general procedure de-

4532

C. Ronco et al. / Bioorg. Med. Chem. 17 (2009) 4523–4536

scribed above using 21 (415 mg, 2.54 mmol), 2a (250 mg,

1.66 mmol). Flash chromatography on a silica gel column (cyclohex-

ane/AcOEt, 7:3)gavethedesiredHuprine8, whichwasrecrystallized

from Et₂O to afford as a pale orange solid (210 mg, 45%); mp: 200 °C;

¹H NMR (300 MHz, CDCl₃): d = 1.52 (s, 3H, H₁₄), 1.77–1.87 (m, 1H,

5.3.1.13. ( )-9-Amino-1-(methylthio)-1H-pyrazolo[3,4-b]-3,4,7,8-

tetrahydro-6-methyl-4,8-methanocycloocta[e]pyridine (10). This

compound was prepared according to the general procedure described

above using 3-amino-5-(methylthio)-1H-pyrazole-4-carbonitrile

(785 mg, 5.09 mmol), 2a (500 mg, 3.33 mmol). Puriﬁcation by ﬂash

chromatography on a silica gel column (AcOEt) afforded the desired

Huprine 10 as a yellow solid (320 mg, 33%, along with 5% of unsepara-

ble undesired isomer); mp: 256 °C (decomposition); ¹H NMR

(300 MHz, CDCl₃): d = 1.55 (s, 3H, H₁₄), 1.83–1.93 (m, 1H, H₁₀), 1.89–

1.99 (m, 1H, H₁₃), 1.95–2.05 (m, 1H, H₁₀), 2.40–2.50 (m, 1H, H₁₃), 2.57

(s, 3H, SMe), 2.65–2.75 (m, 1H, H₇), 3.01 (br d, J = 17.4 Hz, 1H, H₂),

3.07–3.17 (m, 1H, H₁₁), 3.19 (dd, J = 17.4 Hz, J = 5.5 Hz, 1H, H₆), 5.42

(br s, 2H, NH₂), 5.49–5.59 (m, 1H, H₈); ¹³C NMR (75 MHz, CDCl₃):

d = 19.3 (SMe), 23.6 (C₁₄), 26.9 (C₁₁), 28.3 (C₇), 29.4 (C₁₀), 35.9 (C₁₃),

39.5 (C₆), 104.1 (C₁), 111.6 (C_11a), 125.5 (C₈), 132.3 (C₉), 137.2 (C₂),

H₁₀), 1.90 (br d, J = 17.5 Hz, 1H, H₁₃), 2.02–2.12 (m, 3H, H₁₀

CH₂bcyclobutyl), 2.39–2.49 (m, 5H, H₁₃, CH₂bcyclobutyl, CH₂cyclo-

butyl), 2.61–2.71 (m, 1H, H₇), 2.78 (br d, J = 17.5 Hz, 1H, H₆), 3.04 (dd,

J = 17.5 Hz, J = 5.6 Hz, 1H, H₆), 3.04–3.14 (m, 1H, H₁₁), 3.69 (quintd,

J = 8.3 Hz, J = 0.9 Hz, 1H, CHcyclobutyl), 4.73, (br s, 2H, NH₂), 5.45–

5.55 (m, 1H, H₈); ¹³C NMR (75 MHz, CDCl₃): d = 18.8 (CH₂cyclobu-

tyl), 23.5 (C₁₄), 27.0 (CH₂bcyclobutyl), 27.1 (CH₂bcyclobutyl), 27.4

(C₁₁), 28.5 (C₇), 29.0 (C₁₀), 33.88 (CHcyclobutyl), 35.8 (C₁₃), 39.2

(C₆), 116.5 (C_11a), 118.1 (C₁), 125.4 (C₈), 132.0 (C₉), 142.7 (C_4a),

151.6 (C_5a), 158.9 (C₁₂), 165.4 (C₃); IR (KBr):

m = 3332, 2918, 1652,

1472, 1329, 1110 cm^ꢀ1; MS (ESI+): m/z (%): 296 (100) [M+H]⁺; Anal.

Calcd for C₁₈H₂₁N₃O: C, 73.19; H, 7.17; N, 14.23. Found: C, 73.28; H,

7.28; N, 14.19; HPLC: t_R= 18.1 min (purity >95%).

145.9 (C_4a), 153.2 (C₁₂), 157.5 (C_5a); IR (KBr): m = 3364, 2923, 1611,

1582, 1425, 1373, 1278, 1168, 1031 cm^ꢀ1; MS (ESI+): m/z (%): 287

(100) [M+H]⁺; Anal. Calcd for C₁₅H₁₈N₄S: C, 62.91; H, 6.33; N, 19.56;

S, 11.20. Found:C, 62.89; H, 6.46;N, 19.63; S, 11.13; HPLC: t_R= 15.4 min

(purity >95%).

5.3.1.11. ( )-11-Amino-2-thiopropyl-5,6,9,10-tetrahydro-thiaz-

olo[4,5-b]-8-methyl-6,10-methanocycloocta[e]pyridine (9a). This

compound was prepared according to the general procedure de-

scribedaboveusing22 (507 mg, 2.54 mmol), 2a(250 mg,1.66 mmol).

Flash chromatography on a silica gel column (CH₂Cl₂/MeOH, 98:2)

gave the desired Huprine 9a as a pale orange solid (390 mg, 70%);

mp: 205 °C; ¹H NMR (300 MHz, CDCl₃): d = 1.00 (t, J = 7.5 Hz, 3H,

CH₃thioalkyl chain,), 1.49 (s, 3H, H₁₄), 1.84 (quint, J = 7.5 Hz, 2H,

CH₂b thioalkyl chain,), 1.81–1.91 (m, 1H, H₁₃), 1.85–1.95 (m, 1H,

H₁₀), 1.92–2.02 (m, 1H, H₁₃), 2.37–2.47 (m, 1H, H₁₀), 2.63–2.73 (m,

1H, H₈), 2.91 (br d, J = 17.5 Hz 1H, H₆), 3.08 (dd, J = 17.5 Hz,

5.3.1.14. ( )-9-Amino-1H-pyrazolo[3,4-b]-3,4,7,8-tetrahydro-6-

methyl-4,8-methanocycloocta[e]pyridine (11). This compound

was prepared according to the general procedure described above

using 3-amino-4-pyrazolecarbonitrile (550 mg, 5.09 mmol), 2a

(500 mg, 3.33 mmol). Puriﬁcation by ﬂash chromatography on a

silica gel column (AcOEt) afforded the desired Huprine 11 as a

white solid (400 mg, 41% along with 9% of unseparable undesired

isomer); mp: 224 °C (decomposition); ¹H NMR (300 MHz, CD₃OD):

d = 1.54 (s, 3H, H₁₄), 1.83–2.01 (m, 3H, H₁₀, H₁₃, H₁₀), 2.41 (dd,

J = 17.4 Hz, J = 4.3 Hz, 1H, H₁₃), 2.58–2.68 (m, 1H, H₇), 2.74 (br d,

J = 17.4 Hz, 1H, H₆), 3.00 (dd, J = 17.4 Hz, J = 5.5 Hz, 1H, H₆), 3.19–

3.29 (m, 1H, H₁₁), 5.50 (d, J = 4.5 Hz, 1H, H₈), 8.11 (s, 1H, H₂); ¹³C

NMR (75 MHz, CD₃OD): d = 23.7 (C₁₄), 27.8 (C₁₁), 29.7 (C₇), 30.3

(C₁₀), 37.0 (C₁₃), 39.7 (C₆), 105.3 (C₁), 111.9 (C_11a), 125.9 (C₈),

132.3 (C₉), 134.1 (C₂), 149.7 (C_4a), 151.8 (C₁₂), 156.9 (C_5a); IR

J = 5.5 Hz, 1H, H₆), 3.08–3.18(m, 1H, H₁₁), 3.32–3.42(m, 2H, CH₂

oalkyl chain), 4.24 (br s, 2H, NH₂), 5.51 (br d, J = 4.5 Hz, 1H, H₈); ¹³

NMR (75 MHz, CDCl₃): d = 13.4 (CH₃thioalkyl chain), 22.8 (CH₂b thi-

oalkyl chain), 23.4 (C₁₄), 27.3 (C₁₁), 28.4 (C₇), 29 (C₁₃), 35.4 (CH₂thi-

athi-

oalkyl chain), 35.7 (C₁₀), 39.2 (C₆), 111.3 (C₁), 115.8 (C_11a), 125.0 (C₈),

131.8 (C₉), 144.7 (C_4a), 154.9 (C_5a), 163.1 (C₁₂), 168.3 (C₃); IR (KBr):

= 3340, 2921, 1618, 1548, 1452, 1353, 1285 cm^ꢀ1; MS (ESI+): m/z

(KBr):

m = 3342, 3216, 2918, 1654, 1589, 1495, 1435, 1398, 1372,

1333, 1274, 1154, 951, 920, 854, 831, 782 cm^ꢀ1

; HPLC:

(%): 332 (100) [M+H]⁺; Anal. Calcd for C₁₇H₂₁N₃S₂: C, 61.59; H, 6.39;

N, 12.68. Found: C, 61.36; H, 6.32; N, 12.68; HPLC: t_R= 20.4 min (pur-

ity >95%).

t_R= 13.7 min (purity >95%).

5.3.1.15. ( )-2-Chloro-N-(3-chloro-6,7,10,11-tetrahydro-12-ylam-

ido-9-methyl-7,11-methanocyclo octan[b]quinoline)acetamide

(12). To a mixture of ( )-Huprine Y (1 g, 3.96 mmol), Et₃N (1.22 mL,

8.72 mmol) dissolved in THF (40 mL) was added slowly chloroacetyl-

chloride (0.63 mL, 7.93 mmol). The reaction was stirred overnight. To

the mixture was added water (50 mL), aqueous saturated solution of

NaHCO₃(50 mL), and AcOEt (100 mL). The aqueous layer was ex-

tracted once with AcOEt (100 mL), dried over MgSO₄, and evaporated

under vacuumtofurnisha brownresidue. Theproductwaspuriﬁedby

ﬂash chromatography on a silica gel column (cyclohexane/AcOEt,

4:1–7:3) to afford 12 as a yellow solid (350 mg, 24%); mp: 230 °C;

¹H NMR (300 MHz, CDCl₃): d = 1.51 (s, 3H, H₁₄), 1.76 (d, J = 17.2 Hz,

1H, H₁₃), 1.96–2.06 (m, 2H, H₁₀), 2.59 (br dd, J = 17.2 Hz, J = 4.6 Hz,

1H, H₁₃), 2.74–2.84 (m, 1H, H₇), 3.11 (br d, J = 17.7 Hz, 1H, H₆), 3.20

(dd, J = 17.7 Hz, J = 5.1 Hz, 1H, H₆), 3.41–3.51 (m, 1H, H₁₁), 4.38 (s,

2H, H₁₉), 5.51–5.61 (m, 1H, H₈), 7.40 (dd, J = 9.0 Hz, J = 2.0 Hz 1H,

H₂), 7.63 (d, J = 9.0 Hz, 1H, H₁), 8.00 (d, J = 2.0 Hz, 1H, H₄), 8.30 (br s,

1H, NH); ¹³C NMR (75 MHz, CDCl₃): d = 23.4 (C₁₄), 28.2 (C₇), 28.4

(C₁₀), 29.0 (C₁₁), 38.1 (C₁₃), 40.1 (C₆), 43.0 (C₁₉), 122.5 (C_12a), 124.3

(C₁), 125.6 (C₈), 127.3 (C₂), 127.8 (C₄), 132.0 (C₃), 132.6 (C₉), 135.1

(C_11a), 137.3 (C_4a), 147.9 (C₁₂), 160.6 (C_5a), 165.3 (C₁₅); IR (KBr):

5.3.1.12. ( )-11-Amino-2-thiopropyl-5,6,9,10-tetrahydro-thiaz-

olo[4,5-b]-8-ethyl-6,10-methano cycloocta[e]pyridine (9b). This

compound was prepared according to the general procedure de-

scribed above using 22 (507 mg, 2.54 mmol), 2b (250 mg,

1.52 mmol). Flash chromatography on

a silica gel column

(CH₂Cl₂/AcOEt, 4:1) gave the desired Huprine 9b as a white-off so-

lid (290 mg, 56%); mp: 196 °C; ¹H NMR (300 MHz, CDCl₃): d = 1.00

(t, J = 7.5 Hz, 3H, CH₃thioalkyl chain), 1.49 (s, 3H, H₁₄), 1.84 (quint,

J = 7.5 Hz, 2H, CH₂b thioalkyl chain,), 1.81–1.91 (m, 1H, H₁₄), 1.85–

1.95 (m, 1H, H₁₀), 1.92–2.02 (m, 1H, H₁₃), 2.37–2.47 (m, 1H, H₁₀),

2.63–2.73 (m, 1H, H₇), 2.91 (br d, J = 17.5 Hz, 1H, H₆), 3.08 (dd,

J = 17.5 Hz, J = 5.5 Hz, 1H, H₆), 3.11–3.21 (m, 1H, H₁₁), 3.32–3.42

(m, 2H, CH₂athioalkyl chain), 4.24 (br s, 2H, NH₂), 5.51 (br d,

J = 4.5 Hz, 1H, H₈); ¹³C NMR (75 MHz, CDCl₃): d = 12.1 (CH₃thi-

oalkyl chain), 13.4 (C₁₅), 22.8 (CH₂thioalkyl chain), 27.3 (C₁₁),

28.4 (C₇), 29.3 (C₅), 30.0 (C₁₄), 34.2 (C₁₃), 35.4 (CH₂thioalkyl chain),

39.2 (C₆), 111.3 (C₁), 115.9 (C_11a), 123.5 (C₈), 137.2 (C₉), 144.6 (C₁₃),

155.0 (C_5a), 163.1 (C₁₂), 168.3 (C₃); IR (KBr):

m = 3346, 2893, 1619,

1549, 1452, 1353, 1287 cm^ꢀ1; MS (ESI+): m/z (%): 346 (100)

[M+H]⁺; Anal. Calcd for C₁₈H₂₃N₃S₂: C, 62.57; H, 6.71; N, 12.16; S,

18.56. Found: C, 62.73; H, 7.14; N, 11.91; S, 17.68; HPLC:

t_R= 21.9 min (purity 81%).

= 3232, 2836, 1673, 1509, 1404, 1215 cm^ꢀ1; MS (ESI+): m/z (%):

361 (100) [M+H]⁺; HPLC: t_R= 16.7 min (purity 94%).

C. Ronco et al. / Bioorg. Med. Chem. 17 (2009) 4523–4536

4533

5.3.2. General procedure for the preparation of racemic 12-

chloro-Huprines

J = 2.2 Hz, 1H, H₂), 7.89 (d, J = 2.2 Hz, 1H, H₄), 8.04 (d, J = 9.0 Hz,

1H, H₁); ¹³C NMR (75 MHz, CDCl₃): d = 12.1 (C₁₅), 28.3 (C₇), 29.0

(C₁₀), 30.0 (C₁₄), 30.9 (C₁₁), 35.7 (C₁₃), 40.7 (C₆), 123.1 (C₈), 124.2

(C_12a), 125.6 (C₁), 127.5 (C₄), 127.7 (C₂), 133.7 (C_11a), 135.3 (C₃),

mixture of ( )-7-alkylbicyclo[3.3.1]non-6-en-3-one

(1 equiv) and o-aminobenzoic acid derivative (1 equiv) was slowly

added POCl₃(10 equiv) at 0 °C. The resulting reaction mixture was

heated under reﬂux for 3 h, then cooled to room temperature and

poured carefully onto a mixture of ice and aqueous saturated solu-

tion of K₂CO₃. The aqueous layer was extracted twice with AcOEt,

dried over MgSO₄, and concentrated under reduced pressure.

Recrystallization or ﬂash chromatography on a silica gel column

afforded the desired 12-chloro-Huprine.

138.2 (C₉), 140.9 (C₁₂), 147.5 (C_4a), 160.5 (C_5a); IR (KBr):

m = 2908,

1769, 1508, 1452, 1210 cm^ꢀ1; MS (ESI+): m/z (%): 340 (100)

[M+Na]⁺, 342 (30); HPLC: t_R= 26.9 min (purity >95%).

5.3.3. Synthesis of ( )-N-(2-Hydroxyethyl)-3-chloro-6,7,10,11-

tetrahydro-12-ylamino-9-methyl-7,11-methanocyclooctan[b]-

quinoline (15a)

A mixture of 14a (800 mg, 2.6 mmol), ethanolamine (160

lL,

5.3.2.1. ( )-3-Chloro-12-hydroxy-6,7,10,11-tetrahydro-9-methyl-

7,11-ethanocyclooctan[b]quinoline (13). This compound was pre-

pared according to the general procedure described above using 2a

(1.4 g, 9.3 mmol) and 2-amino-4-chlorobenzoic acid (1.6 g,

9.3 mmol). Neutralization of the reaction was carried out in the

presence of 5 M KOH solution (instead of saturated K₂CO₃solu-

tion). Stirring at rt for 30 min completed the S_NAr substitution.

Recrystallization from diethyl ether afforded 13 as a white solid

(720 mg, 27%); mp > 280 °C; ¹H NMR (300 MHz, CD₃OD): d = 1.55

(s, 3H, H₁₄), 1.76–1.80 (m, 1H, H₁₀), 1.91–2.03 (m, 2H, H₁₀, H₁₃),

2.39 (dd, J = 18.0 Hz, J = 4.5 Hz, 1H, H₁₃), 2.61 (d, J = 18.0 Hz 1H,

H₆), 2.67 (br s, 1H, H₇), 3.03 (dd, J = 18.0 Hz, J = 5.5 Hz 1H, H₅),

3.46 (br s, 1H, H₁₁), 5.51 (d, J = 4.5 Hz, 1H, H₈), 7.29 (dd,

J = 9.0 Hz, J = 1.9 Hz, 1H, H₂), 7.48 (d, J = 1.9 Hz, 1H, H₄), 8.19 (d,

J = 9.0 Hz, 1H, H₁); ¹³C NMR (75 MHz, CD₃OD): d = 23.7 (C₁₄), 26.9

(C₇), 28.9 (C₁₁), 29.8 (C₁₀), 35.6 (C₁₃), 37.1 (C₆), 117.9 (C₄), 122.7

(C_12a), 124.8 (C₂), 125.0 (C₈), 128.4 (C₁), 135.4 (C_11a), 138.7 (C₃),

2.6 mmol), phenol (1.14 g, 12 mmol), and NaI (55 mg, 370 mol)

was carefully heated at 150 °C under Ar for 2.5 h and then cooled

to room temperature. The mixture was diluted with AcOEt

(50 mL), H₂O (40 ml), and basiﬁed (pH 14) with NaOH (5 M) solu-

tion. The aqueous phase was extracted with AcOEt (2 ꢁ 50 mL)

then the combined organic layers were washed with brine, dried

over MgSO₄, and concentrated under reduced pressure to give a

pale brown solid. The product was puriﬁed by ﬂash chromatogra-

phy on a silica gel column (AcOEt) to afford 15a as a white solid

(670 mg, 77%); mp: 80 °C (decomposition); ¹H NMR (300 MHz,

CDCl₃): d = 0.81 (t, J = 7.5 Hz, 3H, H₁₅), 1.75 (q, J = 7.5 Hz, 2H,

H₁₄), 1.78–1.88 (m, 1H, H₁₃), 1.83–1.93 (m, 1H, H₁₀), 1.94–2.04

(m, 1H, H₁₀), 2.54 (br d, J = 17.4 Hz, 1H, H₁₃), 2.71 (br s, 1H, H₇),

2.97 (br d, J = 17.6 Hz, 1H, H₆), 3.12 (dd, J = 17.6 Hz, J = 5.5 Hz, 1H,

H₆), 3.38–3.48 (m, 1H, H₁₁), 3.59–3.69 (m, 2H, H₁₉), 3.83–3.93

(m, 2H, H₂₀), 4.91 (br t, J = 5.3 Hz, 1H, NH or OH,), 5.47 (m, 1H,

H₈), 7.25 (dd, J = 9.0 Hz, J = 2.2 Hz, 1H, H₂), 7.87 (d, J = 2.2 Hz, 1H,

H₄), 7.95 (d, J = 9.0 Hz, 1H, H₁); ¹³C NMR (75 MHz, CDCl₃):

d = 12.0 (C₁₅), 27.4 (C₁₁), 28.2 (C₇), 29.4 (C₁₀), 30.0 (C₁₄),35.7

(C₁₃), 40.1 (C₆), 52.1 (C₁₉), 61.8 (C₂₀), 119.4 (C_12a), 122.4 (C_11a),

123.4 (C₈), 124.5 (C₂), 125.6 (C₁), 127.3 (C₄), 134.2 (C₃), 137.5

(C₉), 148.4 (C₁₂or C_4a), 150.6 (C₁₂or C_4a), 158.8 (C_5a); IR (KBr):

138.9 (C₉), 141.5 (C₁₂), 150.2 (C_4a), 159.4 (C_5a); IR (KBr):

m = 3244,

3096, 2986, 2925, 2820, 1634, 1604, 1555, 1504, 1464, 1410,

1359, 1249, 1172, 1079, 952, 838, 765 cm^ꢀ1; MS (ESI+): m/z (%):

286 (100) [M+H]⁺, 288 (30); HPLC: t_R= 20.4 min (purity 91%).

5.3.2.2. ( )-3,12-Dichloro-6,7,10,11-tetrahydro-9-methyl-7,11-

ethanocyclooctan[b]quinoline (14a). This compound was pre-

pared according to the general procedure described above using

2a (1.4 g, 9.3 mmol) and 2-amino-4-chlorobenzoic acid (1.6 g,

9.3 mmol). Flash chromatography on a silica gel column (CH₂Cl₂),

followed by a recrystallization from acetone afforded 14a as a

white solid (730 mg, 27%); mp: 136 °C; ¹H NMR (300 MHz,

CDCl₃): d = 1.52 (s, 3H, H₁₄), 2.01–2.11 (m, 3H, H₁₀, H₁₃), 2.50–

2.60 (m, 1H, H₁₃), 2.73–2.83 (m, 1H, H₇), 3.09 (d, J = 17.9 Hz,

1H, H₆), 3.19 (dd, J = 17.9 Hz, J = 5.3 Hz, 1H, H₅), 3.69–3.79 (m,

1H, H₁₁), 5.49–5.59 (m, 1H, H₈), 7.47 (dd, J = 8.9 Hz, J = 1.9 Hz,

1H, H₂), 7.97 (d, J = 1.9 Hz, H₄, 1H), 8.12 (d, J = 8.9 Hz, 1H, H₁);

¹³C NMR (75 MHz, CDCl₃): d = 23.5 (C₁₄), 28.4 (C₇), 28.7 (C₁₀),

30.8 (C₁₁), 37.2 (C₁₃), 40.6 (C₆), 124.2 (C_12a), 125.1 (C₈), 125.6

(C₄), 127.4 (C₁or C₂), 127.7 (C₁or C₂), 132.9 (C_11a), 133.7 (C₃),

135.4 (C₉), 141.0 (C₁₂), 147.5 (C_4a), 160.4 (C_5a); IR (KBr):

= 3394, 3148, 2927, 1736, 1558, 1417, 1279, 1076 cm^ꢀ1; MS

(ESI+): m/z (%): 329 (100) [M+H]⁺, 331 (33); HPLC: t_R= 17.4 min

(purity 93%).

5.3.4. Synthesis of ( )-N-(2-Hydroxyethyl)-3-chloro-6,7,10,11-

tetrahydro-12-ylamino-9-ethyl-7,11-methanocyclooctan[b]-

quinoline (15b)

A mixture of 14b (400 mg, 1.25 mmol), ethanolamine (76

1.25 mmol), phenol (544 mg, 5.78 mmol), and NaI (26 mg,

176 mol) was carefully heated at 150 °C under Ar for 2.5 h and

lL,

then cooled to room temperature. The mixture was diluted with

AcOEt (40 mL), H₂O (30 ml), and basiﬁed (pH 14) with 5 M NaOH

solution. The aqueous phase was extracted three times with AcOEt

(40 mL) then the organic layer was washed with brine, dried over

MgSO₄, and concentrated under vacuum to give a pale orange solid.

The product was puriﬁed by ﬂash chromatography on a silica gel

column (AcOEt/cyclohexane, 1:1) to afford 15b as a white solid

(410 mg, 95%); mp: 90 °C (decomposition); ¹H NMR (300 MHz,

CDCl₃): d = 1.43 (s, 3H, H₁₄), 1.77–1.87 (m, 1H, H₁₀), 1.81–1.91

(m, 1H, H₁₃), 1.89–1.99 (m, 1H, H₁₀), 2.45 (br d, J = 17.1 Hz 1H,

H₁₃), 2.59–2.69 (m, 1H, H₇), 2.94 (br d, J = 17.5 Hz, 1H, H₆), 3.10

(dd, J = 17.5 Hz, J = 5.3 Hz 1H, H₆), 3.36–2.46 (m, 1H, H₁₁), 3.61–

3.71 (m, 2H, H₁₅), 3.85–3.95 (m, 2H, H₁₉), 4.90 (br t, J = 5.3 Hz,

1H, OH or NH), 5.37–5.47 (m, 1H, H₈), 7.20 (dd, J = 9.0 Hz,

J = 2.1 Hz 1H, H₂), 7.85 (d, J = 2.1 Hz, 1H, H₄), 7.92 (d, J = 9.0 Hz,

1H, H₁); ¹³C NMR (75 MHz, CDCl₃): d = 23.4 (C₁₄), 27.2 (C₁₁), 28.1

(C₇), 29.0 (C₁₀), 36.8 (C₁₃), 39.6 (C6), 52.0 (C₁₅), 61.2 (C₁₉), 119.1

(C_12a), 121.7 (C_11a), 124.3 (C₂), 125.1 (C₈), 125.7 (C₁), 126.6 (C₄),

132.2 (C₉), 134.3 (C₃), 148.1 (C_4a), 150.1 (C₁₂), 158.3 (C_5a); IR

= 2927, 1609, 1544, 1476, 1301, 1195, 1072 cm^ꢀ1; MS (ESI+):

m/z (%): 304 (100) [M+H]⁺, 306 (25); HPLC: t_R= 24.6 min (purity

>95%).

5.3.2.3.

( )-3,12-Dichloro-6,7,10,11-tetrahydro-9-ethyl-7,11-

ethano-cyclooctan[b]quinoline (14b). This compound was pre-

pared according to the general procedure described above using

2b (800 mg, 4.9 mmol) and 2-amino-4-chlorobenzoic acid

(836 mg, 9.3 mmol). Flash chromatography on a silica gel column

(CH₂Cl₂), followed by recrystallization from acetone afforded 14b

as a white solid (720 mg, 46%); mp: 86 °C; ¹H NMR (300 MHz,

CDCl₃): d = 0.78 (t, J = 7.5 Hz, 3H, H₁₅), 1.73 (q, J = 7.5 Hz, 2H,

H₁₄), 1.83–1.93 (m, 1H, H₁₃), 2.02 (br d, J = 17.2 Hz, 2H, H₁₀), 2.50

(br dd, J = 17.4 Hz, J = 4.7 Hz, 1H, H₁₃), 2.68–2.78 (m, J = 17.7 Hz,

1H, H₇), 3.01(br d, J = 17.7 Hz, 1H, H₆), 3.13 (dd, J = 17.7 Hz,

J = 5.3 Hz 1H, H₆), 5.40–5.50 (m, 1H, H₈), 7.40 (dd, J = 9.0 Hz,

(KBr):

= 3204, 2928, 2108, 1608, 1558, 1418, 1076 cm^ꢀ1; MS

(ESI+): m/z (%): 343 (100) [M+H]⁺, 345 (30); HPLC: t_R= 18.9 min

(purity 92%).

4534

C. Ronco et al. / Bioorg. Med. Chem. 17 (2009) 4523–4536

5.3.5. Synthesis of ( )-N-(2-chloroethyl)-3-chloro-6,7,10,11-

tetrahydro-12-ylamino-9-methyl-7,11-methanocyclooctan[b]-

quinoline (16a)

52.1 (C₁₅), 124.9 (C₂), 125.0 (C₁), 125.5 (C₈), 127.7 (C₄), 131.8

(C₉), 134.2 (C₃), 148.3 (C_4a), 149.2 (C₁₂), 159.1 (C_5a); IR (KBr):

= 3340, 2928, 2100, 1607, 1558, 1415, 1281 cm^ꢀ1; MS (ESI+):

A mixture of 15a (450 mg, 1.37 mmol) and SOCl₂(2.1 mL,

29 mmol) was heated at reﬂux for 45 min. The mixture was con-

centrated under reduced pressure and the residue was diluted in

AcOEt (40 mL), H₂O (30 mL), and basiﬁed (pH 12–14) with KOH

(5 M) solution. The aqueous layer was extracted with AcOEt

(2 ꢁ 50 mL), dried over MgSO₄, and concentrated under reduced

pressure to afford 16a as a pale brown solid (460 mg, quantitative).

This product was pure enough to avoid puriﬁcation; ¹H NMR

(300 MHz, CDCl₃): d = 1.51 (s, 3H, H₁₄), 1.81 (d, J = 15.5 Hz, 1H,

H₁₃), 1.88–1.98 (m, 1H, H₁₀), 2.01–2.11 (m, 1H, H₁₀), 2.58 (br dd,

J = 15.5 Hz, J = 4.3 Hz, 1H, H₁₃), 2.70–2.80 (m, 1H, H₇), 3.02 (dt,

J = 1.6 Hz, J = 17.7 Hz, 1H, H₆), 3.16 (dd, J = 17.7 Hz, J = 5.3 Hz 1H,

H₆), 3.42–3.52 (m, 1H, H₁₁), 3.66–3.76 (m, 2H, H₁₉), 3.71–3.81

(m, 2H, H₁₅), 4.48 (br s, 1H, NH), 5.48–5.58 (m, 1H, H₈), 7.30 (dd,

J = 9.0 Hz, J = 2.3 Hz, 1H, H₂), 7.87 (d, J = 9.0 Hz, 1H, H₁), 7.91 (d,

J = 2.3 Hz, 1H, H₄); ¹³C NMR (75 MHz, CDCl₃): d = 23.5 (C₁₄), 27.6

(C₁₁), 28.3 (C₇), 29.1 (C₁₀), 37.5 (C₁₃), 40.0 (C₆), 45.4 (C₁₉), 51.3

(C₁₅), 119.7 (C_12a), 123.6 (C_11a), 125.0 (C₁), 125.1 (C₂), 125.6 (C₈),

127.8 (C₄), 131.9 (C₉), 134.4 (C₃), 148.3 (C4a), 149.1 (C₁₂), 159.1

m/z (%): 354 (100) [M+H]⁺, 356 (30).

5.3.8. Synthesis of ( )-N-(2-azidoethyl)-3-chloro-6,7,10,11-

tetrahydro-12-ylamino-9-ethyl-7,11-methanocyclooctan[b]-

quinoline (17b)

A mixture of 16b (300 mg, 830 lmol,) and NaN₃(216 mg,

3.3 mmol) in DMF (5 mL) was heated overnight at 80 °C. The

mixture was concentrated under reduced pressure and the resi-

due was diluted with AcOEt (20 mL) and water (20 mL). The

aqueous layer was extracted twice with AcOEt (20 mL), dried

over MgSO₄, and evaporated under vacuum to furnish a pale

brown solid. The product was puriﬁed by ﬂash chromatography

on a silica gel column (AcOEt/cyclohexane, 7:3) to afford the de-

sired product 17b as a pale brown solid (210 mg, 69%); mp:

60 °C (decomposition); ¹H NMR (300 MHz, CDCl₃): d = 0.85 (t,

3H, H₁₅, J = 7.5 Hz), 1.73–1.83 (m, 2H, H₁₄), 1.77–1.87 (m, 1H,

H₁₃), 1.90–2.00 (m, 1H, H₁₀), 2.04–2.14 (m, 1H, H₁₀), 2.62 (br

dd, 1H, H₁₃, J = 17.7 Hz, J = 4.6 Hz), 2.73–2.83 (m, 1H, H7), 3.01

(br d, 1H, H₆, J = 17.7 Hz), 3.20 (dd, 1H, H₆, J = 17.7 Hz,

J = 5.5 Hz), 3.37–3.47 (m, 1H, H₁₁), 3.54–3.64 (m, 4H, H₁₉, H₂₀),

5.47–5.57 (m, 1H, H₈), 7.31 (dd, 1H, H₂, J = 9.0 Hz, J = 2.0 Hz),

7.88 (d, 1H, H₁, J = 9.0 Hz), 7.90 (d, 1H, H₄, J = 2.0 Hz); ¹³C NMR

(75 MHz, CDCl₃): d = 12.1 (C₁₅), 27.7 (C₁₁), 28.2 (C₇), 29.5 (C₁₀),

30.0 (C₁₄), 36.1 (C₁₃), 40.3 (C₆), 49.1 (C₂₀), 52.2 (C₁₉), 119.7

(C_11a), 123.6 (C₈), 125.1 (C₁, C₂), 128.0 (C₄), 134.3 (C_12a, C₃),

137.2 (C₉), 148.6 (C₁₂or C_4a), 149.2 (C₁₂or C_4a), 159.4 (C_5a); IR

(C_5a); IR (KBr):

m = 2929, 2108, 1607, 1557, 1486, 1412, 1346 cm

^ꢀ1; MS (ESI+): m/z (%): 347 (100) [M+H]⁺, 349.

5.3.6. Synthesis of ( )-N-(2-chloroethyl)-3-chloro-6,7,10,11-

tetrahydro-12-ylamino-9-ethyl-7,11-methanocyclooctan[b]quin-

oline (16b)

A mixture of 15b (300 mg, 836 lmol) and SOCl₂(1.3 mL,

17 mmol) was heated at reﬂux for 45 min. The mixture was con-

centrated under reduced pressure and the residue was diluted with

AcOEt (30 mL), H₂O (20 mL), and basiﬁed (pH 9–10) with 5 M KOH

solution. The aqueous layer was extracted with AcOEt (3 ꢁ 30 mL),

dried over MgSO₄, and concentrated under vacuum to afford 16b as

a pale brown solid (320 mg, 98%). This product was pure enough to

avoid puriﬁcation; ¹H NMR (300 MHz, CDCl₃): d = 0.85 (t, J = 7.3 Hz,

3H, H₁₅), 1.79 (br q, J = 7.3 Hz, 2H, H₁₄), 1.81–1.91 (m, 1H, H₁₃),

1.90–2.00 (m, 1H, H₁₀), 2.02–2.12 (m, 1H, H₁₀), 2.62 (br dd,

J = 17.4 Hz, J = 4.8 Hz, 1H, H₁₃), 2.73–2.83 (m, 1H, H₇), 3.03 (br d,

J = 17.6 Hz, 1H, H₆), 3.18 (dd, J = 17.6 Hz, J = 5.3 Hz, 1H, H₆), 3.43–

3.53 (m, 1H, H₁₁), 3.69–3.79 (m, 2H, H₁₉), 3.74–3.84 (m, 2H, H₂₀),

5.47–5.57 (m, 1H, H₈), 7.30 (dd, J = 9.0 Hz, J = 2.0 Hz, 1H, H₂), 7.88

(d, J = 9.0 Hz, 1H, H₁), 7.93 (d, J = 2.0 Hz, 1H, H₄); ¹³C NMR

(75 MHz, CDCl₃): d = 12.0 (C₁₅), 27.6 (C₁₁), 28.1 (C₇), 29.4 (C₁₀),

30.0 (C₁₄), 35.9 (C₁₃), 39.9 (C₆), 45.3 (C₂₀), 51.2 (C₁₉), 119.5 (C_12a),

123.4 (C_11a), 123.5 (C₈), 125.1 (C₁or C₂), 125.2 (C₁or C₂), 127.5

(C₄), 134.6 (C₃), 137.3 (C₉), 148.0 (C₁₂or C_4a), 149.4 (C₁₂or C_4a),

(KBr): m

= 3369, 2929, 2100, 1607, 1557, 1415, 1280 cm^ꢀ1. MS

(ESI+): m/z (%): 368 (100) [M+H]⁺, 370 (33).

5.3.9. Analyses for 1-(3-nitrophenyl)-6,7-dimethoxy-2-(oct-7-

ynyl)-1,2,3,4-tetrahydroisoquinoline (22)

¹H NMR (CDCl₃) d = 1.27 (m, 4H, H₁₈, H₁₉), 1.46 (m, 4H, H₁₇

H₂₀), 1.88 (t, J = 2.7 Hz, 1H, H₂₃), 2.05 (td, J = 2.7 Hz, J = 7.0 Hz,

2H, H₂₁), 2.39 (m, 2H, H₁₆), 2.55 (m, 1H, H₈), 2.75 (dt,

J = 4.1 Hz, J = 16.0 Hz, 1H, H₉), 2.96 (m, 1H, H₉), 3.12 (dt,

J = 4.1 Hz, J = 16.0 Hz, 1H, H₈), 3.61 (s, 3H, OMe), 3.85 (s, 3H,

OMe), 4.60 (s, 1H, H₁), 6.12 (s, 1H, H₁₄), 6.60 (s, 1H, H₁₁), 7.44

(t, J = 7.8 Hz, 1H, H₆), 7.60 (dt, J = 1.3 Hz, J = 7.8 Hz, 1H, H₇),

8.10 (ddd, J = 1.3 Hz, J = 2.3 Hz, J = 7.8 Hz, 1H,H₅), 8.12 (t_app

J = 1.3 Hz, 1H, H₃). ¹³C NMR (CDCl₃) d = 18.4 (C₂₁), 26.7 (C₁₈or

C₁₉), 26.8 (C₁₈or C₁₉), 28.0 (C₉), 28.4 (C₁₇or C₂₀), 28.6 (C₁₇or

C₂₀), 46.5 (C₈), 54.1 (C₁₆), 55.9 (OMe), 56.0 (OMe), 67.3 (C₁),

68.2 (C₂₃), 84.7 (C₂₂), 111.2 (C₁₁), 111.4 (C₁₄), 122.3 (C₅), 124.3

(C₃), 127.4 (C₁₀), 128.4 (C₆), 129.1 (C₇), 135.6 (C₁₅), 147.3 (C₁₂

or C₁₃), 147.4 (C₄), 147.8 (C₁₂or C₁₃), 148.2 (C₂);); IR (KBr):

158.9 (C_5a); IR (KBr):

m .

= 2829, 1622, 1557, 1422, 1348 cm^ꢀ1

= 3275, 2932, 1609, 1537, 1450, 1353, 1254, 1135 cm^ꢀ1MS

5.3.7. Synthesis of ( )-N-(2-azidoethyl)-3-chloro-6,7,10,11-

tetrahydro-12-ylamino-9-methyl-7,11-methanocyclooctan[b]-

quinoline (17a)

A mixture of 16a (350 mg, 1 mmol) and NaN₃(262 mg, 4 mmol)

in DMF (5 mL) was heated overnight at 80 °C. The mixture was

concentrated under reduced pressure and puriﬁed by ﬂash chro-

matography on a silica gel column (AcOEt/cyclohexane, 1:1) to af-

(ESI+): m/z (%): 423 (100) [M+H]⁺.

5.3.10. Analyses for 1-(4-nitrophenyl)-6,7-dimethoxy-2-(oct-7-

ynyl)-1,2,3,4-tetrahydroisoquinoline (23)

¹H NMR (CDCl₃) d = 1.30 (m, 4H, H₁₈, H₁₉), 1.45 (m, 4H, H₁₇

H₂₀), 1.96 (t, 1H, J = 2.7 Hz, H₂₃), 2.12 (td, 2H, J = 2.7 Hz,

J = 7.0 Hz, H₂₁), 2.39 (m, 2H, H₁₆), 2.58 (m, 1H, H₈), 2.77 (dt,

J = 4.1 Hz, J = 15.9 Hz, 1H, H₉), 2.96 (m, 1H, H₉), 3.17 (dt,

J = 4.1 Hz, J = 15.9 Hz, 1H, H₈), 3.61 (s, 3H, OMe), 3.85 (s, 3H,

OMe), 4.58 (s, 1H, H₁), 6.10 (s, 1H, H₁₄), 6.62 (s, 1H, H₁₁), 7.45

(dt, J = 1.9 Hz, J = 8.8 Hz, 2H, H₃, H₇), 8.15 (dt, J = 1.9 Hz,

J = 8.8 Hz, 2H, H₄, H₆). ¹³C NMR (CDCl₃) d = 18.5 (C₂₁), 26.8 (C₁₈

or C₁₉), 26.9 (C₁₈or C₁₉), 28.2 (C₉), 28.5 (C₁₇or C₂₀), 28.7 (C₁₇

or C₂₀), 46.8 (C₈), 54.4 (C₁₆), 55.9 (OMe), 55.9 (OMe), 67.5 (C₁),

68.3 (C₂₃), 84.7 (C₂₂), 111.2 (C₁₁or C₁₄), 111.3 (C₁₁or C₁₄),

123.5 (C₄, C₆), 127.3 (C₁₀), 128.5 (C₁₅), 130.3 (C₃, C₇), 147.2

(C₅), 147.3 (C₁₂or C₁₃), 147.8 (C₁₂or C₁₃), 152.8 (C₂); IR (KBr):

ford 17a as

a pale brown solid (280 mg, 78%); mp: 74 °C

(decomposition); ¹H NMR (300 MHz, CDCl₃): d = 1.48 (s, 3H, H₁₄),

1.77 (br d, J = 15.5 Hz, 1H, H₁₃), 1.85–1.95 (m, 1H, H₁₀), 1.99–2.09

(m, 1H, H₁₀), 2.56 (br dd, J = 15.5 Hz, J = 4.0 Hz, 1H, H₁₃), 2.67–

2.77 (m, 1H, H₇), 2.99 (br d, J = 17.7 Hz, 1H, H₆), 3.13 (dd,

J = 17.7 Hz, J = 5.5 Hz, 1H, H₆), 3.32–3.42 (m, 1H, H₁₁), 3.49–3.59

(m, 4H, H₁₅, H₁₉), 4.26 (br s, 1H, NH), 5.45–5.55 (m, 1H, H₈), 7.27

(dd, J = 8.9 Hz, J = 2.0 Hz, 1H, H₂), 7.85 (d, J = 8.9 Hz 1H, H₁), 7.88

(d, J = 2.0 Hz, 1H, H₄); ¹³C NMR (75 MHz, CDCl₃): d = 23.4 (C₁₄),

27.5 (C₁₁), 28.2 (C₇), 29.0 (C₁₀), 37.4 (C₁₃), 39.9 (C₆), 48.9 (C₁₉),

C. Ronco et al. / Bioorg. Med. Chem. 17 (2009) 4523–4536

4535

= 3307, 2927, 1609, 1518, 1450, 1348, 1221, 1137 cm^ꢀ1MS

Acknowledgments

(ESI+): m/z (%): 421 (100) [M+H]⁺.

We acknowledge the french MRES (‘Ministère de l’Enseignement

Supérieur et de la Recherche’) for Ph.D. Fellowships to C.R. and G.S.

FRM(‘Fondationpourla RechercheMédicale’), and IUF (‘InstitutUni-

versitaire de France’) and the Région Haute Normandie for ﬁnancial

support via the CRUNCh program (CPER 2007-2013). We thank Elis-

abeth Roger (INSA de Rouen) for IR measurements, Annick Leboisse-

lier (INSA de Rouen) for the determination of elemental analyses and

QUIDD Company for the open access to its HPLC instrument.

5.3.11. Synthesis of ( )-3-chloro-6,7,10,11-tetrahydro-N-(2-(4-

(6-(3,4-dihydro-6,7-dimethoxy-1-(3-nitrophenyl)isoquinolin-

2(1H)-yl)hexyl)-1H-1,2,3-triazol-1-yl)-9-methyl)-12-amino-6,10-

methanocycloocta[b]quinoline (24)

A mixture of 17a (22.8 mg, 64

lmol), tetrahydroisoquinoline 22

(27.2 mg, 64 mol), and CuI (0.85 mg, 4.5

lmol) in dry MeCN

(1 mL) was stirred at room temperature for 15 h. THF (1 mL) was

then added to the solution and the mixture was concentrated un-

der vacuum. The product was puriﬁed by ﬂash chromatography on

a silica gel column (CH₂Cl₂/MeOH, 100:0–7:3) to afford the desired

product 24 as a yellow solid (24 mg, 48%); ¹H NMR (300 MHz,

CDCl₃): d = 1.23 (m, 6H, H₁₇, H₁₈, H₁₉), 1.46 (s, 3H, H₄₂), 1.57 (m,

2H, H₂₀), 1.65 (d, J = 15.7 Hz, 1H, H₄₁), 1.83 (m, 1H, H₃₈), 1.97 (m,

1H, H₃₈), 2.40 (m, 2H, H₁₆), 2.52 (m, 2H, H₈, H₄₁), 2.65 (t,

J = 7.5 Hz, 2H, H₂₁), 2.73 (m, 1H, H₉), 2.79 (m, 1H, H₃₅), 2.97 (m,

2H, H₉, H₃₄), 3.09 (m, 1H, H₃₄), 3.12 (m, 2H, H₃₄, H₃₉), 3.61 (s, 3H,

OMe), 3.85 (s, 3H, OMe), 3.94 (m, 2H, H₂₅), 4.56 (m, 3H, H₁, H₂₄),

4.65 (br s, 1H, NH), 5.49 (m, 1H, H₃₆), 6.11 (s, 1H, H₁₄), 6.62 (s,

1H, H₁₁), 7.28 (m, 2H, H₂₃, H₂₉), 7.45 (t, J = 7.7 Hz, 1H, H₆), 7.58

(br d, J = 7.7 Hz, 1H, H₇), 7.76 (d, J = 8.7 Hz, 1H, H₂₈), 7.88 (d,

J = 1.9 Hz, 1H, H₃₁), 8.09 (br d, J = 7.7 Hz, 1H, H₇), 8.15 (br s, 1H,

H₃). ¹³C NMR (CDCl₃) d = 23.5 (C₄₂), 25.6 (C₂₁), 26.9 (C₁₇), 27.0

(C₁₈), 27.5 (C₃₉), 28.1 (C₃₅), 28.3 (C₉), 29.1 (C₁₉), 29.2 (C₃₈), 29.6

(C₁₀), 37.4 (C₄₁), 40.1 (C₃₄), 46.6 (C₈), 49.3 (C₂₅), 50.8 (C₂₄), 55.9

(OMe), 56.0 (OMe), 67.4 (C₁), 111.2 (C₁₁), 111.5 (C₁₄), 119.6 (C₂₇),

121.5 (C₂₉), 122.3 (C₅), 123.7 (C₄₀), 124.3 (C₃), 124.8 (C₂₈), 125.0

(C₂₃), 125.4 (C₃₆), 127.4 (C₁₀), 128.0 (C₃), 128.4 (C₁₅), 129.1 (C₆),

132.0 (C₃₇), 134.2 (C₃₀), 135.7 (C₇), 147.3 (C₁₂or C₁₃), 147.5 (C₄),

147.9 (C₁₂or C₁₃), 148.3 (C₂₂), 148.5 (C₂), 148.9 (C₂₆or C₃₃),

149.0 (C₂₆or C₃₃), 159.3 (C₃₂). MS (ESI+): m/z (%): 776 (100)

[M+H]⁺, 778 (35).

Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.bmc.2009.05.005.

References and notes

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5.3.12. Synthesis of ( )-3-chloro-6,7,10,11-tetrahydro-N-(2-(4-

(6-(3,4-dihydro-6,7-dimethoxy-1-(4-nitrophenyl)isoquinolin-

2(1H)-yl)hexyl)-1H-1,2,3-triazol-1-yl)-9-ethyl)-12-amino-6,10-

methanocycloocta[b]quinoline (25)

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A mixture of 17b (18.4 mg, 50

mol), tetrahydroisoquinoline 23

(21.2 mg, 50 mol), and CuI (1.1 mg, 5.5

lmol) in dry MeCN (1 mL)

was stirred at room temperature for 15 h. THF (1 mL) was added to

the solution and the mixture was concentrated under vacuum. The

product was puriﬁed by ﬂash chromatography on a silica gel col-

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yellow solid (39 mg, 98%); ¹H NMR (300 MHz, CDCl₃): d = 0.83 (t,

J = 7.4 Hz, 3H, H₄₃), 1.24 (m, 4H, H₁₈, H₁₉), 1.46 (m, 2H, H₁₇), 1.59

(m, 2H, H₂₀), 1.74 (m, 3H, H₄₁, H₄₂), 1.84 (m, 1H, H₃₈), 2.00 (m,

1H, H₃₈), 2.37 (m, 2H, H₁₆), 2.55 (m, 2H, H₄₁, H₈), 2.66 (t,

J = 7.5 Hz, 2H, H₂₁), 2.75 (m, 2H, H₉, H₃₅), 2.94 (m, 1H, H₉), 3.02

(m, 1H, H₃₄), 3.13 (m, 3H, H₈, H₃₄, H₃₉), 3.60 (s, 3H, OMe), 3.84

(s, 3H, OMe), 3.95 (m, 2H, H₂₅), 4.57 (m, 3H, H₁, H₂₄), 4.74 (br s,

1H, NH), 5.47 (m, 1H, H₃₆), 6.08 (s, 1H, H₁₄), 6.61 (s, 1H, H₁₁),

7.26 (m, 2H, H₂₃, H₂₉), 7.44 (d, J = 8.7 Hz, 2H, H₃, H₇), 7.76 (d,

J = 9.0 Hz, 1H, H₂₈), 7.93 (d, J = 1.9 Hz, 1H, H₃₁), 8.13 (d, J = 8.7 Hz,

2H, H₄, H₆). ¹³C NMR (CDCl₃) d = 12.0 (C₄₃), 25.7 (C₂₁), 26.9 (C₁₇),

30.0 (C₁₈), 27.4 (C₃₉), 28.1 (C₃₅), 28.2 (C₉), 29.1 (C₁₉), 29.3 (C₃₈),

29.6 (C₂₀), 29.9 (C₄₂), 35.8 (C₄₁), 40.2 (C₃₄), 46.8 (C₈), 49.3 (C₂₅),

50.7 (C₂₄), 54.3 (C₁₆), 55.9 (OMe), 60.0 (OMe), 67.6 (C₁), 111.2

(C₁₁), 111.3 (C₁₄), 119.4 (C₂₇), 121.5 (C₂₉), 123.4 (C₃₆), 123.5 (C₄,

C₆), 123.6 (C₄₀), 124.9 (C₂₈), 125.0 (C₂₃), 127.3 (C₁₀), 127.6 (C₃₁),

128.4 (C₁₅), 130.3 (C₃, C₇), 134.3 (C₃₀), 137.4 (C₃₇), 147.1 (C₅),

147.3 (C₁₂or C₁₃), 147.8 (C₁₂or C₁₃), 148.2 (C₂₂), 148.9 (C₂₆or

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m = 3361,

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4536

C. Ronco et al. / Bioorg. Med. Chem. 17 (2009) 4523–4536

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