Substituted pentacyclic carbazolones as novel muscarinic allosteric agents: Synthesis and structure - Affinity and cooperativity relationships

J . Med. Chem. 2002, 45, 1259-1274

1259

Su bstitu ted P en ta cyclic Ca r ba zolon es a s Novel Mu sca r in ic Alloster ic Agen ts:

Syn th esis a n d Str u ctu r e-Affin ity a n d Coop er a tivity Rela tion sh ip s

Parviz Gharagozloo,*^,†Sebastian Lazareno,^†Masao Miyauchi,^†,‡Angela Popham,^†and Nigel J . M. Birdsall^§

MRC Technology Research Division, 1-3 Burtonhole Lane, Mill Hill, London, NW7 1AD, U.K., and

Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London, NW7 1AA, U.K.

Received J une 7, 2001

Two series of pentacyclic carbazolones, 22 and 23, have been synthesized utilizing a facile

intramolecular Diels-Alder reaction and are allosteric modulators at muscarinic acetylcholine

receptors. Their affinities and cooperativities with acetylcholine and the antagonist N-

methylscopolamine (NMS) at M₁-M₄receptors have been analyzed and compared. All of the

synthesized compounds are negatively cooperative with acetylcholine. In contrast, the majority

of the compounds exhibit positive cooperativity with NMS, particularly at M₂and M₄receptors.

The subtype selectivity, in terms of affinity, was in general M₂> M₁> M₄> M₃. The largest

increases in affinity produced by a single substitution of the core structure were given by the

1-OMe (22b) and 1-Cl (22d ) derivatives. The position of the N in the ring did not appear to be

important for binding affinity or cooperativity. Two compounds 22y and 23i, both trisubstituted

analogues, were the most potent compounds synthesized, with dissociation constants of 30-

100 nM for the M₂NMS-liganded and unliganded receptor, respectively. The results indicate

that the allosteric site, like the primary binding site, is capable of high-affinity interactions

with molecules of relatively low molecular weight.

In tr od u ction

Until recently, it has proven difficult to discover

competitive antagonists that exhibit a substantial se-

lectivity for any one of the five muscarinic receptor

subtypes (M₁-M₅) over all the other subtypes.^1-4In fact,

no M₅selective antagonist and no agonists of substantial

subtype selectivity, at least based on “affinity”, have

been described. This has been disappointing in view of

the importance of muscarinic receptors as therapeutic

targets in a variety of diseases, including asthma,

F igu r e 1. Ternary complex model of allosteric action of a

ligand L with an allosteric agent X at a receptor R.

disorders of intestinal motility, urinary bladder dys-

function, and Alzheimer’s disease, and in the control of

pain.²

on muscarinic receptors. Intrinsic to allosteric agents

is a form of selectivity that is based on differences in

cooperativity between subtypes. This is not available to

antagonists and agonists where affinity and efficacy (in

the case of agonists) are the determinants of relative

receptor subtype selectivity.

The difficulty in designing subtype selective competi-

tive muscarinic agents is probably because of the strong

conservation of sequence in regions considered to bind

agonists.^5,6Muscarinic receptors contain a second, al-

losteric, binding site in addition to the “primary” site,

which binds acetylcholine (ACh), as well as competitive

antagonists such as N-methylscopolamine and atropine

(for recent reviews, see refs 7-12). It is conceivable that

there could be greater sequence differences between the

amino acid residues contributing to the allosteric sites

on different muscarinic receptor subtypes, hence provid-

ing a prospect of developing agents with greater subtype

selectivity, based on affinity. Moreover, ligands binding

to the allosteric site modulate the binding and actions

of ligands, including ACh, at the primary binding site

The simplest mechanism depicting the simultaneous

interactions of two ligands with a receptor is the

allosteric ternary complex model¹³(Figure 1) in which

the affinity constant K_Xof the binding of the ligand X

to the primary site on the receptor R is modified by a

factor R, the cooperativity factor, by the binding of the

allosteric ligand L to the second site on the receptor. In

a complementary manner, the affinity of L, K_L, is

changed by the same factor R by the binding of X to the

receptor. The cooperativity of the interaction of the two

ligands with the receptor is defined by the factor R: if

R > 1, there is positive cooperativity; if R < 1, there is

negative cooperativity. For the special case where R )

1, there is neutral cooperativity and the binding of L to

R does not affect the binding of X. The binding, actions,

and subtype selectivity of the allosteric ligand are

therefore defined by two parameters: its affinity K_L,

* To whom correspondence should be addressed. Address: Compu-

tational, Combinatorial and Medicinal Chemistry, Purdue Pharma, 7

Clarke Drive, Cranbury, NJ 08512. Telephone: 609-860-0700. Fax:

609-860-0781. E-mail: parviz.gharagozloo@pharma.com.

^†MRC Technology Research Division.

^‡Current address: Sankyo Co. Ltd., 2-58 Hiromachi 1-chome,

Shinagawa-ku, Tokyo, 140, J apan.

^§National Institute for Medical Research.

Published on Web 02/19/2002

1260 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6

Gharagozloo et al.

which is just dependent on the nature of R, and R, which

depends on both R and X. The subtype selectivity and

the pharmacological activity of L for modifying the

action of X at different subtypes are therefore manifest

by differences in either or both K_Land R.

Design of Novel Mu sca r in ic Alloster ic Agen ts

The first ligand shown to interact with the allosteric

site on muscarinic receptors was gallamine 1,^13,14which

F igu r e 2. Side view (from the direction of the arrow) of the

solution NMR structure of diasterioisomers 5 and 6.

allosteric enhancer at only M₁receptors, and N-chlo-

romethylbrucine 4 is an enhancer at only M₃recep-

tors.^26,28,29

Our studies on the brucine analogues and also on a

series of strychnine derivatives pointed to the fact that

small changes in structure can produce large changes

in cooperativity and subtype selectivity.^28,30However,

the complex structures of strychnine and brucine were

viewed as a major limitation in their use as a template

for the development of allosteric muscarinic drugs.

Subsequent screening of compounds with fused indole

ring systems, structurally related to brucine, led to the

discovery of other multicyclic agents with allosteric

properties. Among these, the aminotetrahydroisoquino-

linocarbazole 5 was our most interesting hit.³¹This

agent is more potent than brucine at all muscarinic

receptor subtypes. In addition, 5 displays neutral or

positive cooperativity with NMS at all subtypes and low

negative cooperativity with ACh at M₁, M₃, and M₄

receptors, but most interestingly, it has a 3-fold positive

cooperativity with ACh at M₂receptors.³¹This is the

only well-characterized allosteric enhancer of ACh at

M₂receptors identified to date. In contrast, its diaste-

reoisomer 6 exhibits a 8-20-fold higher affinity at M₁,

M₃, and M₄receptors but also has a high negative

cooperativity with both NMS and ACh at all receptor

subtypes.³¹

These stereoisomers have structures simpler than

that of brucine and were regarded as suitable lead

agents for the synthesis of more potent muscarinic

allosteric agents. The reported solution NMR structures

of 5 and 6 (Figure 2) have rings ABCD forming a rigid

and an almost planar structure, with ring E perpen-

dicular to this plane and in a chair conformation.³¹The

only difference between the two isomers is the spatial

orientation of the exocyclic amino group relative to the

pentacyclic ring system; the amino group is equatorial

in 5 and exists in the plane of the ABCD ring structure,

whereas it is axial in 6 and approximately perpendicular

to the ring system. One conclusion is that the spatial

position of the amino group relative to the ABCD ring

plays a major role in the binding at unliganded and

liganded receptors and consequently in their cooperat-

ivity with primary ligands.

exhibits an M₂selectivity as regards K_L. In equilibrium

and kinetic studies gallamine obeyed the predictions of

the allosteric ternary complex model.¹³The R values

were also found to be different (but all much less than

1) for the interaction between gallamine, and a variety

of agonists and antagonists at a given subtype also

differed for a given competitive ligand at different

subtypes.¹³A similar M₂selectivity to gallamine (as

regards K_L) is exhibited by alcuronium 2, and it also

shows a subtype selectivity in R, with positive cooper-

ativity with the antagonist N-methylscopolamine (NMS)

being found at M₂receptors but not at other subtypes.¹⁵

Ligands based around the structure of alcuronium or

with flexible spacers between the two quaternary ni-

trogens have been investigated intensively.^16-24A num-

ber of these molecules are more potent than gallamine

and alcuronium, especially under low ionic strength

conditions. One such ligand, [³H]-dimethyl W-84, has

been used to label an allosteric site on the M₂receptor.²⁵

The quantitative analyses of the allosteric interac-

tions in these studies have involved primarily the

determination of the potency of the ligands in slowing

down the dissociation rate of [³H]-NMS from M₂recep-

tors, i.e., the parameter RK_Lwhere X ) NMS. However,

because our aim is to develop allosteric molecules for

eventual therapeutic use, it is important to estimate the

two parameters that define their actions on the different

receptor subtypes in vivo, namely, K_Land R, where X

is the endogenous ligand ACh. The bis-onium ligands,

where investigated, are strongly negatively cooperative

with ACh at all subtypes.

The first ligands reported to exhibit positive cooper-

ativity with ACh in binding and functional studies at

one or more subtypes were brucine 3 and its quaternized

derivatives.^26-30For this interesting series of com-

pounds, subtype selectivity was primarily based on R

(with ACh) and not K_L; for example, brucine is an

On the basis of these observations, we designed an

analogous pentacyclic ring system (7) that incorporates

Substituted Pentacyclic Carbazolones

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1261

Sch em e 1. Intramolecular Cyclization of 3-THPIs with Subsequent Debenzylation

the ABCD ring structure of brucine and 5 but, in

addition, offers the potential for locating the basic amino

group in a variety of endo- and exocyclic positions. Ring

E of 5 was eliminated (for ease of synthesis), and the

compounds described in this paper do not contain

stereocenters. This enabled both the nature and the

spatial position of the amino group to be readily

investigated in the anticipation that both affinity and

cooperativity might be optimized. We synthesized sev-

eral novel ring scaffolds of type 7 (in which ring C was

reported.³²Taking into consideration the affinity and

cooperativity of these ring systems and their relative

ease of synthesis, the pentacyclic carbazolones 22a and

23a were chosen as templates for the development of

more potent allosteric agents. In this paper we present

our data on the effect of ring substitution in 22a and

23a where the aim was to increase affinity while

maintaining R > 1 or at least close to unity for the

antagonist NMS and for ACh. A preliminary report of

some of the findings has been presented.³³

Ch em istr y

We previously described an efficient four-step syn-

thetic route to a variety of pentacyclic carbazolones via

the intramolecular [4 + 2]π cycloaddition of 3-(tetrahy-

dropyridinyl)indoles (THPIs).³²Following this proce-

dure, we were able to prepare two series of pentacyclic

carbazolones 22a-aa an d 23a-i, illustrated by Schemes

1 and 4 and incorporating a variety of substituents.

The prerequisite 3-THPIs 8a -s and 9a -f were pre-

pared from the one-step condensation of appropriately

both saturated and unsaturated), readily accessible by

an intramolecular [4 + 2]π cycloaddition procedure of

1-substituted-3-(tetrahydropyridinyl)indoles previously

1262 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6

Gharagozloo et al.

Sch em e 2

by the position or the nature of the substituents on the

indole ring (diene) or on the acetylene moiety (dieneo-

phile).

Removal of the benzyl group was achieved by catalytic

hydrogenation of the Diels-Alder adducts, using 5%

Pd/C in MeOH at about 40 °C. This procedure worked

well for most of the benzylated analogues (20 and 21),

and good yields of the target ligands (22 and 23) were

obtained. However, debenzylation of the 3-chloro-

substituted analogue 20o by catalytic hydrogenation

also resulted in extensive dechlorination of the molecule,

and only poor yields of 22p were obtained. This problem

was overcome for the other chloro-substituted analogues

(e.g., 20d and 20i) by simply exchanging the protecting

groups. Thus, reaction of 20d and 20i with vinyl

chloroformate in THF followed by in situ acid hydrolysis

of the corresponding carbamate intermediate furnished

the debenzylated chloro-substituted analogues 22d and

22i in high yields as illustrated in Scheme 4.

Sch em e 3

The analogues incorporating a hydroxyl substituent

(22c, 22o, 22t, 22a a , and 23f) were obtained by the

demethylation of the corresponding methoxy compounds

(e.g., 20c, 20n , 20s, 20z, and 21f, respectively) using

BBr₃in CH₂Cl₂followed by hydrogenolysis to remove

the benzyl group (Scheme 4). The methyl ester 22k was

obtained by acid-catalyzed esterification of the corre-

sponding carboxylic acid 22j in MeOH (Scheme 4).

P h a r m a cology. Ra d ioliga n d Bin d in g Assa ys a n d

Da ta An a lysis

The binding assays and analyses have been described

previously.^28,36,37These provide estimates of the affini-

ties of the allosteric compounds for the unliganded and

NMS-liganded M₁-M₄receptors (Table 1) together with

the cooperativities of the ligands with NMS (shown

symbolically in Table 2). Two types of experimental

design were used: equilibrium assays to measure the

3

effects of allosteric agents on the binding of H-NMS

and ACh, and “off-rate” assays to measure the effects

3

of the allosteric ligands on H-NMS dissociation. The

substituted indole and N-benzylpiperidone under either

acidic or basic conditions according to our previously

published procedures.³⁴In the case of 8k , the starting

5-morpholinomethylindole 12 was obtained from the

LiAlH₄reduction of the corresponding amide 11, which

in turn was prepared from the commercially available

indole-5-carboxylic acid 10 according to Scheme 2.

experimental data were analyzed by nonlinear least-

squares analysis using equations derived from the

allosteric ternary complex model (Figure 1). The com-

pounds in both series in general exhibited Hill coef-

ficients greater than 1 for their binding to both unli-

ganded and NMS-liganded receptors. Possible explana-

tions for this are discussed later under “Apparent

Positive Cooperativity”. Detailed descriptions of the

methodology and the data analysis are provided in the

Experimental Section and in Supporting Information.

It should be noted that the binding measurements are

made in a Na/Mg/Hepes buffer and not in a low ionic

strength buffer where affinities of certain allosteric

agents are considerably higher.^18,38

Depending on the nature of R₁and R₂in the target

molecules, the 3-THPIs were acylated with the ap-

propriate acid chlorides (13, 14, 16a , and 17) under

anhydrous conditions to generate the Diels-Alder pre-

cursors 18a -v and 19a -h , using NaH as base. The

synthesis of the hexynoyl chloride 16a , not previously

described, was achieved starting from 15³⁵followed by

subsequent reduction, acetylation, deprotection, and

oxidation to yield 16, which was converted to 16a upon

treatment with oxalyl chloride in CH₂Cl₂as illustrated

in Scheme 3.

Resu lts a n d Discu ssion

The binding affinities of 36 substituted pentacyclic

carbazolones (22a -a a and 23a -i) at the free and NMS-

liganded human M₁-M₄muscarinic receptors were

measured using our “semiquantitative equilibrium” and

“off-rate” assays and are listed in Table 1. The affinities

of the ligands for the NMS-occupied receptor, estimated

from the equilibrium and kinetic experiments, were in

excellent agreement with each other; in only 3 out of

The thermal cyclization of the Diels-Alder precursors

and the subsequent aromatization by loss of H₂could

only be affected in diphenyl ether at elevated temper-

atures (∼240 °C). This procedure furnished the cycload-

ducts 20a -z and 21a -i in good yields and usually free

from impurities. Cyclization appeared to be unaffected

Substituted Pentacyclic Carbazolones

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1263

Sch em e 4

140 possible comparisons were the differences greater

than 3-fold. Such a concordance is in agreement with

the predictions of the allosteric ternary complex model

if the equilibrium and kinetic measurements reflect the

same binding processes. Because of their higher preci-

sion, the estimates of the affinity at the NMS-occupied

receptor in Table 1 are those from the off-rate experi-

ments.

As discussed earlier, the pentacyclic carbazolones 22a

and 23a were regarded as suitable allosteric templates

for the development of more potent muscarinic allosteric

agents. For our initial attempt, we chose to introduce a

variety of substituents into all available positions of the

indole ring (i.e., positions 1, 2, 3, and 4). The choice of

the substituents was largely dependent on the com-

mercial availability of the starting indoles and included

an array of hydrophilic, hydrophobic, and hydrogen bond

donors and acceptors.

Effect of Su bstitu en ts on Affin ity a t F r ee a n d

NMS-Liga n d ed M₁-M₄Recep tor s. The unsubsti-

tuted pentacyclic carbazolone 22a binds with the loga-

rithm of the affinity of 4.6-5.5 at free and NMS-

liganded muscarinic receptor subtypes. In general, the

results from the substitution study reveal that addition

of substituents to positions 1-4 of the carbazolone ring

system gives rise to an increase in affinity at both free

and NMS-liganded receptors for all subtypes. The only

exception is the 2-morpholinoamido derivative (22l)

where a small but consistent reduction of affinity at all

subtypes is apparent.

At the unliganded M₁receptor the maximum increase

in affinity for a single substituent is about 10-fold and

is found for the 1-OMe substituent (22b). A similar gain

in affinity is observed at free and NMS-liganded M₂-

M₄subtypes. Relocation of this group to positions 2, 3,

and 4 (i.e., 22g, 22n , and 22s, respectively) has little

effect or reduces affinity at all free and NMS-liganded

subtypes with the 4-position being the least tolerated.

Further additions of methoxy groups (i.e., 22u and 22w )

appear to have little further effect on affinity. The SAR

observed for the 1-OMe substituent is also generally

true for the 1-OH substitution (22c), although this agent

binds with slightly lower affinity at most free and

liganded M₂-M₄subtypes.

Substitution with a Cl atom in the 1-position (22d )

results in the highest gain in affinity achieved for a

single substituent at free and liganded M₁-M₄subtypes

(with the exception of free M₁receptors). For example,

the logarithm of the affinity at NMS-liganded M₂

receptors is 7.1, a gain of over 40-fold in affinity over

22a . Relocation of the Cl atom to the 2- or 3-position

(22i and 22p ) slightly lowers the affinity particularly

at the free and NMS-liganded M₃and M₄subtypes.

Introduction of an electron-withdrawing group such as

a COOMe into the 1-position (22e) makes little differ-

ence to the overall binding of the molecule. However,

relocation of this group to either the 2- or 3-position (22k

and 22q) increases affinity at all subtypes particularly

at the unliganded receptors.

Other substituents such as a 2-Me (22f) or 2,3-

methylenedioxy (22v) slightly enhance affinity, whereas

2-COOH (22j), 2-morpholinomethyl (22m ), and 3-CN

(22r ) do not have significant effects on binding. The 2,3-

dimethoxy analogue 22u has good aqueous solubility

and binds with moderate affinities. This agent was

further functionalized with a methyl ester at the 8-posi-

tion (i.e., 22x), but no improvement in affinity was

observed. However, functionalization with an acetoxy-

methyl group (22y) at that position results in an

increase in affinity (up to 5-fold) at all free and NMS-

liganded subtypes. This agent 22y, with a logarithm of

the affinity of 7.2 at NMS-liganded M₂receptors, is the

most potent positive allosteric ligand in this series.

Analogues 22b and 22c were both functionalized at the

7-position with methyl groups (i.e., 22z and 22a a ,

respectively), but no gain in affinity was observed.

Compound 23a has an allosteric binding profile

similar to that of its isomer 22a . The only difference in

the two ring systems is the position of the basic

endocyclic nitrogen atom, and that results in a slight

1264 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6

Gharagozloo et al.

Substituted Pentacyclic Carbazolones

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1265

Ta ble 2. Cooperativity of 22a -a a and 23a -i with NMS at

M₁-M₄Receptors^a

subtype versus all other subtypes. The only marginal

exceptions are the 3-OH substituted analogues 22o and

23f. These isomers display about a 10-fold selectivity

at M₁over the M₂-M₄subtypes, although the observed

selectivity is only apparent at the unliganded receptors.

compd

M₁

M₂

M₃

M₄

22a

+

-

+

22b

22c

22d

22e

-

++

+

++

+

0

++

+

Su btyp e Selectivity. Coop er a tivity w ith NMS.

The pattern of occurrence of positive cooperativity is M₂

> M₄> M₁> M₃, which is somewhat different from that

observed for the subtype selectivity pattern of affinity.

Most of the substituents examined give rise to positive

cooperativity with NMS at M₂and M₄receptors. How-

ever, only a small number of substituents support

positive cooperativity at the M₁and M₃subtypes (Table

2). Two compounds were positively cooperative with

NMS at all subtypes, and two were negatively coopera-

tive at all subtypes. Within this series of compounds

there is an organization in the cooperativity pattern

between subtypes. Compounds that are positive at M₄

are positive at M₂. All compounds that are positive at

M₁are positive at both M₂and M₄, and all compounds

that are positive at M₃are positive at the other three

subtypes. The only exception is the trisubstituted 22w ,

which is positive at M₄and neutral at M₂.

The magnitude and nature of the observed cooperat-

ivity also appear to be highly dependent on the ring

position and electronic character of the substituents as

well as on the receptor subtype. For example, electron-

donating substituents such as a hydroxyl group in ring

positions 1 and 2 (i.e., 22c and 22h , respectively) exhibit

strong positive cooperativities at M₁, M₂, and M₄recep-

tors, whereas the same substituent in the 3- or 4-posi-

tion (i.e., 22o and 22t, respectively) manifest weak

positive, neutral, or low negative cooperativities de-

pending on the receptor subtype. Furthermore, electron-

withdrawing substituents such as a methyl ester (22e,

22k , and 22q) or a nitrile (22r ) tend to mostly support

negative cooperativities.

Su btyp e Selectivity. Coop er a tivity w ith ACh . All

of the synthesized compounds inhibited the binding of

ACh at all muscarinic receptor subtypes as assessed

from our “semiquantitative equilibrium” binding assay.

The cooperativity of (23g) with ACh at M₁receptors

was estimated by a simultaneous analysis of ACh/³H-

NMS competition curves measured in the absence and

presence of three concentrations of 23g. The estimated

value of the cooperativity with ACh (0.10 ( 0.01, n )

2) indicated that 23g is not strongly negatively coopera-

tive with ACh at this subtype. A comparable experiment

with 22b at M₁receptors gave an R value versus ACh

of 0.13.

-

22f

22g

22h

22i

22j

22k

22l

-

+

++

0

-

+

0

++

0

++

0

++

-

0

-

- -

-

- -

-

++

+

22m

22n

22o

22p

22q

22r

+

-

+

-

+

-

0

-

+

++

-

0

22s

22t

-

+

-

+

-

22u

22v

22w

22x

22y

22z

22a a

-

0

+

0

+

-

0

-

0

+

++

0

++

+

-

0

+

23a

23b

23c

23d

23e

23f

23g

23h

23i

-

0

-

0

-

+

-

+

-

0

-

+

-

+

0

+

0

+

0

++

+

-

+

-

+

0

++

+

-

a

Cooperativity estimates are from the primary screens and the

data in Table 1. Cooperativity values are as follows: (- -) <0.2;

(-) 0.2-0.8; (0) 0.81-1.19; (+) 1.2-3; (++) >3.

increase in affinity, particularly at unliganded M₃

receptors. The effect of a small number of substituents

in 23a was also examined. As with the first series, the

majority of substituents incorporated led to an increase

in the overall affinity of the molecule. Analogues

incorporating the following substituents 2-MeO (23b),

2-OH (23c), 2-COOMe (23d ), 3-MeO (23e), and 3-OH

(23f) exhibit binding affinities similar to those of their

corresponding isomers (i.e., 22g, 22h , 22k , 22n , and 22o

respectively). This provides a strong indication that the

position of the basic nitrogen atom does not play a

significant role in the binding of these molecules.

Similarly, the 2,3-dimethoxy analogue 23g binds with

almost equal affinities at the unliganded receptor

subtypes compared to its isomer 22u , although at NMS-

liganded receptors, this agents displays higher affinities

than 22u (up to 4-fold). In contrast, functionalization

of 23g with methyl groups at the 7-position (i.e., 23i)

increases affinity at the unliganded receptors with

virtually no effect at NMS-liganded subtypes. Interest-

ingly, addition of methyl groups at the 7-position of 23a

(i.e., 23h ) has little or no effect on muscarinic binding.

Su btyp e Selectivity. Affin ity. In general, the pat-

tern of affinity found for both series of compounds is

M₂> M₁> M₄> M₃. However, none of the analogues

discussed here exhibit significant selectivity for one

Ap p a r en t P ositive Coop er a tivity. One interesting

and distinguishing feature of this series of compounds

is that in both the equilibrium and kinetic measure-

ments the slope factors required to describe the interac-

tions of the compounds with the receptors were greater

than 1. A possible artifact that could give rise to such a

phenomenon is the loss of the ligand by adsorption to

the plastic tubes during dilution or in the assay or by

depletion of the free ligand concentration by its parti-

tioning into the membranes. Control experiments in-

volving multiple transfers of dilutions between tubes

and the measurement of the optical density of super-

natants from binding assays, as well as the slope factors

not being the same for different receptor subtypes,

1266 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6

Gharagozloo et al.

excluded this possibility. At M₁, M₂, M₃, and M₄recep-

tors, the slope factors in equilibrium studies were 1.70

( 0.31, 1.72 ( 0.22, 1.44 ( 0.29, and 1.76 ( 0.23 (mean

( SD, n ) 36), respectively, with an overall range of

0.9-2.2. The equivalent slope factors estimated from the

“off-rate” assays were 1.78 ( 0.26, 1.76 ( 0.26, 1.66 (

0.31, and 1.77 ( 0.22 with an overall range of 1.0-2.4.

The data with slope factors greater than 2 could also

be fitted satisfactorily if the slope factors were con-

strained to 2. The slope factors at M₃receptors appeared

to be lower than that at the other subtypes. An

analogous result has been observed for the weaker

ligand tacrine.³⁹

Because the slope factors are greater than 1 for the

ligands binding to the unoccupied and NMS-occupied

receptors, it cannot be argued that the apparent positive

cooperativity results from the ligands binding to both

the allosteric site and the competitive site. One expla-

nation is that the ligands are binding in a positively

cooperative fashion to two different allosteric sites on

muscarinic receptors. Indeed, it has recently been

demonstrated that there are two allosteric sites on these

receptors,^12,37and it could be possible, using the meth-

ods described, to test whether these ligands bind to both

sites.

In this paper we have been able to demonstrate,

through substitution effects, that both affinity and

cooperativity with NMS can be altered substantially in

a subtype-dependent manner. Notable compounds, in

terms of affinity, are 22d , 22i, 22w (M₁-M₄), and 22y

(M₂), and these suggest that high-affinity interactions

at the allosteric site should be possible with molecules

of relatively low molecular weight. In terms of receptor

selectivity, 23f binds with a 10-fold higher affinity at

unliganded M₁versus M₂-M₄receptors, although this

agent lacks a comparable selectivity at the NMS-

liganded receptors.

All of the agents are negatively cooperative with the

neurotransmitter molecule acetylcholine, though the

degree of negative cooperativity appears to be low. We

have demonstrated that, through structural manipula-

tion of an allosteric pharmacophore, negative cooperat-

ivity with NMS can be converted to positive cooperat-

ivity and vice versa. In principle, this should apply to

any ligand, including ACh. Therefore, continued syn-

thetic work with this series and other analogous mul-

ticyclic ring systems should enable the identification of

agents with positive cooperativity with acetylcholine.

Exp er im en ta l Section

An alternative explanation is that muscarinic recep-

tors can exist as dimers (or higher oligomers),^40,41and

the binding of these allosteric ligands to its allosteric

site on the receptor either modulates the extent of

receptor dimerization or generates a positive homotropic

interaction transmitted via the receptor-receptor in-

terface. If this were to be true, then these molecules

would have an action additional to their allosteric action

at the competitive site. Such actions could have impor-

tant repercussions on receptor function if the precise

dimerization state of the receptor is crucial for receptor

activation. Because agonist action and binding affinity

are related to the induction or selection of a specific

receptor conformation, one may speculate that the

observed negative cooperativity between acetylcholine

and the ligands described in this paper may be a

consequence of their apparent (or real) positive homo-

tropic cooperativity.

Gen er a l Meth od s. Starting materials were prepared ac-

cording to literature procedures as indicated, and if no refer-

ence is quoted, they are available commercially. Anhydrous

THF and MeOH were obtained according to the procedures

described by Perrin and Armarego.⁴²In the text, petrol refers

to petroleum ether (80-100 °C) and ammonia refers to

concentrated ammonia (d ) 0.88). All reactions were carried

out under a slow stream of argon unless otherwise stated.

Melting points (mp) were carried out in open capillaries on

an Electrothermal digital melting point apparatus (model

IA9100) and are uncorrected. The ¹H NMR spectra were

recorded on Bruker AM 400 WB spectrometer using the

facilities at the MRC Biomedical NMR Centre, National

Institute for Medical Research, Mill Hill. Chemical shifts are

reported in ppm downfield of internal tetramethylsilane (TMS)

or 3-(trimethylsilyl)propionic-2,2,3,3-d₄acid, sodium salt (TSP)

as indicated. Mass spectra were run by fast-atom bombard-

ment on a VG Analytical ZAB-SE double-focusing magnetic

sector mass spectrometer. Elemental analyses were performed

by the microanalytical section of the Department of Chemistry,

University College London, and were within (0.4% of the

calculated values. Column chromatography was carried out

on Merck silica gel 60 (70-230 mesh ASTM).

Su m m a r y a n d Con clu sion s

The existence of allosteric sites on muscarinic recep-

tors raises the possibility of designing highly subtype

selective ligands. This selectivity arises from two pa-

rameters: the binding affinity for the allosteric site and

the cooperativity of interaction with the endogenous

agonist. This latter parameter is not available to directly

acting agonists and antagonists, where affinity oreffi-

cacy is the only determinant of relative receptor subtype

selectivity. Subtype selective cooperativity with ACh has

already been demonstrated for brucine and 5 in both

binding and function.^26,28,29,31However, their low affini-

ties for muscarinic receptors limit their usefulness as

research tools. In an effort to develop high-affinity

allosteric modulators of ACh, key pharmacophoric fea-

tures in the lead molecules were identified and incor-

porated into simpler ring systems. We then probed

different aromatic positions of these novel ring systems

with a variety of substituents to delineate the receptor

requirement for both affinity and cooperativity with

competitive agents.

5-Mor p h olin om eth yl-3-(1-ben zyl-1,2,3,6-tetr a h yd r op y-

r id in -4-yl)-1H-in d ole (8k ). A solution of indole-5-carboxylic

acid 10 (7.2 g, 44.7 mmol) in THF (250 mL) containing oxalyl

chloride (11.3 g, 89.3 mmol) was stirred at room temperature

for 10 h. The reaction mixture was taken to dryness in vacuo,

and the residue was redissolved in anhydrous THF (100 mL)

and cooled to 5 °C. A solution of morpholine (7.8 g, 89.3 mmol)

in THF (50 mL) was added, and the mixture was stirred at

room temperature for 1 h. The mixture was filtered and the

filtrate taken to dryness. The residue was partitioned between

EtOAc and aqueous K₂CO₃. The organic layer was dried over

Na₂SO₄and taken to dryness in vacuo to yield 11 as a white

solid (3.0 g, 28%, yield not maximized). ¹H NMR (DMSO-d₆) δ

3.52 (m, 4H), 3.60 (m, 4H), 6.49 (s, 1H), 7.14 (dd, J ) 1.6 and

8.4 Hz, 1H), 7.39-7.44 (m, 2H), 7.62 (s, 1H), 11.25 (br s, 1H).

A suspension of LiAlH₄(0.93 g, 24.5 mmol) in THF (25 mL)

was added to a solution of 11 (1.87 g, 8.1 mmol) in THF (125

mL) and refluxed for 3 h. The mixture was cooled to 0 °C, and

wet EtOAc (10 mL) was added. The mixture was filtered and

then taken to dryness. The resulting residue was stirred in

dilute HCl. The aqueous layer was filtered, made alkaline with

dilute K₂CO₃, and extracted with EtOAc. The organic layer

Substituted Pentacyclic Carbazolones

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1267

was dried over Na₂SO₄and evaporated in vacuo to give 12 as

a white solid (1.1 g, 63%). H NMR (CDCl₃) δ 2.47 (t, J ) 4.5

Hz, 4H), 3.59 (s, 2H), 3.71 (t, J ) 4.5 Hz, 4H), 6.51 (m, 1H),

7.18 (m, 2H), 7.32 (d, J ) 8.4 Hz, 1H), 7.56 (s, 1H), 8.20 (br s,

1H).

A solution of 12 (1.08 g, 5 mmol), 1-benzyl-4-piperidone (2.84

g, 15 mmol), and sodium methoxide (1.62 g, 30 mmol) in

anhydrous methanol (75 mL) was refluxed under argon for 48

h. The reaction mixture partioned between H₂O and EtOAc.

The organic layer was separated and taken to dryness. The

resulting residue was warmed in 10% MeOH in EtOAc (50

mL). Ether was added until precipitation occurred. The product

8k was collected by filtration and dried in vacuo (0.80 g, 41%,

yield not maximized):

tion, washed successively with THF, H₂O, and ether, and if

necessary recrystallized as stated. Compounds 18a -v and

19a -h were synthesized following the above procedure for

acylation.

1

1-(4-P en tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr a h yd r op yr id in -

4-yl)in d ole Hyd r och lor id e (18a ). 18a was prepared from

the reaction of 8a and 13 according to the general acylation

1

procedure. Yield 76%; mp 263-265 °C; H NMR (DMSO-d₆/

D₂O) δ 2.61 (td, J ) 6.8 and 2.4 Hz, 2H), 2.72 (t, J ) 2.4 Hz,

1H), 2.87 (br s, 2H), 3.29 (t, J ) 6.8 Hz, 2H), 3.45 (br s, 2H),

3.84 (s, 2H), 4.39 (s, 2H), 6.35 (br s, 1H), 7.37 (t, J ) 8 Hz,

1H), 7.43 (t, J ) 8 Hz, 1H), 7.51-7.57 (m, 5H), 7.91 (d, J ) 8

Hz, 1H), 7.96 (s, 1H), 8.44 (d, J ) 8 Hz, 1H).

4-Meth oxy-1-(4-p en tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr a h y-

d r op yr id in -4-yl)in d ole Hyd r och lor id e (18b). 18b was

prepared from the reaction of 8b and 13 according to the

P r ep a r a tion of Acid Ch lor id e 16a . A 1.0 M solution of

diisobutylaluminum hydride in hexane (20 mL) was added

dropwise to a solution of 15 (2.26 g, 10 mmol) in CH₂Cl₂(23

mL) at -70 °C, and the mixture was stirred for 30 min. The

mixture was partitioned between EtOAc and H₂O. The organic

layer was washed with aqueous NaCl, dried over Na₂SO₄, and

removed under reduced pressure. The resulting alcohol was

dissolved in THF (20 mL) and cooled to 0 °C, and triethylamine

(1.2 g, 12 mmol) and acetyl chloride (0.86 g, 11 mmol) were

added successively. The solution was stirred for 30 min, diluted

with H₂O, and extracted with EtOAc. The organic layer was

washed with aqueous NaCl, dried over Na₂SO₄, and removed

under reduced pressure. The product was dissolved in EtOH

(200 mL) containing pyridinium p-toluenesulfonate (0.3 g, 1.2

mmol), and the mixture was stirred at 45 °C for 3 h. The

solution was concentrated under reduced pressure and chro-

matographed on silica, eluting with a mixture of EtOAc/petrol

(1:1). The free alcohol, obtained as a colorless oil, was dissolved

in CH₂Cl₂(50 mL). A solution of DMSO (1.47 g, 19 mmol) in

CH₂Cl₂(1 mL) was added to a 2 M solution of oxalyl chloride

in CH₂Cl₂(7.2 mL) at -75 °C, and the mixture was stirred for

10 min. This was then added to the previous solution, and the

mixture was stirred at -75 °C for 30 min and then at -45 °C

for 40 min and then cooled back to -75 °C before Et₃N (7.5

mL, 53 mmol) was added. The mixture was then allowed to

reach room temperature over a period of 1 h and then was

diluted with a mixture of EtOAc and H₂O. The organic layer

was washed successively with dilute HCl, H₂O, and aqueous

NaHCO₃, dried over anhydrous Na₂SO₄, and evaporated under

reduced pressure. The resulting aldehyde was dissolved in

tBuOH (20 mL) containing 2-methyl-2-butene (2.5 g, 35.5

mmol) and was added to a solution of NaH₂PO₄‚H₂O (1.11 g,

7.1 mmol) in H₂O (10 mL). This was cooled to ∼10 °C, and

NaClO₂(0.83 g, 9.23 mmol) was added in portions over a period

of 10 min. The mixture was stirred at room temperature for

30 min, acidified with dilute HCl, and extracted with EtOAc.

The organic layer was taken to dryness, and the residue was

chromatographed on silica, eluting with EtOAc/petrol (1:1) to

give the acid 16 as a colorless oil that crystallized on cooling

1

general acylation procedure. Yield 73%; H NMR (DMSO-d₆/

D₂O, TSP-salt, 338 K) δ 2.60 (m, 1H), 2.65 (m, 2H), 2.85 (t, J

) 5.8 Hz, 2H), 3.22 (m, 4H), 3.66 (m, 2H), 3.78 (s, 2H), 3.88 (s,

3H), 5.88 (m, 1H), 6.89 (d, J ) 8.2 Hz, 1H), 7.33 (t, J ) 8.2

Hz, 1H), 7.40 (m, 5H), 7.48 (s, 1H), 7.99 (d, J ) 8.2 Hz, 1H).

4-Ch lor o-1-(4-p en t yn oyl)-3-(1-b en zyl-1,2,5,6-t et r a h y-

d r op yr id in -4-yl)in d ole Hyd r och lor id e (18c). 18c was pre-

pared from the reaction of 8c and 13 according to the general

acylation procedure. Yield 83%; ¹H NMR (DMSO-d₆/D₂O, TSP-

salt, 343 K) δ 2.60 (t, J ) 2.6 Hz, 1H), 2.67 (td, J ) 2.6 and

6.7 Hz, 2H), 2.79 (br s, 2H), 3.24 (t, J ) 6.7 Hz, 2H), 3.48 (t,

J ) 6.0 Hz, 2H), 3.84 (br s, 2H), 4.40 (s, 2H), 5.83 (m, 1H),

7.38 (m, 2H), 7.55 (m, 5H), 7.75 (s, 1H), 8.37 (d, J ) 7.6 Hz,

1H).

Meth yl 1-(4-P en tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr a h yd r o-

p yr id in -4-yl)in d ole-4-ca r boxyla te Hyd r och lor id e (18d ).

18d was prepared from the reaction of 8d and 13 according to

the general acylation procedure. Yield 58%; ¹H NMR (DMSO-

d₆/D₂O, TSP-salt, 333 K) δ 2.58 (m, 1H), 2.63 (m, 2H), 3.28 (t,

J ) 6.4 Hz, 2H), 3.44 (br s, 2H), 3.78 (br s, 2H), 3.88 (s, 3H),

3.92 (s, 2H), 4.44 (s, 2H), 5.64 (br s, 1H), 7.49-7.63 (m, 6H),

7.63 (dd, J ) 1 and 7.6 Hz, 2H), 7.91 (s, 1H), 8.66 (dd, J ) 1

and 7.6 Hz, 1H).

5-Met h yl-1-(4-p en t yn oyl)-3-(1-b en zyl-1,2,5,6-t et r a h y-

d r op yr id in -4-yl)in d ole Hyd r och lor id e (18e). 18e was

prepared from the reaction of 8e and 13 according to the

general acylation procedure. Recrystallized from MeOH; yield

1

75%; H NMR (DMSO-d₆/D₂O, TSP-salt, 333 K) δ 2.54-2.64

(m, 3H), 2.85 (br s, 2H), 3.24 (t, J ) 6.7 Hz, 2H), 3.42 (br s,

2H), 3.82 (br s, 2H), 3.91 (s, 3H), 4.35 (s, 2H), 6.31 (br s, 1H),

7.25 (d, J ) 8.6 Hz, 1H), 7.49-7.61 (m, 5H), 7.69 (s, 1H), 7.84

(s, 1H), 8.29 (d, J ) 8.6 Hz, 1H).

5-Meth oxy-1-(4-p en tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr a h y-

d r op yr id in -4-yl)in d ole Hyd r och lor id e (18f). 18f was pre-

pared from the reaction of 8f and 13 according to the general

acylation procedure. Yield 89%; ¹H NMR (DMSO-d₆/D₂O) δ

2.61 (td, J ) 2.4 and 6.7 Hz, 2H), 2.68 (t J ) 2.4 Hz, 1H), 2.86

(br s, 2H), 3.25 (t, J ) 6.7 Hz, 2H), 3.47 (br s, 2H), 3.82 (s,

3H), 3.84 (s, 2H), 4.39 (s, 2H), 6.31 (br s 1H), 7.04 (dd, J ) 2.3

and 9.0 Hz, 1H), 7.32 (d, J ) 2.3 Hz, 1H), 7.52-7.57 (m, 5H),

7.91 (s, 1H), 8.34 (d, J ) 9 Hz, 1H).

1

in the fridge (1.18 g, 69%). H NMR (90 MHz, CDCl₃) δ 2.09

(s, 3H), 2.58 (s, 4H), 4.65 (s, 2H).

To a cold solution of 16 (1.15 g, 6.75 mmol) in CH₂Cl₂(20

mL) was added a 2 M solution of oxalyl chloride (4.05 mL, 8.1

mmol) in CH₂Cl₂. The solution was stirred at room tempera-

ture for 18 h and refluxed for a further hour. The solvent was

removed, and the crude residue was distilled, collecting 16a

as the fraction boiling at 124-125 °C and 5 mmHg (0.65 g,

51%).

5-Ben zyloxy-1-(4-pen tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr ah y-

d r op yr id in -4-yl)in d ole Hyd r och lor id e (18g). 18g was

prepared from the reaction of 8g and 13 according to the

1

general acylation procedure. Yield 76%; H NMR (DMSO-d₆/

D₂O) δ 2.61 (td, J ) 2.4 and 6.8 Hz, 2H), 2.68 (t J ) 2.4 Hz,

1H), 2.83 (br s, 2H), 3.24 (t, J ) 6.8 Hz, 2H), 3.44 (br s, 2H),

3.81 (s, 2H), 4.36 (s, 2H), 5.17 (s, 2H), 6.25 (br s, 1H), 7.11

(dd, J ) 2.2 and 9.1 Hz, 1H), 7.34-7.58 (m, 11H), 7.90 (s, 1H),

8.33 (d, J ) 9.1 Hz, 1H).

Gen er a l P r oced u r e for th e Acyla tion of 3-(1-Ben zyl-

1,2,3,6-tetr a h yd r op yr id in -4-yl)-1H-in d oles. A suspension

of sodium hydride (60% w/v dispersion in mineral oil, 32 mmol)

in THF (30 mL) was added to a cold solution of 3-(tetrahydro-

pyridinyl)indole (30 mmol) in THF (300 mL). The mixture was

stirred at room temperature for 1 h and then cooled to 0-5

°C. A solution of the appropriate acid chloride (32 mmol) in

THF (70 mL) was then added dropwise, and the resulting

mixture was allowed to stir at room temperature for 2 h. The

mixture was concentrated to about a half of its original volume

and then acidified with 2 N hydrochloric acid. Unless otherwise

stated, the resulting white precipitate was collected by filtra-

5-Ch lor o-1-(4-p en t yn oyl)-3-(1-b en zyl-1,2,5,6-t et r a h y-

d r op yr id in -4-yl)in d ole Hyd r och lor id e (18h ). 18h was

prepared from the reaction of 8h and 13 according to the

1

general acylation procedure. Yield 87%; H NMR (DMSO-d₆/

D₂O, TSP-salt, 333 K) δ 2.57 (m, 1H), 2.64 (m, 2H), 2.87 (br s,

2H), 3.28 (t, J ) 6.7 Hz, 2H), 3.48 (br s, 2H), 3.86 (br s, 2H),

4.41 (s, 2H), 6.27 (br s, 1H), 7.44 (dd, J ) 1.8 and 8.9 Hz, 1H),

1268 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6

Gharagozloo et al.

7.51-7.62 (m, 5H), 7.90 (d, J ) 1.8 Hz, 1H), 7.97 (s, 1H), 8.43

(d, J ) 8.9 Hz, 1H).

5,6-Dim eth oxy-1-(4-p en tyn oyl)-3-(1-ben zyl-1,2,5,6-tet-

r a h yd r op yr id in -4-yl)in d ole Hyd r och lor id e (18q). 18q was

prepared from the reaction of 8q and 13 according to the

Ben zyl 1-(4-P en tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr a h yd r o-

p yr id in -4-yl)in d ole-5-ca r b oxyla t e Hyd r och lor id e (18i).

18i was prepared from the reaction of 8i and 13 according to

the general acylation procedure. Yield 75%; ¹H NMR (DMSO-

d₆/D₂O) δ 2.61-2.65 (m, 3H), 2.87 (br s, 2H), 3.29 (t, J ) 6.3

Hz, 2H), 3.48 (br s, 2H), 3.86 (s, 2H), 5.40 (s, 2H), 6.31 (br s,

1H), 7.33-7.58 (m, 10H), 8.02 (s, 1H), 8.05 (d, J ) 8.8 Hz,

1H), 8.48 (s, 1H), 8.53 (d, J ) 8.8 Hz, 1H).

5-Mor p h olin oca r b on yl-1-(4-p e n t yn oyl)-3-(1-b e n zyl-

1,2,5,6-tetr a h yd r op yr id in -4-yl)in d ole (18j). 18j was pre-

pared from the reaction of 8j and 13 according to the general

acylation procedure. The reaction mixture was made alkaline

with aqueous K₂CO₃and extracted with EtOAc. The organic

layer was taken to dryness to give 18j as a light-brown gum

in quantitative yield: ¹H NMR (CDCl₃) δ 2.05 (t, J ) 2.4 Hz,

1H), 2.58 (br s, 2H), 2.73 (m, 4H), 3.18 (m, 2H), 3.22 (m, 2H),

3.65 (s, 2H), 3.4-3.8 (br s, 8H), 6.28 (m, 1H), 7.25-7.42 (m,

7H), 7.91 (s, 1H), 8.51 (d, J ) 8.0 Hz, 1H).

5-Mor ph olin om eth yl-1-(4-pen tyn oyl)-3-(1-ben zyl-1,2,5,6-

tetr a h yd r op yr id in -4-yl)in d ole Dih yd r och lor id e (18k ).

18k was prepared from the reaction of 8k and 13 according to

the general acylation procedure. Yield 76%; ¹H NMR (DMSO-

d₆/D₂O, TSP-salt, 333 K) δ 2.59 (m, 3H), 2.91 (br s, 2H), 3.09

(br s, 4H), 3.31 (t, J ) 7.4 Hz, 2H), 3.51 (br s, 2H), 3.81 (br s,

4H), 3.88 (br s, 2H), 4.29 (s, 2H), 4.42 (s, 2H), 6.36 (br s, 1H),

7.49-7.63 (m, 6H), 7.97 (s, 1H), 7.99 (s, 1H), 8.48 (d, J ) 8.9

Hz, 1H).

1

general acylation procedure. Yield 80%; H NMR (DMSO-d₆/

D₂O) δ 2.61 (td, J ) 2.5 and 6.7 Hz, 2H), 2.68 (t J ) 2.5 Hz,

1H), 2.84 (m, 2H), 3.25 (t, J ) 6.7 Hz, 2H), 3.44 (m, 2H), 3.63

(t, J ) 6.3 Hz, 2H), 3.83 (s, 6H), 4.37 (s, 2H), 6.32 (m 1H),

7.30 (s, 1H), 7.51-7.59 (m, 5H), 7.79 (s, 1H), 8.07 (s, 1H).

5,6-Meth ylen ed ioxy-1-(4-p en tyn oyl)-3-(1-ben zyl-1,2,5,6-

tetr a h yd r op yr id in -4-yl)in d ole (18r ). 18r was prepared

from the reaction of 8r and 13 according to the general

acylation procedure and that described for 18j. Yield 80%; ¹H

NMR (CDCl₃) δ 2.03 (t, J ) 2.6 Hz, 1H), 2.53 (m, 2H), 2.68-

2.76 (m, 4H), 3.13 (t, J ) 7.4 Hz, 2H), 3.22 (m, 2H), 3.66 (s,

2H), 6.00 (m, 2H), 6.15 (m, 1H), 7.17 (s, 1H), 7.21 (s, 1H), 7.26-

7.41 (m, 5H), 8.06 (s, 1H).

5,6,7-Tr im et h oxy-1-(4-p en t yn oyl)-3-(1-b en zyl-1,2,5,6-

tetr a h yd r op yr id in -4-yl)in d ole (18s). 18s was prepared

from the reaction of 8s and 13 according to the general

acylation procedure and that described for 18j. Yield 89%; ¹H

NMR (HCl salt, DMSO-d₆/D₂O, TSP-salt, 338 K) δ 2.50 (t, J

) 2.5 Hz, 1H), 2.65 (m, 2H), 2.73 (m, 2H), 3.20 (t, J ) 6.8 Hz,

2H), 3.27 (t, J ) 5.8 Hz, 2H), 3.63 (m, 2H), 3.83 (s, 3H), 3.84

(s, 3H), 3.89 (s, 3H), 4.20 (s, 2H), 5.99 (m, 1H), 7.49-7.52 (m,

6H), 7.90 (s, 1H).

5,6-Dim eth oxy-1-(5-m eth oxyca r bon yl-4-p en tyn oyl)-3-

(1-ben zyl-1,2,5,6-tetr a h yd r op yr id in -4-yl)in d ole (18t). 18t

was prepared from the reaction of 8q and 14 according to the

general acylation procedure. The reaction mixture was parti-

tioned between EtOAc and aqueous NaHCO₃. The organic

layer was taken to dryness, and the resulting residue was

chromatographed on silica, eluting with EtOAc to give the pure

6-Meth oxy-1-(4-p en tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr a h y-

d r op yr id in -4-yl)in d ole (18l). 18l was prepared from the

reaction of 8l and 13 according to the general acylation

1

product as a white powder in 18% yield (not maximized). H

1

procedure. Yield 82%; H NMR (CDCl₃) δ 2.04 (t, J ) 2.6 Hz,

NMR (CDCl₃) δ 2.56 (m, 2H), 2.77 (t, J ) 5.7 Hz, 2H), 2.86 (t,

J ) 7.8 Hz, 2H), 3.19-3.25 (m, 4H), 3.68 (s, 2H), 3.77 (s, 3H),

3.92 (s, 3H), 3.96 (s, 3H), 6.21 (m, 1H), 7.16 (s, 1H), 7.20 (s,

1H), 7.27-7.41 (m, 5H), 8.11 (s, 1H).

1H), 2.56 (m, 2H), 2.70-276 (m, 4H), 3.15 (t, J ) 7.3 Hz, 2H),

3.23 (m, 2H), 3.67 (s, 2H), 3.88 (s, 3H), 6.26 (m, 1H), 6.91 (dd,

J ) 2.5 and 8.8 Hz, 1H), 7.21 (s, 1H), 7.26-7.41 (m, 5H), 7.66

(d, J ) 8.8 Hz, 1H), 8.12 (d, J ) 2.5 Hz, 1H).

5,6-Dim et h oxy-1-(6-a cet oxy-4-h exyn oyl)-3-(1-b en zyl-

1,2,5,6-tetr a h yd r op yr id in -4-yl)in d ole (18u ). 18u was pre-

pared from the reaction of 8q and 16a according to the general

acylation procedure. The reaction mixture was partitioned

between EtOAc and aqueous NaHCO₃. The organic layer was

concentrated, and the resulting white precipitate was collected

6-Ch lor o-1-(4-p en t yn oyl)-3-(1-b en zyl-1,2,5,6-t et r a h y-

d r op yr id in -4-yl)in d ole Hyd r och lor id e (18m ). 18m was

prepared from the reaction of 8m and 13 according to the

1

general acylation procedure. Yield 83%; H NMR (DMSO-d₆/

D₂O, TSP-salt, 333 K) δ 2.60 (t, J ) 5.9 Hz, 1H), 2.66 (td, J )

2.6 and 6.6 Hz, 2H), 2.87 (br s, 2H), 3.26 (t, J ) 6.6 Hz, 2H),

3.48 (t, J ) 5.9 Hz, 2H), 3.86 (s, 2H), 4.38 (s, 2H), 6.27 (br s,

1H), 7.41 (dd, J ) 2.0 and 8.6 Hz, 1H), 7.55 (m, 5H), 7.86 (m,

2H), 8.44 (d, J ) 2.0 Hz, 1H).

Meth yl 1-(4-P en tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr a h yd r o-

p yr id in -4-yl)in d ole-6-ca r boxyla te Hyd r och lor id e (18n ).

18n was prepared from the reaction of 8n and 13 according

to the general acylation procedure. Yield 83%; ¹H NMR

(DMSO-d₆/D₂O, TSP-salt, 333 K) δ 2.57 (m, 1H), 2.66 (m, 2H),

2.91 (br s, 2H), 3.29 (t, J ) 6.7 Hz, 2H), 3.90 (br s, 2H), 3.92

(s, 2H), 3.93 (s, 3H), 4.43 (s, 2H), 6.34 (br s, 1H), 7.52-7.63

(m, 5H), 7.95-8.04 (m, 2H), 8.09 (s, 1H), 9.05 (s, 1H).

6-Cya n o-1-(4-pen tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr a h ydr o-

p yr id in -4-yl)in d ole Hyd r och lor id e (18o)?? 18o was pre-

pared from the reaction of 8o and 13 according to the general

acylation procedure. Yield 72%; ¹H NMR (DMSO-d₆/D₂O, TSP-

salt, 343 K) δ 2.59 (m, 1H), 2.67 (td, J ) 2.5 and 6.7 Hz, 2H),

2.77 (t, J ) 6.8 Hz, 2H), 3.21 (t, J ) 2.5 Hz, 2H), 3.30 (t, J )

6.8 Hz, 2H), 3.60 (s, 2H), 3.68 (s, 2H), 6.29 (m, 1H), 7.39 (m,

5H), 7.68 (d, J ) 8.0 Hz, 1H), 7.97 (s, 1H), 8.03 (d, J ) 8.0 Hz,

1H), 8.74 (s, 1H).

7-Meth oxy-1-(4-p en tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr a h y-

d r op yr id in -4-yl)in d ole Hyd r och lor id e (18p ). 18p was

prepared from the reaction of 8p and 13 according to the

general acylation procedure. The product was recrystallized

from a mixture of MeOH/Et₂O. Yield 48% (not maximized);

¹H NMR (DMSO-d₆/D₂O, TSP-salt, 333 K) δ 2.56-2.62 (m, 3H),

2.83 (br s, 2H), 3.23 (m, 2H), 3.43 (br s, 2H), 3.83 (br s, 2H),

3.92 (s, 3H), 4.36 (s, 2H), 6.24 (br s, 1H), 7.05 (d, J ) 8.0 Hz,

1H), 7.34 (t, J ) 8.0 Hz, 1H), 7.48 (d, J ) 8.0 Hz, 1H), 7.50-

7.60 (m, 5H), 7.78 (s, 1H).

1

by filtration, Yield 17% (not maximized); H NMR (CDCl₃) δ

2.08 (s, 3H), 2.5-2.9 (br, 2H), 2.76 (m, 2H), 3.14 (t, J ) 7.3

Hz, 2H), 3.2-3.8 (br, 4H), 3.93 (s, 3H), 3.96 (s, 3H), 4.1-4.5

(m, 2H), 4.66 (t, J ) 2.0 Hz, 2H), 6.10 (m, 1H), 7.11 (s, 1H),

7.29 (s, 1H), 7.46-7.71 (m, 5H), 8.12 (s, 1H).

4-Met h oxy-1-(3,3-d im et h yl-4-p en t yn oyl)-3-(1-b en zyl-

1,2,5,6-tetr a h yd r op yr id in -4-yl)in d ole (18v). 18v was pre-

pared from the reaction of 8b and 17 according to the general

acylation procedure. Yield 46%; ¹H NMR (CDCl₃) δ 1.47 (s,

6H), 2.14 (s, 1H), 2.56 (m, 2H), 2.73 (t, J ) 5.6 Hz, 2H), 3.00

(s, 2H), 3.17 (m, 2H), 3.67 (s, 2H), 3.89 (s, 3H), 5.80 (m, 1H),

6.72 (d, J ) 8.0 Hz, 1H), 7.22-7.41 (m, 7H), 8.17 (d, J ) 8.0

Hz, 1H).

1-(4-P en tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr a h yd r op yr id in -

3-yl)in d ole Hyd r och lor id e (19a ). 19a was prepared from

the reaction of 9a and 13 according to the general acylation

1

procedure. Yield 76%; mp 226-227 °C; H NMR (DMSO-d₆/

D₂O) δ 2.60-2.65 (m, 4H), 2.69 (t, J ) 2.5 Hz, 1H), 3.26 (t, J

) 6.7 Hz, 2H), 3.30 (m, 2H), 4.15 (s, 2H), 4.42 (s, 2H), 6.53 (m,

1H), 7.39 (t, J ) 8 Hz, 1H), 7.45 (t, J ) 8 Hz, 1H), 7.50-7.57

(m, 5H), 7.87 (d, J ) 8 Hz, 1H), 7.88 (s, 1H), 8.43 (d, J ) 8 Hz,

1H).

5-Meth oxy-1-(4-p en tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr a h y-

d r op yr id in -3-yl)in d ole Hyd r och lor id e (19b). 19b was

prepared from the reaction of 9b and 13 according to the

1

general acylation procedure. Yield 78%; H NMR (DMSO-d₆/

D₂O) δ 2.57 (t, J ) 2.4 Hz, 1H), 2.60-2.66 (m, 4H), 3.22 (t, J

) 6.4 Hz, 2H), 3.34-3.7 (br, 2H), 3.82 (s, 3H), 4.08 (s, 2H),

4.48 (s, 2H), 6.49 (m, 1H), 7.06 (dd, t, J ) 2.2 and 9.1 Hz, 1H),

7.24 (d, J ) 2.2 Hz, 1H), 7.36-7.60 (m, 5H), 7.77 (s, 1H), 8.32

(d, J ) 9.1 Hz, 1H).

Substituted Pentacyclic Carbazolones

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1269

5-Ben zyloxy-1-(4-pen tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr ah y-

d r op yr id in -3-yl)in d ole Hyd r och lor id e (19c). 19c was pre-

pared from the reaction of 9c and 13 according to the general

acylation procedure. Yield 65%; ¹H NMR (DMSO-d₆/D₂O) δ

2.59-2.65 (m, 5H), 3.20 (t, J ) 6.5 Hz, 2H), 3.40 (br s, 2H),

4.05 (s, 2H), 4.45 (s, 2H), 5.16 (s, 2H), 6.40 (m, 1H), 7.11 (dd,

t, J ) 2.2 and 9.0 Hz, 1H), 7.28 (d, J ) 2.2 Hz, 1H), 7.36-7.58

(m, 10H), 7.77 (s, 1H), 8.32 (d, J ) 9.0 Hz, 1H).

3.61 (br s, 2H), 3.72 (m, 2H), 3.85 (m, 4H), 3.99 (s, 3H), 7.00

(d, J ) 7.7 Hz, 1H), 7.14 (s, 1H), 7.40 (t, J ) 7.7 Hz, 1H), 7.52-

7.61 (m, 5H), 8.12 (d, J ) 7.7 Hz, 1H).

11-Ben zyl-1-h ydr oxy-7,8,10,11,12,13-h exah ydr o-6H-dipy-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r obr om id e (20c).

A solution of 1 N BBr₃in CH₂Cl₂(16 mL) was added with

stirring to a cold solution of 20b (0.8 g, 2 mmol) in CH₂Cl₂(50

mL) under a slow stream of Ar. The reaction mixture was

allowed to stir at room temperature for 8 h and then cooled to

0 °C and carefully diluted with MeOH. The mixture was

heated at 50 °C for 15 min, cooled, filtered, and concentrated,

and if necessary, ether was added until precipitation occurred.

The resulting light-brown precipitate was collected by filtration

and dried in vacuo (0.48 g, 52%).

11-Ben zyl-1-ch lor o-7,8,10,11,12,13-h exa h yd r o-6H-d ip y-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (20d ).

Recrystallized from MeOH; yield 83%; ¹H NMR (DMSO-d₆/

D₂O, TSP-salt, 333 K) δ 3.03 (t, J ) 7.3 Hz, 2H), 3.26 (t, J )

7.3 Hz, 2H), 3.56 (br s, 2H), 3.95 (t, J ) 6.1 Hz, 2H), 4.47 (s,

2H), 4.48 (s, 2H), 7.27 (s, 1H), 7.52-7.64 (m, 7H), 8.60 (d, J )

8.0 Hz, 1H).

Meth yl 1-(4-p en tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr a h yd r o-

p yr id in -3-yl)in d ole Ca r boxyla te (19d ). 19d was prepared

from the reaction of 9d and 13 according to the general

acylation procedure and as described for 18j. Recrystallized

from a mixture of EtOAc/n-hexane; yield 49%; ¹H NMR (CDCl₃)

δ 2.03 (t, J ) 2.5 Hz, 1H), 2.44 (m, 2H), 2.67-2.75 (m, 4H),

3.16 (t, J ) 7.3 Hz, 2H), 3.36 (m, 2H), 3.73 (s, 2H), 3.95 (s,

3H), 6.38 (m, 1H), 7.27-7.42 (m, 6H), 8.05 (dd, J ) 1.5 and

8.8 Hz, 1H), 8.49 (d, J ) 1.5 Hz, 1H), 8.52 (d, J ) 8.8 Hz, 1H).

6-Meth oxy-1-(4-p en tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr a h y-

d r op yr id in -3-yl)in d ole (19e). 19e was prepared from the

reaction of 9e and 13 according to the general acylation

procedure and that described for 18j. Yield 68%; ¹H NMR

(CDCl₃) δ 2.03 (t, J ) 2.6 Hz, 1H), 2.40 (m, 2H), 2.67 (t, J )

5.8 Hz, 2H), 2.72 (td, J ) 2.6 and 7.3 Hz, 2H), 3.14 (t, J ) 7.3

Hz, 2H), 3.34 (m, 2H), 3.71 (s, 2H), 3.87 (s, 3H), 6.31 (m, 1H),

6.92 (dd, J ) 2.4 and 8.7 Hz, 1H), 7.15 (s, 1H), 7.26-7.42 (m,

5H), 7.63 (d, J ) 8.7 Hz, 1H), 8.11 (d, J ) 2.4 Hz, 1H).

5,6-Dim eth oxy-1-(4-p en tyn oyl)-3-(1-ben zyl-1,2,5,6-tet-

r a h yd r op yr id in -3-yl)in d ole Hyd r och lor id e (19f). 19f was

prepared from the reaction of 9f and 13 according to the

Meth yl 11-Ben zyl-7,8,10,11,12,13-h exa h yd r o-6H-d ip y-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e-1-ca r boxyla te Hyd r o-

ch lor id e (20e). Yield 80%; ¹H NMR (DMSO-d₆/D₂O, TSP-salt,

313 K) δ 3.05 (t, J ) 7.5 Hz, 2H), 3.21 (br s, 2H), 3.28 (t, J )

7.5 Hz, 2H), 3.56 (br s, 2H), 3.98 (s, 3H), 4.50 (s, 2H), 4.51 (s,

2H), 7.27 (s, 1H), 7.56-7.63 (m, 6H), 7.69 (t, J ) 8 Hz, 1H),

8.69 (d, J ) 8 Hz, 1H).

11-Ben zyl-2-m eth yl-7,8,10,11,12,13-h exa h ydr o-6H-d ip y-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (20f).

1

general acylation procedure. Yield 73%; H NMR (DMSO-d₆/

1

D₂O) δ 2.6-2.7 (m, 5H), 3.23 (t, J ) 5.7 Hz, 2H), 3.31 (br s,

2H), 3.82 (s, 3H), 3.85 (s, 3H), 3.97 (br s, 2H), 4.42 (s, 2H),

6.48 (br s, 1H), 7.22 (s, 1H), 7.5-7.6 (m, 5H), 7.67 (s, 1H), 8.06

(s, 1H).

Yield 63%; H NMR (DMSO-d₆/D₂O, TSP-salt, 333 K) δ 3.01

(t, J ) 8.0 Hz, 2H), 3.24 (t, J ) 8.0 Hz, 2H), 3.59 (t, J ) 8.0

Hz, 2H), 3.67 (t, J ) 8.0 Hz, 2H), 3.91 (s, 3H), 4.43 (s, 2H),

4.48 (s, 2H), 7.15 (s, 1H), 7.42 (d, J ) 8.6 Hz, 1H), 7.56-7.63

(m, 5H), 7.89 (s, 1H), 8.30 (d, J ) 8.6 Hz, 1H).

1-(3,3-Dim eth yl-4-pen tyn oyl)-3-(1-ben zyl-1,2,5,6-tetr ah y-

d r op yr id in -3-yl)in d ole Hyd r och lor id e (19g). 19g was

prepared from the reaction of 9a and 17 according to the

11-Ben zyl-2-m eth oxy-7,8,10,11,12,13-h exah ydr o-6H-dipy-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (20g).

1

general acylation procedure. Yield 86%; H NMR (DMSO-d₆/

Yield >95%; H NMR (DMSO-d₆/D₂O) δ 2.99 (t, J ) 7.5 Hz,

D₂O) δ 1.40 (s, 6H), 2.63 (s, 1H), 2.64 (br s, 2H), 3.14 (s, 2H),

3.38 (br s, 2H), 4.01 (s, 2H), 4.42 (s, 2H), 6.54 (br s, 1H), 7.39-

7.48 (m, 2H), 7.52-7.58 (m, 5H), 7.81 (s, 1H), 7.86 (d, J ) 8

Hz, 1H), 8.45 (d, J ) 8 Hz, 1H).

2H), 3.22 (t, J ) 7.5 Hz, 2H), 3.58 (br s, 2H), 3.64 (br s, 2H),

3.89 (s, 3H), 4.42 (s, 2H), 4.47 (s, 2H), 7.17 (s, 1H), 7.21 (dd, J

) 2.2 and 9.0 Hz, 1H), 7.52 (d, J ) 2.2 Hz, 1H), 7.54-7.60 (m,

5H), 8.33 (d, J ) 9.0 Hz, 1H).

11-Ben zyl-2-b en zyloxy-7,8,10,11,12,13-h exa h yd r o-6H -

d ip yr id o[3,4-c:1′,2′,3′-lm ]ca r b a zol-6-on e h yd r och lor id e

5,6-Dim eth oxy-1-(3,3-d im eth yl-4-p en tyn oyl)-3-(1-ben -

zyl-1,2,5,6-tetr a h yd r op yr id in -3-yl)in d ole Hyd r och lor id e

(19h ). 19h was prepared from the reaction of 9f and 17

1

(20h ). Yield 92%; H NMR (DMSO-d₆/D₂O) δ 2.98 (t, J ) 7.5

1

Hz, 2H), 3.22 (t, J ) 7.5 Hz, 2H), 3.55 (br s, 2H), 3.63 (br s,

2H), 4.42 (s, 2H), 4.47 (s, 2H), 5.24 (s, 2H), 7.17 (s, 1H), 7.28

(dd, J ) 1.8 and 8.9 Hz, 1H), 7.36-7.61 (m, 11H), 8.32 (d, J )

8.9 Hz, 1H).

according to the general acylation procedure. Yield 57%; H

NMR (DMSO-d₆/D₂O) δ 1.40 (s, 6H), 2.62 (m, 2H), 2.68 (s, 1H),

3.13 (s, 2H), 3.32 (br s, 2H), 3.82 (s, 3H), 3.85 (s, 3H), 3.95 (br

s, 2H), 4.40 (s, 2H), 6.47 (br s, 1H), 7.5-7.6 (m, 5H), 7.69 (s,

1H), 7.81 (s, 1H), 8.08 (s, 1H).

11-Ben zyl-2-ch olr o-7,8,10,11,12,13-h exa h yd r o-6H-d ip y-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (20i).

Yield 60% (not maximized); ¹H NMR (DMSO-d₆/D₂O, TSP-salt,

333 K) δ 3.04 (t, J ) 7.5 Hz, 2H), 3.27 (t, J ) 7.5 Hz, 2H), 3.59

(br s, 2H), 3.65 (br s, 2H), 4.42 (s, 2H), 4.46 (s, 2H), 7.22 (s,

1H), 7.51-7.62 (m, 5H), 7.63 (dd, J ) 2.0 and 8.9 Hz, 1H),

8.09 (d, J ) 2.0 Hz, 1H), 8.90 (d, J ) 8.9 Hz, 1H).

Ben zyl 11-Ben zyl-2-7,8,10,11,12,13-h exah ydr o-6H-dipy-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e-2-ca r boxyla te Hyd r o-

ch lor id e (20j). Yield 94%; ¹H NMR (DMSO-d₆/D₂O) δ 3.04 (t,

J ) 7.5 Hz, 2H), 3.25 (t, J ) 7.5 Hz, 2H), 3.55-3.90 (br s, 2H),

3.63 (br s, 2H), 4.48 (s, 2H), 4.53 (s, 2H), 5.43 (s, 2H), 7.00 (d,

J ) 8.5 Hz, 2H), 7.18 (d, J ) 7.8 Hz, 1H), 7.22 (s, 1H), 7.39-

7.62 (m, 7H), 8.21 (d, J ) 8.6 Hz, 1H), 8.50 (d, J ) 8.6 Hz,

1H), 8.61 (s, 1H).

11-Ben zyl-2-m or ph olin ocar bon yl-7,8,10,11,12,13-h exah y-

d r o-6H-d ip yr id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r o-

ch lor id e (20k ). Yield >95%; ¹H NMR (DMSO-d₆/D₂O) δ 3.06

(t, J ) 7.2 Hz, 2H), 3.28 (t, J ) 7.2 Hz, 2H), 3.49 (m, 2H), 3.64

(m, 6H), 3.79 (m, 4H), 4.49 (s, 2H), 4.53 (s, 2H), 7.22 (s, 1H),

7.48-7.62 (m, 5H), 7.65 (d, J ) 9 Hz, 1H), 8.10 (s, 1H), 8.51

(d, J ) 9 Hz, 1H).

11-Ben zyl-2-m or p h olin om eth yl-7,8,10,11,12,13-h exa h y-

d r o-6H-d ip yr id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Dih yd r o-

ch lor id e (20l). Yield 85%; ¹H NMR (DMSO-d₆/D₂O, TSP-salt,

Gen er a l P r oced u r e for In t r a m olecu la r Diels-Ald er

Rea ction s. The Diels-Alder precursor (0.01 mol) was sus-

pended in diphenyl ether (400 mL) at 230-240 °C for about 2

h or until the starting material was fully consumed under a

slow stream of argon gas. The solution was cooled to room

temperature, and unless otherwise stated, ether containing

hydrogen chloride was added until precipitation occurred. The

resulting precipitate was collected by filtration, washed ex-

haustively with ether, and dried in vacuo. The following Diels-

Alder adducts 20a -z and 21a -i were synthesized using the

above procedure for cyclization.

11-Ben zyl-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[3,4-

c:1′,2′,3′-lm ]ca r ba zol-6-on e (20a ). Diphenyl ether was re-

moved in vacuo, and the resulting residue was chromato-

graphed on silica, eluting with EtOAc/toluene (1:1) to give 20a

1

as a white crystalline solid in 56% yield. Mp 126-127 °C; H

NMR (CDCl₃) δ 2.93 (t, J ) 5.9 Hz, 2H), 3.01 (t, J ) 7.5 Hz,

2H), 3.20 (t, J ) 7.5 Hz, 2H), 3.36 (t, J ) 5.9 Hz, 2H), 3.76 (s,

4H), 6.93 (s, 1H), 7.25-7.45 (m, 6H), 7.50 (t, J ) 8 Hz, 1H),

7.98 (d, J ) 8 Hz, 1H), 8.54 (d, J ) 8 Hz, 1H).

11-Ben zyl-1-m eth oxy-7,8,10,11,12,13-h exah ydr o-6H-dipy-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (20b).

1

Yield ∼50% (not maximized); H NMR (DMSO-d₆/D₂O, TSP-

salt, 343 K) δ 3.00 (t, J ) 7.5 Hz, 2H), 3.20 (t, J ) 7.5 Hz, 2H),

1270 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6

Gharagozloo et al.

333 K) δ 3.05 (m, 4H), 3.28 (t, J ) 7.3 Hz, 2H), 3.60 (br s, 2H),

3.81 (br s, 4H), 3.92 (br s, 4H), 4.31 (br s, 2H), 4.40 (br s, 2H),

4.42 (br s, 2H), 7.22 (s, 1H), 7.48-7.61 (m, 5H), 7.69 (d, J )

8.2 Hz, 1H), 8.22 (s, 1H), 8.49 (d, J ) 8.2 Hz, 1H).

11-Ben zyl-3-m eth oxy-7,8,10,11,12,13-h exah ydr o-6H-dipy-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (20m ).

Recrystallized from MeOH; yield 62%; ¹H NMR (DMSO-d₆/

D₂O) δ 3.00 (t, J ) 7.4 Hz, 2H), 3.01 (t, J ) 7.4 Hz, 2H), 3.52

(br s, 2H), 3.63 (br s, 2H), 3.88 (s, 3H), 4.42 (s, 2H), 4.48 (s,

2H), 7.11 (s, 1H), 7.11 (dd, J ) 2.5 and 8.6 Hz, 1H), 7.53-7.62

(m, 5H), 7.97 (d, J ) 2.5 Hz, 1H), 7.99 (d, J ) 8.6 Hz, 1H).

11-Ben zyl-3-h ydr oxy-7,8,10,11,12,13-h exah ydr o-6H-dipy-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r obr om id e (20n ).

20n was prepared as described for 20c, starting with 20m .

Recrystallized from MeOH; yield 83%; ¹H NMR (DMSO-d₆/

D₂O) δ 2.99 (t, J ) 7.4 Hz, 2H), 3.21 (t, J ) 7.4 Hz, 2H), 3.51

(br s, 2H), 3.65 (br s, 2H), 4.44 (s, 2H), 4.50 (s, 2H), 6.95 (dd,

J ) 0.9 and 8.7 Hz, 1H), 7.08 (s, 1H), 7.53-7.61 (m, 5H), 7.89

(d, J ) 0.9 Hz, 1H), 7.90 (d, J ) 8.7 Hz, 1H).

11-Ben zyl-3-ch lor o-7,8,10,11,12,13-h exa h yd r o-6H-d ip y-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (20o).

Recrystallized from MeOH; yield 81%; ¹H NMR (DMSO-d₆/

D₂O, TSP-salt, 323 K) δ 3.04 (t, J ) 7.4 Hz, 2H), 3.26 (t, J )

7.4 Hz, 2H), 3.56 (t, J ) 6.0 Hz, 2H), 3.67 (t, J ) 6.0 Hz, 2H),

4.44 (s, 2H), 4.48 (s, 2H), 7.20 (s, 1H), 7.50-7.62 (m, 6H), 8.08

(d, J ) 8.5 Hz, 1H), 8.40 (d, J ) 1.8 Hz, 1H).

Meth yl 11-Ben zyl-7,8,10,11,12,13-h exa h yd r o-6H-d ip y-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e-3-ca r boxyla te Hyd r o-

ch lor id e (20p ). Yield 80%; ¹H NMR (DMSO-d₆/D₂O, TSP-salt,

333 K) δ 2.92 (t, J ) 5.6 Hz, 2H), 3.05 (t, J ) 7.5 Hz, 2H), 3.26

(t, J ) 7.5 Hz, 2H), 3.34 (t, J ) 5.6 Hz, 2H), 3.74 (s, 2H), 3.76

(s, 2H), 3.96 (s, 3H), 7.14 (s, 1H), 7.30-7.46 (m, 5H), 8.08 (dd,

J ) 1.5 and 8.2 Hz, 1H), 8.24 (d, J ) 8.2 Hz, 1H), 9.05 (d, J )

1.5 Hz, 1H).

11-Ben zyl-3-cya n o-7,8,10,11,12,13-h exa h yd r o-6H-d ip y-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (20q).

Recrystallized from MeOH; yield 75%; ¹H NMR (DMSO-d₆/

D₂O, TSP-salt, 323 K) δ 3.06 (t, J ) 7.4 Hz, 2H), 3.29 (t, J )

7.4 Hz, 2H), 3.60 (t, J ) 5.4 Hz, 2H), 3.67 (t, J ) 5.4 Hz, 2H),

4.46 (s, 2H), 4.48 (s, 2H), 7.29 (s, 1H), 7.55-7.62 (m, 5H), 7.83

(d, J ) 8.2 Hz, 1H), 8.24 (d, J ) 8.2 Hz, 1H), 8.66 (s, 1H).

(s, 3H), 4.47 (s, 2H), 4.51 (s, 2H), 7.11 (s, 1H), 7.50-7.62 (m,

5H), 7.97 (s, 1H).

Meth yl 11-Ben zyl-2,3-dim eth oxy-7,8,10,11,12,13-h exah y-

d r o-6H-d ip yr id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e-9-ca r box-

yla te (20w ). Yield 41% (not maximized);

¹H NMR (CDCl₃) δ

2.88 (t, J ) 6.0 Hz, 2H), 3.00 (t, J ) 7.5 Hz, 2H), 3.33 (t, J )

7.5 Hz, 2H), 3.34 (t, J ) 6.0 Hz, 2H), 3.75 (s, 2H), 3.86 (s, 2H),

3.89 (s, 3H), 3.98 (s, 3H), 4.03 (s, 3H), 7.26-7.42 (m, 5H), 7.45

(s, 1H), 8.17 (s, 1H).

11-Ben zyl-9-a cet oxym et h yl-2,3-d im et h oxy-7,8,10,11,-

12,13-h exa h yd r o-6H-d ip yr id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-

on e h yd r och lor id e (20x). Yield 66%; ¹H NMR (DMSO-d₆/

D₂O) δ 1.94 (s, 3H), 3.02 (t, J ) 7.4 Hz, 2H), 3.33 (t, J ) 7.4

Hz, 2H), 3.61 (s, 4H), 3.89 (s, 3H), 3.91 (s, 3H), 4.45 (s, 2H),

4.49 (s, 2H), 5.13 (s, 2H), 7.54-7.8 (m, 6H), 8.05 (s, 1H).

11-Be n zyl-8,8-d im e t h yl-1-m e t h oxy-7,8,10,11,12,13-

h exah ydr o-6H-dipyr ido[3,4-c:1′,2′,3′-lm ]car bazol-6-on e Hy-

d r och lor id e (20y). Yield 85%; ¹H NMR (DMSO-d₆/D₂O) δ 1.36

(s, 6H), 2.91 (s, 2H), 3.52 (br s, 2H), 3.81 (br s, 2H), 3.97 (s,

3H), 4.42 (s, 4H), 7.06 (d, J ) 8 Hz, 1H), 7.27 (s, 1H), 7.53-

7.60 (m, 6H), 8.12 (d, J ) 8 Hz, 1H).

11-B e n zy l-8,8-d im e t h y l-1-h y d r o x y -7,8,10,11,12,13-

h exah ydr o-6H-dipyr ido[3,4-c:1′,2′,3′-lm ]car bazol-6-on e Hy-

d r obr om id e (20z). 20z was prepared as described for 20c,

1

starting with 20y. Recrystallized from MeOH; yield 73%; H

NMR (DMSO-d₆/D₂O) δ 1.36 (s, 6H), 2.90 (s, 2H), 3.50 (br s,

2H), 3.90 (br s, 2H), 4.38 (s, 2H), 4.41 (s, 2H), 6.90 (d, J ) 8

Hz, 1H), 7.22 (s, 1H), 7.40 (t, J ) 8 Hz, 1H), 7.52-7.61 (m,

5H), 8.00 (d, J ) 8 Hz, 1H).

12-Ben zyl-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[4,3-

c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (21a ). Yield

94%; mp 291-292 °C; ¹H NMR (DMSO-d₆/D₂O) δ 3.03 (t, J )

7.4 Hz, 2H), 3.20-3.30 (m, 4H), 3.53 (br s, 2H), 4.56 (s, 2H),

4.81 (s, 2H), 7.28 (s, 1H), 7.51 (t, J ) 8 Hz, 1H), 7.56-7.65 (m,

6H), 7.84 (d, J ) 8 Hz, 1H), 8.45 (d, J ) 8 Hz, 1H).

12-Ben zyl-2-m eth oxy-7,8,10,11,12,13-h exah ydr o-6H-dipy-

r id o[4,3-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (21b).

Yield 96%; ¹H NMR (DMSO-d₆/D₂O) δ 2.98 (t, J ) 7.5 Hz, 2H),

δ 3.23 (t, J ) 7.5 Hz, 2H), 3.26 (br s, 2H), 3.65 (br s, 2H), 3.85

(s, 3H), 4.63 (s, 2H), 4.76 (s, 2H), 7.10 (d, J ) 1.8 Hz, 1H),

7.19 (dd, J ) 1.8 and 8.9 Hz, 1H), 7.27 (s, 1H), 7.56-7.71 (m,

5H), 8.31 (d, J ) 8.9 Hz, 1H).

11-Ben zyl-4-m eth oxy-7,8,10,11,12,13-h exah ydr o-6H-dipy-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (20r ).

12-Ben zyl-2-b en zyloxy-7,8,10,11,12,13-h exa h yd r o-6H -

d ip yr id o[4,3-c:1′,2′,3′-lm ]ca r b a zol-6-on e H yd r och lor id e

1

Yield 73%; H NMR (DMSO-d₆/D₂O, TSP-salt, 343 K) δ 3.03

1

(t, J ) 7.4 Hz, 2H), 3.24 (t, J ) 7.4 Hz, 2H), 3.58(br s, 2H),

3.64 (br s, 2H), 3.97 (s, 3H), 4.40 (s, 2H), 4.44 (s, 2H), 7.16 (s,

1H), 7.26 (d, J ) 8 Hz, 1H), 7.43-7.60 (m, 6H), 7.71 (d, J )

8.0 Hz, 1H).

(21c). Yield 83%; H NMR (DMSO-d₆/D₂O) δ 2.98 (t, J ) 7.5

Hz, 2H), δ 3.23 (t, J ) 7.5 Hz, 2H), 3.26 (br s, 2H), 3.66 (br s,

2H), 4.64 (s, 2H), 4.76 (s, 2H), 5.13 (s, 2H), 7.20 (d, J ) 2.3

Hz, 1H), 7.26 (dd, J ) 2.3 and 8.9 Hz, 1H), 7.28 (s, 1H), 7.39-

7.72 (m, 10H), 8.31 (d, J ) 8.9 Hz, 1H).

11-Ben zyl-4-h ydr oxy-7,8,10,11,12,13-h exah ydr o-6H-dipy-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r obr om id e (20s).

20s was prepared as described for 20c, starting with 20r . Yield

75%; ¹H NMR (DMSO-d₆/D₂O, TSP-salt, 323 K) δ 3.11 (t, J )

7.4 Hz, 2H), 3.23 (t, J ) 7.4 Hz, 2H), 3.56 (br s, 2H), 3.80 (br

s, 2H), 4.48 (s, 2H), 4.52 (s, 2H), 7.04 (d, J ) 8 Hz, 1H), 7.21

(s, 1H), 7.42 (t, J ) 8 Hz, 1H), 7.56-7.61 (m, 6H).

11-Ben zyl-2,3-d im et h oxy-7,8,10,11,12,13-h exa h yd r o-

6H-d ip yr id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lo-

r id e (20t). Yield 91%; ¹H NMR (DMSO-d₆/D₂O, TSP-salt, 343

K) δ 3.00 (t, J ) 7.4 Hz, 2H), 3.22 (t, J ) 7.4 Hz, 2H), 3.57(br

s, 2H), 3.62 (br s, 2H), 3.87 (s, 3H), 3.90 (s, 3H), 4.40 (s, 2H),

4.49 (s, 2H), 7.09 (s, 1H), 7.50 (s, 1H), 7.53-7.62 (m, 5H), 7.99

(s, 1H).

Meth yl 12-Ben zyl-7,8,10,11,12,13-h exa h yd r o-6H-d ip y-

r id o[4,3-c:1′,2′,3′-lm ]ca r ba zol-6-on e-2-ca r boxyla te Hyd r o-

1

ch lor id e (21d ). Yield 78%; H NMR (DMSO-d₆/D₂O) δ 3.06

(t, J ) 7.4 Hz, 2H), δ 3.28 (m, 4H), 3.62 (br s, 2H), 3.98 (s,

3H), 4.64 (s, 2H), 4.82 (s, 2H), 7.35 (s, 1H), 7.59-7.73 (m, 5H),

8.20 (dd, J ) 1.6 and 8.6 Hz, 1H), 8.28 (d, J ) 1.6 Hz, 1H),

8.52 (d, J ) 8.6 Hz, 1H).

12-Ben zyl-3-m eth oxy-7,8,10,11,12,13-h exah ydr o-6H-dipy-

r id o[4,3-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (21e).

Yield 78%; ¹H NMR (DMSO-d₆/D₂O) δ 3.02 (t, J ) 7.4 Hz, 2H),

δ 3.19-3.26 (m, 4H), 3.52 (br s, 2H), 3.88 (s, 3H), 4.59 (s, 2H),

4.81 (s, 2H), 7.10 (d, J ) 8.6 Hz, 1H), 7.18 (s, 1H), 7.55-7.67

(m, 5H), 7.76 (d, J ) 8.6 Hz, 1H), 7.99 (s, 1H).

11-Ben zyl-2,3-m eth ylen ed ioxy-7,8,10,11,12,13-h exa h y-

12-Ben zyl-3-h ydr oxy-7,8,10,11,12,13-h exah ydr o-6H-dipy-

r id o[4,3-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r obr om id e (21f).

21f was prepared as described for 20c, starting with 21e.

Recrystallized from MeOH; yield 70%; ¹H NMR (DMSO-d₆/

D₂O) δ 3.00 (t, J ) 7.5 Hz, 2H), δ 3.18-3.24 (m, 4H), 3.45 (br

s, 2H), 4.62 (br s, 2H), 4.82 (s, 2H), 6.94 (dd, J ) 2.3 and 8 Hz,

1H), 7.16 (s, 1H), 7.56-7.68 (m, 6H), 7.91 (d, J ) 2.3 Hz, 1H).

d r o-6H-d ip yr id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r o-

1

ch lor id e (20u ). Yield 81%; H NMR (DMSO-d₆/D₂O) δ 2.99

(t, J ) 7.4 Hz, 2H), 3.20 (t, J ) 7.4 Hz, 2H), 3.50 (br s, 2H),

3.62 (br s, 2H), 4.42 (s, 2H), 4.48 (s, 2H), 6.14 (s, 2H), 7.09 (s,

1H), 7.53-7.61 (m, 5H), 7.57 (s, 1H), 7.93 (s, 1H).

11-Ben zyl-1,2,3-tr im eth oxy-7,8,10,11,12,13-h exa h yd r o-

6H-d ip yr id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lo-

r id e (20v). Yield 36% (not maximized); H NMR (DMSO-d₆/

D₂O, TSP-salt, 318 K) δ 3.00 (t, J ) 7.5 Hz, 2H), 3.21 (t, J )

7.5 Hz, 2H), 3.60-4.05 (m, 4H), 3.86 (s, 3H), 3.93 (s, 3H), 4.00

12-Ben zyl-2,3-d im et h oxy-7,8,10,11,12,13-h exa h yd r o-

6H-d ip yr id o[4,3-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lo-

r id e (21g). Yield 88%; ¹H NMR (DMSO-d₆/D₂O) δ 3.00 (t, J )

7.4 Hz, 2H), δ 3.19 (br s, 2H), 3.24 (t, J ) 7.4 Hz, 2H), 3.53 (br

1

Substituted Pentacyclic Carbazolones

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1271

(22e). Recrystallized twice from a mixture of MeOH/H₂O; yield

s, 2H), 3.85 (s, 3H), 3.87 (s, 3H), 4.64 (s, 2H), 4.72 (s, 2H), 7.05

(s, 1H), 7.19 (s, 1H), 7.5-7.8 (m, 5H), 8.00 (s, 1H).

1

73%; mp >300 °C; H NMR (DMSO-d₆/D₂O, TSP-salt, 313 K)

δ 3.06 (t, J ) 7.5 Hz, 2H), 3.11 (t, J ) 6.3 Hz, 2H), 3.29 (t, J

) 7.5 Hz, 2H), 3.43 (t, J ) 6.3 Hz, 2H), 4.02 (s, 3H), 4.45 (s,

2H), 7.32 (s, 1H), 7.61 (d, J ) 8 Hz, 1H), 7.71 (t, J ) 8 Hz,

1H), 8.72 (d, J ) 8 Hz, 1H); MS m/e 335 (MH⁺, 100). Anal.

(C₂₀H₁₈N₂O₃‚HCl) C, H, N.

2-Meth yl-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[3,4-c:

1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (22f). Yield 63%;

mp 265-268 °C; ¹H NMR (DMSO-d₆/D₂O) δ 2.49 (s, 3H), 2.97

(t, J ) 7.5 Hz, 2H), 3.21 (t, J ) 7.5 Hz, 2H), 3.48 (t, J ) 6 Hz,

2H), 3.63 (t, J ) 6 Hz, 2H), 4.45 (s, 2H), 7.18 (s, 1H), 7.39 (d,

J ) 8 Hz, 1H), 7.44 (s, 1H), 8.22 (d, J ) 8 Hz, 1H); MS m/e

335 (MH⁺, 100). Anal. (C₁₉H₁₈N₂O‚HCl) C, H, N.

12-Ben zyl-8,8-d im eth yl-7,8,10,11,12,13-h exa h yd r o-6H-

d ip yr id o[4,3-c:1′,2′,3′-lm ]ca r b a zol-6-on e H yd r och lor id e

(21h ). Yield 88%; ¹H NMR (DMSO-d₆/D₂O) δ 1.40 (s, 6H), 2.96

(s, 2H), δ 3.26 (br s, 2H), 3.57 (br s, 2H), 4.63 (s, 2H), 4.90 (s,

2H), 7.41 (s, 1H), 7.5-7.7 (m, 7H), 7.88 (d, J ) 8 Hz, 1H), 8.44

(d, J ) 8 Hz, 1H).

12-Ben zyl-8,8-d im eth yl-2,3-d im eth oxy-7,8,10,11,12,13-

h exah ydr o-6H-dipyr ido[4,3-c:1′,2′,3′-lm ]car bazol-6-on e Hy-

d r och lor id e (21i). Yield 91%; ¹H NMR (DMSO-d₆/D₂O) δ 1.38

(s, 6H), 2.92 (s, 2H), 3.28 (br s, 2H), 3.63 (br s, 2H), 3.85 (s,

3H), 3.88 (s, 3H), 4.62 (s, 2H), 4.72 (s, 2H), 7.08 (s, 1H), 7.30

(s, 1H), 7.55-7.72 (m, 5H), 8.00 (s, 1H).

2-Meth oxy-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[3,4-

c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (22g). Recrys-

tallized from a mixture of MeOH/H₂O as colorless needles;

Gen er a l P r oced u r e for Deben zyla tion Rea ction s. A

suspension of the Diels-Alder adduct (1.0 g) and 5% Pd/C (0.5

g) in MeOH (75 mL) was stirred initially under argon (10 min)

and then under hydrogen at atmospheric pressure at 40 °C

for 2 h or until the reaction was complete. The reaction mixture

was filtered, and unless otherwise stated, the filtrate was

concentrated under reduce pressure and the resulting precipi-

tate was collected by filtration. If necessary, further purifica-

tion was carried out as described. Unless otherwise stated, the

following compounds were obtained by the above debenzylation

procedure.

1

yield 83%; mp 285-290 °C; H NMR (DMSO-d₆/D₂O) δ 2.99

(t, J ) 7.4 Hz, 2H), 3.22 (t, J ) 7.4 Hz, 2H), 3.47 (t, J ) 5.4

Hz, 2H), 3.61 (t, J ) 5.4 Hz, 2H), 3.90 (s, 3H), 4.40 (s, 2H),

7.20 (d, J ) 8.9 Hz, 1H), 7.22 (s, 1H), 7.52 (d, J ) 1.8 Hz, 1H),

8.32 (d, J ) 8.9 Hz, 1H); MS m/e 307 (MH⁺, 50). Anal.

(C₁₉H₁₈N₂O₂‚HCl) C, H, N.

2-Hyd r oxy-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[3,4-

c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (22h ). Recrys-

tallized from a mixture of MeOH/H₂O as colorless needles;

7,8,10,11,12,13-Hexa h yd r o-6H-d ip yr id o[3,4-c:1′,2′,3′-lm ]-

ca r ba zol-6-on e Hyd r och lor id e (22a ). Recrystallized from

small quantities of MeOH as colorless needles; yield 64%; mp

1

yield 56%; mp >300 °C; H NMR (DMSO-d₆/D₂O) δ 2.99 (t, J

) 7.5 Hz, 2H), 3.23 (t, J ) 7.5 Hz, 2H), 3.46 (t, J ) 6.2 Hz,

2H), 3.57 (t, J ) 6.2 Hz, 2H), 4.40 (s, 2H), 7.05 (dd, J ) 2.2

and 8.8 Hz, 1H), 7.20 (s, 1H), 7.44 (d, J ) 2.2 Hz, 1H), 8.24 (d,

J ) 8.8 Hz, 1H); MS m/e 293 (MH⁺, 100). Anal. (C₁₈H₁₆N₂O₂‚

HCl‚0.6H₂O) C, H, N.

1

287-290 °C; H NMR (DMSO-d₆/D₂O) δ 2.99 (t, J ) 7.5 Hz,

2H), 3.22 (t, J ) 7.5 Hz, 2H), 3.47 (t, J ) 6.1 Hz, 2H), 3.61 (t,

J ) 6.1 Hz, 2H), 4.44 (s, 2H), 7.21 (s, 1H), 7.49 (t, J ) 8 Hz,

1H), 7.57 (t, J ) 8 Hz, 1H), 8.03 (d, J ) 8 Hz, 1H), 8.34 (d, J

) 8 Hz, 1H); MS m/e 277 (MH⁺, 100). Anal. (C₁₈H₁₆N₂O‚HCl)

C, H, N.

2-Ch lor o-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[3,4-c:

1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (22i). 22i was

prepared as described for 22d , starting with 20i. Recrystallized

from MeOH; yield 55%; mp 289-292 °C; ¹H NMR (DMSO-d₆/

D₂O) δ 2.82 (t, J ) 7.5 Hz, 2H), 3.24 (t, J ) 7.5 Hz, 2H), 3.42

(t, J ) 5.9 Hz, 2H), 3.49 (t, J ) 5.9 Hz, 2H), 4.32 (s, 2H), 7.21

(s, 1H), 7.42 (d, J ) 8 Hz, 1H), 7.90 (s, 1H), 8.21 (d, J ) 8 Hz,

1H); HRMS (C₁₈H₁₆ClN₂O) found m/z 311.0970, calcd 311.0958.

7,8,10,11,12,13-Hexa h yd r o-6H-d ip yr id o[3,4-c:1′,2′,3′-lm ]-

ca r b a zol-6-on e-2-ca r b oxylic Acid H yd r och lor id e (22j).

Recrystallized from MeOH as colorless needles; yield 76%; mp

> 330 °C; ¹H NMR (DMSO-d₆/D₂O) δ 3.03 (t, J ) 7.4 Hz, 2H),

3.25 (t, J ) 7.4 Hz, 2H), 3.48 (t, J ) 6.2 Hz, 2H), 3.64 (t, J )

6.2 Hz, 2H), 4.46 (s, 2H), 7.27 (s, 1H), 8.05 (d, J ) 8.6 Hz,

1H), 8.36 (d, J ) 8.6 Hz, 1H), 8.44 (s, 1H); MS m/e 321 (MH⁺,

100). Anal. (C₁₉H₁₆N₂O₃‚HCl‚1.2H₂O) C, H, N.

1-Meth oxy-7,8,10,11,12,13-Hexa h yd r o-6H-d ip yr id o[3,4-

c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (22b). Recrys-

tallized from small quantities of MeOH; yield 63%; mp 284-

1

286 °C; H NMR (DMSO-d₆/D₂O, TSP-salt) δ 2.99 (t, J ) 7.5

Hz, 2H), 3.21 (t, J ) 7.5 Hz, 2H), 3.60 (t, J ) 6.0 Hz, 2H), 3.75

(t, J ) 6.0 Hz, 2H), 4.02 (s, 3H), 4.51 (s, 2H), 7.04 (d, J ) 8.2

Hz, 1H), 7.20 (s, 1H), 7.55 (t, J ) 8.2 Hz, 1H), 8.03 (d, J ) 8.2

Hz, 1H); MS m/e 307 (MH⁺, 100). Anal. (C₁₉H₁₈N₂O_2.HCl‚

0.1H₂O) C, H, N.

1-Hyd r oxy-7,8,10,11,12,13-Hexa h yd r o-6H-d ip yr id o[3,4-

c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r obr om id e (22c). The prod-

uct was obtained from 20c by the general procedure for

debenzylation and then recrystallized from small quantities

1

of MeOH. Yield 62%; mp > 300 °C; H NMR (DMSO-d₆/D₂O,

TSP-salt, 298 K) δ 2.97 (t, J ) 7.6 Hz, 2H), 3.18 (t, J ) 7.6

Hz, 2H), 3.53 (t, J ) 6.2 Hz, 2H), 3.82 (t, J ) 6.2 Hz, 2H), 4.43

(s, 2H), 6.87 (d, J ) 8 Hz, 1H), 7.12 (s, 1H), 7.37 (t, J ) 8 Hz,

1H), 7.92 (d, J ) 8 Hz, 1H); MS m/e 293 (MH⁺, 100). Anal.

(C₁₈H₁₆N₂O_2.HBr‚0.5H₂O) C, H, N.

Meth yl 7,8,10,11,12,13-Hexa h yd r o-6H-d ip yr id o[3,4-c:

1′,2′,3′-lm ]ca r ba zol-6-on e-2-ca r boxyla te Hyd r och lor id e

(22k ). A solution of 22j (0.32 g) in MeOH (50 mL) containing

catalytic amounts of 98% H₂SO₄was refluxed overnight. The

mixture was cooled, and the white precipitate was collected

by filtration and recrystallized from a mixture of MeOH/H₂O

as colorless needles. Yield 76%; mp >300 °C; ¹H NMR (DMSO-

d₆/D₂O, TSP-salt) δ 3.00 (t, J ) 7.5 Hz, 2H), 3.23 (t, J ) 7.5

Hz, 2H), 3.41 (t, J ) 6 Hz, 2H), 3.63 (t, J ) 6 Hz, 2H), 3.96 (s,

3H), 4.44 (s, 2H), 7.28 (s, 1H), 8.05 (d, J ) 8 Hz, 1H), 8.32 (d,

J ) 8 Hz, 1H), 8.33 (s, 1H); MS m/e 335 (MH⁺, 100). Anal.

(C₂₀H₁₈N₂O₃‚0.5H₂SO₄‚1H₂O) C, H, N.

2-Mor p h olin oca r bon yl-7,8,10,11,12,13-h exa h yd r o-6H-

d ip yr id o[3,4-c:1′,2′,3′-lm ]ca r b a zol-6-on e H yd r och lor id e

(22l). The residue obtained from the reaction was chromato-

graphed on silica, eluting with 4% concentrated ammonia in

MeOH. The product was redissolved in MeOH, and Et₂O

containing HCl was added. The white precipitate was collected

by filtration and dried in vacuo. Yield 88%; mp 250 °C (dec);

¹H NMR (DMSO-d₆/D₂O, TSP-salt) δ 3.09 (t, J ) 7.5 Hz, 2H),

3.31 (t, J ) 7.5 Hz, 2H), 3.57 (m, 4H), 3.66 (m, 4H), 3.83 (m,

4H), 4.45 (s, 2H), 7.31 (s, 1H), 7.69 (d, J ) 8 Hz, 1H), 8.15 (s,

1H), 8.53 (d, J ) 8 Hz, 1H); MS m/e 390 (MH⁺, 100). Anal.

(C₂₃H₂₃N₃O₃‚HCl‚H₂O) C, H, N.

1-Ch lor o-7,8,10,11,12,13-Hexa h yd r o-6H-d ip yr id o[3,4-c:

1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (22d ). To a sus-

pension of 20d (60 mg, 0.14 mmol) in anhydrous THF (25 mL)

and K₂CO₃(39 mg, 0.28 mmol) was added with stirring vinyl

chloroformate (250 mg, 2.35 mmol). The mixture was stirred

at room temperature for 8 h and then filtered. The filtrate was

taken to dryness, and the residue was redissolved in MeOH

(25 mL) containing trifluoroacetic acid (80 mg, 0.7 mmol). The

solution was refluxed for 10 h and cooled to room temperature,

ether containing HCl (10 mL) and charcoal (100 mg) was

added, and the mixture was stirred for 1 h. The mixture was

filtered and the filtrate taken to dryness. The resulting residue

was recrystallized from MeOH to give 22d as a white powder

(35 mg, 73% yield). Mp 281-284 °C; ¹H NMR (DMSO-d₆/D₂O,

TSP-salt, 338 K) δ 3.06 (t, J ) 7.5 Hz, 2H), 3.29 (t, J ) 7.5

Hz, 2H), 3.50 (t, J ) 6.3 Hz, 2H), 3.91 (t, J ) 6.3 Hz, 2H), 4.47

(s, 2H), 7.31 (s, 1H), 7.56 (m, 2H), 8.57 (d, J ) 8 Hz, 1H); MS

m/e 313 and 311 (MH⁺, 33 and 100, respectively). Anal.

(C₁₈H₁₅N₂OCl‚HCl) C, H, N.

Meth yl 7,8,10,11,12,13-Hexa h yd r o-6H-d ip yr id o[3,4-c:

1′,2′,3′-lm ]ca r ba zol-6-on e-1-ca r boxyla te Hyd r och lor id e

2-Mor ph olin om eth yl-7,8,10,11,12,13-h exah ydr o-6H-dipy-

r ido[3,4-c:1′,2′,3′-lm ]car bazol-6-on e Dih ydr och lor ide (22m ).

1272 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6

Gharagozloo et al.

Yield 73%; mp 230 °C (dec); ¹H NMR (DMSO-d₆/D₂O, TSP-

salt, 313 K) δ 3.08 (t, J ) 7.5 Hz, 2H), 3.25 (t, J ) 7.5 Hz, 2H),

3.39 (br s, 4H), 3.65 (t, J ) 6 Hz, 2H), 3.78 (t, J ) 6 Hz, 2H),

3.93 (br s, 4H), 4.52 (s, 2H), 4.58 (s, 2H), 7.26 (s, 1H), 7.67 (d,

J ) 8 Hz, 1H), 8.05 (s, 1H), 8.43 (d, J ) 8 Hz, 1H); MS m/e

376 (MH⁺, 100). Anal. (C₂₃H₂₅N₃O₂‚2HCl‚1H₂O) C, H, N.

3.92 (s, 3H), 4.39 (s, 2H), 7.13 (s, 1H), 7.53 (s, 1H), 8.02 (s,

1H); MS m/e 377 (MH⁺, 100). Anal. (C₂₀H₂₀N₂O₃‚HCl‚1.5H₂O)

C, H, N.

2,3-Meth ylen edioxy-7,8,10,11,12,13-h exah ydr o-6H-dipy-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (22v).

Recrystallized from MeOH as colorless needles; yield 69%; mp

1

294-298 °C; H NMR (DMSO-d₆/D₂O) δ 3.00 (t, J ) 7.4 Hz,

3-Meth oxy-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[3,4-

c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (22n ). Recrys-

tallized from MeOH as colorless needles; yield 77%; mp >300

2H), 3.22 (t, J ) 7.4 Hz, 2H), 3.41 (t, J ) 6.0 Hz, 2H), 3.52 (t,

J ) 6.0 Hz, 2H), 4.36 (s, 2H), 6.15 (s, 2H), 7.12 (s, 1H), 7.60 (s,

1H), 7.95 (s, 1H); MS m/e 321 (MH⁺, 100). Anal. (C₁₉H₁₆N₂O₃‚

HCl‚0.2H₂O) C, H, N.

1

°C; H NMR (DMSO-d₆/D₂O) δ 3.02 (t, J ) 7.4 Hz, 2H), 3.24

(t, J ) 7.4 Hz, 2H), 3.43 (t, J ) 6.0 Hz, 2H), 3.54 (t, J ) 6.0

Hz, 2H), 3.88 (s, 3H), 4.38 (s, 2H), 7.11 (dd, J ) 2.4 and 8.7

Hz, 1H), 7.15 (s, 1H), 7.99 (d, J ) 2.4 Hz, 1H), 8.01 (d, J ) 8.7

Hz, 1H); MS m/e 307 (MH⁺, 100). Anal. (C₁₉H₁₆N₂O₂‚HCl‚

0.5H₂O) C, H, N.

1,2,3-Tr im et h oxy-7,8,10,11,12,13-h exa h yd r o-6H -d ip y-

r id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (22w ).

Recrystallized from MeOH; yield 55%; mp 210 °C (dec); ¹H

NMR (DMSO-d₆/D₂O, TSP-salt, 298 K) δ 2.98 (t, J ) 7.5 Hz,

2H), 3.21 (t, J ) 7.5 Hz, 2H), 3.56 (t, J ) 6 Hz, 2H), 3.64 (t, J

) 6 Hz, 2H), 3.89 (s, 3H), 3.92 (s, 3H), 4.03 (s, 3H), 4.46 (s,

2H), 7.18 (s, 1H), 7.86 (s, 1H); MS m/e 367 (MH⁺, 100). Anal.

(C₂₁H₂₂N₂O₄‚HCl) C, H, N.

Meth yl 2,3-Dim eth oxy-7,8,10,11,12,13-h exa h yd r o-6H-

dipyr ido[3,4-c:1′,2′,3′-lm ]car bazol-6-on e-9-car boxylate Hy-

d r och lor id e (22x). Yield 65%; mp 270 °C (dec); ¹H NMR

(DMSO-d₆/D₂O) δ 3.00 (t, J ) 7.4 Hz, 2H), 3.32 (t, J ) 7.4 Hz,

2H), 3.54 (s, 4H), 3.89 (s, 3H), 3.92 (s, 3H), 3.94 (s, 3H), 4.43

(s, 2H), 7.53 (s, 1H), 8.01 (s, 1H); MS m/e 395 (MH⁺, 100). Anal.

(C₂₂H₂₂N₂O₅‚HCl‚0.6H₂O) C, H, N.

9-Acetoxym eth yl-2,3-dim eth oxy-7,8,10,11,12,13-h exah y-

d r o-6H-d ip yr id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r o-

ch lor id e (22y). Yield 75%; mp 290 °C (dec); ¹H NMR (DMSO-

d₆/D₂O) δ 2.06 (s, 3H), 3.02 (t, J ) 7.4 Hz, 2H), 3.34 (t, J ) 7.4

Hz, 2H), 3.53 (s, 4H), 3.88 (s, 3H), 3.92 (s, 3H), 4.49 (s, 2H),

5.22 (s, 2H), 7.51 (s, 1H), 8.01 (s, 1H); MS m/e 409 (MH⁺, 100).

Anal. (C₂₃H₂₄N₂O₅‚HCl‚1.1H₂O) C, H, N.

3-Hyd r oxy-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[3,4-

c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r obr om id e (22o). The prod-

uct was obtained from 20n following the general procedure

for debenzylation. Recrystallized from MeOH as colorless

1

needles; yield 56%; mp >300 °C; H NMR (DMSO-d₆/D₂O) δ

3.00 (t, J ) 7.4 Hz, 2H), 3.22 (t, J ) 7.4 Hz, 2H), 3.42 (t, J )

6.0 Hz, 2H), 3.52 (t, J ) 6.0 Hz, 2H), 4.38 (s, 2H), 6.94 (dd, J

) 2.1 and 8.7 Hz, 1H), 7.11 (s, 1H), 7.90 (d, J ) 2.1 Hz, 1H),

7.91 (d, J ) 8.7 Hz, 1H); MS m/e 293 (MH⁺, 100). Anal.

(C₁₈H₁₆N₂O₂‚HBr‚0.5H₂O) C, H, N.

3-Ch lor o-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[3,4-c:

1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (22p ). Recrystal-

lized twice from MeOH as colorless needles; yield 19%; mp

295-298 °C; ¹H NMR (DMSO-d₆/D₂O,TSP-salt, 298 K) δ 3.06

(t, J ) 7.5 Hz, 2H), 3.26 (t, J ) 7.5 Hz, 2H), 3.44 (t, J ) 6 Hz,

2H), 3.51 (t, J ) 6 Hz, 2H), 4.35 (s, 2H), 7.26 (s, 1H), 7.57 (d,

J ) 8.5 Hz, 1H), 8.14 d, J ) 8.5 Hz, 1H), 8.44 (s, 1H); HRMS

(C₁₈H₁₆ClN₂O) found m/e 311.0980, calcd 311.0955.

8,8-Dim eth yl-1-m eth oxy-7,8,10,11,12,13-h exa h yd r o-6H-

d ip yr id o[3,4-c:1′,2′,3′-lm ]ca r b a zol-6-on e H yd r och lor id e

(22z). Recrystallized from MeOH as colorless needles; yield

50%; mp 275-278 °C; ¹H NMR (DMSO-d₆/D₂O) δ 1.39 (s, 6H),

2.90 (s, 2H), 3.44 (t, J ) 6.4 Hz, 2H), 3.85 (t, J ) 6.4 Hz, 2H),

3.98 (s, 3H), 4.39 (s, 2H), 6.91 (d, J ) 8 Hz, 1H), 7.29 (s, 1H),

7.40 (t, J ) 8 Hz 1H), 8.0 (d, J ) 8 Hz, 1H); MS m/e 335 (MH⁺,

100); HRMS (C₂₁H₂₂N₂O₂) found m/z 335.1790, calcd 335.1772.

8,8-Dim eth yl-1-h yd r oxy-7,8,10,11,12,13-h exa h yd r o-6H-

d ip yr id o[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r ob r om id e

(22a a ). The product was obtained from 20z following the

general procedure for debenzylation. Recrystallized from

Meth yl 7,8,10,11,12,13-Hexa h yd r o-6H-d ip yr id o[3,4-c:

1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e-3-ca r boxyla te

Hyd r och lor id e (22q). The reaction mixture was filtered, and

ether containing HCl was added. The resulting precipitate was

recrystallized twice from MeOH/H₂O. Yield 64%; mp >300 °C;

¹H NMR (DMSO-d₆/D₂O, 323 K) δ 3.04 (t, J ) 7.5 Hz, 2H),

3.26 (t, J ) 7.5 Hz, 2H), 3.42 (br s, 2H), 3.63 (t, J ) 6 Hz, 2H),

3.96 (s, 3H), 4.43 (s, 2H), 7.23 (s, 1H), 7.93-8.02 (m, 2H), 8.71

(s, 1H); MS m/e 335 (MH⁺, 100). Anal. (C₂₀H₁₈N₂O₃‚HCl) C,

H, N.

3-Cya n o-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[3,4-c:

1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (22r ). Recrystal-

lized from a mixture of MeOH/H₂O; yield 64%; mp 282-285

1

MeOH as colorless needles; yield 50%; mp >330 °C; H NMR

(DMSO-d₆/D₂O) δ 1.38 (s, 6H), 2.91 (s, 2H), 3.44 (t, J ) 6.4

Hz, 2H), 3.83 (t, J ) 6.4 Hz, 2H), 4.40 (s, 2H), 6.91 (d, J ) 8

Hz, 1H), 7.30 (s, 1H), 7.40 (t, J ) 8 Hz 1H), 8.0 (d, J ) 8 Hz,

1H); MS m/e 321 (MH⁺, 100). Anal. (C₂₀H₂₀N₂O₂‚HBr‚0.2H₂O)

C, H, N.

1

°C; H NMR (DMSO-d₆/D₂O, TSP-salt, 298 K) δ 2.86 (t, J )

7.3 Hz, 2H), 3.25 (t, J ) 7.3 Hz, 2H), 3.61 (s, 2H), 3.63 (s, 2H),

4.43 (s, 2H), 7.23 (s, 1H), 7.63 (d, J ) 8 Hz, 1H), 8.07 (s, 1H),

8.33 (d, J ) 8 Hz, 1H); MS m/e 302 (MH⁺, 100). Anal.

(C₁₉H₁₅N₃O‚HCl) C, H, N.

7,8,10,11,12,13-Hexa h yd r o-6H-d ip yr id o[4,3-c:1′,2′,3′-lm ]-

ca r ba zol-6-on e Hyd r och lor id e (23a ). Recrystallized from

MeOH/H₂O as colorless needles (97% yield before recrystalli-

zation); mp 295-298 °C; ¹H NMR (DMSO-d₆/D₂O) δ 3.02 (t, J

) 7.4 Hz, 2H), 3.21 (t, J ) 6.0 Hz, 2H), 3.26 (t, J ) 7.4 Hz,

2H), 3.53 (t, J ) 6.0 Hz, 2H), 4.77 (s, 2H), 7.28 (s, 1H), 7.52 (t,

J ) 8 Hz, 1H), 7.61 (t, J ) 8 Hz, 1H), 7.93 (t, J ) 8 Hz, 1H),

8.40 (t, J ) 8 Hz, 1H); MS m/e 277 (MH⁺, 100). Anal.

(C₁₈H₁₆N₂O‚HCl‚0.6H₂O) C, H, N.

4-Meth oxy-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[3,4-

c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (22s). Recrys-

tallized from MeOH/H₂O; yield 67%; mp 288-290 °C; ¹H NMR

(DMSO-d₆/D₂O, 323 K) δ 3.03 (t, J ) 7.3 Hz, 2H), 3.25 (t, J )

7.3 Hz, 2H), 3.53 (br s, 2H), 3.61 (br s, 2H), 3.96 (s, 3H), 4.44

(s, 2H), 7.23 (s, 1H), 7.29 (d, J ) 8 Hz, 1H), 7.53 (t, J ) 8 Hz,

1H), 7.75 (d, J ) 8 Hz, 1H); MS m/e 307 (MH⁺, 100). Anal.

(C₁₉H₁₈N₂O₂‚HCl) C, H, N.

4-Hyd r oxy-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[3,4-

c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r obr om id e (22t). The prod-

uct was obtained from 20s following the general procedure for

debenzylation and then recrystallized from MeOH/H₂O. Yield

90%; mp >300 °C; ¹H NMR (DMSO-d₆/D₂O) δ 3.11 (t, J ) 7.5

Hz, 2H), 3.24 (t, J ) 7.5 Hz, 2H), 3.50 (t, J ) 6 Hz, 2H), 3.65

(t, J ) 6 Hz, 2H), 4.47 (s, 2H), (d, J ) 8 Hz, 1H), 7.23 (s, 1H),

7.41 (t, J ) 8 Hz, 1H), 7.56 (d, J ) 8 Hz, 1H); MS m/e 293

(MH⁺, 100). Anal. (C₁₈H₁₆N₂O₂‚HBr) C, H, N.

2-Meth oxy-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[4,3-

c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (23b). Recrys-

tallized from MeOH/H₂O as colorless needles (91% yield before

1

recrystallization); mp 288-295 °C; H NMR (DMSO-d₆/D₂O)

δ 2.98 (t, J ) 7.4 Hz, 2H), 3.18 (t, J ) 5.9 Hz, 2H), 3.23 (t, J

) 7.4 Hz, 2H), 3.49 (t, J ) 5.9 Hz, 2H), 3.90 (s, 3H), 4.75 (s,

2H), 7.19 (dd, J ) 2.4 and 8.9 Hz, 1H), 7.26 (s, 1H), 7.35 (d, J

) 2.4 Hz, 1H), 8.31 (d, J ) 8.9 Hz, 1H), 8.40 (t, J ) 8 Hz, 1H);

MS m/e 307 (MH⁺, 100). Anal. (C₁₉H₁₈N₂O₂‚HCl) C, H, N.

2-Hyd r oxy-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[4,3-

c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (23c). Recrys-

tallized from MeOH/H₂O as colorless needles (83% yield before

recrystallization); mp >300 °C; ¹H NMR (DMSO-d₆/D₂O) δ 2.96

(t, J ) 7.5 Hz, 2H), 3.19 (t, J ) 7.5 Hz, 2H), 3.21 (t, J ) 6.0

2,3-Dim eth oxy-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o-

[3,4-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (22u ). Re-

crystallized from MeOH as colorless needles; yield 60%; mp

1

288-290 °C; H NMR (DMSO-d₆/D₂O) δ 3.00 (t, J ) 7.4 Hz,

2H), 3.23 (t, J ) 7.4 Hz, 2H), 3.49-3.55 (m, 4H), 3.88 (s, 3H),

Substituted Pentacyclic Carbazolones

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1273

Hz, 2H), 3.52 (t, J ) 6.0 Hz, 2H), 4.69 (s, 2H), 7.00 (dd, J )

1.9 and 8.8 Hz, 1H), 7.21 (d, J ) 1.9 Hz, 1H), 7.24 (s, 1H),

8.16 (d, J ) 8.8 Hz, 1H); MS m/e 293 (MH⁺, 100). Anal.

(C₁₈H₁₆N₂O₂‚HCl‚H₂O) C, H, N.

Meth yl 7,8,10,11,12,13-Hexa h yd r o-6H-d ip yr id o[4,3-c:

1′,2′,3′-lm ]ca r ba zol-6-on e-2-ca r boxyla te Hyd r och lor id e

(23d ). Recrystallized from MeOH as colorless needles; yield

83%; mp >300 °C; ¹H NMR (DMSO-d₆/D₂O) δ 3.06 (t, J ) 7.3

Hz, 2H), 3.18 (t, J ) 6.0 Hz, 2H), 3.28 (t, J ) 7.3 Hz, 2H), 3.48

(t, J ) 6.0 Hz, 2H), 3.94 (s, 3H), 4.84 (s, 2H), 7.33 (s, 1H), 8.20

(dd, J ) 1.5 and 8.6 Hz, 1H), 8.47 (d, J ) 1.5 Hz, 1H), 8.52 (d,

J ) 8.6 Hz, 1H); MS m/e 335 (MH⁺, 100). Anal. (C₂₀H₁₈N₂O₃‚

HCl‚0.2H₂O) C, H, N.

overnight in scintillation fluid (ReadySafe, Beckman), and

counted for radioactivity in Beckman LS6000 scintillation

counters. Membrane protein concentrations (5-50 µg/mL)

were adjusted so that not more than about 15% of added

radioligand was bound. Nonspecific binding was measured in

the presence of 10^-6M QNB (an antagonist with picomolar

potency) and accounted for 1-5% of total binding. Test

compounds were dissolved in DMSO that, at the highest final

concentration of 1%, had no effect on binding.

The equilibrium assays utilized an incubation time of at

least 2 h in the presence of GTP (2 × 10^-4M) and ³H-NMS

concentrations of 0.1-0.7 nM, depending on receptor subtype.

In most assays binding of ³H-NMS in the absence and

presence of an IC₅₀concentration of ACh was measured alone

and in the presence of three or more concentrations of test

agent. Selected compounds were studied further with an

equilibrium assay that measured inhibition curves with ACh

alone and in the presence of three concentrations of test agent

and/or with a nonequilibrium assay in which the receptors in

3-Meth oxy-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[4,3-

c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (23e). Recrys-

tallized from MeOH as colorless needles; yield 65%; mp >300

1

°C; H NMR (DMSO-d₆/D₂O) δ 3.02 (t, J ) 7.4 Hz, 2H), 3.14

(t, J ) 5.8 Hz, 2H), 3.24 (t, J ) 7.4 Hz, 2H), 3.45 (t, J ) 5.8

Hz, 2H), 3.89 (s, 3H), 4.72 (s, 2H), 7.11 (dd, J ) 2.3 and 8.6

Hz, 1H), 7.18 (s, 1H), 7.88 (d, J ) 8.6 Hz, 1H), 7.99 (d, J ) 2.3

Hz, 1H); MS m/e 307 (MH⁺, 100); HRMS (C₁₉H₁₉N₂O₂) found

m/z 307.1470, calcd 307.1460.

3

half the tubes were equilibrated with H-NMS before addition

of ACh and test agent, while the receptors in the remaining

tubes were exposed to ACh and test agent before exposure to

³H-NMS. In all cases the data were fitted to the allosteric

model using nonlinear regression analysis (SigmaPlot, SPSS,

Erkrath, Germany) to provide quantitative estimates of the

affinity of the agent at the unliganded receptor and its

cooperativity with ³H-NMS and ACh. When the analysis

3-Hyd r oxy-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o[4,3-

c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (23f). The prod-

uct was obtained from 21f following the general procedure for

debenzylation and then recrystallized from MeOH/H₂O as

1

colorless needles; yield 75%; mp >300 °C; H NMR (DMSO-

3

required that kinetic effects on H-NMS binding be taken into

d₆/D₂O) δ 3.00 (t, J ) 7.5 Hz, 2H), 3.13 (t, J ) 5.7 Hz, 2H),

3.22 (t, J ) 7.5 Hz, 2H), 3.45 (t, J ) 5.7 Hz, 2H), 4.69 (s, 2H),

6.95 (dd, J ) 2.3 and 8.6 Hz, 1H), 7.14 (s, 1H), 7.78 (d, J ) 8.6

Hz, 1H), 7.90 (d, J ) 2.3 Hz, 1H); MS m/e 293 (MH⁺, 100).

Anal. (C₁₈H₁₆N₂O₂‚HBr‚0.2H₂O) C, H, N.

account, values of 0.075, 0.361, 0.064, and 0.066 min^-1were

used for the dissociation rate constants of ³H-NMS at M₁, M₂,

M₃, and M₄receptors, respectively.²⁸

In the off-rate assay, a high concentration of membranes

(2-4 mg protein/mL) was incubated with a high concentration

of ³H-NMS (5 nM) for about 15 min. Then 10 µL aliquots were

distributed into tubes that were empty or contained 1 mL of

10^-6M QNB alone and in the presence of at least four

concentrations of allosteric agent. Nonspecific binding was

measured in separately prepared tubes containing 10 µL of

membrane and 2 mL of ³H-NMS + QNB. Sometime later,

about 2.5 dissociation half-lives, the samples were filtered. The

data were transformed to dissociation rate constants, ex-

2,3-Dim eth oxy-7,8,10,11,12,13-h exa h yd r o-6H-d ip yr id o-

[4,3-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (23g). Re-

crystallized from MeOH as colorless needles; yield 29%; mp

1

285-287 °C; H NMR (DMSO-d₆/D₂O) δ 3.00 (t, J ) 7.4 Hz,

2H), 3.15 (t, J ) 5.8 Hz, 2H), 3.24 (t, J ) 7.4 Hz, 2H), 3.47 (t,

J ) 5.8 Hz, 2H), 3.86 (s, 3H), 3.93 (s, 3H), 4.80 (s, 2H), 7.16 (s,

1H), 7.40 (s, 1H), 8.02 (s, 1H); MS m/e 337 (MH⁺, 100). Anal.

(C₂₀H₂₀N₂O₃‚HCl‚H₂O) C, H, N.

8,8-Dim et h yl-7,8,10,11,12,13-h exa h yd r o-6H -d ip yr id o-

[4,3-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lor id e (23h ). Re-

crystallized from MeOH as colorless needles; yield 89%; mp

3

pressed as the percentage inhibition of the “true” H-NMS

dissociation rate constant measured in the absence of allosteric

agent, and fitted to a logistic function using nonlinear regres-

sion analysis. This curve corresponds theoretically to the

occupancy curve of the allosteric agent at the ³H-NMS-

occupied site, and the regression analysis provides a quantita-

tive estimate of the affinity of the agent for the ³H-NMS-

occupied receptor.

1

292-296 °C; H NMR (DMSO-d₆/D₂O) δ 1.41 (s, 6H), 2.96 (s,

2H), 3.20 (t, J ) 5.9 Hz, 2H), 3.50 (t, J ) 5.9 Hz, 2H), 4.81 (s,

2H), 7.39 (s, 1H), 7.52 (t, J ) 8 Hz, 1H), 7.63 (t, J ) 8 Hz, 1H),

8.01 (d, J ) 8 Hz, 1H), 8.44 (d, J ) 8 Hz, 1H); MS m/e 305

(MH⁺, 100). Anal. (C₂₀H₂₀N₂O‚HCl‚0.5H₂O) C, H, N.

8,8-Dim eth yl-2,3-d im eth oxy-7,8,10,11,12,13-h exa h yd r o-

6H-d ip yr id o[4,3-c:1′,2′,3′-lm ]ca r ba zol-6-on e Hyd r och lo-

r id e (23i). Recrystallized from MeOH as colorless needles;

Ack n ow led gm en t. This work was supported by

Sankyo Co. Ltd., Tokyo, J apan, and by the Medical

Research Council. We also gratefully acknowledge the

ULIRS mass spectrometry facility at the School of

Pharmacy for providing the MS data and the microana-

lytical section of the Department of Chemistry, Univer-

sity College London for elemental analysis.

1

yield 77%; mp 285-288 °C; H NMR (DMSO-d₆/D₂O) δ 1.39

(s, 6H), 2.93 (s, 2H), 3.18 (t, J ) 5.7 Hz, 2H), 3.48 (t, J ) 5.7

Hz, 2H), 3.89 (s, 3H), 3.93 (s, 3H), 4.82 (s, 2H), 7.28 (s, 1H),

7.42 (s, 1H), 8.02 (s, 1H); MS m/e 365 (MH⁺, 100). Anal.

(C₂₂H₂₄N₂O₃‚HCl‚0.2H₂O) C, H, N.

Ra d ioliga n d Bin d in g. CHO cells stably expressing cDNAs

encoding human muscarinic M₁-M₄receptors were grown to

confluence in R-MEM medium (GIBCO) containing 10% (v/v)

newborn calf serum, 50 U/mL penicillin, 50 mg/mL strepto-

mycin, and 2 mM glutamine at 37 °C under 5% CO₂and

harvested by scraping in a hypotonic medium (20 mM Hepes

+ 10 mM EDTA, pH 7.4). Membranes were prepared at 0 °C

by homogenization with a Polytron followed by centrifugation

(40 000g, 15 min), were washed once in 20 mM Hepes + 0.1

mM EDTA, pH 7.4, and were stored at -70 °C in the same

buffer at protein concentrations of 2-5 mg/mL.

The binding assays and analyses have been described

previously.^28,36,37In general, binding of ³H-NMS was con-

ducted in a buffer containing 20 mM Hepes + 100 mM NaCl

+ 10 mM MgCl₂(pH 7.4) at 30 °C in a volume of 1 mL.

Membranes were collected by filtration over glass fiber filters

(Whatman GF/B) presoaked in 0.1% polyethylenimine, using

a Brandel cell harvester (Semat, Herts, U.K.), extracted

Su p p or t in g In for m a t ion Ava ila b le: Additional infor-

mation on the methodology for performing and analyzing the

equilibrium and nonequilibrium binding assays. This

material is available free of charge via the Internet at http://

pubs.acs.org.

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J M010946Z

Article Doi

Substituted pentacyclic carbazolones as novel muscarinic allosteric agents: Synthesis and structure - Affinity and cooperativity relationships

DOI: 10.1021/jm010946z

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Full text of DOI:10.1021/jm010946z

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