Optimization of alkylidene hydrazide based human glucagon receptor antagonists. Discovery of the highly potent and orally available 3-cyano-4-hydroxybenzoic acid [1-(2,3,5,6-tetramethylbenzyl)-1h-indol-4ylmethylene]hydrazide

J . Med. Chem. 2002, 45, 5755-5775

5755

Op tim iza tion of Alk ylid en e Hyd r a zid e Ba sed Hu m a n Glu ca gon Recep tor

An ta gon ists. Discover y of th e High ly P oten t a n d Or a lly Ava ila ble

3-Cya n o-4-h yd r oxyben zoic Acid [1-(2,3,5,6-Tetr a m eth ylben zyl)-1H-in d ol-4-

ylm eth ylen e]h yd r a zid e

Peter Madsen,*^,†Anthony Ling,^‡Michael Plewe,^‡Christian K. Sams,^†Lotte B. Knudsen,^§Ulla G. Sidelmann,^|

Lars Ynddal,^|Christian L. Brand, Birgitte Andersen,^#Douglas Murphy,^‡Min Teng,^‡Larry Truesdale,^[

Dan Kiel,⁺J ohn May,⁺Atsuo Kuki,^×Shenghua Shi,^×Michael D. J ohnson,^‡Kimberly Ann Teston,^‡J un Feng,^‡

J ames Lakis,⁺Kenna Anderes,⁺Vlad Gregor,^‡and J esper Lau^†

Departments of Medicinal Chemistry, Molecular Pharmacology, Exploratory ADME, Pharmacological Research, and

Biochemistry and Metabolism, Novo Nordisk A/ S, Novo Nordisk Park, DK-2760 Måløv, Denmark, and Departments of

Medicinal Chemistry, Computational Chemistry, Combinatorial Chemistry Technology, and Pharmacology,

Pfizer Global Research and Development, 10770 Science Center Drive, San Diego, California 92121

Received February 25, 2002

Highly potent human glucagon receptor (hGluR) antagonists have been prepared employing

both medicinal chemistry and targeted libraries based on modification of the core (proximal)

dimethoxyphenyl group, the benzyl ether linkage, as well as the (distal) benzylic aryl group of

the lead 2, 3-cyano-4-hydroxybenzoic acid (3,5-dimethoxy-4-isopropylbenzyloxybenzylidene)-

hydrazide. Electron-rich proximal aryl moieties such as mono- and dimethoxy benzenes,

naphthalenes, and indoles were found to be active. The SAR was found to be quite insensitive

regarding the linkage to the distal aryl group, since long and short as well as polar and apolar

linkers gave highly potent compounds. The presence of a distal aryl group was not crucial for

obtaining high binding affinity to the hGluR. In many cases, however, the affinity could be

further optimized with substituted distal aryl groups. Representative compounds have been

tested for in vitro metabolism, and structure-metabolism relationships are described. These

efforts lead to the discovery of 74, NNC 25-2504, 3-cyano-4-hydroxybenzoic acid [1-(2,3,5,6-

tetramethylbenzyl)-1H-indol-4-ylmethylene]hydrazide, with low in vitro metabolic turnover.

74 was a highly potent noncompetitive antagonist of the human glucagon receptor (IC₅₀) 2.3

nM, K_B) 760 pM) and of the isolated rat receptor (IC₅₀) 430 pM, K_B) 380 pM). Glucagon-

stimulated glucose production from isolated primary rat hepatocytes was inhibited competitively

by 74 (K_i) 14 nM). This compound was orally available in dogs (F_po) 15%) and was active in

a glucagon-challenged rat model of hyperglucagonemia and hyperglycemia.

In tr od u ction

rats, in normal and alloxan-induced diabetic rabbits,

and in ob/ob-mice.^8-11Thus, the glucagon receptor is a

potential target for a new drug class for treatment of

type 2 diabetes. Concerns have been raised that gluca-

gon antagonism poses a risk of hypoglycemia. However,

hypoglycemia was never observed in any of the immu-

noneutralization studies.^8-11Furthermore, a patient

with hyperglucagonemia has been described¹²to have

a nonfunctional glucagon receptor and yet has no

symptoms of either the glucagonoma syndrome or

hypoglycemia. Very recently, glucagon receptor gene

knock-out mice have been published¹³and their phe-

notype was strikingly similar to that of this patient.¹²

Glucagon, a 29 amino acid hormone, secreted from

the R-cells of the pancreas, stimulates hepatic glucose

output via increasing glycogenolysis and gluconeogen-

esis. The human glucagon receptor was cloned in 1993,¹

and the use of the receptor has been patented.²

A

glucagon receptor antagonist is suggested to decrease

the hepatic glucose output and thus lower hyperglyce-

mia in type 2 diabetic patients.^3-7Previous studies have

shown, in both acute and chronic models, that in vivo

immunoneutralization of glucagon does lead to lowered

blood glucose levels in normal and STZ-induced diabetic

* To whom correspondence should be addressed. E-mail: pem@

novonordisk.com. Phone: +45 44 43 48 94. Fax: +45 44 66 34 50.

^†Department of Medicinal Chemistry, Novo Nordisk A/S.

^‡Department of Medicinal Chemistry, Pfizer Global Research and

Development.

A few classes of non-peptide glucagon receptor an-

tagonists have recently been published, primarily by

scientists from Merck, Bayer, and Novo Nordisk.^14-17

Previously we have published¹⁸the discovery of a new

class of human glucagon receptor (hGluR) antagonists,

alkylidene hydrazides (1), and the subsequent optimiza-

tion leading to 2¹⁹(see Figure 1). In the present

publication, further optimization of these two com-

pounds are reported leading to series of high-affinity

hGluR antagonists. Among these series, one promising

compound that is orally available in dogs has been

^§Department of Molecular Pharmacology, Novo Nordisk A/S.

^|Department of Exploratory ADME, Novo Nordisk A/S.

Department of Pharmacological Research, Novo Nordisk A/S.

#

Department of Biochemistry and Metabolism, Novo Nordisk A/S.

^[Department of Combinatorial Chemistry Technology, Pfizer Global

Research and Development.

⁺Department of Pharmacology, Pfizer Global Research and Devel-

opment.

^×Department of Computational Chemistry, Pfizer Global Research

and Development.

Published on Web 11/20/2002

5756 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26

Madsen et al.

Sch em e 1. Preparation Method A

F igu r e 1. First examples of alkylidene hydrazide hGluR

antagonists.

The objective for the studies presented here was

consequently to optimize the pharmacological/pharma-

cokinetic profile of this alkylidene hydrazide class of

glucagon receptor antagonists. It was speculated that

the inadequate pharmacokinetic profiles primarily were

due to the physicochemical properties of the compounds,

their high lipophilic character, and low aqueous solu-

bilities. The strategy adopted was consequently to

improve on these two parameters through incorporation

of more polar motifs in the molecule, guided by in vitro

metabolism screening in rat liver microsomes (i.e., to

generate structure-metabolism relationships (SMRs).

Thus, in parallel to assaying the compounds for receptor

binding, the compounds were assayed for in vitro

metabolism in rat liver microsomes or primary rat

hepatocytes, and for selected compounds the metabolic

profile (i.e., the structures of metabolites formed in vitro)

was determined. These data provided structural feed-

back for the design of new types of compounds and

targeted libraries.

Primarily high-throughput synthesis of targeted li-

braries and also traditional medicinal synthesis of single

compounds were employed to obtain ligands with the

desired properties. All data presented herein are based

on either purified library hits or resynthesized pure

compounds. Consequential to the utilization of combi-

natorial chemistry, the inactive compounds were neither

analyzed nor resynthesized and the SAR was primarily

developed around the positively identified hGluR an-

tagonists.

F igu r e 2. Definition of structural components of alkylidene

hydrazide hGluR antagonists.

tested further and was found to be active in vivo in a

glucagon-challenged rat model of hyperglucagonemia

and hyperglycemia.

During the discovery¹⁸and optimization¹⁹of 1, the

most important SAR and hypothesis of receptor binding

interactions became apparent (see Figure 2). An acidic

phenol group in the benzoic acid part of the molecule

was found to be an important pharmacophore, and

electronegative ortho substituents (such as chloro, nitro,

and cyano) gave access to very potent compounds. All

attempts to replace the alkylidene hydrazide moiety

resulted in compounds with low affinity, and it was

found that the electron-rich proximal aryl moieties (such

as 3,5-dimethoxyphenyl, indolyl, and naphthyl) gave

compounds with high affinity to the hGluR.

The SAR of the series of alkylidene hydrazides was

investigated further, and here, we describe the influence

of combinations of various substituted distal aromatic

groups through several linkages with the proximal aryl

groups for binding to the hGluR. The improvement of

the ADME properties was performed in parallel to the

optimization of the binding affinity to the hGluR.

Compounds 1 and 2 were found to possess inadequate

pharmacokinetic properties in rats characterized by very

short in vivo half-lives and no oral bioavailabilities. In

vitro investigations in rat liver microsomes suggested

that the inadequate PK properties primarily were due

to a high metabolic turnover, oxidative metabolism in

the distal aromatic ring (phase I metabolism), and

glucuronidation of the phenol moiety (phase II metabo-

lism).

Ch em istr y

The hydroxymethyl analogues (3-6 and 8) and the

naphthalene 1,5-regioisomer 7 were prepared through

condensation of the corresponding hydrazides (I-IV)

with the naphthaldehydes (V-VII) as shown in Scheme

1 (preparation method A).

Compounds that were further derivatized through the

hydroxymethyl group were prepared as shown in Scheme

2, preparation method B. Benzylation of the hydroxy-

Optimization of Glucagon Receptor Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5757

Sch em e 2. Preparation Method B

Sch em e 3. Preparation Method C

methyl naphthaldehydes V and VI with benzyl halides

followed by condensation with I afforded 9 and 10. The

amines (12-15) were made from the bromomethyl-

naphthalene (VIII) using amines as nucleophiles fol-

lowed by condensation with I. The sulfones (11 and 16-

19) were similarly made using the corresponding thiols

as nucleophiles, and the resulting sulfides were oxidized

to sulfones prior to hydrazone formation.

Compounds with a 2-aminoethoxy linker between the

proximal and the distal aromatic rings were prepared

either using a solution-phase route as shown in Scheme

3 (preparation method C) or using a solid-phase ap-

proach as shown in Scheme 4 (preparation method D).

In the solution-phase route, the 4-hydroxynaphthalene-

1-carbaldehyde was O-alkylated with 1,2-dichloroethane

and the 2-chloroethoxynaphthaldehyde was then con-

densed with the hydrazide (I). Subsequent nucleophilic

displacement of the chloro group with amines furnished

the desired products (20 and 21). In the solid-phase

method, preparation method D, resin-bound hydrazide

(I) was prepared by attachment of 3-chloro-4-hydroxy-

benzoic acid methyl ester to a PS-Wang resin via

nucleophilic substitution of PS-Wang mesylate. The

resulting resin-bound ester was subsequently hydro-

lyzed using potassium trimethylsilanolate followed by

acetic acid. Activation of the resin-bound carboxylic acid

using PyBoP followed by reaction with hydrazine af-

forded the resin-bound hydrazide (I). Methoxy-substi-

5758 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26

Sch em e 4. Preparation Method D

Madsen et al.

Sch em e 5. Preparation Method E

tuted 4-(2-bromoethoxy)benzaldehydes or 4-(2-bromo-

ethoxy)-1-naphthaldehydes were then condensed with

the resin-bound hydrazide (I), and subsequent nucleo-

philic substitution with amines gave, after TFA cleav-

age, the desired compounds. This procedure was used

in a library approach on a 50 mg scale (of resin-bound

hydrazide I) for preparation of a targeted library (eight

different 2-bromoethoxy-substituted aldehydes × 48

amines, 384 compounds) as well as for scale-up of

representative hits (23 and 24, using 3 g of resin-bound

hydrazide I). The solid-phase method was found to be

better than the solution-phase method because in solu-

tion products coming from overalkylation of the em-

ployed amines with the 2-chloroethoxy-substituted hy-

drazide were difficult to remove by chromatography.

This problem was not encountered on solid support (i.e.,

no cross-linking was observed).

Large libraries of amides were prepared as shown in

Scheme 5 (preparation method E). Compounds 27-51

also including heteroaromatic proximal rings such as

indoles were made. Targeted libraries were made by

activation of the carboxylic acids (such as IX-XI and

the corresponding indole, XII, eight carboxylic acids in

total) by carbonyldiimidazole followed by reaction with

a large set of amines. In all, >2300 amides were made

and >50 hits were found when screened at 100 nM

Optimization of Glucagon Receptor Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5759

Sch em e 6. Preparation Method F

Sch em e 7. Preparation Method G

concentration. Scale-up of selected compounds was done

according to Scheme 5. Here, the naphthaldehydes,

appended with a carboxylic acid functionality, were

converted into the corresponding amides prior to hy-

drazone formation.

Sch em e 8. Preparation Method H

Compounds where the amide was reversed compared

to the amides described in Scheme 5 were prepared as

described in Scheme 6 (preparation method F) by

reacting 4-amino-1-naphthaldehyde (XIII) (or indole

carboxaldehydes, not shown in scheme) with carboxylic

acid chlorides. The resulting amides were in turn

condensed with either I or IV to afford the correspond-

ing amides (52-57).

Another series of amides with a methoxy-substituted

phenyl ring as the proximal aryl group (58-66) was

made in library format on solid support and as single

compounds in solution. In Scheme 7 (preparation method

G), the solid-phase synthesis is described. The resin-

bound hydrazide I was condensed with methoxy-

substituted Fmoc-protected 4-aminobenzaldehydes. Af-

ter deprotection of the amine, coupling of carboxylic

acids to afford the desired amides was done using

symmetric anhydrides. The targeted libraries were

prepared from 50 mg of resin-bound hydrazide I. In all,

eight different Fmoc-protected aminobenzaldehydes

were used and >250 compounds were made. The Fmoc-

protected 4-aminobenzaldehydes were prepared from

the corresponding 4-aminobenzoic acid esters via Fmoc

protection, DIBAL reduction, and MnO₂oxidation.

Resynthesis of hits (scale-up) was done either on solid

support by employing more resin or in solution as shown

in Scheme 8 using preparation method H.

Here, coupling of the desired carboxylic acid (using

the symmetric anhydride) with methoxy-substituted

4-aminobenzoic acid methyl esters followed by DIBAL

reduction of the ester and MnO₂oxidation furnished the

appropriately substituted benzaldehydes. These were

condensed with the 3-chloro- or 3-cyano-4-hydroxyben-

zoic acid hydrazides (I and IV, respectively) to afford

the desired compounds.

Sch em e 9. Preparation Method I

Finally, a library of benzylated indoles (73-77) was

prepared as described in Scheme 9 (preparation method

I). Various substituted indolecarbaldehydes were depro-

tonated with NaH in DMF, and the corresponding anion

was benzylated using benzyl chlorides or bromides. The

resulting aldehydes were then condensed with hy-

drazide I or IV essentially as described earlier for

phenol alkylation.¹⁹

Biologica l Meth od s

The biological methods used are described in detail

in the Experimental Section.

5760 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26

Madsen et al.

Ta ble 1. Structures, Binding Affinities, Rates of in Vitro

Metabolism, and Physical Properties of Synthesized

Compounds

Recep tor Assa ys. Receptor assays were carried out

using plasma membranes from BHK cells expressing

the cloned human glucagon receptor. The functional

assays were carried out as earlier described,²⁰whereas

the binding assay was slightly modified.

P r im a r y Hep a tocyte Assa ys. As a model system

for hepatic glucose production, rat hepatocytes were

isolated and cultured as described earlier.²¹The hepa-

tocytes were loaded with glycogen, and the effect of the

glucagon antagonists on glucagon-induced glycogenoly-

sis was studied.

In Vitr o Dr u g Meta bolism . It had initially been

found that the present series of compounds exhibited

fast metabolic turnover with resulting poor in vivo PK

profiles; therefore, optimization with respect to binding

potency and DMPK properties was performed in paral-

lel. Metabolic rates were estimated from incubations

with rat liver microsomes in order to rank compounds

in terms of their metabolic stability in the liver. Analysis

was performed by means of LC-MS. Selected com-

pounds were profiled with respect to the major metabo-

lites formed in rat liver microsomes. In this manner,

potential weak spots (from a metabolic point of view)

in the molecules could be identified. Metabolic profiles

were analyzed by means of LC-MS-MS.

P h a r m a cok in etics. For pharmacokinetic experi-

ments, four purpose-bred male beagle dogs weighing

11-19 kg and that were 15-18 months of age were

used. Blood sampling was done at predetermined time

points from predose to 4 h postdose. The blood samples

were analyzed using LC-MS-MS.

Glu ca gon -Ch a llen ged Ra t. To demonstrate acute

in vivo efficacy, the effect of the compounds on glucagon-

stimulated hyperglycemia was studied in rats. Non-

fasted male Sprague-Dawley rats (200 g) were main-

tained in the anesthetized state during the test by

subcutaneous administration of a 1:1 mixture of hyp-

norm (fentanyl, 0.05 mg/mL, and fluanizone, 2.5 mg/

mL, J anssen Pharma Ltd., Copenhagen, Denmark) and

dormicum (midazolam, 1.25 mg/mL, Roche, Basel, Swit-

zerland). A catheter was inserted in a jugular vein for

administration of compounds. Approximately 60 min

after initiation of anesthesia, test compounds (0, 3, and

10 mg/kg) and glucagon (3 µg/kg) were each adminis-

tered in a 5 min interval. Samples for determination of

blood glucose concentrations were taken from the tail

tip 25 and 5 min prior to administration of the com-

pound to represent average basal values and again 10

min after administration of glucagon (time for peak

response to glucagon). The results were expressed as

the change (∆) in values, which is the value obtained

10 min after glucagon administration minus the average

of the two basal values. This method was adapted from

the method previously published.⁸

a

Binding affinity of compounds bound to the recombinant

human glucagon receptor in BHK cells. Data are expressed as the

mean IC₅₀( SD, n ) 3. N: calcd, 7.90; found, 7.22.

b

increased binding affinity, 41 nM for 3 as shown in

Table 1. The substitution pattern of the naphthalene

moiety had influence on the affinity because the 1,5-

substituted naphthalene (7) was less potent than the

1,4-analogues. Replacing the 3-chloro substituent of 3

with other electron-withdrawing groups such as fluorine

(4) and cyano (6) did not alter the binding affinity

significantly, but an impact on in vitro metabolism was

observed. The microsomal turnover of 6 with a 3-cyano

group was reduced compared to 3 with a 3-chloro

substituent, 37 vs 75 pmol min^-1mg^-1protein, respec-

tively. This trend was quite general for this and other

subclasses described in the present publication and was

the result of a diminished rate of glucuronidation of the

phenolic group when the chloro substituent was re-

placed with a cyano group. Interestingly, it was possible

to diminish the in vitro metabolic turnover in parallel

to optimization of the hGluR affinity because by a

change in the substitution pattern in the naphthalene

moiety, e.g., the less potent 1,5 analogue, 7 also had the

fastest metabolic turnover within this series, 225 pmol

min^-1mg^-1protein, whereas the 1,4-analogue 6 was

much more potent and had lower metabolic turnover

compared to 7.

The encouraging results obtained for the hydroxy-

methylnaphthalenes prompted further exploration and

optimization of this region by alkylation of the hy-

droxymethyl group. The hydrogen bond donor property

of the hydroxymethyl group of 3 was not essential for

hGluR binding because the corresponding methoxy

analogue 8 was only about 2-fold less potent, as can be

seen in Table 2. As experienced in the former naphthol

series,¹⁸it was found that extension of the hydroxy-

methylnaphthalenes with lipophilic moieties (O-benzyl-

ation, 9 and 10) resulted in improvement of the binding

affinity of the compounds. Interestingly, the extension

with lipophilic moieties improved the potency for both

the 1,4- and the 1,5-substituted naphthalenes, and the

effect on binding of this extra lipophilic moiety was more

pronounced for the 1,5-arrangement (10) than for to 1,4-

arrangement (9).

Discu ssion

The original lead compound 1 contained both a phenol

and a naphthol functionality, and it was speculated that

these groups were causing the fast metabolic turnover

due to glucoronidation. The phenolic group was earlier

shown to be important for binding.¹⁸The naphthol group

was, however, not the optimal moiety because the

homologous hydroxymethylnaphthalene resulted in an

The importance of the ether linkage between the

proximal naphthyl group and the distal aryl group was

also investigated, and it was found that other functional

Optimization of Glucagon Receptor Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5761

Ta ble 2. Structures, Binding Affinities, Rates of in Vitro Metabolism, and Physical Properties of Synthesized Compounds

a

Binding affinity of compounds bound to the recombinant human glucagon receptor in BHK cells. Data are expressed as the mean

IC₅₀( SD, n ) 3. ND: not determined. N: calcd, 9.99; found, 9.32. ^cN: calcd, 5.82; found, 5.38.

b

groups in the linker could give access to very potent

compounds. Thus, a strong hydrogen bond acceptor (a

sulfone, 11) and a positively charged group (a secondary

amine, 12) in this linker were both well tolerated,

indicating that these linkers did not form direct contacts

with the receptor. It was also concluded that compounds

that were lacking the distal aryl group had much less

affinity to hGluR as exemplified with compounds 16-

19. On the basis of these results, it was hypothesized

that the role of the linker was to orientate the distal

lipophilic moiety in a correct position relative to the

proximal naphthyl group, thereby adding extra lipophilc

receptor interactions. This hypothesis was further con-

firmed by the preparation of two chiral compounds 14

and 15 with (R)- and (S)-phenylalanine carboxamide as

distal moieties, respectively. The (R) and (S) stereo-

chemistry of these two compounds controls the orienta-

tion of the distal liphophilic benzylic moiety, which

resulted in significant impact on the binding affinities,

since the (R)-isomer (14) was almost 10-fold more potent

than the (S)-isomer (15). It is noteworthy that these

highly polar and charged linkers without any loss of

affinity could replace the lipophilic benzyl ether linkages

in 2 and in 9.

(51 pmol min^-1mg^-1protein). It was found that this

was due to the fact that the piperazine linker was more

stable toward oxidative N-dealkylation than the second-

ary amine in 12. As in the series of substituted hy-

droxymethylnaphthalenes (Table 1), in this series of

compounds it was also possible to optimize the receptor

affinity in parallel with the in vitro metabolic stability.

The best compound (11) had exceptional low metabolic

turnover (14 pmol min^-1mg^-1protein) and was also a

high-affinity ligand (6.8 nM). Unfortunately, further in

vivo studies with this compound showed that the PK

profile was still inappropriate with a fast in vivo

clearance, which was concluded not to be driven by

biotransformations.

On the basis of these results, it was decided to

continue with the strategy of improving binding affinity

and the rate of in vitro metabolism in parallel by further

modifications of the substitution pattern of the distal

aryl group and to combine these with other types of

linkers to the proximal aryl moiety. Again, it was

hypothesized that the inappropriate PK profile of the

compounds could be improved by an increased aqueous

solubility, and accordingly, it was decided to prepare a

series of compounds wherein a polar 2-aminoethoxy

linker (Table 3) was examined with respect to SAR and

SMR. The binding affinities of the substituted benzyl-

amines prepared showed a weak preference for para

substitution as seen by comparison of the affinities of

20 and 21. No tight distance constraints were observed,

since 20 and 22 were almost equipotent (28 and 20 nM,

respectively). For further improvement of the hydro-

The in vitro metabolic turnovers of some of the

compounds from this series were investigated. Most of

the compounds tested had a very fast metabolic turn-

over, and 12 with a basic secondary amine as linker was

found to be metabolically very labile with a turnover of

304 pmol min^-1mg^-1protein. Interestingly, the other

basic analogue 13 had a much lower metabolic turnover

5762 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26

Madsen et al.

Ta ble 3. Structures, Binding Affinities, Rates of in Vitro Metabolism, and Physical Properties of Synthesized Compounds

a

Binding affinity of compounds bound to the recombinant human glucagon receptor in BHK cells. Data are expressed as the mean

IC₅₀( SD, n ) 3. ND: not determined. H: calcd, 4.53; found 3.97. ^cN: calcd, 7.78; found 8.35. H: calcd. 4.75; found. 5.17. N: calcd,

7.08; found, 7.77.

b

d

philicity of the compounds, the proximal lipophilic

naphthyl moiety was replaced with monocyclic aryl

groups. Replacing the proximal naphthalene moiety

with substituted 1,4-phenylene systems, such as 2-meth-

oxyphenylene (24 and 25) and 3-chloro-5-methoxyphen-

ylene (26) resulted, however, in less potent compounds.

The SMR of this series of compounds was also investi-

gated. The general trend of this subclass of compounds

was that the in vitro metabolic turnover was very high.

This was primarily due to phase I metabolism, oxidative

N-dealkylations in the linker, or in the case of 24 and

25, oxidations leading to dihydroisoquinolines and iso-

quinolinium salts.

The conclusion from these results confirmed the

hypothesis of the large tolerance of the receptor toward

the linker. Also, the previous finding¹⁹that the proximal

aromatic ring needs to be electron-rich was substanti-

ated. However, compounds with highly polar linkers,

such as the charged amines (e.g., 12 and 23) were

generally rapidly metabolized (due to oxidative N-

dealkylations) and were thus not considered to be worth

pursuing further.

For a functionalized linkage that could be metaboli-

cally more stable, it was decided to follow up on the

previous finding¹⁹that a compound with a diethyl amide

in the linker region was quite active. The amide group

is not charged at physiological pH and has a different

geometry and polarity compared to amines. It was

hypothesized that the metabolically driven N-dealky-

lation of the compounds containing an amine linkage

might be avoided using an amide linker and thereby

give access to potent compounds with reduced metabolic

turnover. Among the compounds prepared, it was found

that a distal aromatic ring was not needed in order to

obtain high-affinity compounds as seen, for example, for

the series of diethyl amides 27-30 (Table 4). The

importance of the proximal naphthalene ring was

emphasized by the low affinity of the corresponding

indole 31. The potency of 28 could be further optimized

to a single digit nanomolar range by a combination of

constraint of the flexibility of the alkyl groups in a

piperidine ring and by an increase of the lipophilic size

of the piperidine ring with alkyl groups (32-34). It was

also possible to obtain compounds with similar high

affinity by appending an aromatic residue to the piper-

idine ring (e.g., as phenylpiperidine (35-37) or as a

1,2,3,4-tetrahydroisoquinoline (38)). The affinities were,

however, not further increased compared to the affini-

ties of the methyl- and ethylpiperidine systems in 32-

34. Exchange of the neutral piperidine amide system

with a basic methylpiperazine moiety in 39 resulted in

a substantial loss in affinity. Increasing the distal

lipohilicity of the piperazine compounds with aromatic

groups such as benzyl groups (40 and 41) or with phenyl

groups (42 and 43) gave compounds with regained

binding affinity. The presence of a distal aromatic

residue was similarly important for potency in the much

more flexible ring-opened analogues as seen by com-

parison of, for example, the methyl analogue 44 (441

nM) with the corresponding benzyl analogue 45 (6.2

nM). The in vitro metabolic stability of this series of

amides was in general low, and the high in vitro

metabolic turnover was mainly due to oxidative N-

dealkylations as observed in the previous amine series.

In a few cases it was, however, possible to improve the

metabolic stability by a combination of substitutions

Optimization of Glucagon Receptor Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5763

Ta ble 4. Structures, Binding Affinities, Rates of in Vitro Metabolism, and Physical Properties of Synthesized Compounds

5764 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26

Ta ble 4 (Continued)

Madsen et al.

a

Binding affinity of compounds bound to the recombinant human glucagon receptor in BHK cells. Data are expressed as mean IC₅₀

(

SD, n ) 3. ND: not determined.

that gave compounds with diminished metabolic turn-

over without losing potency. Thus, the in vitro metabolic

turnover of 32 was quite high but was markedly reduced

by exchanging the 3-chloro substituent with a 3-cyano

(33) without loosing potency (267 vs 65 pmol min^-1mg^-1

protein). Further investigations showed that the re-

duced metabolic turnover of 33 was mainly due to

decreased glucuronidation of the phenol moiety. This

example demonstrates the importance of improving

potency and metabolic stability in parallel in order to

get access to druglike compounds.

Another series of amide compounds where the amide

linker was reversed were investigated (Table 5), since

it was hypothesized that reversing the amide function-

ality might improve the metabolic stability because

N-acylindoles and anilides cannot be oxidatively me-

tabolized by the same mechanism as the previous

amides. Also, in this series of reversed amides, high-

affinity ligands could be obtained without aromatic

distal groups. The aliphatic amides 54 and 57 had

moderate affinities (39 and 33 nM, respectively) and

increasing the lipophilicity and size of the distal moiety

by an extra N-acyl substituent improved the affinity to

low nanomolar levels for 55 and 56 (3.4 and 2.8 nM,

respectively).

had IC₅₀) 29 nM. Homologation to the 3-phenylpro-

pionanilides 60 and 62 and to the phenylthioacetanilide

63 gave similarly potent compounds (21, 34, and 15 nM,

respectively). Compounds included in the libraries with

methyl substituents on the proximal phenyl ring or with

a 1,3-branched substitution pattern did not result in hits

when screened in the binding assay. These results

indicated the preference for electron-rich 1,4-branched

proximal aromatic rings. Additionally, it was found that

for these reversed amides an indole could replace the

proximal naphthalene moiety to give high-affinity com-

pounds, 52 and 53 with IC₅₀) 6 and 19 nM, respec-

tively. Unfortunately, none of these amides had suitable

pharmacokinetic properties because of high metabolic

turnovers (oxidative metabolisms).

As mentioned above, an indole was a good naphtha-

lene replacement in the series of “reversed amides” (52)

and not in the amide series (31). This was intriguing

and prompted a reinvestigation of compounds with

indole as the proximal aromatic ring and lipophilic

linkers tethering the distal aromatic ring (N-benzylin-

doles, Table 6). It was found that substituted 1,4-

branched indoles gave potent compounds and that

compounds with multiple methyl substituents on the

distal phenyl group resulted in very potent compounds

(73, 5.3 nM; 72, 5.8 nM). It was also found that the

branching of the indole ring was very important because

the 1,4- was much more potent than the 1,5- and 1,3-

branched analogues (71, 75, and 76, respectively). The

importance of the substituents in the benzyl part of the

molecules regarding affinity was evidenced by the

reduced potency of the unsubstituted 77. As observed

in the other series of compounds in this publication, the

ortho chlorophenol moiety could be replaced by an ortho

During this optimization of the compounds, it was

decided to replace the lipophilic naphthalene core for

improvement of the aqueous solubility, thereby decreas-

ing the oxidative metabolic turnover. Previously, it was

found that this core must be very electron-rich. Conse-

quently, incorporation of monomethoxyaniline systems

as alternatives to the aminonaphthalene system was

investigated, and such compounds were found to possess

good binding properties; e.g., the phenylacetanilide 58

Optimization of Glucagon Receptor Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5765

Ta ble 5. Structures, Binding Affinities, Rates of in Vitro Metabolism, and Physical Properties of Synthesized Compounds

a

Binding affinity of compounds bound to the recombinant human glucagon receptor in BHK cells. Data are expressed as the mean

IC₅₀( SD, n ) 3. ND: not determined. N: calcd, 11.03; found, 10.09. ^cN: calcd, 10.34; found, 9.81.

b

cyanophenol moiety without a decrease in receptor

affinity, and by this minor structural change, the rate

of in vitro metabolism was significantly reduced (73 vs

74 (105 vs 34 pmol min^-1mg^-1protein) and 67 vs 68

(116 vs 59 pmol min^-1mg^-1protein)). The metabolic

oxidation of the methyl groups on the distal aromatic

ring; also observed was conjugation of the parent

compound, resulting in both the glucuronide and the

sulfate. N-Debenzylation of the indole ring was a minor

metabolic route. The high binding affinity and low rate

of in vitro metabolism of 74 prompted a more detailed

in vitro and in vivo evaluation of this compound.

In Vitr o P h a r m a cology. A functional in vitro char-

acterization of 74 was carried out (see Figures 4 and

profile obtained in rat hepatocytes of 74, with IC₅₀

)

2.3 ( 0.6 nM, the most potent and in vitro metabolically

stable compound from this indole series, was investi-

gated (see Figure 3). The major metabolic route was

5766 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26

Madsen et al.

Ta ble 6. Structures, Binding Affinities, Rates of in Vitro Metabolism, and Physical Properties of Synthesized Compounds

a

Binding affinity of compounds bound to the recombinant human glucagon receptor in BHK cells. Data are expressed as the mean

IC₅₀( SD, n ) 3. ND: not determined.

5). It was shown to be a mixed competitive and non-

competitive antagonist of the human receptor because

it right-shifted the glucagon dose-response curve and

also lowered the maximal response (Figure 4). K_Bwas

calculated²²for the individual experiments and found

to be 0.76 ( 0.18 nM. Similarly, by use of isolated rat

liver membranes, 74 was shown to be a highly potent,

truly noncompetitive antagonist of the rat receptor with

IC₅₀) 0.43 ( 0.1 nM and K_B) 0.38 ( 0.26 nM. The

maximal response in the glucagon dose-response assay

was lowered with only a slight right-shift (Figure 5).

This species difference may be explained by the fact that

there are more spare receptors in the BHK cells

expressing the human receptor than in the isolated rat

liver.

rat hepatocytes. 74 inhibited glucagon-induced (0.5 nM)

glycogenolysis (glycogen breakdown) dose-dependently

with an IC₅₀value of 160 ( 90 nM (data not shown).

Likewise, the glucose production (glycogenolysis) from

hepatocytes was inhibited with an IC₅₀value of 120 (

20 nM (data not shown). These two IC₅₀values are not

significantly different, and therefore, glucose production

was used as a measure of hepatic glycogenolysis in the

following study. To characterize the mode of inhibition

in rat hepatocytes, a functional K_iwas determined

(Figure 6). The mode of inhibition was found to be

competitive with a right-shift of the dose-response

curve and no effect on the maximal response. K_iwas

calculated from four individual experiments and found

to be 14.1 ( 3.9 nM. The higher K_ivalue in this

experiment compared to the functional data on the

isolated rat liver glucagon receptor is possibly due to

P r im a r y Hep a tocytes. The effect of 74 on glucagon-

induced glycogenolysis was studied in primary cultured

Optimization of Glucagon Receptor Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5767

F igu r e 3. Metabolic profile of 74 in rat hepatocytes analyzed by LC-MS-MS.

F igu r e 4. Inhibition of glucagon-stimulated cAMP by 74

using the recombinant human glucagon receptor in BHK cells.

F igu r e 5. Inhibition of glucagon-stimulated cAMP by 74

Shown is the observed shift in the glucagon dose-response

curve by addition of 74: glucagon (9), glucagon + 25 nM 74

(0), glucagon + 250 nM 74 (b), glucagon + 2500 nM 74 (O).

Shown are data from one representative experiment out of

three (mean ( SD of triplicate samples). Inserted is the K_B

plot, where the 2500 nM 74 curve was used. K_Bwas deter-

mined to be 0.76 ( 0.18 nM.

using the isolated rat liver glucagon receptor. Shown is the

observed shift in the glucagon dose-response curve by addition

of 74: glucagon (9), glucagon + 1 nM 74 (2), glucagon + 10

nM 74 (1), glucagon + 100 nM 74 ([). Shown are data from

one representative experiment out of three (mean ( SD of

triplicate samples). Inserted is the K_Bplot, where the 1 nM

74 curve was used. K_Bwas determined to be 0.38 ( 0.26 nM.

the presence of 0.1% human serum albumin in the

incubation buffer, since, as previously mentioned,²³this

compound is able to bind to albumin. A further differ-

ence from isolated membranes is that primary hepato-

cytes are metabolically active. The discrepancy in the

mode of inhibition, noncompetitive in isolated rat liver

membranes versus competitive in the primary rat

hepatocytes, is not obvious.

P h a r m a cok in etics. To assess the pharmacokinetic

properties of 74, the compound was dosed to purpose-

bred male dogs. The PK profiles (Figure 7A) after

intravenous administration of 0.5 mg/kg 74 gave uni-

form profiles with a harmonic mean elimination half-

life of 1.11 h. After per oral administration by gavage

(2.0 mg/kg) and use of the same dosing solution as used

for intravenous administration (Figure 7B), three pro-

files were obtained with very fast absorption (t_max) 30-

45 min) and one dog showed some delay with respect

to absorption (t_max) 75 min). The elimination half-life

after per oral administration was slightly longer than

after intravenous administration with a harmonic mean

half-life of 1.4 h. The bioavailability was 15%. The PK

analysis was performed using noncompartmental meth-

ods.

In Vivo P h a r m a cology. Results from the in vivo test

of 74 in the glucagon-challenged rat model are shown

in Figure 8. At 3 mg/kg, 74 was inactive. However, at

10 mg/kg, the hyperglycaemic effect of the exogenously

administered glucagon was completely abolished. In

fact, the change (∆) in blood glucose value after admin-

istration of 10 mg/kg was negative (below basal level),

which suggests that the compound may also, at least

partly, have inhibited the action of the endogenous

glucagon responsible for maintenance of euglycemia.

5768 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26

Madsen et al.

F igu r e 6. Inhibition of glucagon-stimulated glucose produc-

tion in primary rat hepatocytes by 74. The hepatocytes were

incubated with glucagon and increasing concentrations of 74:

0 nM (9), 10 nM (2), 30 nM (1), 100 nM ([), 300 nM (b), and

1000 nM (0). Results are presented as the mean ( SEM of

four independent experiments (n is defined as the number of

separate hepatocytes preparations). Inserted is the Schild plot.

F igu r e 8. Test of 74 in glucagon-challenged Sprague-Dawley

rats. Dots represents individual changes (∆) in the values, and

bars represent the corresponding mean values (n ) 5-6 per

dose). The asterisk (/) represents p < 0.0005 vs vehicle

(0 mg/kg).

The SAR was quite insensitive toward structural

changes in the linker region. High-affinity compounds

could be obtained with both long and short linkers as

well as with polar and apolar linkers. One noteworthy

exception was the activity of the enantiomers 14 and

15 with (R) and (S) configurations, respectively, where

the (R)-isomer was 10-fold more potent than the (S)-

isomer.

One promising compound (74) with low in vitro

metabolic turnover was a highly potent noncompetitive

antagonist of the human glucagon receptor (K_B) 760

pM) and of the isolated rat receptor (K_B) 380 pM).

Glucagon-stimulated glucose production from isolated

primary rat hepatocytes was inhibited competitively by

74 (K_i) 14 nM). This compound was orally available

in dogs (F_po) 15%) and was active in a glucagon-

challenged rat model of hyperglucagonemia and hyper-

glycemia.

Exp er im en ta l Section

Gen er a l. ¹H NMR spectra were recorded in deuterated

solvents at 200, 300, and 400 MHz (DRX 200, DRX 300, and

AMX2 400 from Bruker Instruments, respectively). Chemical

shifts are reported as δ values (ppm) relative to internal

tetramethylsilane (δ ) 0 ppm). Elemental analyses were

performed by the microanalytical laboratories at Novo Nordisk

A/S, Denmark, and at Numega and Gailbraith. Column

chromatography²⁴was performed on silica gel 60 (40-63 µm).

Melting points were determined in open capillary tubes on a

Bu¨chi 535 apparatus and are uncorrected. Chemicals and

solvents used were commercially available and were used

without further purification. Yields refer to pure materials and

are not optimized. The preparation methods are illustrated

by single representative experimental procedures. Further

experimental details (including NMR data) have been pub-

lished in the patent applications.^25,26

F igu r e 7. Plasma concentration vs time plots of 74 after (A)

intravenous dosing of 0.5 mg/kg to dogs and (B) peroral

administration of 2.0 mg/kg by gavage to dogs. Legends

identify dogs. Mean half-lives were 1.11 and 1.40 h after iv

and po administration, respectively. The mean oral bioavail-

ability was 15%.

Con clu sion s

3-Ch lor o-4-h yd r oxyben zoic Acid Hyd r a zid e (I). To

methyl 3-chloro-4-hydroxybenzoate (2 g, 11 mmol) dissolved

in EtOH (50 mL) was added hydrazine (1.8 mL). The reaction

mixture was refluxed overnight under nitrogen. When the

mixture was cooled, the desired product crystallized. The white

solid was isolated by filtration. Recrystallization from hot

EtOH afforded 1.2 g (60%) of 3-chloro-4-hydroxybenzoic acid

Optimization of the lead structures 1 and 2 led to

series of high-affinity human glucagon receptor antago-

nists, addressing pharmacokinetic issues with in vitro

metabolism screening and metabolite profiling while

optimizing hGluR affinity. The SAR of these series

revealed that it was not necessarily a requirement to

have a distal aromatic ring to obtain high-affinity

glucagon receptor antagonists, since, for example, the

diethyl acetamide (29) and the 2-ethylpiperidyl acet-

amide (34) had IC₅₀values of 17 and 7.4 nM, respec-

tively. The presence of a distal aromatic ring, however,

often increased the affinity further.

1

hydrazide (I). H NMR (DMSO-d₆): δ 4.49 (bs, 2H), 7.05 (dd,

1H), 7.71 (dd, 1H), 7.89 (d, 1H), 9.69 (s, 1H), 10.72 (bs, 1H).

3-F lu or o-4-h yd r oxyben zoic Acid Hyd r a zid e (II). ¹H

NMR (DMSO-d₆): δ 9.45 (bs, 1H), 7.5 (d, 1H), 7.43 (d, 1H),

6.85 (t, 1H), 5.55 (bs, 3H).

2,3-Dich lor o-4-h yd r oxyben zoic Acid Hyd r a zid e (III).

4-Amino-2-chlorobenzoic acid (5 g, 29 mmol) was dissolved in

Optimization of Glucagon Receptor Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5769

H₂SO₄(12 N, 120 mL) with heating. After the solution was

cooled in an ice bath, aqueous NaNO₂(2.5 M, 25 mL) was

added dropwise such that the internal temperature remained

at 5 °C. Urea was added to the mixture after stirring for 15

min to destroy excess NaNO₂(monitored by starch iodine test).

CuSO₄(100-200 mg) was added, and the mixture was heated

to 90 °C until evolution of gas stopped. After cooling, the

mixture was extracted with ethyl ether (3×). The combined

organic fractions were extracted with 3 N NaOH (3×). The

combined aqueous layer was acidified with concentrated HCl,

and the product was extracted with ethyl ether (3×). The

organic fractions were washed with water and brine, dried over

MgSO₄, and concentrated in vacuo. The residue 2-chloro-4-

hydroxybenzoic acid was dissolved in anhydrous MeOH and

added thionyl chloride (1.5 equiv). After the solution was

stirred at room temperature for 16 h, the solvent was evapo-

rated. The residue was taken up in EtOAc and washed with

saturated aqueous NaHCO₃, water, and brine, dried over

MgSO₄, and concentrated in vacuo to afford 3.5 g (65%) of

combined organic extracts were washed with water (3×) and

brine, dried (MgSO₄), and concentrated in vacuo. The residue

was recrystallized from MeOH/H₂O to afford 2.1 g (84%) of

3-cyano-4-hydroxybenzoic acid. ¹H NMR (DMSO-d₆): δ 7.09

(d, 1H), 8.00 (dd, 1H), 8.07 (d, 1H), 12.50 (bs, 2H).

3-Cyano-4-hydroxybenzoic acid (1.88 g, 11.5 mmol) was

dissolved in a 1:1 mixture of DCM/DMF (20 mL) and cooled

in an ice bath. Diisopropylethylamine (12 mL, 69 mmol), tert-

butyl carbazate (1.76 g, 13.3 mmol), and PyBroP (bromotri-

spyrrolidinophosphonium hexafluorophosphate, 6 g, 12.9 mmol)

were added, and the mixture was stirred to form a clear

solution. The solution was left at 5 °C for 16 h. Additional

diisopropylethylamine (22 mL, 127 mmol), tert-butyl carbazate

(0.85 g, 6.4 mmol), and PyBroP (3.0 g, 6.4 mmol) were added.

After 8 h at 0 °C, the mixture was concentrated in vacuo. The

residue was diluted with EtOAc (100 mL) and washed with

several portions of 0.1 M HCl (until the wash remained acidic

to litmus paper). The EtOAc layer was further washed with

brine (three portions), dried (MgSO₄), filtered, and concen-

trated in vacuo. The residue was purified by chromatography

on silica gel (6:4 hexane/EtOAc) to afford 1.8 g (56%) of tert-

butoxycarbonyl (3-cyano-4-hydroxy)benzoic acid hydrazide as

a white solid. ¹H NMR (DMSO-d₆): δ 1.42 (s, 9H), 7.09 (d, 1H),

7.98 (m, 1H), 8.11 (bs, 1H), 8.92 (s, 1H), 10.15 (s, 1H), 11.73

(bs, 1H).

tert-Butoxycarbonyl(3-cyano-4-hydroxy)benzoic acid hy-

drazide (0.554 g, 2 mmol) was suspended in cold water (10

mL) and cooled in an ice bath. Concentrated HCl (10 mL) was

added, and the mixture was stirred at room temperature. Upon

being stirred, the suspension became a solution and then

turned back into a suspension. The reaction vessel was

warmed until a solution was obtained. Upon sitting overnight,

the product crystallized as white needles. This afforded 0.24

g (68%) of 3-cyano-4-hydroxybenzoic acid hydrazide (IV). ¹H

NMR (DMSO-d₆): δ 7.16 (d, 1H), 8.00 (dd, 1H), 8.14 (d, 1H),

10.47 (bs, 5H).

P r ep a r a tion Meth od A. 3-Ch lor o-4-h yd r oxyben zoic

Acid (4-Hydr oxym eth yln aph th alen -1-ylm eth ylen e)h ydr a-

zid e (3). 1,4-Naphthalenedicarboxylic acid (25 g, 116 mmol)

was added in portions to a mixture of LiAlH₄(15 g, 395 mmol)

in anhydrous THF (600 mL) and refluxed for 2 days. The

mixture was cooled in an ice bath, and excess LiAlH₄was

decomposed by the slow addition of MeOH followed by ice

chips. THF was removed in vacuo, and the residue was

acidified with 1 N HCl. The aqueous phase was extracted with

EtOAc (3×), and the combined organic phases were washed

with aqueous NaHCO₃(3×), water, and brine, dried (MgSO₄),

and evaporated in vacuo to afford 15.3 g (70%) of 1,4-

bishydroxymethylnaphthalene as a solid. This product can be

used in the subsequent oxidation step without further puri-

fication. ¹H NMR (DMSO-d₆): δ 5.19 (s, 4H), 7.77 (m, 4H),

8.32 (m, 2H).

To a solution of 1,4-bishydroxymethylnaphthalene (12 g, 65

mmol) in EtOAc (300 mL) was added MnO₂(28 g, 325 mmol).

After being stirred for 45 min, the mixture was passed through

a bed of Celite and eluted with additional volumes of EtOAc.

The solvent was evaporated in vacuo, and the residue was

purified by column chromatography using hexane/EtOAc (80:

20 to 75:25) to afford 6.0 g (50%) of 4-hydroxymethyl-1-

naphthaldehyde (V). ¹H NMR (DMSO-d₆): δ 5.19 (s, 2H), 5.71

(brd s, 1H), 7.73 (t, 1H), 7.78 (t, 1H), 7.95 (d, 1H), 8.26 (m,

2H), 9.34 (d, 1H), 10.46 (s, 1H).

1

methyl 2-chloro-4-hydroxybenzoate. H NMR (CDCl₃): δ 3.91

(s, 3H), 6.67 (bs, 1H), 6.80 (dd, 1H), 6.97 (d, 1H), 7.83 (d, 1H).

To a solution of methyl 2-chloro-4-hydroxybenzoate (13.6 g,

73.1 mmol) in acetic acid (300 mL) was added N-chlorosuc-

cinimide (9.8 g, 73.7 mmol). The solution was refluxed for 24

h, and the solvent was evaporated in vacuo. The residue was

taken up in chloroform, washed with water and brine, dried

over MgSO₄, filtered, and concentrated. Methyl 2,3-dichloro-

4-hydroxybenzoate precipitated out of EtOAc. Chromatography

of the residue using EtOAc/hexane (1:9 to 3:7) afforded methyl

2,5-dichloro-4-hydroxybenzoate (1.4 g, 9%) as well as an

additional crop of methyl 2,3-dichloro-4-hydroxybenzoate (total

yield 8.4 g, 52%). ¹H NMR (DMSO-d₆): δ 3.81 (s, 3H), 7.02 (d,

1H), 7.70 (d 1H), 11.52 (s, 1H).

2,3-Dichloro-4-hydroxybenzoic acid hydrazide was prepared

from the methyl 2,3-dichloro-4-hydroxybenzoate as described

above with the exception that pentanol was used as the

solvent. The product was purified via silica gel column chro-

matography using DCM/MeOH (95:5 to 80:20) to afford 2,3-

dichloro-4-hydroxybenzoic acid hydrazide (III) (50%). ¹H NMR

(DMSO-d₆): δ 4.41 (bs, 2H), 6.99 (1, 1H), 7.37 (s, 1H), 9.46 (s,

1H), 11.04 (s, 1H).

3-Cya n o-4-h yd r oxyben zoic Acid Hyd r a zid e (IV). Meth-

yl 4-hydroxybenzoate (35.5 g, 0.233 mol) was dissolved in warm

(65 °C) HOAc (200 mL). A solution of iodine monochloride (37.8

g, 0.233 mol) in HOAc (50 mL) was added over 40 min while

maintaining a temperature of 65 °C and vigorous stirring. The

product crystallized from solution upon cooling to room tem-

perature and standing overnight. The crystals were filtered,

washed with water, and dried in vacuo to afford 28.6 g (44%)

of methyl 4-hydroxy-3-iodobenzoate. ¹H NMR (DMSO-d₆): δ

3.79 (s, 3H), 6.95 (d, 1H), 7.81 (dd, 1H), 8.22 (d, 1H).

Methyl 4-hydroxy-3-iodobenzoate (2.00 g, 7.2 mmol) was

dissolved in dry DMF (5 mL). CuCN (0.72 g, 8.0 mmol) and a

small crystal of NaCN were added. The mixture was flushed

with nitrogen and stirred at 100-110 °C for 16 h. The mixture

was cooled and filtered. The solids were extracted with DMF

(3 mL). The combined filtrate and washings were dissolved in

EtOAc (100 mL) and washed with brine (3×). The solids and

aqueous washings were combined and extracted with

a

mixture of EtOAc (50 mL) and an FeCl₃solution (4 g of

hydrated FeCl₃in 7 mL of concentrated HCl). The EtOAc

phases were combined, washed with brine containing sodium

metabisulfite, dried (Na₂SO₄), filtered, and evaporated in

vacuo. The residue was purified by flash chromatography on

silica gel (20% EtOAc/ hexane) to afford 0.93 g (73%) of methyl

3-cyano-4-hydroxybenzoate. ¹H NMR (DMSO-d₆): δ 3.79 (s,

3H), 7.07 (d, 1H), 8.02 (dd, J ) 8.7, 1.9, 1H), 8.10 (d, 1H).

Methyl 3-cyano-4-hydroxybenzoate (2.71 g, 15.3 mmol) was

dissolved in THF (50 mL). The solution was cooled in an ice

bath, and 2.0 M KOH (17 mL, 34 mmol) was added dropwise.

The resulting mixture was stirred at room temperature

overnight. The organic solvent was removed by rotary evapo-

ration in vacuo, and the aqueous residue was acidified with 1

N HCl. The product was extracted with EtOAc (3×), and the

3-Chloro-4-hydroxybenzoic acid hydrazide (I) (186 mg, 1

mmol) was dissolved in DMSO (1 mL) and added to a solution

of 4-hydroxymethyl-1-naphthaldehyde (V) (186 mg, 1 mmol)

in DMSO (0.5 mL) followed by HOAc (50 µL). The mixture

was stirred at 25 °C for 16 h. The mixture was diluted with

EtOAc (10 mL) and washed with brine (10 mL) and water (5

mL) and dried (MgSO₄). As the solvent was evaporated, the

product (276 mg, 78%) 3-chloro-4-hydroxybenzoic acid (4-

hydroxymethylnaphthalen-1-ylmethylene)hydrazide (3) pre-

cipitated out of solution. Mp >250 °C. ¹H NMR (DMSO-d₆): δ

5.02 (s, 2H), 5.43 (t, 1H), 7.10 (d, 1H), 7.66 (m, 3H), 7.80 (d,

1H), 7.90 (d, 1H), 8.02 (s, 1H), 8.15 (d, 1H), 8.87 (d, 1H), 9.08

5770 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26

Madsen et al.

(s, 1H), 10.98 (s, 1H), 11. 79 (s, 1H). Anal. (C₁₉H₁₅ClN₂O₃): C,

H. N: calcd, 7.90; found, 7.21.

oxymethyl-1-naphthaldehyde (0.15 g, 0.70 mmol) followed by

a catalytic amount of acetic acid. The reaction was stirred at

room temperature for 16 h. The mixture was diluted with

EtOAc, washed with aqueous NaHCO₃(3×) and brine (3×),

dried (MgSO₄), and filtered. Upon concentration of the solvent

in vacuo, 69 mg (23%) of 3-chloro-4-hydroxybenzoic acid (4-

methoxymethylnaphthalen-1-ylmethylene)hydrazide (8) pre-

cipitated out of solution and was isolated by filtration, mp

204-206.5 °C. ¹H NMR (DMSO-d₆): δ 3.43 (s, 3H), 4.91 (s,

2H), 7.10 (d, 1H), 7.62-7.71 (m, 3H), 7.82 (d, 1H), 7.91 (d, 1H),

8.01 (s, 1H), 8.13 (d. 1H), 8.85 (d, 1H), 9.09 (s, 1H), 11.0 (brd

s, 1H), 11.82 (s, 1H). Anal. (C₂₀H₁₇ClN₂O₃‚0.25H₂O) C, H, N.

5-Hyd r oxym eth yl-1-n a p h th a ld eh yd e (VI). To a suspen-

sion of 5-bromo-1-naphthoic acid (5 g, 20 mmol) in anhydrous

MeOH (200 mL) was added concentrated H₂SO₄(5 mL), and

the resulting mixture was refluxed overnight. The mixture was

cooled and concentrated in vacuo to one-third the volume. The

residue was diluted with water and extracted with Et₂O. The

organic phase was washed with water (2×), dried (MgSO₄),

and concentrated in vacuo. Silica gel column chromatography

using hexane/EtOAc (2:1) afforded 4.91 g (92%) of methyl

1

5-bromo-1-naphthylcarboxylate. H NMR (CDCl₃): δ 4.01 (s,

3H), 7.44 (dd, 1H), 7.61 (dd, 1H), 7.85 (dd, 1H), 8.22 (dd, 1H),

8.51 (d, 1H), 8.90 (d, 1H).

P r ep a r a tion Meth od B. 3-Ch lor o-4-h yd r oxyben zoic

Acid [5-(4-Tr iflu or om eth oxyben zyloxym eth yl)n a p h th a l-

en e-1-ylm eth ylen e]h yd r a zid e (10). To a solution of 5-hy-

droxymethyl-1-naphthaldehyde (VI) (0.5 g, 2.1 mmol), 4-tri-

fluoromethoxybenzyl bromide (0.64 g, 2.5 mmol), and 100 mg

of n-Bu₄NCl (100 mg) in DCM (20 mL) was added aqueous

5% KOH (20 mL). The reaction mixture was refluxed for 16

h, and the two phases were separated. The organic phase was

washed with water and brine, dried (MgSO₄), and concentrated

in vacuo. The residue was purified by silica gel column

chromatography using hexane/EtOAc to afford 0.32 g (89%)

of 5-(4-trifluoromethoxybenzyloxy)methyl-1-naphthaldehyde.

¹H NMR (CDCl₃): δ 4.54 (s, 2H), 5.05 (s, 2H), 7.21 (d, 2H),

7.39 (d, 2H), 7.59-7.74 (m, 3H), 8.01 (d, 1H), 8.45 (d, 1H), 9.30

(d, 1H), 10.43 (s, 1H).

A mixture of methyl 5-bromo-1-naphthylcarboxylate (5.2 g,

19 mmol) and CuCN (3.4 g, 38 mmol) in anhydrous DMF (100

mL) was refluxed overnight. After the mixture was cooled to

70 °C, a solution of NaCN (2 g) in water (50 mL) was added.

The mixture was extracted with EtOAc. The organic phase was

washed with brine, dried (MgSO₄), and concentrated in vacuo.

Column chromatography on silica gel using hexane/EtOAc (5:

1) afforded 3.8 g (95%) of methyl 5-cyano-1-naphthylcarbox-

ylate. ¹H NMR (CDCl₃): δ 4.01 (s, 3H), 7.60-7.80 (m, 3H),

7.98 (d, 1H), 8.30 (d, 1H), 8.45 (d, 1H), 9.21 (d, 1H).

To a cooled (0 °C) solution of methyl 5-cyano-1-naphthyl-

carboxylate (1 g, 5 mmol) in anhydrous THF (20 mL) was

added DIBAL (1 M in hexane, 20 mL, 20 mmol) via syringe.

The mixture was stirred at 50-60 °C overnight. The mixture

was then cooled to room temperature and poured into cold (0

°C) 2 N HCl (100 mL). The mixture was extracted with Et₂O

(2×), and the organic phase was washed with brine, dried

(MgSO₄), and concentrated in vacuo. Silica gel column chro-

matography using hexane/EtOAc (2:1) afforded 0.85 g (91%)

of 5-hydroxymethyl-1-naphthaldehyde (VI). ¹H NMR (CD-

Cl₃): δ 4.95 (s, 2H), 7.45-7.58 (m, 3H), 7.82 (dd, 1H), 8.24 (d,

1H), 9.03 (dd, 1H), 10.21 (s, 1H).

3-Ch lor o-4-h yd r oxyben zoic Acid (4-Meth oxym eth yl-

n a p h th a len -1-ylm eth ylen e)h yd r a zid e (8). 4-Hydroxymeth-

yl-1-naphthaldehyde (V) (7 g, 37 mmol) was suspended in

aqueous HBr (50 mL, 48%) and cooled in an ice bath. Con-

centrated H₂SO₄(50 mL) was added, and the mixture was

stirred for 4 h, diluted with EtOAc, and washed with brine

(3×) and aqueous NaHCO₃(3×), dried (MgSO₄), and concen-

trated in vacuo. The residue was purified though a bed of silica

gel using ethyl acetate/hexane (5-50%). Two recrystallizations

from EtOAc afforded 7 g (75%) of 4-bromomethyl-1-naphthal-

dehyde (VIII). ¹H NMR (DMSO-d₆): δ 5.27 (s, 2H), 7.76 (m,

2H), 7.92 (d, 1H), 8.13 (d, 1H), 8.15 (m, 1H), 9.20 (, 1H), 10.39

(s, 1H).

Hydrazone formation was done as described in preparation

method A to afford 3-chloro-4-hydroxybenzoic acid [5-(4-

trifluoromethoxybenzyloxymethyl)naphthalene-1-ylmethylene]-

1

hydrazide (10), mp 183-185 °C. H NMR (DMSO-d₆): δ 4.67

(s, 2H), 5.05 (s, 2H), 7.11 (d, 1H), 7.35 (d, 2H), 7.50 (d, 2H),

7.57-7.75 (m, 3H), 7.82 (d, 1H), 7.95-8.08 (m, 2H), 8.22 (d,

1H), 8.78 (s, 1H), 9.14 (s, 1H), 11.01 (s, 1H), 11.85 (s, 1H). Anal.

(C₂₇H₂₀ClF₃N₂O₄) C, H, N.

(S)-2-({4-[(3-Ch lor o-4-h ydr oxyben zoyl)h ydr azon om eth -

yl]n aph th -1-ylm eth yl}am in o)-3-ph en ylpr opion am ide (15).

4-Bromomethyl-1-naphthaldehyde (VIII) (5.7 g, 23 mmol) was

suspended in Et₂O (125 mL). To this suspension was added

neopentylglycol (3.6 g, 34.58 mmol), trimethyl orthoformate

(3.66 g, 34.53 mmol), and p-TsOH (20 mg). The reaction

mixture was refluxed for 12 h. The solvent was evaporated in

vacuo, the residue was dissolved in EtOAc, and the solution

was washed with H₂O (3×) and brine (2×), dried (MgSO₄), and

concentrated in vacuo. The residue was purified by silica gel

column chromatography using 10% EtOAc/hexane. Recrystal-

lization from MeCN afforded 5 g (65%) of 2-[4-(bromomethyl)-

1-naphthyl]-5,5-dimethyl-1,3-dioxane. ¹H NMR (DMSO-d₆): δ

0.81 (s, 3H), 1.27 (s, 3H), 3.79 (m, 4H), 5.22 (s, 2H), 6.03 (s,

1H), 7.67 (m, 4H), 8.22 (d, 1H), 8.30 (d, 1H).

(S)-Phenylalanine amide hydrochloride (0.45 g, 2.25 mmol)

was suspended in CH₃CN (3 mL), and saturated aqueous

NaHCO₃(2 mL) was added. To this mixture, 2-[4-(bromometh-

yl)-1-naphthyl]-5,5-dimethyl-1,3-dioxane (0.25 g, 0.75 mmol)

dissolved in MeCN (3 mL) was added. The reaction mixture

was refluxed for 48 h and cooled to room temperature. The

solid formed was collected by filtration and washed with H₂O.

Drying in vacuo afforded 0.25 g (80%) of N-{[4-(5,5-dimethyl-

1,3-dioxan-2-yl)-1-naphthyl]methyl}-(S)-phenylalaninamide. ¹H

NMR (DMSO-d₆): δ 0.8 (s, 3H), 1.25 (s, 3H), 2.27 (m, 1H), 2.75

(dd, 1H), 2.87 (dd, 1H), 3.77 (brd s, 4H), 3.92 (dd, 1H), 4.12

(dd, 1H), 5.97 (s, 1H), 7.12 (s, 1H), 7.23 (m, 5H), 7.34 (d, 1H),

7.51 (m, 4H), 8.03 (d, 1H), 8.23 (d, 1H).

To a solution of 4-bromomethyl-1-naphthaldehyde (1 g, 4

mmol) in toluene (70 mL) was added ethylene glycol (2.2 mL,

40 mmol) followed by a catalytic amount of p-TsOH. The

mixture was refluxed overnight and diluted with EtOAc. The

mixture was washed with brine (3×), dried (MgSO₄), and

concentrated in vacuo. The residue was purified via silica gel

column chromatography using ethyl acetate/hexane (5:95) to

afford 0.51 g (44%) of 2-(4-bromomethylnaphthalen-1-yl)-[1,3]-

1

dioxolane. H NMR (DMSO-d₆): δ 4.10 (m, 4H), 5.22 (s, 2H),

6.38 (s, 1H), 7.59-7.71 (m, 4H), 8.24 (t, 2H).

To a solution of 2-(4-bromomethylnaphthalen-1-yl)-[1,3]-

dioxolane (0.2 g, 0.68 mmol) in anhydrous DMF (20 mL) was

added sodium methoxide (0.75 mmol). After the mixture was

stirred for 1 h, the mixture was diluted with EtOAc and

washed with brine (3×), dried (MgSO₄), filtered, and concen-

trated in vacuo. The residue was dissolved in acetone (7 mL),

concentrated HCl (2 mL) was added, and the mixture was

stirred for 16 h. The mixture was diluted with EtOAc, washed

with brine (3×), dried (MgSO₄), and concentrated in vacuo to

afford 0.15 g (100%) of 4-methoxymethyl-1-naphthaldehyde

N-{[4-(5,5-Dimethyl-1,3-dioxan-2-yl)-1-naphthyl]methyl}-S-

phenylalaninamide (0.25 g, 0.6 mmol) was dissolved in acetone

(25 mL) by gentle warming. Water (5 mL) and pyridinium

p-toluenesulfonic acid (0.05 g) were added, and the reaction

mixture was refluxed for 4 days. The mixture was concentrated

in vacuo and dissolved in EtOAc. The organic solution was

washed with H₂O (3×) and brine (2×), dried (MgSO₄), and

concentrated in vacuo. The residue was purified by column

chromatography using 35% EtOAc/hexane to afforded 66 mg

(33%) of N-[(4-formyl-1-naphthyl)methyl]-(S)-phenylalanin-

1

(VII). H NMR (DMSO-d₆): δ 3.52 (s, 3H), 4.97 (s, 2H), 7.62-

7.72 (m, 3H), 7.96 (d, 1H), 8.10 (d, 1H), 9.32 (d, 1H), 10.37 (s,

1H).

To a solution of 4-hydroxy-3-chlorobenzoic acid hydrazide

(I) (0.15 g, 0.8 mmol) in DMSO (10 mL) was added 4-meth-

1

amide. H NMR (DMSO-d₆): δ 3.26 (m, 2H), 4.16 (brd s, 1H),

Optimization of Glucagon Receptor Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5771

4.59 (m, 2H), 7.12 (d, 1H), 7.31 (m, 5H), 7.49 (d, 1H), 7.80 (m,

4H), 8.24 (m, 2H), 9.24 (d, 1H), 10.44 (s, 1H).

column chromatography using ethyl acetate/hexane (1:9 to 2:8)

afforded 0.21 g (41%) of 4-(2-chloroethoxy)-1-naphthaldehyde.

¹H NMR (DMSO-d₆): δ 3.99 (t, 2H), 4.51 (t, 2H), 6.90 (d, 1H),

7.26 (s, 1H), 7.63 (t, 1H), 7.72 (t, 1H), 7.91 (d, 1H), 8.39 (d,

1H), 9.30 (d, 1H), 10.22 (s, 1H).

A solution of 4-(2-chloroethoxy)-1-naphthaldehyde (2.35 g,

10 mmol), 3-chloro-4-hydroxybenzoic acid hydrazide (I) (1.87

g, 10 mmol), HOAc (0.2 mL), and DMSO (15 mL) were stirred

at room temperature overnight. Ethyl acetate (100 mL) was

added, and the solution was treated with water and brine,

which induced precipitation. Filtration afforded 3.1 g (77%)

of 3-chloro-4-hydroxybenzoic acid [4-(2-chloroethoxy)-1-naph-

thylmethylene]hydrazide. ¹H NMR (DMSO-d₆): δ 11.5 (s, 1H),

10.7 (s, 1H), 8.7 (bs, 2H), 8.1 (m, 1H), 7.8 (s, 1H), 7.6-7.3 (m,

2H), 7.0 (m, 2H), 4.3 (t, 2H), 3.7 (t, 2H).

To 4-trifluoromethoxybenzylamine (0.29 g, 1 mmol) in DMF

(5 mL) was added 3-chloro-4-hydroxybenzoic acid [4-(2-chlo-

roethoxy)-1-naphthylmethylene]hydrazide (0.403 g, 1 mmol)

and triethylamine (0.1 g, 1 mmol). The reaction mixture was

heated at 80 °C for 16 h. Removal of most of the solvent in

vacuo followed by flash chromatography (10:1 CHCl₃/MeOH)

on silica gel afforded 0.15 g (27%) of 3-chloro-4-hydroxybenzoic

acid {4-[2-(4-trifluoromethoxy)benzylaminoethoxy]-1-naphthyl-

methylene}hydrazide (20) as a solid, mp 125-127 °C. ¹H NMR

(DMSO-d₆): δ 11.6 (s, 1H), 9.0 (m, 2H), 8.3 (m, 1H), 8.0 (m,

1H), 7.8 (s, 2H), 7.7 (m, 1H), 7.6 (m, 1H), 7.5 (m, 3H), 7.3 (m,

2H), 7.1 (m, 2H), 4.3 (t, 2H), 3.9 (s, 2H), 3.0 (t, 2H). Anal.

(C₂₈H₂₃ClF₃N₃O₄‚1.75H₂O): C, N. H: calcd, 4.53; found, 3.97.

3-Chloro-4-hydroxybenzoic acid hydrazide (I) (41 mg, 0.22

mmol) and N-[(4-formyl-1-naphthyl)methyl]-S-phenylalanin-

amide (66 mg, 0.2 mmol) were combined and dissolved in

DMSO (1.5 mL). AcOH (0.2 mL) was added, and the mixture

was stirred at room temperature overnight. The reaction

mixture was diluted with EtOAc (30 mL) and washed with

H₂O (2×) and brine (2×), and upon concentration in vacuo to

about 10 mL a white solid precipitated. The solid was filtered,

washed with EtOAc, and dried in vacuo to afford 10 mg

(10%) of (S)-2-({4-[(3-chloro-4-hydroxybenzoyl)hydrazonometh-

1

yl]naphth-1-ylmethyl}amino)-3-phenylpropionamide (15). H

NMR (DMSO-d₆): δ 2.76 (m, 1H), 2.91 (m, 1H), 3.97 (d, 1H),

3.36 (brd s, 1H), 4.17 (d, 1H), 7.12 (m, 2H), 7.25 (m, 5H), 7.49

(m, 3H), 7.65 (t, 1H), 7.80 (m, 2H), 8.02 (d, 1H), 8.1 (d, 1H),

8.82 (d, 1H), 9.07 (brd s, 1H), 11.04 (brd s, 1H), 11.8 (s, 1H).

Anal. (C₂₈H₂₅ClN₄O₃‚1¹/₂H₂O) C, H, N.

3-Ch lor o-4-h yd r oxyben zoic Acid (4-Cyclop en ta n esu l-

fon ylm eth yln a p h th a len -1-ylm eth ylen e)h yd r a zid e (19). A

mixture of 4-bromomethyl-1-naphthaldehyde (VIII) (120 mg,

0.48 mmol), cyclopentylthiol (0.06 mL, 0.48 mmol), and K₂-

CO₃(130 mg) was stirred at room temperature for 12 h. After

filtration, the mixture was concentrated in vacuo and the

residue was purified by column chromatography using EtOAc/

hexane (1:5) to afford 96 mg (74%) of 4-cyclopentylthiomethyl-

1

1-naphthaldehyde as an oil. H NMR (CDCl₃): δ 13.1 (s, 1H),

9.3 (d, 1H), 8.2 (d, 1H), 7.9 (d, 1H), 7.7 (m, 3H), 7.6 (d, 1H),

4.2 (s, 2H), 3.1 (m, 1H), 1.9 (m, 2H), 1.7 (m, 2H), 1.5 (m, 4H).

P r ep a r a tion Meth od D. 3-Ch lor o-4-h yd r oxyben zoic

Acid {4-[2-(1,2,3,4-Tetr a h yd r oisoqu in olin -2-yl)eth oxy]-2-

m eth oxyben zylid en e}h yd r a zid e (24). Polystyrene resin

loaded with the Wang linker (15 g, 0.92 mmol/g) was succes-

sively washed with DMF (3 × 40 mL) and DCM (3 × 40 mL).

The resin was suspended in DCM (80 mL), and diisopropyl-

ethylamine (60 mL) was added. The mixture was cooled to 0

°C, and methanesulfonyl chloride (5.8 mL) dissolved in DCM

(30 mL) was added dropwise while maintaining the temper-

ature below 5 °C. When addition was complete, the mixture

was stirred at 0 °C for 30 min and at room temperature for 30

min. The resin was successively washed with DCM (3 × 80

mL) and N-methylpyrrollidone (NMP) (3 × 80 mL). This resin

and Cs₂CO₃(12.3 g) were added to ethyl 3-chloro-4-hydroxy-

benzoate (15 g) dissolved in NMP (200 mL), and the mixture

was stirred at 80 °C for 4 h. After cooling, the resin was

successively washed with NMP (3 × 80 mL) and MeOH (3 ×

80 mL). The resin was washed with THF (3 × 300 mL) and

treated with NaOSiMe₃in THF (1 M, 300 mL) for 4 h at 25

°C. After filtration, the resin was washed with THF (2 × 300

mL), and added THF (300 mL) and HOAc (75 mL) were added.

The mixture was stirred overnight at 25 °C. The resulting

resin-bound carboxylic acid was washed with THF (2 × 300

mL) and DCM (300 mL). To the resin was added a solution of

PyBoP (46.8 g, 90 mmol) in DCM (300 mL) followed by

N-methylmorpholine (30 mL). The mixture was stirred for 45

min at 25 °C, and hydrazine hydrate (30 mL) was added. After

the mixture was shaken for 4 h at 25 °C, the resin was filtered

and washed with DCM (2 × 300 mL) and DMF (300 mL) to

afford the resin-bound 3-chloro-4-hydroxybenzoic acid hy-

drazide (I).

1,2-Dibromoethane (57 mL, 0.66 mol) was added to a

mixture of 4-hydroxy-2-methoxybenzaldehyde (10 g, 66 mmol)

and K₂CO₃(45 g, 0.33 mol) in DMF (130 mL), and the resulting

mixture was stirred vigorously at room temperature for 16 h.

The mixture was poured into water (0.8 L) and extracted with

EtOAc (3 × 300 mL). The combined organic phases were

washed with brine (400 mL), dried (MgSO₄), and evaporated

in vacuo to afford 17.4 g (99%) of 4-(2-bromoethoxy)-2-meth-

oxybenzaldehyde, mp 78-79 °C.

The resin-bound 3-chloro-4-hydroxybenzoic acid hydrazide

(I) (3 g, ∼3 mmol) was swelled in DMF (35 mL) for 30 min.

Then 4-(2-bromoethoxy)-2-methoxybenzaldehyde (2.33 g, 9

mmol) and triethyl orthoformate (18 mL) were added and the

mixture was shaken at room temperature for 16 h. The resin

was repeatedly swelled in DMF (4 × 35 mL), DCM (6 × 35

To a solution of 4-cyclopentylthiomethyl-1-naphthaldehyde

(96 mg) in DCM (3 mL) at room temperature was added

mCPBA (226 mg, 48.7%). The reaction mixture was stirred at

room temperature for 1 h and was washed with 0.5 M of

NaHSO₃followed by 0.5 M of K₂CO₃. The solution was dried

(MgSO₄) and concentrated in vacuo to give 4-cyclopentylsul-

fonylmethyl-1-naphthaldehyde as an oil that was used without

characterization in the next step.

Hydrazone formation was done as described in preparation

method A to afford 3-chloro-4-hydroxybenzoic acid (4-cyclo-

pentanesulfonylmethylnaphthalen-1-ylmethylene)hydrazide

(19), mp 245-247 °C. ¹H NMR (DMSO-d₆): δ 1.60 (4H, m),

1.95 (4H, m), 3.71 (1H, m), 5.04 (2H, s), 8.31 (1H, d), 7.67 (3H,

m), 7.79 (1H, d), 7.94 (1H, d), 8.01 (1H, s), 8.31 (1H, d), 8.78

(1H, d), 9.12 (1H, s), 11.0 (1H, s). Anal. (C₂₄H₂₃ClN₂O₄S‚¹/₄H₂O)

C, H, N.

3-Ch lor o-4-h yd r oxyben zoic Acid [4-(4-Tr iflu or om eth -

oxyph en ylm eth an esu lfon ylm eth yl)n aph th alen -1-ylm eth -

ylen e]h ydr azide (11). To a mixture of 4-bromomethyl-1-naph-

thaldehyde (VIII) (113 mg, 0.45 mmol) and 4-trifluoromethoxy-

benzylmercaptan (99 mg, 0.47 mmol) in DMF (2 mL) was

added K₂CO₃(200 mg), and the resulting mixture was stirred

at room temperature for 2 h. Then mCPBA (281 mg, 55% pure,

0.9 mmol) was added to the mixture. The reaction mixture was

stirred at room temperature for another 2 h and poured into

a cold 10% K₂CO₃solution. The aqueous phase was extracted

with EtOAc (3 × 20 mL), dried (MgSO₄), and concentrated in

vacuo. The residue (54% crude yield) was reacted with 3-chloro-

4-hydroxybenzoic acid hydrazide (I) as described in preparation

method A to afford 61 mg (23%) of 3-chloro-4-hydroxybenzoic

acid [4-(4-trifluoromethoxyphenylmethanesulfonylmethyl)-

naphthalen-1-ylmethylene]hydrazide (11), mp 240-241 °C. ¹H

NMR (DMSO-d₆): δ 11.7 (b, 1H), 10.4 (b, 1H), 9.2 (s, 1H), 8.6

(d, 1H), 8.1 (m, 2H), 8.0 (s, 1H), 7.9 (d, 1H), 7.6 (m, 2H), 7.5

(d, 2H), 7.2 (d, 2H), 7.0 (d, 1H), 4.9 (s, 2H), 4.5 (s, 2H). HR-

MS: calcd for C₂₇H₂₀ClF₃N₂O₅S, 577.08063; found, 577.0800.

P r ep a r a tion Meth od C. 3-Ch lor o-4-h yd r oxyben zoic

Acid {4-[2-(4-Tr iflu or om eth oxyben zylam in o)eth oxy]n aph -

th a len -1-m eth ylen e}h yd r a zid e (20). A solution of 4-hy-

droxy-1-naphthaldehyde (0.37 g, 2.15 mmol) and 1-chloro-2-

bromoethane (0.3 g, 2.15 mmol) in DMF (3 mL) containing

K₂CO₃(0.3 g, 2.15 mmol) was stirred at 80 °C for 12 h. The

solvent was evaporated in vacuo, and the residue was diluted

with ethyl acetate. The organic phase was washed with water,

dried (MgSO₄), filtered, and concentrated in vacuo. Silica gel

5772 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26

Madsen et al.

mL), and N-methyl-2-pyrrolidinone (NMP) (2 × 35 mL) and

filtered. The resin was swelled in NMP (40 mL), and 1,2,3,4-

tetrahydroisoquinoline (3.75 mL, 30 mmol) and KI (1.0 g, 6

mmol) were added. The resin was shaken at room temperature

for 16 h and filtered. The resin was repeatedly washed with

DMF (5 × 40 mL) and DCM (10 × 40 mL) and filtered. The

compound was cleaved off the resin by shaking for 1 h at room

temperature with a 50% solution of TFA in DCM (40 mL). The

mixture was filtered, and the resin was extracted with DCM

(2 × 40 mL). The combined DCM extracts were concentrated

in vacuo. The residue was dissolved in DCM (40 mL) and

concentrated in vacuo. The residue was dissolved in MeOH

(40 mL) and concentrated in vacuo. The residue was parti-

tioned between EtOAc (50 mL) and saturated NaHCO₃(50

mL). The aqueous phase was extracted with EtOAc (50 mL),

and the combined organic extracts were dried over (MgSO₄)

and concentrated in vacuo. The residue was purified by column

chromatography over silica gel (200 mL), eluting with a

mixture of DCM and MeOH (9:1). This afforded 280 mg of

3-chloro-4-hydroxybenzoic acid {4-[2-(1,2,3,4-tetrahydroiso-

quinolin-2-yl)ethoxy]-2-methoxybenzylidene}hydrazide (24),

The precipitate was filtered, washed with hexane, and dried

to afford 3.0 g (quantitative) of 4-[(3-chloro-4-hydroxybenzoyl)-

hydrazonomethyl]naphthoic acid (IX) as a white solid. ¹H NMR

(DMSO-d₆): δ 4.70 (d, 1H), 7.70 (m, 2H), 7.83 (d, 1H), 8.03

(m, 2H), 8.18 (d, 1H), 8.72 (s, 1H), 8.90 (d, 1H), 9.17 (s, 1H),

11.0 (bs, 1H), 11.94 (s, 1H), 13.4 (bs, 1H).

{4-[(3-Ch lor o-4-h yd r oxyb en zoyl)h yd r a zon om et h yl]-

n a p h th -1-yl}a cetic Acid (X). 4-Formylnaphthalene-1-ylace-

tic acid methyl ester was prepared from the reduction of

4-cyano-1-naphthylacetic acid in the presence of 85% formic

acid and Raney alloy as described in the literature.²⁷By

condensation of 4-formylnaphthalene-1-ylacetic acid and 3-chlo-

ro-4-hydroxybenzoic acid hydrazide (I) as described above,

{4-[(3-chloro-4-hydroxybenzoyl)hydrazonomethyl]naphth-1-yl}-

acetic acid (X) was prepared. ¹H NMR (DMSO-d₆): δ 4.1 (s,

2H), 7.1 (d, 1H), 7.5 (d, 1H), 7.7 (qt, 2H), 7.8 (d, 1H), 7.9 (d,

1H), 8.0 (s, 1H), 8.1 (d, 1H), 8.8 (d, 1H), 9.1 (s, 1H), 11.0 (bs,

1H), 11.8 (s, 1H), 12.2 (bs, 1H).

{4-[(3-Ch lor o-4-h yd r oxyb en zoyl)h yd r a zon om et h yl]-

n a p h th -1-yloxy}a cetic Acid (XI). 4-Hydroxy-1-naphthal-

dehyde (10 g, 58 mmol), K₂CO₃(16 g, 110 mmol), and methyl

bromoacetate (16 g, 100 mmol) were refluxed in acetone (120

mL) for 16 h. The reaction mixture was poured into ice (500

mL). Filtration and drying in vacuo afforded 13 g (86%) of

4-formylnaphthalene-1-yloxyacetic acid methyl ester. ¹H NMR

(CDCl₃): δ 3.86 (s, 3H), 4.93 (s, 2H), 6.80 (d, 1H), 7.61 (t, 1H),

7.72 (t, 1H), 7.90 (d, 1H), 8.42 (d, 1H), 9.29 (d, 1H), 10.22 (s,

1H).

1

mp 170-171.9 °C. H NMR (DMSO-d₆): δ 2.80 (4H, m), 2.90

(2H, t), 3.69 (2H, s), 3.86 (3H, s), 4.25 (2H, t), 6.68 (2H, m),

7.04 (1H, d), 7.07-7.14 (5H, m), 7.75 (1H, dd), 7.80 (1H, bs),

7.96 (1H, d), 8.58 (1H, s), 11.6 (1H, s). HR-MS: calcd for C₂₆H₂₆

ClN₃O₄, 479.1611; found, 479.1604.

-

P r ep a r a tion Meth od E. Libr a r y Meth od . 4-[(3-Ch lor o-

4-h ydr oxyben zoyl)h ydr azon om eth yl]n aph th oic Acid (IX).

A mixture of 4-methyl-1-naphthoic acid (10 g, 54 mmol), NBS

(10 g, 56 mmol), and AIBN (100 mg) in CCl₄(250 mL) was

refluxed for 3 h. The reaction mixture was concentrated in

vacuo and dissolved in EtOAc. The organic solution was

washed with water and brine and dried (MgSO₄). Evaporation

of the solvent in vacuo afforded 16 g, (80%) of 4-bromomethyl-

1-naphthoic acid. ¹H NMR (DMSO-d₆): δ 5.24 (s, 2H), 7.73

(m, 3H), 8.03 (d, 1H), 8.28 (d, 1H), 8.86 (d, 1H), 13.29 (bs, 1H).

4-Bromomethyl-1-naphthoic acid (16 g, 60 mmol) in an

aqueous solution of K₂CO₃(10%, 100 mL) was stirred at 70

°C for 30 min. The reaction mixture was cooled and made

acidic with concentrated HCl. The resulting precipitate was

filtered off and dried to afford 12 g (100%) of 4-hydroxymethyl-

1-naphthoic acid. ¹H NMR (DMSO-d₆): δ 5.01 (s, 2H), 5.96 (s,

1H), 7.70 (m, 3H), 8.10 (m, 2H), 8.90 (d, 1H).

A mixture of 4-hydroxymethyl-1-naphthoic acid (10 g, 50

mmol), MeOH (300 mL), and concentrated H₂SO₄(2 mL) was

refluxed overnight. Insoluble material was filtered off, and the

filtrate was concentrated. The residue was dissolved in EtOAc

and washed with aqueous NaHCO₃(2×) and brine, dried

(MgSO₄), and concentrated in vacuo. Column chromatography

on silica gel using EtOAc/hexane (1:3) afforded 3.3 g (35%) of

methyl 4-hydroxymethyl-1-naphthoate as an oil. ¹H NMR

(CDCl₃): δ 2.05 (t, 1H), 4.01 (s, 3H), 5.22 (s, 2H), 7.66 (m, 3H),

8.09 (d, 1H), 8.16 (d, 1H), 8.96 (d, 1H).

To a solution of methyl 4-hydroxymethyl-1-naphthoate (3.3

g, 15.3 mmol) in DCM (20 mL) was added MnO₂(6.6 g, 76

mmol). After the mixture was stirred for 16 h, the mixture

was filtered through a bed of Celite. Evaporation of the solvent

in vacuo afforded 3.3 g (quantitative) of methyl 4-formyl-1-

naphthoate as a white solid. ¹H NMR (CDCl₃): δ 4.06 (S, 3H),

7.75 (m, 2H), 8.03 (d, 1H), 8.20 (d, 1H), 8.80 (d, 1H), 9.27 (d,

1H), 10.50 (s, 1H).

4-Formylnaphthalene-1-yloxyacetic acid methyl ester (13 g,

50 mmol) was dissolved in MeOH (100 mL), and 2 M NaOH

(40 mL) was added. The reaction mixture was stirred for 16 h

and concentrated in vacuo to approximately 100 mL. The

residue was poured into ice (500 mL), and the mixture was

acidified with the addition of 3 N HCl. Filtration, washing with

water, and drying afforded 9 g (78%) of 4-formylnaphthalene-

1-yloxyacetic acid, which was used in the next step without

characterization. To a solution of 3-chloro-4-hydroxybenzoic

acid hydrazide (I) (2 g, 10.7 mmol) in DMSO (20 mL) was

added 4-formylnaphthalene-1-yloxyacetic acid (3 g, 13 mmol)

and a catalytic amount of acetic acid (10 drops). The solution

was stirred for 16 h and diluted with EtOAc. The solution was

washed with water (3×) and brine and dried (MgSO₄). The

volume was reduced to approximately 100 mL by evaporation

in vacuo and cooled to 0 °C. The resulting solid was filtered

and washed with cold EtOAc to afford 4 g (99%) of {4-[(3-

chloro-4-hydroxybenzoyl)hydrazonomethyl]naphth-1-yloxy}-

1

acetic acid (XI). H NMR (DMSO-d₆): δ 4.91 (s, 2H), 6.95 (d,

1H), 7.02 (d, 1H), 7.55 (t, 1H), 7.64 (t, 1H), 7.74 (d, 1H), 7.92

(d, 1H), 8.27 (d, 1H), 8.90 (m, 2H), 10.92 (bs, 1H), 11.63 (s,

1H), 13.14 (bs, 1H).

{4-[(3-Ch lor o-4-h yd r oxyb en zoyl)h yd r a zon om et h yl]-

in d ol-1-yl}a cetic Acid (XII). Under a nitrogen atmosphere,

methyl indole-4-carboxylate (2 g, 11.4 mmol) was dissolved in

anhydrous THF (20 mL). The solution was cooled in an ice

bath, and LiAlH₄(12 mL, 1 M/THF, 12 mmol) was added

dropwise. The reaction mixture was stirred for 2 h at room

temperature, diluted with Et₂O, and washed with water (3×)

and brine, dried (MgSO₄), and filtered. Evaporation of the

solvent gave the crude product, which was purified by column

chromatography using 25% ethyl acetate/hexanes. This af-

forded 1.6 g (97%) of 4-(hydroxymethyl)indole. The reaction

was repeated with 6 g of the ester, and 5.6 g (78%) of the

product was isolated. ¹H NMR (DMSO-d₆): δ 4.98 (s, 2H), 6.66

(m, 1H), 7.13 (d, 1H), 7.18 (m, 1H), 7.21 (d, 1H), 7.33 (d, 1H,),

8.50 (s, 1H).

4-Hydroxymethylindole (5.6 g, 38.6 mmol) was dissolved in

a minimum volume of EtOAc (125 mL). To this, MnO₂(10 g,

116 mmol) was added, and the reaction mixture was refluxed

for 10 h. After cooling, the reaction mixture was filtered and

concentrated in vacuo to afford 4.9 g (89%) 4-formylindole. ¹H

NMR (DMSO-d₆): δ 7.10 (s, 1H), 7.30 (t, 1H), 7.64 (m, 2H),

7.76 (d, 1H), 10.18 (s, 1H), 11.61 (s, 1H).

A mixture of methyl 4-formyl-1-naphthoate (2.3 g, 1 mmol)

and Na₂CO₃(1.25 g, 12 mmol) in water (30 mL) was heated

at 100 °C until a clear solution was obtained (approximately

2 h). The solution was cooled and filtered, and the filtrate was

acidified with concentrated HCl. The solid was isolated by

filtration and dried for 16 h to afford 1.86 g (87%) of 4-formyl-

1-naphthoic acid. ¹H NMR (DMSO-d₆): δ 7.76 (m, 2H), 8.22

(m, 2H), 8.71 (d, 1H), 9.20 (d, 1H), 10.49 (s, 1H).

To a solution of 3-chloro-4-hydroxybenzoic acid hydrazide

(I) (1.53 g, 8.23 mmol) in DMSO (20 mL) was added a solution

of 4-formyl-1-naphthoic acid (1.65 g, 8.23 mmol) in DMSO (2

mL). After the solution was stirred for 16 h, the reaction

mixture was diluted with EtOAc (30 mL) and water (30 mL).

To a solution of 4-formylindole (1 g, 7 mmol) and ethyl

bromoacetate (1.75 g, 10.5 mmol) in DMSO (20 mL) was added

Optimization of Glucagon Receptor Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5773

K₂CO₃(2 g, 14.4 mmol). The reaction mixture was stirred at

room temperature for 12 h, and water (100 mL) was added.

The mixture was extracted with EtOAc (3 × 50 mL), dried

(MgSO₄), and concentrated in vacuo. The residue was purified

by column chromatography using hexane/ethyl acetate (2:1)

as eluent to afford 1.55 g (96%) of ethyl (4-formylindol-1-yl)-

acetate. ¹H NMR (CDCl ₃): δ 1.13 (t, 3H), 4.15 (q, 2H), 4.86 (s,

2H), 7.22-7.35 (m, 3H), 7.49 (d, 1H), 7.60 (d, 1H), 10.20 (s,

1H).

Ethyl (4-formylindol-1-yl)acetate (1 g, 4.9 mmol) was dis-

solved in MeOH (50 mL), 5% aqueous KOH (10 mL) was added,

and the resulting mixture was stirred at room temperature

for 4 h. The reaction mixture was concentrated in vacuo to

one-third of the original volume, diluted with aqueous KOH

(20 mL, 10%), and washed with Et₂O (2 × 30 mL). The aqueous

layer was acidified by the addition of concentrated HCl. This

afforded, after filtration and drying, 0.9 g (90%) of (4-

formylindol-1-yl)acetic acid. ¹H NMR (DMSO-d₆): δ 5.15 (s,

2H), 7.12 (d, 1H), 7.36 (d, 1H), 7.61 (d, 1H), 7.71 (d, 1H), 7.82

(d, 1H), 10.20 (s, 1H), 12.94 (brd s, 1H).

tography using EtOAc/hexane (5:95) and recrystallized from

MeOH to afford 1.75 g (27%) of 4-nitro-1-methylnaphthalene

as yellow needles. ¹H NMR (CDCl₃): δ 2.79 (s, 3H), 7.38 (d,

1H), 7.65-7.73 (m, 2H), 8.10 (d, 1H), 8.14 (d, 1H), 8.61 (d, 1H).

To a stirred refluxing solution of sulfur (3.7 g) in 12%

aqueous NaOH (50 mL) was added a solution of 4-nitro-1-

methylnaphthalene (8 g, 43 mmol) in EtOH (50 mL). After the

mixture was refluxed for 1 h, the mixture was diluted with

EtOAc (500 mL) and washed with water and brine, dried

(MgSO₄), and concentrated in vacuo. The residue was purified

via silica gel column chromatography using EtOAc/hexane (5:

95 to 10:90) to afford 2.54 g (34%) of 4-amino-1-naphthalde-

hyde (XIII). The product was stored at -78 °C. ¹H NMR

(DMSO-d₆): δ 6.55 (d, 1H), 6.95 (bs, 2H), 7.25 (t, 1H), 7.45 (t,

1H), 7.60 (d, 1H), 8.05 (d, 1H), 9.10 (d, 1H), 9.68 (s, 1H).

To a solution of 4-amino-1-naphthaldehyde (210 mg, 1.12

mmol) and 4-(dimethylamino)pyridine (410 mg, 3.36 mmol) in

EtOAc (3 mL) was added cyclopentanecarbonyl chloride (2 mL,

15 mmol). After the mixture was stirred for 12 h at room

temperature, the mixture was diluted with EtOAc and washed

with 1 N HCl (3×) and brine (3×), dried (MgSO₄), filtered, and

concentrated in vacuo. The residue was purified by column

chromatography on silica gel using EtOAc/hexane (1:9) to

afford 147 mg (45%) of N-(4-formyl-1-naphthyl)cyclopentane-

carboxamide. ¹H NMR (CDCl₃): δ 1.71-1.74 (m, 2H), 1.84-

1.89 (m 2H), 2.01-2.10 (m, 4H), 2.91 (quintet, 1H), 7.64-7.73

(m, 2H), 7.89 (d, 1H), 8.00 (d, 1H), 8.46 (d, 1H), 9.44 (d, 1H),

10.29 (s, 1H).

Condensation of (4-formylindol-1-yl)acetic acid with 3-chloro-

4-hydroxybenzoic acid hydrazide (I) according to the general

procedure for the synthesis of alkylidene hydrazides afforded

4-[(3-chloro-4-hydroxybenzoyl)hydrazonomethyl]indol-1-yl ace-

tic acid (XII). ¹H NMR (DMSO-d₆): δ 5.09 (s, 2H), 7.09 (d, 1H),

7.16-7.25 (m, 2H), 7.32 (d, 1H), 7.45-7.55 (m, 2H), 7.81 (d,

1H), 8.01 (d, 1H), 8.68 (s, 1H), 10.96 (s, 1H), 11.71 (s, 1H),

12.90 (b, 1H).

Libr a r y P r ep a r a tion P r oced u r e. To a solution of a

derivative of alkylidene hydrazide carboxylic acid (IX-XII) in

DMSO was added carbonyldiimidazole (1.2 equiv). The solution

was agitated for 5 min and diluted with DMSO to a concentra-

tion of 50 mM. The solution was then dispensed into 88 deep-

well plates containing solutions of amines in DMSO (50 mM).

The plates were covered and agitated for 16 h. The products

were purified by HPLC.

Scale-Up P r ocedu r e. 2-{4-[(3-Cyan o-4-h ydr oxyben zoyl)-

h yd r a zon om et h yl]n a p h t h a len -1-yl}-N,N-d iet h yla cet a -

m id e (29). To a solution of 4-formylnaphthalene-1-ylacetic acid

(2.5 g, 11.6 mmol) in DMF (10 mL) was added CDI (2.5 g, 15.4

mmol). The mixture was stirred at room temperature for 1 h,

and freshly distilled diethylamine (1.3 mL, 12.5 mmol) was

added. After the mixture was stirred at room temperature for

16 h, EtOAc (100 mL) was added and the mixture was washed

with H₂O (2 × 50 mL), 0.5 N HCl (50 mL) and brine (2 × 50

mL), dried (MgSO₄), and concentrated in vacuo. Flash chro-

matography (hexane/EtOAc 1:1) afforded 1.6 g (50%) of N,N-

diethyl-4-formylnaphthalene-1-ylacetamide. ¹H NMR (CDCl₃):

δ 1.20 (t, 6H), 3.37 (q, 2H), 3.47 (q, 2H), 4.19 (s, 2H), 7.53 (d,

1H), 7.71 (m, 2H), 7.94 (d, 1H), 8.01 (d, 1 H), 9.33 (d, 1H),

10.36 (s, 1H).

N,N-Diethyl-4-formylnaphthalene-1-ylacetamide (1.6 g, 5.92

mmol) was dissolved in DMSO (10 mL), and 3-cyano-4-

hydroxybenzoylhydrazide (1.05 g, 5.92 mmol) in DMSO (5 mL)

and a catalytic amount of HOAc were added. After the mixture

was stirred at room temperature for 16 h, water was added

and the precipitate was filtered and washed with water (3 ×

10 mL) and DCM (3 × 10 mL) to afford 2.33 g (92%) of 2-{4-

[(3-cyano-4-hydroxybenzoyl)hydrazonomethyl]naphthalen-1-

yl}-N,N-diethylacetamide (29), mp 265-268 °C. ¹H NMR

(DMSO-d₆): δ 1.08 (3H, t), 1.18 (3H, t), 3.35 (2H, q), 3.49 (2H,

q), 4.20 (2H, s), 7.16 (1H, d), 7.45 (1H, d), 7.65 (3H, m), 7.89

(1H, d), 8.01 (1H, d), 8.12 (1H, d), 8.30 (1H, s), 8.97 (1H, d),

9.10 (1H, s), 11.9 (1H, bs). Anal. (C₂₅H₂₄N₄O₃‚0.5H₂O) C,

H, N.

P r ep a r a tion Meth od F . Cyclop en ta n eca r boxylic Acid

{4-[(3-Ch lor o-4-h yd r oxyb e n zoyl)h yd r a zon om e t h yl]-

n a p h th a len -1-yl}a m id e (54). To a cold (0 °C) suspension of

1-methylnaphthalene (5 g, 35 mmol) in HNO₃was added H₂-

SO₄(5 mL) dropwise. After the reaction mixture was stirred

for 1 h at room temperature, the solution was diluted with

EtOAc and washed with water (3×), aqueous saturated

NaHCO₃(2×), and brine, dried (MgSO₄), and concentrated in

vacuo. The residue was purified by silica gel column chroma-

To a solution of 4-hydroxy-3-chlorobenzoic acid hydrazide

(I) (100 mg, 0.55 mmol) in DMSO (3 mL) was added N-(4-

formyl-1-naphthyl)cyclopentanecarboxamide (147 mg, 0.55

mmol) followed by a catalytic amount of acetic acid. After the

mixture was stirred for 16 h, the mixture was diluted with

ethyl acetate and washed with water (2×), dried (MgSO₄), and

concentrated in vacuo to about 10 mL. The mixture was

filtered and dried to afford 190 mg (36%) of cyclopentanecar-

boxylic acid {4-[(3-chloro-4-hydroxybenzoyl)hydrazonomethyl]-

naphthalen-1-yl}amide (54), mp >250 °C. ¹H NMR (DMSO-

d₆): δ 1.61 (m, 2H), 1.71 (m 2H), 1.82 (m, 2H), 1.90 (m, 2H),

3.06 (quintet, 1H), 7.10 (d, 1H), 7.65 (quintet, 2H), 7.83 (qt,

2H), 7.90 (d, 1H), 8.02 (s, 1H), 8.18 (d, 1H), 8.90 (d, 1H), 9.04

(s, 1H), 10.01 (s, 1H), 10.99 (s, 1H), 11.78 (s, 1H). Anal. (C₂₄H₂₂

ClN₃O₃) C, H, N.

-

P r ep a r a tion Meth od G. N-{4-[(3-Ch lor o-4-h yd r oxyben -

zoyl)h yd r a zon om et h yl]-3-m et h oxyp h en yl}-2-(4-t r iflu -

or om eth ylp h en yl)p r op ion a m id e (60). Methyl 4-amino-2-

methoxybenzoate (14.7 g, 81 mmol) and Fmoc-OSu (26.1 g, 77.3

mmol) were stirred in a mixture of MeCN and water (1:1, 320

mL) at reflux for 16 h. The reaction mixture was concentrated

in vacuo to half the volume, and the precipitate was isolated

by filtration. The isolated solid was dissolved in EtOAc (300

mL) and washed with 0.4 N HCl (200 mL), 0.2 N HCl (200

mL), water (200 mL), and a 20% saturated solution of sodium

chloride (200 mL). After the mixture was dried (MgSO₄), the

organic phase was evaporated in vacuo. The residue was

washed with MeOH and dried. The crude product (12 g) was

dissolved in DCM (1 L) under nitrogen, and a solution of

diisobutylaluminum hydride (90 mL, 1.2 M in toluene) was

added dropwise at 0-5 °C. The reaction mixture was stirred

at 20 °C for 16 h and quenched by dropwise addition of water

(58 mL) at 0-5 °C. The reaction mixture was stirred at 20 °C

for 3 h and filtered. The filtrate was concentrated in vacuo.

The crude product (6.8 g) was suspended in DCM (400 mL),

and MnO₂(15.6 g, 180 mmol) was added. The mixture was

stirred for 16 h at 20 °C and filtered. The filtrate was

concentrated in vacuo to afford 5.1 g (17%) of (4-formyl-3-meth-

oxyphenyl)carbamic acid 9H-fluoren-9-ylmethyl ester, mp

187-188 °C. Anal. (C₂₃H₁₉NO₄) C, H, N.

A sample of 0.75 g of Wang resin loaded with 3-chloro-4-

hydroxybenzoic acid hydrazide (I) was swelled in DMF (6 mL)

for 30 min and filtered. (4-Formyl-3-methoxyphenyl)carbamic

acid 9H-fluoren-9-ylmethyl ester (0.5 g, 1.36 mmol) dissolved

in DMF (3 mL) was added followed by addition of triethyl

orthoformate (1.5 mL). The mixture was shaken for 16 h at

5774 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26

Madsen et al.

20 °C and drained. The resin was successively washed with

DMF (5 × 4 mL), DCM (5 × 4 mL), and DMF (5 × 4 mL). The

coupling of the aldehyde was repeated once. The resin was

swelled in DMF (5 mL), and piperidine (1.25 mL) was added.

After being shaken for 30 min, the resin was drained and

successively washed with DMF (5 × 4 mL), NMP (5 × 4 mL),

and DMF (5 × 4 mL). The resin-bound 3-chloro-4-hydroxy-

benzoic acid (4-amino-2-methoxybenzylidene)hydrazide (0.5 g)

was swelled in DMF (2.5 mL) and filtered. 3-(4-Trifluorometh-

ylphenyl)propionic acid (0.5 g, 2.3 mmol) was dissolved in DMF

(2 mL) together with diisopropylcarbodiimide (DIC) (1.13

mmol), and after 10 min, this mixture was added to the

drained resin. After 30 min of shaking, a 1 M solution of DMAP

in DMF (0.32 mL) was added and the mixture was shaken for

16 h and filtered. The resin was successively washed with DMF

(5 × 4 mL) and DCM (5 × 4 mL). Coupling with the acid was

repeated once. The resin was swelled in DCM (2 mL), and TFA

(2 mL) was added. After being shaken for 30 min, the resin

was filtered. The filtrate was collected and concentrated in

vacuo. The residue was crystallized from MeOH to afford

64 mg (27%) of N-{4-[(3-chloro-4-hydroxybenzoyl)hydrazono-

methyl]-3-methoxyphenyl}-2-(4-trifluoromethylphenyl)pro-

with EtOAc (125 mL), and washed with water (100 mL). The

aqueous phase was extracted with EtOAc (100 mL), and the

combined organic phases were dried (MgSO₄) and concentrated

in vacuo. The crude product was crystallized from MeOH/DCM

(1:9) to afford 0.5 g (50%) of N-{4-[(3-cyano-4-hydroxybenzo-

yl)hydrazonomethyl]-3-methoxyphenyl}-2-(3-trifluoromethyl-

phenyl)acetamide (59), mp 257-260 °C. ¹H NMR (DMSO-

d₆): δ 11.8 (s, 1H), 11.7 (s, 1H), 10.5 (s, 1H), 8.7 (s, 1H), 8.2 (s,

1H), 8.05 (dd, 1H), 7.8 (d, 1H), 7.7 (s, 1H), 7.65-7.5 (m, 4H),

7.15 (d, 1H), 7.1 (d, 1H), 3.8 (s, 5H).

P r ep a r a tion Meth od I. Libr a r y P r oced u r e. 4-Formylin-

dole (1.45 g, 10 mmol) was dissolved in dry DMF (8.6 mL).

With an atmosphere of nitrogen or argon over the solution,

NaH (0.27 g of 95% dry reagent, 1.1 equiv) was transferred to

the indole solution. The mixture was stirred for 15 min. Amber

glass vials (for preparing stock solutions) were dried for at least

4 h at 110 °C, then were allowed to cool under an argon

atmosphere in a desiccator. Halide solutions (1.0 M) were

prepared in anhydrous DMF in the dried vials. Each halide

solution (100 µL) was added to its corresponding well of a deep-

well plate. An amount of 100 µL of the 1.0 M indole salt

solution was quickly delivered to each halide in the deep-well

plates. The plates were vortexed briefly to mix, then allowed

to stand for 2 h. 3-Substituted 4-hydroxybenzoic acid hy-

drazides (10 mmol) were dissolved in 5 mL of dry DMSO,

followed by TFA (0.77 mL). The resulting solutions were

diluted to final volumes of 10.0 mL. The hydrazide solutions

(100 µL) were added to each well of the deep-well plate. The

plates were vortexed for 1 min to mix and then allowed to

stand for 30 min. The products were purified by chromatog-

raphy on silica gel with EtOAc/hexane eluent.

1

pionamide (60), mp 233-235 °C. H NMR (DMSO-d₆): δ 2.72

(2H, t), 3.03 (2H, t), 3.84 (3H, s), 7.07 (1H, d), 7.18 (1H, d), 7.5

(3H, m), 7.69 (2H, d), 7.78 (2H, m), 7.99 (1H, s), 8.70 (1H, s),

10.2 (1H, s), 10.9 (1H, s), 11.7 (1H, s). Anal. (C₂₅H₂₁ClF₃N₃O₄‚

¹/₄H₂O) C, H, N.

P r ep a r a tion Meth od H. N-{4-[(3-Cya n o-4-h yd r oxy-

b en zoyl)h yd r a zon om et h yl]-3-m et h oxyp h en yl}-2-(3-t r i-

flu or om eth ylp h en yl)a ceta m id e (59). DIC (8.1 g, 42 mmol)

was added to a solution of 3-(trifluoromethyl)phenylacetic acid

(17 g, 83 mmol) in DCM (50 mL). After 10 min, methyl

4-amino-2-methoxybenzoate (5.0 g, 28 mmol) was added and

the mixture was stirred at reflux for 4 h and at 20 °C for 16 h.

The mixture was diluted with DCM (100 mL) and extracted

with a saturated solution of sodium hydrogen carbonate (3 ×

50 mL) and water (3 × 50 mL). The organic phase was dried

(MgSO₄), filtered, and evaporated in vacuo. The residue was

purified by column chromatography on silica gel using hep-

tane/EtOAc (3:2) as eluent to afford 8 g (78%) of 2-methoxy-

4-[2-(3-trifluoromethylphenyl)acetetylamino]benzoic acid meth-

yl ester. ¹H NMR (DMSO-d₆): δ 10.5 (s, 1H), 7.7-7.5 (m, 6H),

Sca le-Up P r oced u r e. 3-Cya n o-4-h yd r oxyben zoic Acid

[1-(2,3,5,6-Tetr a m eth ylben zyl)-1H-in d ol-4-ylm eth ylen e]-

h yd r a zid e (74). To a solution of 4-formylindole (4 g, 28 mmol)

in DMF at 0 °C was added sodium hydride (1.2 g, 60%, 30

mmol). The mixture was stirred for 30 min, and 2,3,5,6-

tetramethylbenzyl chloride (6.13 g, 33.6 mmol) was added. The

mixture was stirred for 2 h, diluted with EtOAc, washed with

H₂O (3×) and brine (3×), dried (MgSO₄), filtered, and concen-

trated in vacuo. The residue was purified via column chroma-

tography using ethyl acetate/hexane (1:9) to afford 5.9 g (71%)

1

of 4-formyl-1-(2,3,5,6-tetramethylbenzyl)-1H-indole. H NMR

7.2 (d, 1H), 3.85 (s, 2H), 3.75 (s, 3H), 3.7 (s, 3H). Anal. (C₁₈H₁₆

NO₄) C, H, N.

-

(CDCl₃): δ 2.15 (s, 6H), 2.30 (s, 6H), 5.35 (s, 2H), 6.84 (d, 1H),

7.08 (s, 1H), 7.20 (s, 1H), 7.42 (t, 1H), 7.69 (d, 1H), 7.81 (d,

1H), 10.27 (s, 1H).

2-Methoxy-4-[2-(3-trifluoromethylphenyl)acetylamino]ben-

zoic acid methyl ester (2.0 g, 5.4 mmol) was dissolved in dry

DCM (100 mL) under nitrogen and cooled to -20 °C. Diisobu-

tylaluminum hydride (1.2 M in toluene, 16 mL, 18.9 mmol)

was added dropwise over 40 min. The reaction mixture was

warmed to 20 °C and stirred at this temperature for 2 h. After

dilution with DCM (100 mL), the reaction mixture was

quenched by dropwise addition of water (10 mL) at 20-25 °C.

After 16 h, the mixture was filtered and the organic phase

was dried (MgSO₄), filtered, and concentrated in vacuo. The

residue was purified by column chromatography on silica gel

using heptane/EtOAc (3:2) as eluent to afford 0.7 g (37%) of

N-(4-hydroxymethyl-3-methoxyphenyl)-2-(3-trifluoromethyl-

To a solution of 3-cyano-4-hydroxybenzoic acid hydrazide

(IV) (0.84 g, 4.7 mmol) in a minimum volume of DMSO was

added 4-formyl-1-(2,3,5,6-tetramethylbenzyl)-1H-indole (1.2 g,

4.2 mmol) and a catalytic amount of HOAc (five drops). The

mixture was stirred overnight under nitrogen and diluted with

EtOAc. The organic phase was washed with saturated NaH-

CO₃, water, and brine and dried (MgSO₄). Upon concentration

of the solvent in vacuo, 1.7 g (83%) of 3-cyano-4-hydroxybenzoic

acid [1-(2,3,5,6-tetramethylbenzyl)-1H-indol-4-ylmethylene]hy-

drazide (74) crystallized out of solution, mp >250 °C. ¹H NMR

(DMSO-d₆): δ 2.08 (s, 6H), 2.21 (s, 6H), 5.37 (s, 2H), 6.78 (s,

1H), 7.05 (m, 3H), 7.26 (t, 1H), 7.33 (d, 1H), 7.76 (d, 1H), 8.01

(d, 1H), 8.19 (s, 1H), 8.64 (s, 1H), 11.68 (s, 1H). Anal.

(C₂₈H₂₆N₄O₂‚¹/₄H₂O) C, H, N.

Biologica l Assa ys. Bin d in g a n d F u n ction a l Assa ys. Re-

ceptor binding was carried out using membranes from BHK

cells expressing the cloned human glucagon receptor. The

assay was carried out as earlier described²⁰with some modi-

fications. The buffer was 50 mM HEPES, 5 mM EGTA, 5 mM

MgCl₂, 0.005% Tween 20, pH 7.4, and compounds were diluted

in 100% DMSO. A total of 140 (for standard curve) or 165 µL

of buffer was added to wells. A total of 10 µL samples in DMSO

or DMSO (for standard curve) were added, and 25 µL of

different glucagon concentrations were added. The 50.000 CPM

tracer was added in 25 µL, and 4 µg of plasma membrane was

added in 25 µL. Incubation was for 2 h at 30 °C. The functional

assay was carried out as previously described.²⁰

1

phenyl)acetamide. H NMR (DMSO-d₆): δ 10.2 (s, 1H), 7.7-

7.5 (m, 4H), 7.35 (s, 1H), 7.25 (d, 1H), 7.1 (d, 1H), 4.9 (t, 1H),

4.4 (d, 2H), 3.8 (s, 2H), 3.7 (s, 3H).

N-(4-Hydroxymethyl-3-methoxyphenyl)-2-(3-trifluorometh-

ylphenyl)acetamide (0.7 g, 2 mmol) was dissolved in EtOAc

(40 mL), and MnO₂(3 g, 34 mmol) was added. The reaction

mixture was stirred at 20 °C for 3 h and filtered. The organic

phase was concentrated in vacuo to give crude 0.67 g (99%) of

N-(4-formyl-3-methoxyphenyl)-2-(3-trifluoromethylphenyl)ac-

etamide, which was used for the next step without further

purification. ¹H NMR (DMSO-d₆): δ 10.65 (s, 1H), 10.2 (s, 1H),

7.7-7.5 (m, 6H), 7.2 (d, 1H), 3.85 (s, 5H).

N-(4-Formyl-3-methoxyphenyl)-2-(3-trifluoromethylphenyl)-

acetamide (0.67 g, 2 mmol) was dissolved in DMSO (10 mL).

3-Cyano-4-hydroxybenzoic acid hydrazide (IV) (0.35 g, 2 mmol)

was added followed by addition of glacial acetic acid (0.3 mL).

The reaction mixture was stirred for 16 h at 20 °C, diluted

P r im a r y Hep a tocytes Assa ys. Hepatocytes were isolated

from male Sprague-Dawly rats fed ad libitum using a two-

Optimization of Glucagon Receptor Antagonists

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step perfusion technique and cultured essentially as previously

described.²¹To study glycogenolysis, hepatocytes were incu-

bated with 15 mM glucose and 10 nM insulin for 24 h to induce

glycogen synthesis. The hepatocytes were washed twice in

prewarmed buffer A (117.6 mM NaCl, 5.4 mM KCl, 0.82 mM

Mg₂SO₄, 1.5 mM KH₂PO₄, 20 mM HEPES, 9 mM NaHCO₃,

0.1% w/v HSA, and 2.25 mM CaCl₂, pH 7.4 at 37 °C) and

incubated with increasing concentrations of 74 (0-1000 nM)

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concentration of glucagon (0-100 nM) in buffer A for 70 min.

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In Vitr o Meta bolism . Meta bolic Ra te of Disa p p ea r -

a n ce. Test compounds were screened in rat liver microsomes

in order to determine the metabolic rate of disappearance.

Incubation times were 0, 5, 10, 10, 30 min (n ) 3), total

incubation volume was 150 µL, protein content was 0.331 mg/

mL, the incubation mixture contained 1 mM UDP-GA, 1 mM

NADPH, KH₂PO₄(pH 7.4) buffer up to 150 µL, and the

compound concentration was 10 µM. Incubation temperature

was 37 °C. All incubations were performed in a 96-well plate

format, and a Packard liquid handler was applied for incuba-

tions and liquid handling in general. Microsomal incubations

were terminated by addition of 150 µL of MeCN. The metabolic

rates were determined via LC-MS analysis (disappearance

of parent).

Meta bolic P r ofilin g. Incubation times were 0 and 60 min

(n ) 3), total incubation volume was 1000 µL, protein content

was 1 mg/mL, compound concentration was 25 µM, and

incubation temperature was 37 °C. The incubation mixture

contained 1 mM UDP-GA, 1 mM NADPH, and KH₂PO₄(pH

7.4) buffer up to 1000 µL. Microsomal incubations were

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SP E Meth od . SPE column: SPEC (C₈) SPE cartridge.

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Sample volume: 1000 µL. Washing: 2 × 1000 µL of NaHPO₄

(pH 7.4). Eluate: 1000 µL of MeOH. The three samples (LC-

MS) at each time point were pooled, and the solvent was

evaporated under N₂gas at 40 °C. A total of 250 µL of mobile

phase was then added to the evaporated sample and analyzed

by LC-MS.

Ch r om a togr a p h y. A reversed-phase chromatographic col-

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mL/min. Eluent A consisted of 5% MeCN in ammonium acetate

(20 mM). Eluent B consisted of 21% MeCN in ammonium

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A, which was increazed to 100% B in 5 min.

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pump 200LC and a UV detector APB 785A (operating at 254

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running with a turbo Ionspray interphase (Thornhill, Canada)

was also used.

Ack n ow led gm en t. The skillful technical assistance

of Annette Nielsen, Paw Bloch, Dorthe E. J ensen, Sanne

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Karin H. Albrechtsen, J ørgen S. J ensen, Lone Rise-

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J M0208572

Article Doi

Optimization of alkylidene hydrazide based human glucagon receptor antagonists. Discovery of the highly potent and orally available 3-cyano-4-hydroxybenzoic acid [1-(2,3,5,6-tetramethylbenzyl)-1h-indol-4ylmethylene]hydrazide

DOI: 10.1021/jm0208572

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Authors:

Article abstract of DOI:10.1021/jm0208572

Full text of DOI:10.1021/jm0208572

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