Discovery and development of a new class of potent, selective, orally active oxytocin receptor antagonists

7882

J. Med. Chem. 2005, 48, 7882-7905

Discovery and Development of a New Class of Potent, Selective, Orally Active

Oxytocin Receptor Antagonists

Anna Quattropani,^†Je´roˆme Dorbais,^†David Covini,^†Pierre-Andre´ Pittet,^†Ve´ronique Colovray,^†

Russell J. Thomas,^|,XRichard Coxhead,^|Serge Halazy,^†Alexander Scheer,^‡Marc Missotten,^‡Guidon Ayala,^‡

Charles Bradshaw,^‡Anne-Marie De Raemy-Schenk,^‡Anthony Nichols,^‡Rocco Cirillo,^§Enrico Gillio Tos,^§

Claudio Giachetti,^#Lucia Golzio,^#Paolo Marinelli,^#,3Dennis J. Church,^†Claude Barberis, Andre´ Chollet,^†and

Matthias K. Schwarz*^,†

Serono Pharmaceutical Research Institute, Departments of Chemistry and Biochemical Pharmacology, 14 Chemin des Aulx,

1228 Plan-Les-Ouates, Geneva, Switzerland; Istituto di Ricerche Biomediche “A. Marxer”, Departments of Pharmacology and

DMPK, Colleretto Giacosa, Italy; Evotec OAI, Milton Park, Abingdon, Oxon OX14 4SD, U.K; and Institut National de la Sante´

et de la Recherche Me´dicale U469, Montpellier, France

Received July 7, 2005

We report a novel chemical class of potent oxytocin receptor antagonists showing a high degree

of selectivity against the closely related vasopressin receptors (V1a, V1b, V2). An initial

compound, 7, was shown to be active in an animal model of preterm labor when administered

by the intravenous but not by the oral route. Stepwise SAR investigations around the different

structural elements revealed one position, the arenesulfonyl moiety, to be amenable to structural

changes. Consequently, this position was used to introduce a variety of substituents to improve

the physicochemical properties. Some of the resulting analogues were found to be superior to

7 both in terms of potency in vitro and aqueous solubility, which translated into significantly

improved efficacy in the animal model after intravenous and oral administration. The best

compound, 73, potently inhibited oxytocin-induced uterine contractions in nonpregnant rats

and reduced spontaneous uterine contractions in late-term pregnant rats.

Introduction

manufacturer. The peptide hormone oxytocin, OT (1)

(Figure 1), is a potent contractor of the human uterus,

mediating its effect through activation of the G protein-

coupled oxytocin receptor, OT-R, that is expressed in

myometrial cells. Despite some controversial experi-

mental evidence,^8,9oxytocin is generally considered to

be a key mediator of uterine contractile activity in the

initiation and maintenance of term and preterm labor.

Consequently, strategies aimed at inhibiting oxytocin

action by blockade of its receptor have emerged as a

promising new approach to maintain uterine quiescence.

Clinical validation of this concept has come from the

peptidic OT-R antagonist atosiban (2),^10-12shown to be

effective in the treatment of imminent preterm birth,^13,14

and later approved in several European countries under

the tradename of Tractocile. Compared to ritodrine,

atosiban shows an improved maternal and fetal side-

effect profile, but its peptidic nature requiring constant

infusion,¹⁵as well as its poor selectivity toward the

closely related vasopressin (AVP) receptor V1a, limits

its use to short-term treatment of the acute phase of

preterm labor.

In an attempt to improve on the shortcomings of the

first generation peptide antagonists, several companies

started small molecule drug discovery programs aimed

at identifying orally active OT-R antagonists with a

higher degree of selectivity toward the AVP receptors

(V1a, V1b, V2). As a result, a number of distinct

chemical series of selective OT-R antagonists with

single-digit nanomolar potencies have emerged over the

past decade, and these have recently been reviewed by

us in some detail.¹⁶The most successful campaign,

reported by Merck, has produced advanced compounds

Premature birth is a major problem in obstetrics,

affecting about 10% of all pregnancies and constituting

the largest cause of perinatal morbidity and mortality.

The impact on society is significant, due to the high costs

associated with the perinatal care of preterm babies and

the long-term health complications often encountered

at a later stage. Although the detailed etiology of

preterm (and term) labor remains to be elucidated, the

final expression of the pathology is a dramatic increase

in uterine contractions. Current therapeutic strategies

for the pharmaceutical management of preterm labor

thus focus primarily on maintaining uterine quiescence

(tocolysis). Among the tocolytic agents historically used

in the clinic, only the â2-adrenergic agonist ritodrine

was ever approved by the FDA for the therapeutic

treatment of preterm labor. However ritodrine suffers

from moderate effectiveness^1-3and lack of uterine

selectivity, causing important fetal and maternal side

effects.^4-7Consequently, it has been withdrawn by its

* To whom correspondence should be addressed. Phone:

+41227069820. Fax: +41227069565. E-mail: Matthias.Schwarz@

serono.com.

^†Department of Chemistry, Serono Pharmaceutical Research In-

stitute.

^‡Department of Biochemical Pharmacology, Serono Pharmaceutical

Research Institute.

^§Departments of Pharmacology, Istituto di Ricerche Biomediche “A.

Marxer”.

^#Department of DMPK, Istituto di Ricerche Biomediche “A. Marx-

er”.

^|Evotec OAI.

Institut National de la Sante´ et de la Recherche Me´dicale U469.

^XCurrent address: Sienabiotech S.p.A Via Fiorentina 1, Siena,

53100, Italy.

³Current address: RCC Ltd., Zelgliweg 1, CH-4452 Itingen.

Published on Web 11/01/2005

Oxytocin Receptor Antagonists

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7883

Figure 1. Structures of oxytocin, the peptide oxytocin receptor antagonist Atosiban, the nonpeptide oxytocin receptor antagonists

L-368,899 and L-372,662, the arginine vasopressin receptor V1a antagonist SR-49059, and a new sulfanilide compound identified

as a primary screening hit.

such as L-368,899 (3)^17-19and L-372,662 (4),^20,21which

have set the standard for all competitor compounds by

combining excellent potency and selectivity in vitro with

remarkable physicochemical and ADME-properties, as

well as in vivo efficacy in rat and rhesus monkey models

of preterm labor. The structural variety of OT-R an-

tagonists reported in the literature facilitates the design

of screening sets enriched with structural motifs likely

to confer activity at the OT-R, using, e.g., the compu-

tational approaches we have reported on previously.^22-25

In addition, there are a number of companies who, in

the context of their antagonist programs directed at the

AVP receptors, have identified compounds that show

residual activity at the OT-R, such as Sanofi’s potent

V1a antagonist SR-49059 (5) (hOTR: K_i) 130 nM;

hV1a: K_i) 2 nM),²⁶and these provide further struc-

tural information useful in the context of OT-R directed

set design. As an illustration, Sanofi has recently

reported on a structural analogue of 5 showing selectiv-

ity toward the OT-R.²⁷

We set out to develop a new class of oxytocin receptor

antagonists with the aim of achieving a superior selec-

tivity profile against the vasopressin receptors, com-

bined with oral efficacy in animal models of preterm

labor. The results of this study are detailed in the

present report.

Primary Screening Results and Initial SAR

Information. In-house screening of a primary library

designed to have a general bias toward G protein-

coupled receptors, with an additional emphasis on the

OT-R, revealed a number of compounds that potently

inhibited radioligand binding to the receptor, among

them the hydrazone sulfanilide compound 6 (hOT-R: K_i

) 90 nM), presenting a remote similarity to the Sanofi

V1a antagonist 5. In contrast to the latter, however,

compound 6 appeared to be selective for the OT-R (hOT-

R: K_i) 90 nM; hV1a: < 50% inhib @ 10 µM).

Preliminary SAR information, summarized in Table 1,

was obtained by means of a secondary, focused screen-

ing campaign against hOT-R and V1a involving around

900 commercial analogues of 6, which was to serve as a

basis for the subsequent medicinal chemistry efforts. As

can be seen from Table 1, a variety of substituent types

are accepted in the Eastern part (R1), giving rise to

three distinct subseries, namely, hydrazones (6-10),

secondary anilides (11-16), and tertiary amides (17-

20), characterized by similar potencies when comparing

equivalent compounds with the same substitution pat-

tern in terms of R2 and R3 (e.g., 8a, 12a, 17b).

Importantly, however, within a given subseries, the

potency is found to vary considerably as a function of

R1, as illustrated by the comparison between the

hydrazone compounds 6-8 versus 9a-c, which indi-

cates that the presence of a hydrogen bond donor (HBD)

within the arylhydrazone moiety is beneficial to activity.

A similar observation can be made for the secondary

amide subseries, in which the potency is strongly

influenced by the aromatic substitution pattern. Thus,

compound 11, derived from an unsubstituted aniline

(likewise for benzylamine, phenethylamine, and simple

alkylamines, data not shown), inhibits the binding of

oxytocin to its receptor by less than 50% at a concentra-

tion of 10 µM. Introduction of a methyl group in the

ortho position or of a chloro substituent in the meta

position increases the affinity to an IC₅₀value of 1200

nM (data not shown), and the combination of both

substituents produces analogues, such as 12a-c, that

are equipotent to the archetype compound 6. Of note,

moving the chloro atom to the para position produces

analogues with a higher affinity to V1a, as illustrated

by compound 14 (hOT-R: K_i) 800 nM; hV1a: K_i) 70

nM, compared to hOT-R: K_i) 100 nM; hV1a: K_i) 300

7884 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Table 1. Preliminary SAR Based on Commercial Analogues

Quattropani et al.

^aValues are reported as means ( SEM for three and more determinations. If number of experiments was less than three, it is shown

in parentheses.

nM for compound 12c). In this report, we focus our

attention on the medicinal chemistry efforts around the

hydrazone subseries.

(as in 13) produces a marked reduction in potency

against both OT-R and V1a. The introduction of R2-

groups other than phenyl, such as phenethyl or even

alkyl, abolishes activity, as shown by the tertiary amide

compounds 18 or 19, respectively, and confirmed by

similar compounds of the other subseries (data not

shown). Consequently, the medicinal chemistry strategy

was built around compounds having para-substituted

phenyl rings at the R2-position.

With respect to the R3-position, the structural diver-

sity present in the set of commercial analogues was

rather limited. Even so, inspection of such compounds

as 7 and 15 indicated that the tolerance in terms of the

As can further be seen from Table 1, the Western part

of the molecule (R2) appears to be less permissive

toward structural changes. In all three subseries, small

groups are accepted in the para-position of the phenyl

ring only (7, 8a,b, 12a-c, 17), whereas increasing the

size of the substituent reduces the affinity toward both

OT-R and V1a (12c,d). Of interest, the introduction of

a substituent in the meta-position reduces the affinity

toward OT-R, but increases it toward V1a (10a,b),

whereas the presence of a para and a meta substituent

Oxytocin Receptor Antagonists

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7885

Scheme 1^a

^aReagents: (a) pyridine, 0 °C to r.t. or DIEA, DCM, r.t.; (b) X ) Br: NaH, DMF, r.t.; then 24a,c,d, DMF, r.t. or 24a,c,d, K₂CO₃, DMF,

60 °C; X ) OH: PPh₃, DEAD, THF, 0 °C to r.t.; then 24b, r.t. (c) Hydrazine hydrate, MeOH, r.t. (d) 27, EtOH/5% AcOH or 100% AcOH,

reflux.

size and position of the aromatic substituents was likely

to be higher than in the R2-position, although nonaro-

matic sulfonyl groups still appeared to be detrimental

to the affinity for both hOT-R and hV1a, as illustrated

by compound 16. Of interest, the introduction of an

ethoxy group in the para-position of the arylsulfonyl

moiety (as in 7) led to an improvement in binding

affinity, rather than a reduction (as previously observed

for 12d). Early on, we therefore considered this position

as a potential point of intervention for modulating the

physicochemical and pharmacokinetic properties of the

series by the introduction of suitable substituents. With

respect to the central, aliphatic moiety of the com-

pounds, no information could be extracted from the set

of commercial analogues, hence the synthesis of a few

cornerstone analogues with varying chain lengths and

different substituents at the alpha-position of the car-

bonyl group constituted an important objective of the

medicinal chemistry campaign.

Compound 7 was considered to be a reasonable

starting point for further optimization work and thus

was characterized in some more detail in order to

identify its major deficiencies and define the objectives

for the medicinal chemistry campaign. The compound

was found to have similar binding affinities to both

human and rat OT-R (hOT-R: K_i) 14 ( 8 nM, rOT-R:

K_i) 25 ( 10 nM), with an encouraging degree of

selectivity over the closely related AVP receptors

(hV1a: K_i) 320 nM, hV1b: K_i> 10000 nM, hV2: K_i)

2500 nM, n ) 2). To ascertain functional antagonism,

the compound was tested in hOT-R transfected HEK293-

cells and shown to inhibit OT-induced Ca²⁺-mobilization

with an IC₅₀value of 10 nM, without any signs of

residual agonist activity up to a concentration of 10 µM.

The major deficiency of compound 7 was soon recognized

to be its very poor solubility in aqueous solvents (<1

µg/mL in H₂O and HBSS buffer), hampering its evalu-

ation in early ADME assays in vitro. As a likely

consequence of this limiting solubility, the oral bioavail-

ability was found to be negligible in rats (F_z/po ) 6%).

Encouragingly though, when slowly infused by the iv

route, compound 7 was found to potently inhibit OT-

induced uterine contractions in anaesthetized, nonpreg-

nant rats, with an ED₅₀value of around 10 mg/kg. With

all of this in mind, a medicinal chemistry program was

launched with the main focus on improving the solubil-

ity, and, as was hoped, concomitantly, the pharmaco-

kinetic properties and efficacy in vivo of the archetype

sulfanilide compound 7.

Chemical Methods. Analogues of the hydrazone

sulfanilide derivative 7 were synthesized following the

general route detailed in Scheme 1. Thus, a substituted

aniline 21 was reacted with an arylsulfonyl chloride 22

in neat pyridine or in DCM in the presence of DIEA, to

afford the corresponding sulfonamide 23. Most of the

anilines 21 and sulfonyl chlorides 22 were commercially

available, or else synthesized according to literature

protocols. To gain access to analogues containing as the

central core an unsubstituted one-carbon spacer (glycine

derivatives), 23 was treated with a strong base, such

as NaH, followed by reaction with 2-bromoacetic acid

methyl ester 24a. The resulting intermediate, 25a (R4

) H, n ) 1), could alternatively be obtained by Mit-

sunobu reaction of 23 with methyl glycolate 24b. Methyl

ester 25a was then converted into the corresponding

hydrazide 26a (R4 ) H, n ) 1) using aqueous hydrazine.

Finally, acid-catalyzed condensation of 26a with carbo-

nyl compounds 27, such as aryl ketones and aldehydes,

afforded the desired acylhydrazones 28a (R4 ) H, n )

1). Carbonyl reagents 27 were either commercially

available or synthesized following procedures described

in the literature. The reaction conditions of each step

were sufficiently optimized to render the whole process

amenable to parallel synthesis.

The same strategy was used to obtain derivatives with

central aliphatic moieties other than glycine, such as

alanine or serine, by replacing 24a with methyl 2-bro-

moproprionate 24c or methyl 2-bromo-3-tert-butoxy-

propionate 24d, respectively. Compound 24c is com-

mercially available, while 24d was prepared from

L-serine tert-butyl ether 29, following the procedure

described in Scheme 2.²⁸Thus, the R-amino acid 29 was

transformed into the corresponding R-bromo acid 30

using sodium nitrite in 0.75 N HBr in the presence of

7886 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Quattropani et al.

Scheme 2^a

acids 34 were coupled to Rink resin 33 using DIC as

coupling agent, followed by removal of the Fmoc pro-

tecting group, and subsequent coupling with bromoace-

tic acid in the presence of DIC, affording the resin-bound

R-bromo acetamides 37. Quantitative substitution of the

bromo group was achieved by treating the resin for 12

h with an excess of anilines 21 in DMSO at room

temperature.^31,32The resulting intermediates 38 were

reacted at 60 °C with sulfonyl chlorides 22 (5 equiv) in

DCE containing N-methyl morpholine. Acid-catalyzed

cleavage afforded the final primary carboxamide deriva-

tives 40, which were tested without further purification.

^aReagents: (a) KBr, NaNO₂, 0.75 M HBr, -7° to -4°C; (b)

TMSCH₂N₂, CHCl₃/MeOH 3:1

KBr, with retention of configuration at the R-carbon

atom²⁸The R-bromo acid 30 was converted into the

corresponding methyl ester 24d using trimethylsilyl

diazomethane. Efficient coupling of both 24c and 24d

with the secondary sulfonamide intermediates 23 was

achieved in the presence of K₂CO₃at 60 °C in DMF, to

afford the corresponding tertiary R-substituted sulfona-

mides 25c (R4 ) Me, n ) 1) and 25d (R4 ) CH₂OtBu,

n ) 1), respectively. The remaining transformations

leading to the final R-substituted products 28c (R4 )

Me, n ) 1) and 28d (R4 ) CH₂OtBu, n ) 1) were

identical to those described in Scheme 1 above.

Derivatives with a two-carbon central aliphatic spacer,

such as 28e (R4 ) H, n ) 2), were obtained via

â-addition of anilines 21 to methyl acrylate 31 (Scheme

3). The resulting secondary amines 32 were reacted with

sulfonyl chlorides 22, to afford the corresponding two-

carbon chain sulfanilides 25e (R4 ) H, n ) 2). The final

two-carbon chain analogues 28e (R4 ) H, n ) 2) were

obtained according to the standard protocols outlined

in Scheme 1 above.

As the inspection of the preliminary SAR had pointed

to a relatively high degree of freedom with respect to

substituents at the para-position of the arylsulfonyl

moiety (R3), a number of analogues substituted at this

position were made with a view to addressing the poor

physicochemical properties of the archetype compound

7. The substituents were introduced at the level of the

secondary sulfonamide intermediates 23, because the

tertiary sulfonamides, such as 25, were found to be

prone to â-elimination of the sulfonyl moiety under basic

conditions. Thus, S_NAr reaction of substituted sodium

alcoholate or lithium amides with para-fluorophenyl

sulfonamides 23a afforded the corresponding O- or

N-substituted sulfonamide intermediates, 23c or 23d,

respectively (Scheme 4).^29,30To gain access to C-substi-

tuted sulfonamide intermediates, such as 23f and 23h,

anilines 21 were reacted with methyl 3-(4-chlorosulfo-

nyl)phenylpropionate 22b. The resulting methyl ester

intermediates 23b were then reduced to the correspond-

ing alcohols 23e using LAH, and protected as the

corresponding silyl ethers 23f. Alternatively, the methyl

ester intermediates 23b were transformed into the

corresponding amides 23h, via a reaction sequence

consisting of saponification, activation with isobutyl

chloroformate, and coupling with amines. All para-

substituted secondary sulfanilide intermediates 23c,

23d, 23f, and 23h were transformed into the corre-

sponding final products 28 following the standard

synthetic protocol outlined in Scheme 1.

Results and Discussion

The primary and secondary focused screening cam-

paigns had revealed the sulfanilide 7 to be a potent and

selective antagonist of the human oxytocin receptor. In

a rat model of preterm labor, the compound was able to

efficiently inhibit oxytocin-induced uterine contractions

after intravenous, but not oral, administration, confirm-

ing the poor oral bioavailability of 6% determined in a

separate PK-study in rats. A 10-fold higher bioavail-

ability was obtained after intraperitoneal administra-

tion in the same vehicle, suggesting that, in addition to

the generally very low solubility, the barrier to oral

bioavailability was either low stability at acidic pH, and/

or an important first-pass effect due to oxidative me-

tabolism. While the first hypothesis could be ruled out

by stability studies at pH 1, any meaningful evaluation

of the microsome metabolism (and membrane perme-

ation) in vitro was prevented by the extremely low

solubility of the compound in aqueous buffer systems.

With this in mind, and since the low solubility was, in

itself, considered to be another factor limiting the oral

bioavailability, the initial focus of the lead optimization

campaign consisted in improving the aqueous solubility,

while maintaining or even improving the overall ac-

ceptable profile in terms of potency and selectivity.

The preliminary SAR information extracted from the

focused set of analogues (see Table 1) had pointed to

the Eastern part (R1) and the para-position of the

arylsulfonyl moiety (R3) as being potentially more

amenable to structural changes than other positions.

Accordingly, these were considered to be prime targets

for the introduction of substituents conducive to improv-

ing the physicochemical properties. However, before

initiating the SAR studies around those peripheral parts

of the molecule, we decided to ascertain whether the

central, glycine-derived part of the compound series was

indeed the optimal core scaffold with a view to binding

affinity against OT-R. To this end, the corresponding

analogues of 7 derived from alanine, serine, and â-ala-

nine, 41, 42, and 43, respectively, were prepared. On

the basis of the marked loss in activity observed for all

three analogues (see Table 2), it was decided to build

the compound optimization program entirely on glycine-

based derivatives.

Thus, starting from the archetype compounds 7 and

two closely related analogues, 44a and 44b, a first series

of derivatives was made to probe the tolerance of the

OT-R toward changes in the Eastern part, isatin-

derived, hydrazone moiety (see Table 3). Omitting the

isatin hydrazone moiety, as in the ester 45 and the

hydrazide 46, completely abolished the binding affinity

Based on the results of the primary screen (12a-c

and 17a,b) and on considerations related to the struc-

tural similarity with SR49059 (5), a primary carboxa-

mide library was designed and produced via solid-phase

chemistry using a Rink linker strategy (Scheme 5). To

this end, a variety of Fmoc-protected R-, â-, and γ-amino

Oxytocin Receptor Antagonists

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7887

Scheme 3^a

^aReagents: (a) MeOH, reflux; (b) DIEA, DCM, r.t.

Scheme 4^a

^aReagents: (a) pyridine, 0 °C to r.t. or DIEA, DCM, r.t.; (b) R6OH, NaH, dioxane, r.t.; then 23a, reflux; or R6R7NH, BuLi, THF,

-78°C to -40 °C; then 23a, -40 °C to 60 °C; (c) LiAlH₄, THF, 0 °C then r.t.; (d) ClSiMe₂tBu, imidazole, DMF, r.t.; (e) NaOH, dioxane/

water, r.t.; (f) isobutyl chloroformate, N-methyl morpholine, THF, -25 °C; then R8R9NH, -25°C to r.t.

toward hOT-R. Similarly, introducing substituents at

position 5 of the isatin ring, as in 47a-c, was not

tolerated. In contrast, replacing the isatin with a

5-azaisatin moiety, as in 48, was found to entail a lesser

reduction in potency, while markedly improving the

solubility of the final product by offering the possibility

to form a salt (pK_a) 4.4). Consequently, the azaisatin

building block was retained as a viable potential alter-

native and reused for the preparation of a more ad-

vanced molecule (76, see Table 5). Based on the previous

results with benzaldehyde and acetophenone-derived

hydrazones, such as 8a and 10a (see Table 1), a series

of further monocyclic derivatives was then synthesized,

again with a view to potentially improving the solubility

by an appropriate choice of substituents. From the

selected results shown in Table 3 (compounds 49-56),

it can be seen that apart from the nonsubstituted ortho-

hydroxy phenylhydrazones 49a and 49b, none of these

derivatives showed any appreciable affinity to OT-R.

The replacement of the phenol moiety with an nonsub-

stituted or substituted aniline, a 2-pyridine, and an

imidazole group, as in 51, 52, and 53, respectively, was

found to be detrimental for activity, and the same was

observed for further substitutions of the phenol ring at

position 4, as in 50 and 54, and in other positions

(results not shown). The phenol compounds 49a and

49b, despite their good affinity against hOT-R, were not

pursued any further, because they were not found to

be superior to the isatin-derived analogues in terms of

solubility or permeability, nor in terms of selectivity

toward V1a. In addition, the phenol moiety was consid-

ered to be a potential liability due to its known propen-

sity toward glucuronidation. In a final effort, a focused

library of amide derivatives of general structure 40 (see

Scheme 5) was synthesized and tested against hOT-R,

based on the previous results with amide compounds,

such as 17a (see Table 1), and on considerations related

to the structural comparison with the Sanofi V1a

antagonist 5. The majority of compounds did not show

any significant activity against OT-R or V1a, except for

close analogues to 17a, such as 57-59, that displayed

moderate levels of activity at both OT and V1a receptors

(Table 3). Again, however, no improvement in terms of

solubility was noted, and since none of the compounds

7888 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Quattropani et al.

Scheme 5^a

^aReagents: (a) 2 equiv of Fmoc amino acid, DIC, HOBT, DMF overnight; (b) piperidine, DMF, r.t.; (c) 5 equiv of bromoacetic acid, DIC,

DMF, r.t., overnight; (d) 21 (10 equiv), DMSO, r.t., overnight; (e) 22 (5 equiv), NMM, DCE, 60 °C, overnight; (f) 50% TFA/DCM, r.t., 1 h.

Table 2. SAR of Central Core Variants

identified as a second potential candidate for the

introduction of solubilizing groups, based on the pre-

liminary SAR obtained from the focused set of com-

mercial analogues (Table 1). A first series of analogues,

60-64 (see Table 4), confirmed this initial impression

in that both disubstituted, heteroaryl, and even benzo-

fused variants, such as 60, 61, and 62a/b, respectively,

retained good levels of affinity toward the OT-R, as well

as selectivity toward V1a. Good binding affinities were

also noted for 4-monosubstituted phenylsulfonyl groups

(as in 63a-f and 64a-e), with a clear preference toward

slim, electron-rich substituents, such as elongated alkyl

and alkoxy chain substituents (63b-d, 64a/b), as well

as a trend toward decreased affinity with increasing size

of the substituents (63e/f, 64d/e). Electron-withdrawing

substituents, albeit small, as in 64c, were equally found

to decrease the binding affinity for the receptor. On the

basis of this, a second, more refined series of analogues

was prepared, in which the 4-alkyl or -alkoxy substit-

uents contained additional groups thought to be con-

ducive to improving solubility (65-73, see Table 5).

Gratifyingly, all of these analogues were found to not

only retain, but even improve the binding affinity

toward OT-R, in some cases down to sub-nanomolar K_i

values (68, 72, 73), while maintaining excellent selectiv-

ity toward V1a, V1b, and V2 (e.g. 73: hV1a/hOT-R )

65, hV1b/hOT-R ) 23 000, hV2/hOT-R ) 245). In view

of this, it was deemed worthwhile to revisit the SAR

around the Eastern part of the molecules in conjunction

with one of the newly identified alkoxyphenylsulfonyl

groups. Thus, several analogues were made containing

1- and 7-substituted isatins (74, 75), the previously

identified 5-azaisatin (76), and the bioisosteric 1,3,4-

(2H)-isoquinolinetrione (77). Among these, only com-

pound 77 was found to retain a good binding affinity

toward OT-R and a high selectivity toward V1a. The

^aValues are reported as means ( SEM for three and more

determinations. If number of experiments was less than three, it

is shown in parentheses.

was more active against OT-R and/or more selective

against V1a than the best hydrazone compounds, the

amide subseries was abandoned. In aggregate, our

investigations into the SAR around the Eastern part of

the molecules had thus indicated an unexpectedly low

tolerance in terms of structural changes, with the

archetype isatin hydrazone remaining the best moiety

in terms of binding affinity toward OT-R and selectivity

against V1a, and with the 5-azaisatin group being the

only replacement that was tolerated and at the same

time conducive to improving the physicochemical prop-

erties, in particular the solubility, of the original

compounds, such as 7.

With this in mind, we turned our attention to the

arylsulfonyl moiety (R3), which had previously been

Oxytocin Receptor Antagonists

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7889

Table 3. SAR around the Eastern Part

^aValues are reported as means ( SEM for three and more determinations. If number of experiments was less than three, it is shown

in parentheses.

7890 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Table 4. SAR around the Southern Part

Quattropani et al.

^aValues are reported as means ( SEM for three and more determinations. If number of experiments was less than three, it is shown

in parentheses.

5-azaisatin derivative 76 was again found to have

markedly improved solubility compared to the corre-

sponding isatin equivalent 69 (see Table 6), but the loss

in hOT-R binding affinity and hV1a selectivity was more

pronounced compared to the previous case (44b and 48,

see Table 3).

values of the binding affinity by factors of 10 to 20 (but

are still better than the antagonistic potency of Atosiban

determined in the same assay). Initially, we speculated

that this lack of correlation between the binding and

the functional assay could be ascribed to the high extent

of protein binding shown by most compounds in the

series, but this hypothesis could be ruled out by running

the cellular assays in the absence of serum (see Experi-

mental Section). Furthermore, in this particular func-

tional assay, no intrinsic partial agonist activity was

seen for any of the compounds when administered alone

at concentrations up to 10 µM. Further investigations

have since shed light on the potential reasons for the

lower potency of this series in the functional assay, and

these will be communicated in a separate article; (iv)

the intrinsic aqueous solubility of a number of deriva-

tives was found to be superior to that of the original

compound 7, allowing them to be evaluated in early

ADME assays in vitro. For those analogues containing

an ionisable group, different salts were made, of which

some were found to be highly soluble in water and

saline; (v) all compounds showed an acceptable, albeit

not optimal, ADME-profile in vitro, characterized by a

relatively low susceptibility toward oxidative metabo-

lism, and no inhibition of any of the cytochrome P₄₅₀

isoforms tested, notable exceptions being compounds 65,

71, and 77.

To select the most appropriate compounds for follow-

up experiments in vivo, several aspects were taken into

account. Those included the binding affinity toward both

human and rat OT-R, the selectivity toward V1a, the

potency in a functional cellular assay (OT-induced

intracellular Ca²⁺-mobilization in hOT-R transfected

HEK293-EBNA cells), the solubility in aqueous media,

the stability toward oxidative metabolism by rat and

human liver microsomes, and the inhibitory potential

against five cytochrome P₄₅₀isoforms. From the results

shown in Table 6, the following conclusions could be

drawn: (i) the affinity toward the rat OT-R parallels

that against the human receptor, with a consistent

small loss of around 2- to 5-fold. This is not entirely

obvious, considering that several examples of GPCR-

antagonists showing marked inter-species differences

have been noted, e.g., in the field of chemokine receptor

antagonists,³³preventing an evaluation in rodent mod-

els of disease, and severely hampering an expedient

progression toward the clinic; (ii) all analogues, except

for 76, display a high degree of selectivity toward V1a,

in contrast to Atosiban (2), which preferentially binds

to V1a; (iii) the functional antagonistic activities as

determined in the cell assay, particularly of the ana-

logues containing an ionizable group, lag behind the

On the basis of a balanced interpretation of these

results, six compounds were finally selected for testing

in a rodent model of preterm labor looking at the

inhibition of OT-induced uterine contractions in anaes-

Oxytocin Receptor Antagonists

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7891

Table 5. Introduction of Solubilizing Groups at the Para Position of the Arylsulfonyl Moiety

^aValues are reported as means ( SEM for three and more determinations. If number of experiments was less than three, it is shown

in parentheses.

thetized, nonpregnant rats (see Table 7).³⁴The com-

pounds were administered in solution either intrave-

nously by infusion or orally by esophagus cannulation.

The results obtained after intravenous administration

revealed three analogues, 68, 69, and 73, to be equally

or more efficacious than the original compound 7, with

ED₅₀values of 9.4, 7.5, and 1.4 mg/kg, respectively,

reflecting their superior potency in vitro in combination

with moderate to good solubility and low intrinsic

clearance (oxidative metabolism). For comparison, the

â2-agonist ritodrine, the only agent ever approved by

the FDA for the treatment of preterm labor, was found

7892 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Quattropani et al.

Table 6. Comparison of Advanced Lead Compounds with Respect to Potency, Selectivity, and Select Physicochemical/ADME

Properties

K_i(nM)^aK_i(nM)^aK_i(nM)^aselectivity IC₅₀(nM)^a

solubility

%MET %MET IC₅₀(nM)

compound

hOT-R

rOT-R

hV1a

V1a/OT hOT-R(cell)

salt

(mg/mL) H₂O saline (human) (rat)

CYP450

Atosiban (2)

27 ( 5

14 ( 8

3 ( 1

76 (2)

25 ( 10

12(2)

0.63 ( 0.2

1410(2)

511 ( 270

1180 ( 960

176 ( 81

133 ( 45

0.02

101

176

171

84

148

59

59 ( 19

30(2)

n/a^b

n/a

n.d.^b

<0.001

n.d.

0.127

0.18

n.d.

<0.001

n.d.

<0.001

n.d.

n.e.^b

79

n.d.

n.e.

89

n.d.

n.e.

2C9:330

n.d.

7

65

66

67

68

69

70

71

7 ( 3

parent

n.d.

7 ( 4

33(2)

8 ( 5

59 ( 10

7 ( 1

14 ( 4

49 ( 20

32(2)

n.d.

57

n.d.

21

2.1 ( 0.7

0.9 ( 0.6

15 ( 6

8 ( 2

HCl

0.0025

0.007

n.d.

>1000

3A4:310

2C19:50

2C9:345

n.d.

1.1 ( 0.4 4.6 ( 1.8 65 ( 19

HCl

0

26

7 ( 2

4.1 ( 1.2

n.d.

582(2)

615(1)

84

150

n.d.

73

40

parent

<0.001

34

31

72

73

0.9 ( 0.3

0.65 ( 0.16 0.67(2)

n.d.

94(1)

42 ( 18

102

65

n.d.

14 ( 5

parent

TFA

fumarate

TFA

<0.001

20

0.58

<0.001

10

0.002

n.d.

18

n.d.

0

>1000

76

77

115 ( 28

1.6 ( 0.1

140 (2)

2.7 (2)

618 (1)

730 (1)

5

456

840 (2)

15 (2)

20

91

41

89

>1000

^aValues are reported as means ( SEM for three and more determinations. If number of experiments was less than three, it is shown

in parentheses. ^bn.d.: not determined; n/a: not applicable; n.e.: not evaluable due to low solubility in assay buffer.

Table 7. Potency in Vitro and Efficacy in Vivo of Advanced Lead Compounds

efficacy in vivo: inhibition of OT-induced contractions

potency in vitro

intravenous administration

oral administration

compd K_i(nM)^ahOT-R K_i(nM)^arOT-R IC₅₀(nM)^ahOT-R (cell) vehicle^binhib @ 10 mg/kg^cED₅₀(mg/kg) vehicle^binhib @ 30 mg/kg^d

7

14 pd( 8

3 ( 1

0.9 ( 0.6

1.1 ( 0.4

115 ( 28

1.6 ( 0.1

0.65 ( 0.16

25 ( 10

12 (2)

30 (2)

7 ( 3

PEG/saline

NP3S

49.9 ( 3.6%

23.3 ( 3.3%

43.0 ( 12.5%

67.4 ( 7.1%

23.8 ( 4.6%

39.8 ( 7.6%

72.0 ( 4.5%

10

n.d.

9.4

n.d.^e

n.d.

NP3S

n.d.

30%

22%

n.d.

35%

65

68

69

76

77

73

8 ( 2

7 ( 1

NP3S

4.6 ( 2

140 (2)

2.7 (2)

0.67 (2)

14 ( 4

840 (2)

15 (2)

14 ( 5

NP3S

7.5

saline

n.d.

1.4

NP3S

n.d.

NP3S

^aValues are reported as means ( SEM for three and more determinations. If number of experiments was less than three, it is shown

in parentheses. ^bVehicles used are NP3S (5% N-methylpyrrolidone, 25% poly(ethylene glycol) 200, 30% poly(ethylene glycol) 400, 20%

propylene glycol, and 20% saline), PEG/saline (50% PEG400, 50% saline) or saline. ^cInhibition of contractions relative to vehicle values

measured at 5 min after OT-administration. ^dMaximum inhibition of contractions relative to vehicle values measured in a 3 h time

interval after OT-administration. ^en.d.: not determined.

to have an ED₅₀value of 45 mg/kg in the same model.

Other analogues having similar potencies in vitro as the

three top compounds, such as 65 and 77, were found to

be less active in vivo, which was ascribed to their low

intrinsic solubility and/or high susceptibility toward

oxidative metabolism. Conversely, the low efficacy of

compound 76, which stands out in terms of solubility

and in vitro ADME profile, was explained by its much

inferior potency in vitro. On the basis of the good results

obtained after intravenous administration, compounds

68, 69, and 73 were then tested via oral route in the

same animal model. Gratifyingly, all three compounds

produced significant reductions in uterine contractility

at a dose of 30 mg/kg, in sharp contrast to the original

compound 7, previously found to lack oral bioavailabil-

ity. The best compound (73) was finally tested in a more

advanced animal model of preterm labor³⁵and shown

to equally inhibit spontaneous uterine contractions in

late-term pregnant rats when administered by oral

route (29.3 ( 7.0% inhibition at 30 mg/kg).

treatment of preterm labor (ritodrine). However, com-

pound 7 was found to lack oral bioavailability, which

was ascribed mainly to its very low solubility in aqueous

media. Stepwise SAR investigations around the differ-

ent structural elements of the chemical series revealed

one position to be fairly permissive to structural changes.

Consequently, this position was used to introduce a

variety of substituents to improve the physicochemical

properties. Some of the resulting analogues were found

to be superior to 7 in terms of both potency in vitro and

aqueous solubility, which translated into significantly

improved efficacy in the animal model after intravenous

and, of note, oral administration. The best compound,

73, was found to inhibit oxytocin-induced uterine con-

tractions in nonpregnant rats by 72% at 10 mg/kg (iv)

and by 35% at 30 mg/kg (po), and to reduce spontaneous

uterine contractions in late-term pregnant rats by 30%

at 30 mg/kg (po).

Experimental Section

General Experimental Methods. Procedures. All chemi-

cals were purchased from Fluka-Aldrich, Buchs (CH) unless

otherwise stated. Melting points were measured with an

apparatus Bu¨chi Melting Point B-545 and were uncorrected.

NMR spectra were recorded on a Bruker DPX-300 MHz

spectrometer. Data were reported as follows: chemical shift

in ppm using either residual DMSO (2.49 ppm), CHCl₃(7.24

ppm), or MeOH (3.35 ppm) as internal standards on the δ

scale, multiplicity (s ) singlet, br s ) broad singlet, d )

doublet, t ) triplet, q ) quadruplet, m ) multiplet), coupling

Conclusion

We have identified a novel chemical class of potent

oxytocin receptor antagonists showing a high degree of

selectivity toward the closely related vasopressin recep-

tors (V1a, V1b, V2). An initial compound, 7, was shown

to be active in an animal model of preterm labor when

administered by intravenous route, comparing favorably

to a reference compound used in the clinic for the

Oxytocin Receptor Antagonists

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7893

constants (J) in hertz, and integration. MS data provided were

obtained using a mass spectrometer Perkin-Elmer API 150 EX

(ESI or APCI). Analytical HPLC method A: Waters Symmetry

C8 50 × 4.6 mm column, 0.1% TFA in H₂O (solvent A), 0.1%

TFA in MeCN (solvent B), linear gradient 5% to 100% B (10

min, total flux 1 mL/min), UV detection 230-400 nm. Analyti-

cal HPLC method B: Zorbax extend C18 150 × 4.6 mm

column, 0.1% TFA in H₂O (solvent A), 0.1% TFA in MeCN

(solvent B), linear gradient 5% to 100% B (20 min, total flux

1 mL/min), UV detection 230-400 nm. Elemental analyses

were performed on an Erba Science 11108 CHN analyzer

7.03 (m, 2H, H arom.), 7.09 (m, 2H, H arom.), 7.15 (m, 2H, H

arom.), 7.63 (m, 2H, H arom.); MS (APCI) m/z ) 378 (M + H);

m/z ) 376 (M - H); HPLC purity ) 91.1% (method A).

4-Ethoxy-N-(2-hydrazino-1-methyl-2-oxoethyl)-N-(4-meth-

ylphenyl)benzenesulfonamide (138 mg, 0.37 mmol) was dis-

solved in EtOH/5% AcOH (8 mL). 1H-Indole-2,3-dione (54 mg,

0.37 mmol) was added. The reaction mixture was stirred

overnight at 76 °C. Solvents were evaporated and the crude

mixture was purified by flash chromatography, using a

mixture of cyclohexane/ethyl acetate 6:4 as eluent. The title

compound, 41 (86 mg, 46%), was isolated as a yellow solid. ¹H

NMR (300 MHz, CDCl₃): δ 1.28 (d, J ) 7.2 Hz, 3H), 1.41 (m,

3H, OCH₂CH₃), 4.04 (m, 2H, OCH₂CH₃), 5.05 (m, 1H, NCHCO,

major conformer (65%)), 6.11 (m, 1H, NCHCO, minor con-

former (35%)), 6.72-6.95 (m, 3H, H arom.), 6.98-7.22 (m, 5H,

H arom.), 7.29 (m, 1H, H arom.), 7.52-7.80 (m, 3H, H arom.),

8.23 (br s, 1H, CONHN, minor conformer (35%)), 8.67 (br s,

1H, CONHN, major conformer (65%)), 12.40 (br s, 1H, CONHN,

minor conformer (35%)), 13.83 (br s, 1H, CONHN, major

conformer (65%)); MS (APCI) m/z ) 507 (M + H); m/z ) 505

(M - H); HPLC purity ) 89% (method A). Anal. (C₂₆H₂₆N₄O₅S‚

0.5H₂O) C, H, N.

For most acylhydrazone derivatives described below, the ¹H

NMR spectra recorded at room temperature indicated the

presence of two isomers, while only one species was detected

by HPLC. In an exemplary study involving compound 7, the

two isomers were shown to be in rapid equilibrium, by

1

recording the H NMR spectrum in DMSO at 80 °C. At this

temperature, complete coalescence of the signals was reached,

and the reversibility of these changes was verified, indicating

the presence of conformational isomers. Spectroscopic resolu-

tion of conformational isomers by ¹H NMR has previously been

reported for similar types of acylhydrazones.³⁶

4-Ethoxy-N-{1-methyl-2-oxo-2-[(2Z)-2-(2-oxo-1,2-dihy-

dro-3H-indol-3-ylidene)hydrazino]ethyl}-N-(4-methylphen-

yl)-benzene-sulfonamide (41). Ethoxybenzene (6.108 g, 50

mmol, 6.3 mL) was dissolved in DCM (200 mL). The solution

was cooled to -5 °C. Chlorosulfonic acid (24.5 g, 210 mmol,

14 mL) in DCM (50 mL) was added over 45 min. The mixture

was stirred additional 60 min at r.t.. The mixture was poured

on ice and the organic phase was separated, washed with Na₂-

CO₃5%, NaHCO₃sat., water and brine. It was finally dried

over Na₂SO₄, filtered and evaporated, affording in 4-ethoxy-

benzenesulfonyl chloride (8.180 g, 74%). MS (ESI) m/z ) 221

(M + H).

1-Amino-4-methylbenzene (1.821 g, 17.0 mmol) was dis-

solved in DCM (25 mL). N,N-Diisopropylethylamine (3.1 mL,

18.1 mmol) and 4-ethoxy-benzenesulfonyl chloride (2.50 g, 11.3

mmol) were added successively. The reaction mixture was

stirred at room-temperature overnight and washed with 10%

HCl (2 × 20 mL) and brine (1 × 20 mL). The organic phase

was dried over sodium sulfate before filtering and removal of

solvent. 4-Ethoxy-N-(4-methylphenyl)benzenesulfonamide (3.087

g, 94%) was obtained as a colorless solid. ¹H NMR (300 MHz,

CDCl₃): δ 1.39 (t, J ) 7.0 Hz, 3H, OCH₂CH₃), 2.24 (s, 3H, CH₃),

4.02 (q, J ) 7.0 Hz, 2H, OCH₂CH₃), 6.35 (br s, 1H, NH), 6.84

(m, 2H, H arom.), 6.91 (m, 2H, H arom.), 7.01 (m, 2H, H arom.),

7.63 (m, 2H, H arom.); MS (ESI) m/z ) 292 (M + H); m/z )

290 (M - H); HPLC purity ) 99.2% (method A).

4-Ethoxy-N-{1-(hydroxymethyl)-2-oxo-2-[(2Z)-2-(2-oxo-

1,2-dihydro-3H-indol-3-ylidene)hydrazino]ethyl}-N-(4-

methylphenyl)benzenesulfonamide (42). A mixture of

potassium bromide (6.830 g, 57.39 mmol) and 0.75 M HBr

solution (91 mL, 68.24 mmol) was cooled to -10 °C. Sodium

nitrite (2.093 g, 29.47 mmol) and ^L-serine tert-butyl ether 29

(Bachem, 2.50 g, 15.51 mmol) were added sequentially and

the mixture was stirred 3 h at room temperature. The mixture

was cooled to 0 °C and was extracted with ethyl acetate (3 ×

100 mL). Combined organic phases were washed with brine

(2 × 180 mL), dried over MgSO₄, filtered and evaporated.

2-Bromo-3-tert-butoxypropanoic acid 30 (3.481 g, quantitative

yield) was isolated as a light yellow oil. This intermediate was

1

used in the next step without further purification. H NMR

(300 MHz, MeOH-d₄): δ 1.21 (s, 9H), 3.68 (m, 1H), 3.84 (m,

1H), 4.22 (m, 1H); MS (APCI) m/z ) 168.4 (M + H).

Compound 30 (1.276 g, 5.67 mmol) was dissolved in a 3:1

chloroform/MeOH mixture (81 mL). Trimethylsilyl diaz-

omethane (8.505 mL, 17.01 mmol) was added dropwise. The

resulting yellow mixture was stirred overnight at room tem-

perature. The solvents were evaporated at atmospheric pres-

sure, affording methyl 2-bromo-3-tert-butoxypropanoate 24c

(1.482 g), in quantitative yield. It was used in the next step

without further purification. ¹H NMR (300 MHz, CDCl₃): δ

1.01 (s, 9H), 3.51 (m, 1H), 3.67 (s, 3H, OCH₃), 3.69 (m, 1H),

4.05 (m, 1H); MS (APCI) m/z ) 240 (M + H).²⁸

4-Ethoxy-N-(4-methylphenyl)benzenesulfonamide (204 mg,

0.70 mmol) was dissolved in DMF (5 mL). Methyl-2-bromo-

propionate (0.117 mL, 1.05 mmol) and potassium carbonate

(193 mg, 1.40 mmol) were added. The mixture was heated

overnight at 60 °C. Solvents were evaporated. The crude

residue was suspended in ethyl acetate (10 mL) and washed

with 1 N HCl solution (7 mL) and brine (7 mL). Organic phase

was dried over MgSO₄, filtered and evaporated. The desired

product, methyl 2-{[(4-ethoxyphenyl)sulfonyl]-4-methylanilino}-

propanoate (276 mg, quantitative crude yield) was isolated as

a light yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 1.42 (t, 3H, J

) 7.1 Hz), 2.30 (s, 3H), 3.65 (s, 3H), 4.01-4.17 (m, 3H), 6.85

(m, 2H, H arom.), 6.98-7.10 (m, 4H, H arom.), 7.58 (m, 2H, H

arom.); MS (APCI) m/z ) 378 (M + H); HPLC purity ) 94.3%

(method A).

The crude carboxylic acid methyl ester, methyl 2-{[(4-

ethoxyphenyl)sulfonyl]-4-methylanilino}propanoate (264 mg,

0.70 mmol), was dissolved in MeOH (2 mL). Hydrazine hydrate

was added (0.44 mL). The reaction mixture was stirred

overnight at 60 °C. Solvents were evaporated. The crude mass

was redissolved in MeOH, and solvents were evaporated again.

This process was repeated three times. 4-Ethoxy-N-(2-hydra-

zino-1-methyl-2-oxoethyl)-N-(4-methylphenyl)benzenesulfon-

amide (138 mg, 52%) was isolated as a colorless solid. ¹H NMR

(300 MHz, CDCl₃): δ 1.13 (d, 3H, J ) 7.2 Hz), 1.45 (t, 3H, J

) 7.0 Hz), 2.36 (s, 3H), 3.34 (m, 1H), 4.15 (q, 2H, J ) 7.0 Hz),

4-Ethoxy-N-(4-methylphenyl)benzenesulfonamide (388 mg,

1.33 mmol, preparation described in synthesis of 41) was

dissolved in DMF (5 mL). 24c (478 mg, 2 mmol) and potassium

carbonate (368 mg, 2.66 mmol) were added. The mixture was

heated overnight at 60 °C. Solvents were evaporated. The

crude residue was suspended in ethyl acetate (10 mL) and was

washed with 1 N HCl solution (7 mL) and with brine (7 mL).

Organic phase was dried over MgSO₄, filtered and evaporated.

The crude mixture was purified by flash chromatography,

using an 8:2 mixture cyclohexane/ethyl acetate as eluent.

Methyl 3-tert-butoxy-2-{[(4-ethoxyphenyl)sulfonyl]-4-methyl-

anilino}propanoate (157 mg, 26.3%) was isolated as a light

yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 1.28 (s. 9H), 1.61 (t,

3H, J ) 7.1 Hz), 2.49 (s, 3H), 3.50 (t, 1H, J ) 9.4 Hz), 3.85

(dd, 1H, J ) 4.9, 9.4 Hz), 3.92 (s, 3H), 4.25 (q, 2H, J ) 7.1

Hz), 5.33 (dd, 1H, J ) 4.9, 9.4 Hz), 7.03 (m, 2H, H arom.),

7.23 (m, 2H, H arom.), 7.28 (m, 2H, H arom.), 7.87 (m, 2H, H

arom.); MS (APCI) m/z ) 450 (M + H); HPLC purity ) 96.4%

(method A).

Methyl 3-tert-butoxy-2-{[(4-ethoxyphenyl)sulfonyl]-4-methyl-

anilino}propanoate (157 mg, 0.35 mmol) was dissolved in

MeOH (1 mL). Hydrazine hydrate was added (0.11 mL). The

reaction mixture was stirred for 3 days at 60 °C. On cool-

ing the reaction mixture, N-[1-(tert-Butoxymethyl)-2-hydra-

zino-2-oxoethyl]-4-ethoxy-N-(4-methylphenyl)benzenesulfon-

amide (40 mg, 25%) crystallized. It was isolated by filtration

7894 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Quattropani et al.

as a colorless solid. ¹H NMR (300 MHz, MeOH-d₄): δ 1.21 (s.

9H), 1.60 (t, 3H, J ) 7.1 Hz), 2.51 (s, 3H), 3.43 (m, 1H), 3.53

(s, 2H), 3.62 (m, 1H), 3.79 (m, 1H), 4.29 (q, 2H, J ) 7.1 Hz),

7.15 (m, 2H, H arom.), 7.29 (m, 4H, H arom.), 7.82 (m, 2H, H

arom.); MS (APCI) m/z ) 450.2 (M + H); HPLC purity ) 86%

(method A).

CH₂CH₂N), 3.92 (m, 2H, COCH₂CH₂N), 4.06 (q, 2H, J ) 9.0

Hz, CH₃CH₂O), 6.88 (m, 3H, H arom.), 6.97 (m, 2H, H arom.),

7.09 (m, 3H, H arom.), 7.32 (m, 1H, H arom.), 7.53 (m, 3H, H

arom.), 7.66 (br s, 1H, NH), 12.31 (br s, 1H, NH); MS (ESI)

m/z ) 507 (M + H); m/z ) 505 (M - H); HPLC purity ) 97%

(method A); HPLC purity ) 90.9% (method B). Anal. (C₂₆H₂₆-

N₄O₅S‚1.2H₂O) C, H; N: calcd 10.61; found 9.82.

N-[1-(tert-Butoxymethyl)-2-hydrazino-2-oxoethyl]-4-ethoxy-

N-(4-methylphenyl)benzenesulfonamide obtained above (40

mg, 0.09 mmol) was dissolved in EtOH/5% AcOH (2 mL). 1H-

Indole-2,3-dione (13 mg, 0.09 mmol) was added. The reaction

mixture was stirred overnight at 76 °C. Solvents were evapo-

rated and the crude mixture was dissolved in DCM (1 mL).

Trifluoroacetic acid (0.5 mL) was added at 0 °C. The reaction

mixture was stirred 3 days at room temperature and the

solvents were evaporated. The crude product was purified by

flash chromatography, using a mixture cyclohexane/ethyl

acetate 1:1 as eluent. The title compound 42 (17 mg, 36%) was

isolated as an orange solid. ¹H NMR (300 MHz, MeOH-d₄): δ

1.16 (m, 3H, OCH₂CH₃), 2.20 (s, 3H, CH₃), 3.56 (m, 1H), 3.64-

3.81 (m, 1H), 3.84-4.02 (m, 2H), 4.86 (m, 1H, NCHCO, major

conformer (68%)), 5.22 (m, 1H, NCHCO, minor conformer

(32%)), 6.77-6.87 (m, 3H, H arom.), 7.93-7.04 (m, 5H, H

arom.), 7.26 (m, 1H), 7.44-7.72 (m, 3H, H arom.); MS (APCI)

m/z ) 521 (M - H); HPLC purity ) 92% (method A); HPLC

purity ) 90.8% (method B).

4-Ethoxy-N-(4-methylphenyl)-N-{3-oxo-3-[2-(2-oxo-

1,2-dihydro-3H-indol-3-ylidene)hydrazino]propyl}-

benzenesulfonamide (43). Toluidine (1.072 g, 10 mmol) was

dissolved in MeOH (10 mL) and methyl acrylate (0.99 mL, 11

mmol) was added. The resulting mixture was stirred at 60 °C

for 2 days. The desired product crystallized in the reaction

mixture at -20 °C. It was filtered off, washed with cold EtOH

and dried under vacuo, affording methyl 3-(4-toluidino)-

propanoate as white-off powder (719 mg, 37%). ¹H NMR (300

MHz, CDCl₃):δ 2.22 (s, 3H, PhCH₃), 2.60 (t, 2H, J ) 6.0 Hz,

C(O)CH₂CH₂N), 3.41 (t, 2H, J ) 6.0 Hz, C(O)CH₂CH₂N), 3.68

(s, 3H, OCH₃), 6.54 (m, 2H, H arom.), 6.98 (m, 2H, H arom.);

HPLC purity ) 100% (method A).

4-Ethoxy-N-(4-methylphenyl)-N-{2-oxo-2-[2-(2-oxo-

1,2-dihydro-3H-indol-3-ylidene)hydrazino]ethyl}benzene-

sulfonamide (7). 1-Amino-4-methylbenzene (1.821 g, 17.0

mmol) was dissolved in DCM (25 mL). N,N-Diisopropylethy-

lamine (3.1 mL, 18.1 mmol) and 4-ethoxy-benzenesulfonyl

chloride (preparation described above, synthesis of 41, 2.50 g,

11.3 mmol) were added successively. The reaction mixture was

stirred at room-temperature overnight and then washed with

10% HCl (2 × 20 mL) and brine (1 × 20 mL). Organic phase

was dried over sodium sulfate before filtering and removal of

solvent. 4-Ethoxy-N-(4-methylphenyl)benzenesulfonamide (3.087

g, 94%) was obtained as a colorless solid. ¹H NMR (300 MHz,

CDCl₃): δ 1.39 (t, J ) 7.0 Hz, 3H, OCH₂CH₃), 2.24 (s, 3H, CH₃),

4.02 (q, J ) 7.0 Hz, 2H, OCH₂CH₃), 6.35 (br s, 1H, NH), 6.84

(m, 2H, H arom.), 6.91 (m, 2H, H arom.), 7.01 (m, 2H, H arom.),

7.63 (m, 2H, H arom.); MS (ESI) m/z ) 292 (M + H); m/z )

290 (M - H); HPLC purity ) 99.2% (method A).

4-Ethoxy-N-(4-methylphenyl)benzenesulfonamide (3.087 g,

10.6 mmol) was dissolved in DMF (10 mL) and was added to

a suspension of NaH (13.6 mmol, 55-65% in oil) in DMF (30

mL). The mixture was stirred 45 min at room temperature.

2-Bromoacetic acid methyl ester (1.45 mL, 15.9 mmol) was

added dropwise. The resulting mixture was stirred at room-

temperature overnight. The solvents were evaporated, afford-

ing methyl {[(4-ethoxyphenyl)sulfonyl]-4-methylanilino}acetate

45 (3.816 g, quantitative crude yield) as a light yellow solid.

¹H NMR (300 MHz, CDCl₃): δ 1.42 (t, J ) 7.0 Hz, 3H,

OCH₂CH₃), 2.29 (s, 3H, CH₃), 3.67 (s, 3H, OCH₃), 4.06 (q, J )

7.0 Hz, 2H, OCH₂CH₃), 4.36 (s, 2H, NCH₂CO), 6.87 (m, 2H, H

arom.), 7.05 (br s, 4H, H arom.), 7.58 (m, 2H, H arom.); MS

(APCI) m/z ) 364 (M + H); m/z ) 362 (M - H); HPLC purity

) 96.1% (method A); HPLC purity ) 94.8% (method B).

Methyl 3-(4-toluidino)propanoate was dissolved in DCM (7.5

mL). 4-Ethoxy-benzenesulfonyl chloride (preparation described

above, synthesis of 41) and DIEA were added and the mixture

was stirred 10 h at r.t. Solvents were evaporated affording

methyl 3-{[(4-ethoxyphenyl)sulfonyl]-4-methylanilino}propan-

oate (557 mg, 40%). This intermediate was used in the next

step without further purification. ¹H NMR (300 MHz, CDCl₃):

δ 1.41 (t, 3H, J ) 6.0 Hz, CH₃CH₂O), 2.29 (s, 3H, PhCH₃),

2.50 (t, 2H, J ) 8.0 Hz, C(O)CH₂CH₂N), 3.56 (s, 3H, OCH₃),

3.77 (t, 2H, J ) 8.0 Hz, C(O)CH₂CH₂N), 4.05 (q, 2H, J ) 6.0

Hz, CH₃CH₂O), 6.87 (m, 4H, H arom.), 7.07 (m, 2H, H arom.),

7.49 (m, 2H, H arom.); HPLC purity ) 85.4% (method A).

Hydrazine hydrate (3.3 mL) was added to a solution of 45

(3.816 g, 10.5 mmol) in MeOH (30 mL). The reaction mixture

was stirred overnight at room temperature. A white precipitate

was formed. It was isolated by filtration and rinsed with cold

MeOH, affording 4-ethoxy-N-(2-hydrazino-2-oxoethyl)-N-(4-

methylphenyl)benzenesulfonamide (46) (2.745 g, 72%) as a

colorless solid. M.p. 117.5-118.5 °C; IR (neat) ν 3326, 1595,

1

1344 cm^-1; H NMR (300 MHz, DMSO-d₆): δ 1.34 (t, J ) 7.0

Hz, 3H, OCH₂CH₃), 2.26 (s, 3H, CH₃), 4.10 (q, J ) 7.0 Hz, 2H,

OCH₂CH₃), 4.11 (s, 2H, NCH₂CO), 4.18 (br s, 2H, N-NH₂),

6.95-7.15 (m, 6H, H arom.), 7.51 (m, 2H, H arom.); MS (APCI)

m/z ) 362 (M - H); HPLC purity ) 99.8% (method A). Anal.

(C₁₇H₂₁N₃O₄S‚0.7H₂O) C, H, N.

Methyl 3-{[(4-ethoxyphenyl)sulfonyl]-4-methylanilino}propan-

oate (279 mg, 0.74 mmol) was dissolved in MeOH (2 mL).

Hydrazine hydrate was added (0.22 mL) and the mixture was

stirred overnight at r.t. Solvents were evaporated. MeOH was

added and evaporated again. This process was repeated three

times, affording 4-ethoxy-N-(3-hydrazino-3-oxopropyl)-N-(4-

methylphenyl)benzene sulfonamide (213 mg, 76.3%). This

intermediate was used in the next step without further puri-

Compound 46 (73 mg, 0.2 mmol) was dissolved in EtOH/

5% AcOH (4 mL). 1H-Indole-2,3-dione (29 mg, 0.2 mmol) was

added. The reaction mixture was stirred overnight at 76 °C.

A precipitate was formed. It was isolated by filtration, washed

with cold EtOH and dried under vacuo at 40 °C, affording a

bright yellow solid 7 (197 mg, 58%). M.p. 130-131 °C; IR (neat)

1

ν 3170, 1682, 1598, 1335 cm^-^{1 1}H NMR (300 MHz, DMSO-

;

fication. H NMR (300 MHz, CDCl₃): δ 1.43 (t, 3H, J ) 6.0

Hz, CH₃CH₂O), 2.31 (s, 3H, PhCH₃), 2.36 (t, 2H, J ) 8.0 Hz,

COCH₂CH₂N), 3.78 (t, 2H, J ) 8.0 Hz, COCH₂CH₂N), 4.07 (q,

2H, J ) 6.0 Hz, CH₃CH₂O), 6.88 (m, 4H, H arom.), 6.93 (br s,

1H, NH), 7.08 (m, 2H, H arom.), 7.48 (m, 2H, H arom.); MS

(APCI) m/z ) 400 (M + Na); HPLC purity ) 84.1% (method

A).

4-Ethoxy-N-(3-hydrazino-3-oxopropyl)-N-(4-methylphenyl)-

benzenesulfonamide (93 mg, 0.25 mmol) was dissolved in

AcOH (6 mL). 1H-Indole-2,3-dione (37 mg, 0.25 mmol) was

added and the mixture was stirred at 75 °C for 6 h. Solvents

were evaporated and the crude product was purified by flash

chromatography, using a mixture cyclohexane/EtOAc 7:3 as

eluent. The title compound 43 (22 mg, 17%) was isolated as a

yellow solid. ¹H NMR (DMSO-d₆, 300 MHz) δ 1.43 (t, 3H, J )

9.0 Hz, CH₃CH₂O), 2.31 (s, 3H, PhCH₃), 3.08 (m, 2H, C(O)-

d₆): δ 1.16 (t, J ) 7.0 Hz, 3H, OCH₂CH₃), 2.07 (s, 3H, CH₃),

3.93 (q, J ) 7.0 Hz, 2H, OCH₂CH₃), 4.29 (br s, 2H, NCH₂CO,

major conformer (67%)), 4.78 (br s, 2H, NCH₂CO, minor

conformer (33%)), 6.77 (m, 1H, H arom.), 7.01-7.20 (m, 7H,

H arom.), 7.20 (m, 1H, H arom.), 7.43-7.68 (m, 3H, H arom.),

11.09 (s, 1H, NH), 12.31 (br s, 1H, CONHN, minor conformer

1

(33%)), 13.46 (br s, 1H, CONHN, major conformer (67%)); H

NMR (300 MHz, DMSO-d₆, 80 °C): δ 1.16 (t, J ) 7.0 Hz, 3H,

OCH₂CH₃), 2.07 (s, 3H, CH₃), 3.93 (q, J ) 7.0 Hz, 2H, OCH₂-

CH₃), 4.7 (br s, 2H, NCH₂CO), 6.77 (m, 1H, H arom.), 7.01-

7.20 (m, 7H, H arom.), 7.20 (m, 1H, H arom.), 7.43-7.68 (m,

3H, H arom.), 11.09 (s, 1H, NH), 13.05 (br s, 1H, CONHN);

MS (APCI) m/z ) 493 (M + H); m/z ) 491 (M - H); HPLC

purity ) 98.9% (method A). Anal. (C₂₅H₂₄N₄O₅S‚1H₂O) C, H,

N.

Oxytocin Receptor Antagonists

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7895

The following compounds were made in analogy with

compound 7, utilizing the appropriate sulfonyl chloride and

aniline in the first step and carbonyl compounds 27 in the last

condensation step, with the exceptions as shown and yields

quoted for the final step.

conformer (45%)), 13.58 (br s, 1H, CONHN, major conformer

(55%)); MS (APCI) m/z ) 522 (M - H); HPLC purity ) 98.9%

(method A). Anal. (C₂₄H₂₁N₅O₇S) C, H, N.

3-[({[(4-Ethoxyphenyl)sulfonyl]-4-methylanilino}-

acetyl)hydrazono]-2-oxo-2,3-dihydro-1H-indole-5-sulfon-

ic Acid (47c). The title compound was prepared using

4-ethoxybenzene-sulfonyl chloride (preparation described above,

synthesis of 41) and aniline in the first step and isatin-5-

sulfonic acid sodium salt dihydrate in the last condensation

step. It precipitated in the reaction mixture, affording a yellow

powder (38 mg, 32%). M.p. 318-319 °C, decomposition; IR

4-Ethoxy-N-{2-oxo-2-[2-(2-oxo-1,2-dihydro-3H-indol-

3-ylidene)hydrazino]-ethyl}-N-phenylbenzenesulfonam-

ide (44a). The title compound was prepared using 4-ethoxy-

benzene-sulfonyl chloride (preparation described above, syn-

thesis of 41) and aniline in the first step and 1H-indole-2,3-

dione in the last condensation step. It was precipitated with

the addition of water and recristallized from MeOH, affording

a bright yellow solid (74 mg, 62%). M.p. 135.5-136.5 °C; IR

(neat) ν 2988, 1698, 1595, 1354 cm^-^{1 1}H NMR (300 MHz,

;

DMSO-d₆): δ 1.42 (t, J ) 7.0 Hz, 3H, OCH₂CH₃), 2.33 (s, 3H,

CH₃), 4.20 (m, 2H, OCH₂CH₃), 4.55 (br s, 2H, NCH₂CO, minor

conformer (45%)), 4.55 (br s, 2H, NCH₂CO, major conformer

(55%)), 6.95 (m, 1H, H arom.), 7.06-7.30 (m, 6H, H arom.),

7.50-7.90 (m, 4H, H arom.), 11.38 (s, 1H, NH), 12.54 (br s,

1H, CONHN, major conformer (55%)), 13.64 (br s, 1H, CONHN,

minor conformer (45%)); MS (APCI) m/z ) 571 (M - H); HPLC

purity ) 98.4% (method A). Anal. (C₂₅H₂₄N₄O₈S₂Na‚0.5H₂O)

C, H, N.

(neat) ν 3168, 1688, 1595, 1337 cm^-^{1 1}H NMR (300 MHz,

;

DMSO-d₆): δ 1.34 (t, J ) 7.0 Hz, 3H, OCH₂CH₃), 4.11 (q, J )

7.0 Hz, 2H, OCH₂CH₃), 4.51 (br s, 2H, NCH₂CO, major

conformer (65%)), 5.00 (br s, 2H, NCH₂CO, minor conformer

(35%)), 6.95 (m, 1H, H arom.), 7.03-7.17 (m, 3H, H arom.),

7.19-7.43 (m, 6H, H arom.), 7.43-7.70 (m, 3H, H arom.), 11.27

(s, 1H, NH), 12.50 (br s, 1H, CONHN, minor conformer (35%)),

13.66 (br s, 1H, CONHN, major conformer (65%)); MS (APCI)

m/z ) 477 (M - H); HPLC purity ) 99.6% (method A). Anal.

(C₂₄H₂₂N₄O₅S‚1H₂O) C, H, N.

N-(4-Chlorophenyl)-4-ethoxy-N-{2-oxo-2-[(2Z)-2-(2-oxo-

1,2-dihydro-3H-pyrrolo[3,2-c]pyridin-3-ylidene)hydrazino]-

ethyl}benzenesulfonamide (48). The title compound was

prepared using 4-ethoxybenzene-sulfonyl chloride (preparation

described above, synthesis of 41) and 4-chloroaniline in the

first step and 5-azaisatin³⁷in the last condensation step. It

was isolated by evaporation of the solvents and purified by

flash chromatography, using a mixture DCM/MeOH 20:1 as

eluent. A light yellow solid was obtained (32 mg, 22%). ¹H

NMR (DMSO-d₆, 300 MHz): δ 1.12 (t, 3H, J ) 6.0 Hz), 3.88

(q, 2H, J ) 6.0 Hz), 4.35 (s, 2H, NCH₂CO, major conformer

(63%)), 4.80 (br s, 2H, NCH₂CO, minor conformer (37%)), 6.79

(m, 1H), 6.87 (m, 2H), 7.03 (m, 2H), 7.19 (m, 2H), 7.32 (m,

2H), 8.24 (d, 1H, J ) 3.0 Hz), 8.40 (br s, 1H), 11.45 (s, 1H,

NH), 12.15 (br s, 1H, CONHN, minor conformer (37%)), 13.28

(br s, 1H, CONHN, major conformer (63%)); MS (ESI) m/z )

514 (M + H); m/z ) 511.8 (M - H); HPLC purity ) 87%

(method A). Anal. (C₂₃H₂₀ClN₅O₅S‚0.1H₂O) C, H, N.

N-(4-Chlorophenyl)-4-ethoxy-N-{2-oxo-2-[(2Z)-2-(2-

oxo-1,2-dihydro-3H-indol-3-ylidene)hydrazino]ethyl}ben-

zenesulfonamide (44b). The title compound was prepared

using 4-ethoxybenzene-sulfonyl chloride (preparation described

above, synthesis of 41) and 4-chloroaniline in the first step

and 1H-indole-2,3-dione in the last condensation step. It was

isolated by evaporation of the solvents and purified by flash

chromatography, using cyclohexane/ethyl acetate 3:1 mixture

as eluent, affording an orange powder (60 mg, 59%). ¹H NMR

(DMSO-d₆, 300 MHz): δ 1.34 (t, 3H, J ) 6.8 Hz), 4.10 (q, 2H,

J ) 6.8 Hz), 4.52 (s, 2H, NCH₂CO, major conformer (60%)),

5.01 (br s, 2H, NCH₂CO, minor conformer (40%)), 6.94 (m, 1H),

7.02-7.14 (m, 3H), 7.26 (m, 2H), 7.33-7.71 (m, 6H), 11.28 (s,

1H, NH), 12.52 (br s, 1H, CONHN, minor conformer (40%)),

13.61 (br s, 1H, CONHN, major conformer (60%)); MS (APCI)

m/z ) 513 (M + H); m/z ) 511 (M - H); HPLC purity ) 95.5%

(method A). Anal. (C₂₄H₂₁ClN₄O₅S) calcd C: 56.19; H: 4.13;

N: 10.92, found C: 56.72; H: 4.31; N: 10.46.

N-(4-Chlorophenyl)-4-ethoxy-N-{2-[(2E)-2-(2-hydroxy-

benzylidene)hydrazino]-2-oxoethyl}benzenesulfonamide

(49a). The title compound was prepared using 4-ethoxyben-

zene-sulfonyl chloride (preparation described above, synthesis

of 41) and 4-chloroaniline in the first step and salicylaldehyde

in the last condensation step. It was isolated by evaporation

of the solvents and purified by crystallization in EtOH,

affording a colorless powder (64 mg, 66.2%). IR (neat) ν 2988,

4-Ethoxy-N-{2-[2-(5-iodo-2-oxo-1,2-dihydro-3H-indol-

3-ylidene)hydrazino]-2-oxoethyl}-N-phenylbenzenesul-

fonamide (47a). The title compound was prepared using

4-ethoxybenzene-sulfonyl chloride (preparation described above,

synthesis of 41) and aniline in the first step and 5-iodo-1H-

indole-2,3-dione (Lancaster) in the last condensation step. It

was precipitated with the addition of water and recristallized

in EtOH, affording a yellow solid (65 mg, 47%). M.p. 233-234

1632, 1351 cm^-^{1 1}H NMR (DMSO-d₆, 300 MHz): δ 1.34 (t,

;

3H, J ) 7.1 Hz), 4.10 (q, 2H, J ) 7.1 Hz), 4.37 (s, 2H, NCH₂-

CO, major conformer (55%)), 4.82 (br s, 2H, NCH₂CO, minor

conformer (45%)), 6.81-7.14 (m, 4H), 7.16-7.32 (m, 3H), 7.35-

7.44 (m, 2H), 7.46-7.73 (m, 3H), 8.24 (s, 1H, minor conformer

(45%)), 8.40 (s, 1H, major conformer (55%)), 9.99 (s, 1H, minor

conformer (45%)), 10.90 (s, 1H, major conformer (55%)), 11.42

(s, 1H, minor conformer (45%)), 11.74 (s, 1H, major conformer

(55%)). MS (APCI) m/z ) 488 (M + H); m/z ) 486 (M - H);

HPLC purity ) 92.2% (method A). Anal. (C₂₃H₂₂ClN₃O₅S) C,

H, N.

1

°C; IR (neat) ν 3371, 1769, 1693, 1594, 1338 cm^-1; H NMR

(300 MHz, DMSO-d₆): δ 1.13 (t, J ) 7.0 Hz, 3H, OCH₂CH₃),

3.89 (q, J ) 7.0 Hz, 2H, OCH₂CH₃), 4.31 (br s, 2H, NCH₂CO,

major conformer (55%)), 1.80 (br s, 2H, NCH₂CO, minor

conformer (45%)), 6.79 (m, 1H, H arom.), 7.07 (m, 2H, H arom.),

7.19-7.41 (m, 5H, H arom.), 7.41-7.92 (m, 4H, H arom.), 11.35

(s, 1H, NH), 12.42 (br s, 1H, CONHN, minor conformer (45%)),

13.58 (br s, 1H, CONHN, major conformer (55%)); MS (APCI)

m/z ) 603 (M - H); HPLC purity ) 95.1% (method A). Anal.

(C₂₄H₂₁IN₄O₅S‚0.1H₂O) C, H, N.

4-Ethoxy-N-{2-[2-(2-hydroxybenzylidene)hydrazino]-

2-oxoethyl}-N-(4-methylphenyl)benzenesulfonamide(49b).

The title compound was prepared using 4-ethoxybenzene-

sulfonyl chloride (preparation described above, synthesis of 41)

and p-toluidine in the first step and 2-hydroxybenzaldehyde

in the last condensation step. It precipitated in the reaction

mixture and was collected by filtration, washed with cold

EtOH and dried under vacuo at 40 °C, affording a colorless

4-Ethoxy-N-{2-[2-(5-nitro-2-oxo-1,2-dihydro-3H-in-

dol-3-ylidene)hydrazino]-2-oxoethyl}-N-phenylbenzenesul-

fonamide (47b). The title compound was prepared using

4-ethoxybenzene-sulfonyl chloride (preparation described above,

synthesis of 41) and aniline in the first step and 5-nitro-1H-

indole-2,3-dione in the last condensation step. It was precipi-

tated with the addition of water and recrystallized in THF/

H₂O, affording a yellow powder (48 mg, 40%). M.p. 252-253

1

powder (63 mg, 68%). H NMR (300 MHz, DMSO-d₆): δ 1.34

1

°C; IR (neat) ν 3107, 1734, 1721, 1624, 1337 cm^-1; H NMR

(t, J ) 7.0 Hz, 3H, OCH₂CH₃), 2.24 (s, 3H, CH₃), 4.10 (q, J )

7.0 Hz, 2H, OCH₂CH₃), 4.32 (s, 2H, NCH₂CO, major conformer

(63%)), 4.77 (s, 2H, NCH₂CO, minor conformer (37%)), 6.81-

7.16 (m, 8H, H arom.), 7.18-7.31 (m, 1H, H arom.), 7.45-7.72

(m, 3H, H arom.), 8.23 (s, 1H, CHdN, minor conformer (37%)),

8.39 (s, 1H, CHdN, major conformer (63%)), 9.98 (s, 1H, OH,

minor conformer (37%)), 10.92 (s, 1H, OH, major conformer

(300 MHz, DMSO-d₆): δ 1.34 (t, J ) 7.0 Hz, 3H, OCH₂CH₃),

4.20 (q, J ) 7.0 Hz, 2H, OCH₂CH₃), 4.68 (br s, 2H, NCH₂CO,

major conformer (55%)), 5.14 (br s, 2H, NCH₂CO, minor

conformer (45%)), 7.12-7.27 (m, 3H, H arom.), 7.30-7.48 (m,

5H, H arom.), 7.52-7.77 (m, 2H, H arom.), 8.23-8.46 (m, 2H,

H arom.), 11.95 (s, 1H, NH), 12.48 (br s, 1H, CONHN, minor

7896 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Quattropani et al.

(63%)), 11.37 (s, 1H, CONHN, minor conformer (37%)), 11.68

(s, 1H, CONHN, major conformer (63%)); MS (APCI) m/z )

466 (M - H); HPLC purity ) 83% (method A). Anal.

(C₂₄H₂₅N₃O₅S) C, H, N.

was stirred for 20 min and acetic anhydride (100 g, 0.98 mol)

was added slowly. After 12 h stirring at room temperature,

the reaction mixture was washed with water, brine and dried

over MgSO₄. The solvents were removed under vacuum to give

the crude N-acetyl-O-acetyl-2-aminobenzyl alcohol (55 g, 88%

yield), which was used without further purification.

To a solution of N-acetyl-O-acetyl-2-aminobenzyl alcohol (55

g, 0.25 mol) in MeOH (550 mL) was added K₂CO₃(200 g, 1.42

mol). After 2 h at room temperature, the resulting solid was

filtered and concentrated. The crude product was dissolved in

DCM (250 mL), washed with water, brine and dried over

MgSO₄. The solvent was removed under vacuum, affording

N-acetyl-2-aminobenzyl alcohol (20 g, 47% yield). It was

oxidized into the corresponding aldehyde without further

purification.

N-(2-{2-[4-(Diethylamino)-2-hydroxybenzylidene]hydra-

zino}-2-oxoethyl)-4-ethoxy-N-(4-methylphenyl)benzenesul-

fonamide (50). The title compound was prepared using

4-ethoxybenzene-sulfonyl chloride (preparation described above,

synthesis of 41) and p-toluidine in the first step and 4-(di-

ethylamino)-2-hydroxy-benzaldehyde in the last condensation

step. It precipitated in the reaction mixture and was collected

by filtration, washed with cold EtOH and dried under vacuo

at 40 °C. A peach powder was obtained (91 mg, 85%). M.p.

227-228 °C; IR (neat) ν 3338, 1687, 1633, 1592, 1338 cm^-1

;

¹H NMR (300 MHz, DMSO-d₆): δ 1.00-1.17 (m, 6H, NCH₂CH₃),

1.34 (t, J ) 7.0 Hz, 3H, OCH₂CH₃), 2.24 (s, 3H, CH₃), 3.20-

3.43 (m, 4H, NCH₂CH₃), 4.10 (q, J ) 7.0 Hz, 2H, OCH₂CH₃),

4.27 (s, 2H, NCH₂CO, major conformer (75%)), 4.68 (s, 2H,

NCH₂CO, minor conformer (25%)), 6.06 (s, 1H, H arom, major

conformer (75%)), 6.10 (s, 1H, H arom, minor conformer (25%)),

6.22 (m, 1H, H arom.), 6.95-7.40 (m, 7H, H arom.), 7.45-7.65

(m, 2H, H arom.), 8.02 (s, 1H, CHdN, minor conformer (25%)),

8.15 (s, 1H, CHdN, major conformer (75%)), 9.75 (s, 1H, OH,

minor conformer (25%)), 11.06 (s, 1H, OH, major conformer

(75%)), 11.10 (s, 1H, CONHN, minor conformer (25%)), 11.36

(s, 1H, CONHN, major conformer (75%)); MS (APCI) m/z )

537 (M - H); HPLC purity ) 98% (method A). Anal.

(C₂₈H₃₄N₄O₅S) C, H, N.

To a stirred solution of N-acetyl-2-amino-benzyl alcohol (20

g) in dry CHCl₃(500 mL) was added MnO₂(160 g, 8

equivalents) and allowed to stir at room temperature for 12

h. The reaction mixture was filtered through Celite and

concentrated. The crude product was recrystallized from

CHCl₃/petroleum ether to give N-acetyl-2-aminobenzaldehyde

1

(14 g, 73%) which was used in the synthesis of 51b. H NMR

(300 MHz, DMSO-d₆): δ 2.01 (s, 3H), 7.30 (m, 2H, H arom.),

7.65 (m, 2H, H arom.), 7.85 (m, 2H, H arom.), 8.09 (m, 2H, H

arom.), 9.95 (s, 1H, CHO), 10.70 (s, 1H, NH).

The title compound 51b was prepared using 4-ethoxyben-

zene-sulfonyl chloride (preparation described above, synthesis

of 41) and 4-toluidine in the first step N-acetyl-2-aminoben-

zaldehyde in the last condensation step. It was isolated by

evaporation of the solvents and purified by flash chromatog-

raphy, using cyclohexane/ethyl acetate 4:1 mixture as eluent.

A light yellow solid was obtained (18 mg, 18%). ¹H NMR

(DMSO-d₆, 300 MHz): δ 1.44 (t, 3H, J ) 6.0 Hz), 2.26 (s, 3H),

2.32 (s, 3H), 4.08 (q, 2H, J ) 6.0 Hz), 4.29 (s, 2H, NCH₂CO),

6.81-7.16 (m, 7H), 7.24 (m, 1H), 7.38 (m, 1H), 7.50 (m, 2H),

8.13 (s, 1H), 8.69 (d, 1H, J ) 6.0 Hz), 9.82 (s, 1H), 11.59 (s,

1H); MS (ESI) m/z ) 507 (M - H); HPLC purity ) 93.6%

(method A); HPLC purity ) 91.6% (method B). Anal. (C₂₆H₂₈-

N₄O₅S‚0.46H₂O) calcd C: 60.42; H: 5.64; N: 10.84, found C:

60.85; H: 5.40; N: 10.39.

N-(4-Chlorophenyl)-4-ethoxy-N-{2-oxo-2-[(2E)-2-(2-pyridin-

ylmethylene)hydrazino]ethyl}benzenesulfonamide (52).

The title compound was prepared using 4-ethoxybenzene-

sulfonyl chloride (preparation described above, synthesis of 41)

and 4-chloroaniline in the first step and 2-pyridinecarboxal-

dehyde in the last condensation step. It was isolated by

evaporation of the solvents and purified by crystallization in

EtOH/5% AcOH, affording a colorless powder (26 mg, 57%).

¹H NMR (DMSO-d₆, 300 MHz): δ 1.34 (t, 3H, J ) 6.0 Hz),

4.11 (q, 2H, J ) 6.0 Hz), 4.38 (s, 2H, NCH₂CO, minor

conformer (35%)), 4.88 (br s, 2H, NCH₂CO, major conformer

(65%)), 7.01-7.66 (m, 9H), 7.80-8.22 (m, 3H), 8.58 (m, 1H),

11.71 (m, 1H); MS (APCI) m/z ) 473 (M + H); m/z ) 471 (M

- H); HPLC purity ) 96.4% (method A); HPLC purity ) 90.9%

(method B).

N-{2-[(2E)-2-(2-Aminobenzylidene)hydrazino]-2-oxoeth-

yl}-4-ethoxy-N-(4-methylphenyl)benzenesulfonamide(51a).

4-Ethoxy-N-[2-((2E)-2-{2-[hydroxy(oxido)amino]benzylidene}-

hydrazino)-2-oxoethyl]-N-(4-methylphenyl)benzenesulfonamide

was prepared using 4-ethoxybenzene-sulfonyl chloride (prepa-

ration described above, synthesis of 41) and 4-toluidine in the

first step and 2-nitrobenzaldehyde in the last condensation

step. It was isolated by evaporation of the solvents and purified

by crystallization in AcOH, afforing a colorless powder (418

1

mg, 84%). H NMR (DMSO-d₆, 300 MHz): δ 1.34 (t, 3H, J )

6.0 Hz), 2.24 (s, 3H), 4.10 (q, 2H, J ) 6.0 Hz), 4.34 (s, 2H,

NCH₂CO, minor conformer (38%)), 4.78 (br s, 2H, NCH₂CO,

major conformer (62%)), 6.88-7.20 (m, 6H), 7.50-7.70 (m, 3H),

7.77 (m, 1H), 7.95-8.10 (m, 2H), 8.29 (s, 1H, major conformer

(62%)), 8.58 (s, 1H, minor conformer (38%)), 11.74 (s, 1H, major

conformer (62%)), 11.81 (s, 1H, minor conformer (38%)), 11.93

(s, 1H); MS (APCI) m/z ) 496.8 (M + H); m/z ) 494.8 (M -

H); HPLC purity ) 98.6% (method A).

4-Ethoxy-N-(4-methylphenyl)-N-{2-[(2E)-2-(2-nitrobenzyli-

dene)hydrazino]-2-oxoethyl}benzenesulfonamide (99 mg, 0.2

mmol) was dissolved in DCM. Palladium 5% on charcoal was

added (10 mol %). The mixture was stirred under H₂at

atmospheric pressure at room-temperature overnight. It was

filtered on Celite and solvents were evaporated. The title

compound 51a (71 mg, 76%) was obtained as a light yellow

1

solid. H NMR (300 MHz, CDCl₃): δ 1.38 (t, J ) 6.0 Hz, 3H,

OCH₂CH₃), 2.26 (s, 3H, CH₃), 3.5 (br s, 2H), 4.01 (q, J ) 6.0

Hz, 2H, OCH₂CH₃), 4.14 (s, 2H), 6.62 (m, 1H), 6.76-6.91 (m,

5H, H arom.), 6.93-7.07 (m, 4H, H arom.), 7.40 (m, 2H, H

arom.), 7.85 (br s, 1H); MS (APCI) m/z ) 467 (M + H); HPLC

purity ) 90.9% (method A). Anal. (C₂₄H₂₆N₄O₄S‚2.45H₂O) C,

H; N: calcd 10.97; found 10.44.

N-(4-Chlorophenyl)-4-ethoxy-N-{2-[(2E)-2-(1H-imida-

zol-2-ylmethylene)hydrazino]-2-oxoethyl}benzenesulfon-

amide (53). The title compound was prepared using 4-ethoxy-

benzene-sulfonyl chloride (preparation described above, syn-

thesis of 41) and 4-chloroaniline in the first step and 2-imi-

dazolecarboxaldehyde in the last condensation step. It was

isolated by evaporation of the solvents and purified by crystal-

lization in EtOH/5% AcOH, affording a colorless powder (34

N-(2-{(E)-[2-(2-{[(4-Ethoxyphenyl)sulfonyl]-4-methyl-

anilino}acetyl)hydrazono]methyl}phenyl)acetamide(51b).

To a solution of 2-nitrobenzyl alcohol (500 g, 3.2 mol) in MeOH

(2.5 L) was added 10% Pd/C (40 g) under N₂and refluxed for

30 min. Hydrazine hydrate (500 mL) was added slowly with

stirring. The resulting mixture was stirred under reflux for

another 2 h. The solid formed was filtered, concentrated and

the crude residue was extracted with ethyl acetate (2 × 500

mL). The organic layer was washed with brine and dried over

MgSO₄. After evaporation of the solvents, 2-aminobenzyl

alcohol (357 g, 89% yield) was isolated as pale yellow solid. It

was used in the next step without further purification.

1

mg, 74%). H NMR (DMSO-d₆, 300 MHz): δ 1.34 (t, 3H, J )

6.0 Hz), 4.10 (q, 2H, J ) 6.0 Hz), 4.35 (s, 2H, NCH₂CO, minor

conformer (35%)), 4.89 (br s, 2H, NCH₂CO, major conformer

(65%)), 6.98-7.44 (m, 8H), 7.54 (m, 2H, minor conformer

(35%)), 7.64 (m, 2H, major conformer (65%)), 7.82 (s, 1H, major

conformer (65%)), 8.07 (s, 1H, minor conformer (35%)), 11.50

(s, 1H, NH), 12.55 (br s, 1H, CONHN, major conformer (65%)),

12.72 (br s, 1H, CONHN, minor conformer (35%)); MS (APCI)

m/z ) 462 (M + H); m/z ) 459.8 (M - H); HPLC purity 98.7%

(method A). Anal. (C₂₀H₂₀ClN₅O₄S) C, H, N.

Et₃N (132 g, 1.3mol) was added to a solution of 2-aminoben-

zyl alcohol (40 g, 0.33 mol) in dry DCM (400 mL). The mixture

Oxytocin Receptor Antagonists

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7897

N-(2-{2-[1-(2,4-dihydroxyphenyl)ethylidene]hydrazino}-

2-oxoethyl)-4-ethoxy-N-(4-methylphenyl)benzenesulfona-

mide (54). The title compound was prepared using 4-ethox-

ybenzene-sulfonyl chloride (preparation described above,

synthesis of 41) and p-toluidine in the first step and 2,4-

dihydroxyacetophenone in the last condensation step. It was

isolated by evaporation of the solvents and purified by recrys-

tallization in MeOH, affording a light yellow powder (33 mg,

mixture as eluent. A yellow powder was obtained (21 mg, 23%).

¹H NMR (DMSO-d₆, 300 MHz): δ 4.59 (s, 2H, NCH₂CO, major

conformer (60%)), 5.03 (br s, 2H, NCH₂CO, minor conformer

(40%)), 6.94 (m, 1H), 7.09 (m, 1H), 7.21-7.59 (m, 7H), 7.64

(m, 1H), 8.05 (m, 1H), 11.28 (s, 1H, NH), 12.52 (br s, 1H,

CONHN, minor conformer (40%)), 13.62 (br s, 1H, CONHN,

major conformer (60%)); MS (APCI) m/z ) 475 (M + H); m/z

) 473 (M - H); HPLC purity ) 99.2% (method A). Anal.

(C₂₀H₁₅ClN₄O₄S₂) H, N; C: calcd 50.58; found 51.44.

1

33%). H NMR (300 MHz, DMSO-d₆): δ 1.34 (t, J ) 7.0 Hz,

3H, OCH₂CH₃), 2.21 (s, 3H, CH₃), 2.25 (s, 3H, CH₃), 4.10 (q, J

) 7.0 Hz, 2H, OCH₂CH₃), 4.45 (s, 2H, NCH₂CO), 6.20 (m, 1H,

H arom.), 6.28 (m, 1H, H arom.), 6.95-7.18 (m, 6H, H arom.),

7.35 (m, 1H, H arom.), 7.55 (m, 2H, H arom.), 9.81 (s, 1H, OH),

10.79 (s, 1H, OH), 13.12 (s, 1H, CONHN); MS (APCI) m/z )

496 (M - H); HPLC purity ) 93% (method A). Anal.

(C₂₅H₂₇N₃O₆S‚0.9H₂O) C, H, N.

N-(4-Methylphenyl)-N-{2-oxo-2-[2-(2-oxo-1,2-dihydro-

3H-indol-3-ylidene)hydrazino]ethyl}-1-naphthalenesul-

fonamide (62a). The title compound was prepared using

1-naphthalene-sulfonyl chloride and p-toluidine in the first

step and 1H-indole-2,3-dione in the last condensation step. It

was isolated by evaporation of the solvents and purified by

recrystallization in MeOH, affording a yellow powder (52 mg,

52%). M.p. 138-139 °C; IR (neat) ν 3431, 1698, 1621, 1336

cm^-1; ¹H NMR (300 MHz, DMSO-d₆): δ 2.27 (s, 3H, CH₃), 4.66

(br s, 2H, NCH₂CO, major conformer (60%)), 5.14 (br s, 2H,

NCH₂CO, minor conformer (40%)), 6.98 (m, 1H, H arom.),

6.99-7.15 (m, 5H, H arom.), 7.30-7.70 (m, 5H, H arom.),

8.02-8.19 (m, 2H, H arom.), 8.21-8.45 (m, 2H, H arom.), 11.30

(s, 1H, NH), 12.54 (br s, 1H, CONHN, minor conformer (40%)),

13.56 (br s, 1H, CONHN, major conformer (60%)); MS (APCI)

m/z ) 497 (M - H); HPLC purity ) 98.5% (method A). Anal.

(C₂₇H₂₂N₄O₄S‚0.5H₂O) C, H, N.

N-(4-Methylphenyl)-N-{2-oxo-2-[2-(2-oxo-1,2-dihydro-

3H-indol-3-ylidene)hydrazino]ethyl}-2-naphthalenesul-

fonamide (62b). The title compound was prepared using

2-naphthalene-sulfonyl chloride and p-toluidine in the first

step and 1H-indole-2,3-dione in the last condensation step. It

was isolated by evaporation of the solvents and purified by

recrystallization in MeOH, affording an orange powder (52 mg,

55%). ¹H NMR (300 MHz, DMSO-d₆): δ 2.24 (s, 3H, CH₃), 4.60

(br s, 2H, NCH₂CO, major conformer (65%)), 5.06 (br s, 2H,

NCH₂CO, minor conformer (35%)), 6.82-7.19 (m, 6H, H

arom.), 7.38 (m, 1H, H arom.), 7.46-7.61 (m, 2H, H arom.),

7.61-7.79 (m, 2H, H arom.), 8.02-8.20 (m, 3H, H arom.), 8.38

(s, 1H, H arom.), 11.27 (s, 1H, NH), 12.51 (br s, 1H, CONHN,

minor conformer (35%)), 13.66 (br s, 1H, CONHN, major

conformer (65%)); MS (APCI) m/z ) 497 (M - H); HPLC purity

) 94.7% (method A). Anal. (C₂₇H₂₂N₄O₄S‚0.5H₂O) C, H, N.

N-(4-Chlorophenyl)-N-{2-oxo-2-[(2Z)-2-(2-oxo-1,2-dihy-

dro-3H-indol-3-ylidene)hydrazino]ethyl}benzenesulfon-

amide (63a). The title compound was prepared using benzene-

sulfonyl chloride and 4-chloroaniline in the first step and 1H-

indole-2,3-dione in the last condensation step. It was isolated

by evaporation of the solvents and purified by flash chroma-

tography, using cyclohexane/ethyl acetate 3:1 mixture as

eluent. A yellow powder was obtained (15 mg, 16.5%). ¹H NMR

(DMSO-d₆, 300 MHz): δ 4.59 (s, 2H, NCH₂CO, major con-

former (55%)), 5.06 (br s, 2H, NCH₂CO, minor conformer

(45%)), 6.94 (m, 1H), 7.09 (m, 1H), 7.26 (m, 2H), 7.33-7.47

(m, 3H), 7.49-7.78 (m, 6H), 11.28 (s, 1H, NH), 12.53 (br s,

1H, CONHN, minor conformer (45%)), 13.58 (br s, 1H,

CONHN, major conformer (55%)); MS (APCI) m/z ) 469 (M +

H); m/z ) 467 (M - H); HPLC purity ) 98% (method A); HPLC

purity ) 91.1% (method B).

N-(4-Chlorophenyl)-N-{2-oxo-2-[(2Z)-2-(2-oxo-1,2-dihy-

dro-3H-indol-3-ylidene)hydrazino]ethyl}-4-propoxyben-

zenesulfonamide (63b). The title compound was prepared

using 4-n-propoxy-1-benzenesulfonyl chloride and 4-chloroa-

niline in the first step and 1H-indole-2,3-dione in the last

condensation step. It was isolated by evaporation of the

solvents and purified by flash chromatography, using cyclo-

hexane/ethyl acetate 3:1 mixture as eluent. A yellow powder

was obtained (46 mg, 44.4%). ¹H NMR (DMSO-d₆, 300 MHz):

δ 0.98 (t, 3H, J ) 7.5 Hz), 1.75 (m, 2H), 4.02 (t, 2H, J ) 6.4

Hz), 4.53 (s, 2H, NCH₂CO, major conformer (65%)), 5.02 (br s,

2H, NCH₂CO, minor conformer (35%)), 6.95 (m, 1H), 7.05-

7.16 (m, 3H), 7.28 (m, 2H), 7.35-7.70 (m, 6H), 11.28 (s, 1H,

NH), 12.52 (br s, 1H, CONHN, minor conformer (35%)), 13.61

(br s, 1H, CONHN, major conformer (65%)); MS (APCI) m/z )

4-Ethoxy-N-(2-{2-[(2-hydroxy-1-naphthyl)methylene]-

hydrazino}-2-oxoethyl)-N-(4-methylphenyl)benzenesul-

fonamide (55). The title compound was prepared using

4-ethoxybenzene-sulfonyl chloride (preparation described above,

synthesis of 41) and p-toluidine in the first step and 2-hydroxy-

1-naphthaldehyde in the last condensation step. It precipitated

in the reaction mixture. It was collected by filtration, washed

with cold EtOH and dried under vacuo at 40 °C, affording a

bright yellow powder (71 mg, 68%). ¹H NMR (300 MHz,

DMSO-d₆): δ 1.34 (t, J ) 7.0 Hz, 3H, OCH₂CH₃), 2.25 (s, 3H,

CH₃), 4.11 (q, J ) 7.0 Hz, 2H, OCH₂CH₃), 4.39 (s, 2H, NCH₂-

CO, major conformer (76%)), 4.81 (s, 2H, NCH₂CO, minor

conformer (24%)), 7.05-7.65 (m, 11H, H arom.), 7.82-7.95 (m,

2H, H arom.), 8.25 (m, 1H, H arom., major conformer (76%)),

8.64 (m, 1H, H arom., minor conformer (24%)), 8.78 (s, 1H,

CHdN, minor conformer (24%)), 9.26 (s, 1H, CHdN, major

conformer (76%)), 10.70 (s, 1H, OH, minor conformer (24%)),

11.41 (s, 1H, CONHN, minor conformer (24%)), 11.77 (s, 1H,

OH, major conformer (76%)), 11.33 (s, 1H, CONHN, major

conformer (76%)); MS (APCI) m/z ) 516 (M - H); HPLC purity

) 92% (method A). Anal. (C₂₉H₂₉N₃O₅S‚0.9H₂O) C, H, N.

4-Ethoxy-N-(2-{(2E)-2-[1-(1-hydroxy-2-naphthyl)eth-

ylidene]hydrazino}-2-oxoethyl)-N-(4-methylphenyl)ben-

zenesulfonamide (56). The title compound was prepared

using 4-ethoxybenzene-sulfonyl chloride (preparation described

above, synthesis of 41) and 4-toluidine in the first step and

1′-hydroxy-2′-acetophenone in the last condensation step. It

was isolated by evaporation of the solvents and purified by

crystallization in EtOH/AcOH 5%, affording a light beige solid

(18 mg, 18%). ¹H NMR (DMSO-d₆, 300 MHz): δ 1.37 (t, 3H, J

) 6.8 Hz), 2.22 (s, 3H), 2.43 (s, 3H), 4.14 (q, 2H, J ) 6.8 Hz),

4.56 (s, 2H), 7.05-7.18 (m, 6H), 7.39 (m, 1H), 7.47-7.70 (m,

5H), 7.85 (m, 1H), 8.29 (m, 1H), 11.1 (s, 1H, OH); MS (APCI)

m/z ) 532.2 (M + H); m/z ) 530.2 (M - H); HPLC purity )

98.2% (method A). Anal. (C₂₉H₂₉N₃O₅S‚0.1H₂O) C, H, N.

N-(4-Chlorophenyl)-3,4-dimethoxy-N-{2-oxo-2-[(2E)-2-

(2-oxo-1,2-dihydro-3H-indol-3-ylidene)hydrazino]ethyl}-

benzenesulfonamide (60). The title compound was prepared

using 3,4-dimethoxybenzenesulfonyl chloride and 4-chloroa-

niline in the first step and 1H-indole-2,3-dione in the last

condensation step. It was isolated by evaporation of the

solvents and purified by crystallization in MeOH, affording a

yellow powder (91.0 mg, 86%). M.p. 132-133°C; IR (neat) ν

1

3188, 1714, 1622, 1337 cm^-1; H NMR (DMSO-d₆, 300 MHz):

δ 3.74 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 4.55 (br s, 2H, NCH₂-

CO, major conformer (60%)), 5.03 (br s, 2H, NCH₂CO, minor

conformer (40%)), 6.90-7.60 (m, 11H, H arom.), 11.27 (s, 1H,

NH), 12.52 (br s, 1H, CONHN, minor conformer (40%)), 13.62

(br s, 1H, CONHN, major conformer (60%)); MS (APCI) m/z )

529 (M + H); HPLC purity ) 97.6% (method A). Anal. (C₂₄H₂₁-

ClN₄O₆S‚1.8H₂O) C, H, N.

N-(4-Chlorophenyl)-N-{2-oxo-2-[(2Z)-2-(2-oxo-1,2-dihy-

dro-3H-indol-3-ylidene)hydrazino]ethyl}-2-thiophene-

sulfonamide (61). The title compound was prepared using

2-thiophenesulfonyl chloride and 4-chloroaniline in the first

step and 1H-indole-2,3-dione in the last condensation step. It

was isolated by evaporation of the solvents and purified by

flash chromatography, using cyclohexane/ethyl acetate 3:1

7898 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Quattropani et al.

527 (M + H); m/z ) 525 (M - H); HPLC purity ) 98.9%

(method A). Anal. (C₂₅H₂₃ClN₄O₅S) H, N; C: calcd 56.97; found

57.67.

described in 63b synthesis) and 4-toluidine in the first step

and 1H-indole-2,3-dione in the last condensation step. It was

isolated by evaporation of the solvents and purified by flash

chromatography, using cyclohexane/ethyl acetate 3:1 mixture

4-Butoxy-N-(4-Chlorophenyl)-N-{2-oxo-2-[(2Z)-2-(2-

oxo-1,2-dihydro-3H-indol-3-ylidene)hydrazino]ethyl}benzene-

sulfonamide (63c). The title compound was prepared using

4-(n-butoxy)benzenesulfonyl chloride (Lancaster) and 4-chlo-

roaniline in the first step and 1H-indole-2,3-dione in the last

condensation step. It was isolated by evaporation of the

solvents and purified by crystallization in MeOH, affording a

1

as eluent. A yellow powder was obtained (46 mg, 46.3%). H

NMR (DMSO-d₆, 300 MHz): δ 0.97 (t, 3H, J ) 7.3 Hz), 1.74

(m, 2H), 2.25 (s, 3H), 4.01 (t, 2H, J ) 6.4 Hz), 4.47 (s, 2H,

NCH₂CO, major conformer (70%)), 4.95 (br s, 2H, NCH₂CO,

minor conformer (30%)), 6.95 (m, 1H), 7.03-7.18 (m, 7H), 7.38

(m, 1H), 7.44-7.68 (m, 6H), 11.28 (s, 1H, NH), 12.49 (br s,

1H, CONHN, minor conformer (30%)), 13.65 (br s, 1H,

CONHN, major conformer (70%)); MS (APCI) m/z ) 507 (M +

H); m/z ) 505 (M - H); HPLC purity ) 99.6% (method A).

Anal. (C₂₆H₂₆N₄O₅S) H, N; C: calcd 61.65; found 62.08.

4-Cyano-N-(4-methylphenyl)-N-{2-oxo-2-[2-(2-oxo-1,2-

dihydro-3H-indol-3-ylidene)hydrazino]ethyl}benzene-

sulfonamide (64c). The title compound was prepared using

4-cyanobenzene-sulfonyl chloride and p-toluidine in the first

step and 1H-indole-2,3-dione in the last condensation step. It

was isolated by evaporation of the solvents and purified by

recrystallization in MeOH, affording a yellow powder (59 mg,

63%). ¹H NMR (300 MHz, DMSO-d₆): δ 2.25 (s, 3H, CH₃), 4.62

(br s, 2H, NCH₂CO, major conformer (57%)), 5.04 (br s, 2H,

NCH₂CO, minor conformer (43%)), 6.94 (m, 1H, H arom.),

7.03-7.22 (m, 5H, H arom.), 7.38 (m, 1H, H arom.), 7.46-7.62

(m, 1H, H arom.), 7.72-7.92 (m, 2H, H arom.), 8.08 (m, 2H, H

arom.), 11.27 (s, 1H, NH), 12.49 (br s, 1H, CONHN, minor

conformer (43%)), 13.52 (br s, 1H, CONHN, major conformer

(57%)); MS (APCI) m/z ) 472 (M - H); HPLC purity ) 93%

(method A). Anal. (C₂₄H₁₉N₅O₄S‚0.2H₂O) C, H, N.

1

yellow powder (26 mg, 25%). H NMR (DMSO-d₆, 300 MHz):

δ 0.93 (t, 3H, J ) 7.5 Hz), 1.43 (sex., 2H, J ) 7.5 Hz), 1.71 (m,

2H), 4.05 (t, 2H, J ) 6.4 Hz), 4.52 (s, 2H, NCH₂CO, major

conformer (65%)), 5.01 (br s, 2H, NCH₂CO, minor conformer

(35%)), 6.95 (m, 1H), 7.03-7.14 (m, 3H), 7.22-7.68 (m, 8H),

11.28 (s, 1H, NH), 12.51 (br s, 1H, CONHN, minor conformer

(35%)), 13.60 (br s, 1H, CONHN, major conformer (65%)); MS

(APCI) m/z ) 541 (M + H); m/z ) 539 (M - H); HPLC purity

) 96.1% (method A). Anal. (C₂₆H₂₅ClN₄O₅S‚1.0H₂O) C, H, N.

N-(4-Chlorophenyl)-N-{2-oxo-2-[(2Z)-2-(2-oxo-1,2-dihy-

dro-3H-indol-3-ylidene)hydrazino]ethyl}-4-propylbenzene-

sulfonamide (63d). The title compound was prepared using

4-n-propylbenzenesulfonyl chloride and 4-chloroaniline in the

first step and 1H-indole-2,3-dione in the last condensation step.

It was isolated by evaporation of the solvents and purified by

flash chromatography, using cyclohexane/ethyl acetate 3:1

mixture as eluent. A yellow powder was isolated (32 mg, 32%).

MS (APCI) m/z ) 511 (M + H); m/z ) 509 (M - H); HPLC

purity ) 67.9% (method A).

4-tert-Butyl-N-(4-chlorophenyl)-N-{2-oxo-2-[(2E)-2-(2-

oxo-1,2-dihydro-3H-indol-3-ylidene)hydrazino]ethyl}ben-

zenesulfonamide (63e). The title compound was prepared

using 4-tert-butylbenzenesulfonyl chloride and 4-chloroaniline

in the first step and 1H-indole-2,3-dione in the last condensa-

tion step. It was isolated by evaporation of the solvents and

purified by crystallization in MeOH, affording a yellow powder

(72 mg, 69%). M.p. 194-195 °C; IR (neat) ν 1698, 1620, 1366

N-(4-Methylphenyl)-N-{2-oxo-2-[2-(2-oxo-1,2-dihydro-

3H-indol-3-ylidene)hydrazino]ethyl}[1,1′-biphenyl]-4-sul-

fonamide (64d). The title compound was prepared using

4-phenylbenzene-sulfonyl chloride and p-toluidine in the first

step and 1H-indole-2,3-dione in the last condensation step. The

resulting precipitate was collected by filtration, washed with

cold EtOH and dried under vacuo at 40 °C, affording a yellow-

orange powder (76 mg, 70%). M.p. 246-247 °C; IR (neat) ν

1

cm^-1; H NMR (DMSO-d₆, 300 MHz): δ 1.30 (s, 9H), 4.57 (br

1

s, 2H, NCH₂CO, major conformer (60%)), 5.04 (br s, 2H, NCH₂-

CO, minor conformer (40%)), 6.94 (m, 1H), 7.09 (m, 1H), 7.25-

7.67 (m, 10H), 11.27 (s, 1H, NH), 12.52 (br s, 1H, CONHN,

minor conformer (40%)), 13.56 (br s, 1H, CONHN, major

conformer (60%)); MS (ESI) m/z ) 523 (M - H); HPLC purity

) 99.4% (method A). Anal. (C₂₆H₂₅ClN₄O₄S‚0.5H₂O) C, H, N.

3098, 1698, 1616, 1317 cm^-1; H NMR (300 MHz, DMSO-d₆):

δ 2.26 (s, 3H, CH₃), 4.57 (br s, 2H, NCH₂CO, major conformer

(65%)), 5.04 (br s, 2H, NCH₂CO, minor conformer (35%)), 6.94

(m, 1H, H arom.), 7.08 (m, 1H, H arom.), 7.16 (m, 4H, H arom.),

7.34-7.60 (m, 5H, H arom.), 7.60-7.83 (m, 4H, H arom.), 7.90

(m, 2H, H arom.), 11.27 (s, 1H, NH), 12.52 (br s, 1H, CONHN,

minor conformer (35%)), 13.64 (br s, 1H, CONHN, major

conformer (65%)); MS (APCI) m/z ) 523 (M - H); HPLC purity

) 99% (method A). Anal. (C₂₉H₂₄N₄O₄S) C, H, N.

N-(4-Chlorophenyl)-N-{2-oxo-2-[(2Z)-2-(2-oxo-1,2-dihy-

dro-3H-indol-3-ylidene)hydrazino]ethyl}-4-tert-pentylben-

zenesulfonamide (63f). The title compound was prepared

using 4-tert-amylbenzenesulfonyl chloride (Lancaster) and

4-chloroaniline in the first step and 1H-indole-2,3-dione in the

last condensation step. It was isolated by evaporation of the

solvents and purified by crystallization in EtOH, affording a

brown-orange powder (59 mg, 55%). MS (APCI) m/z ) 539 (M

+ H); m/z ) 537 (M - H); HPLC purity ) 76% (method A).

4-Methoxy-N-(4-methylphenyl)-N-{2-oxo-2-[2-(2-oxo-

1,2-dihydro-3H-indol-3-ylidene)hydrazino]ethyl}ben-

zenesulfonamide (64a). The title compound was prepared

using 4-methoxybenzene-sulfonyl chloride and p-toluidine in

the first step and 1H-indole-2,3-dione in the last condensation

step. It was isolated by evaporation of the solvents and purified

by recrystallization in MeOH, affording a yellow powder (60

mg, 63%). M.p. 134-135 °C; IR (neat) ν 3353, 1691, 1613, 1334

cm^-1; ¹H NMR (300 MHz, DMSO-d₆): δ 2.25 (s, 3H, CH₃), 3.84

(s, 3H, OCH₃), 4.47 (br s, 2H, NCH₂CO, major conformer

(67%)), 4.96 (br s, 2H, NCH₂CO, minor conformer (33%)), 6.84-

7.01 (m, 1H, H arom.), 7.06-7.18 (m, 7H, H arom.), 7.38 (m,

1H, H arom.), 7.44-7.68 (m, 3H, H arom.), 11.27 (s, 1H, NH),

12.49 (br s, 1H, CONHN, minor conformer (33%)), 13.65 (br s,

1H, CONHN, major conformer (67%)); MS (APCI) m/z ) 479

(M + H); m/z ) 477 (M - H); HPLC purity ) 97% (method A).

Anal. (C₂₄H₂₂N₄O₅S‚1.5H₂O) C, H, N.

4′-Methoxy-N-(4-methylphenyl)-N-{2-oxo-2-[2-(2-oxo-

1,2-dihydro-3H-indol-3-ylidene)hydrazino]ethyl}[1,1′-bi-

phenyl]-4-sulfonamide (64e). The title compound was pre-

pared using 4′-methoxy[1,1′-biphenyl]-4-sulfonyl chloride (Array)

and p-toluidine in the first step and 1H-indole-2,3-dione in the

last condensation step. The resulting precipitate was collected

by filtration, washed with cold EtOH and dried under vacuo

at 40 °C, affording a yellow-orange powder (106 mg, 92%). M.p.

235-236 °C; IR (neat) ν 3204, 1717, 1688, 1595, 1337 cm^-1

;

¹H NMR (300 MHz, DMSO-d₆): δ 2.26 (s, 3H, CH₃), 3.81 (s,

3H, OCH₃), 4.55 (br s, 2H, NCH₂CO, major conformer (63%)),

5.03 (br s, 2H, NCH₂CO, minor conformer (37%)), 6.95 (m, 1H,

H arom.), 7.01-7.22 (m, 7H, H arom.), 7.38 (m, 1H, H arom.),

7.46-7.78 (m, 5H, H arom.), 7.85 (m, 2H, H arom.), 11.27 (s,

1H, NH), 12.51 (br s, 1H, CONHN, minor conformer (37%)),

13.64 (br s, 1H, CONHN, major conformer (63%)); MS (APCI)

m/z ) 553 (M - H); HPLC purity ) 98% (method A). Anal.

(C₃₀H₂₆N₄O₅S) C, H, N.

N-(4-Chlorophenyl)-4-(2-methoxyethoxy)-N-{2-oxo-2-

[(2Z)-2-(2-oxo-1,2-dihydro-3H-indol-3-ylidene)hydrazino]-

ethyl}benzenesulfonamide (66). 4-Chloroaniline (3.827 g,

30 mmol) was dissolved in pyridine (100 mL). The resulting

mixture was cooled to 0 °C. 4-Fluoro-benzenesulfonyl chloride

(3.892 g, 20 mmol) was added in portions. The mixture was

stirred between 0 °C and room-temperature overnight. Sol-

vents were evaporated to dryness. The crude oil was dissolved

in ethyl acetate (75 mL) and washed with 10% HCl (2 × 40

N-(4-Methylphenyl)-N-{2-oxo-2-[(2Z)-2-(2-oxo-1,2-dihy-

dro-3H-indol-3-ylidene)hydrazino]ethyl}-4-propoxyben-

zenesulfonamide (64b). The title compound was prepared

using 4-n-propoxy-1-benzenesulfonyl chloride (preparation

Oxytocin Receptor Antagonists

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7899

mL) and brine (1 × 40 mL). Organic phase was dried over

magnesium sulfate before filtering and removal of solvent. The

resulting solid was recrystallized in cyclohexane/ethyl acetate

9:1. The desired product, 4-fluoro-N-(4-chlorophenyl)benzene-

sulfonamide (3.652 g, 64%) was obtained as a colorless solid.

¹H NMR (300 MHz, CDCl₃): δ 7.05 (m, 2H, H arom.), 7.15 (m,

2H, H arom.), 7.24 (m, 2H, H arom.), 7.81 (m, 2H, H arom.);

MS (ESI) m/z ) 284 (M - H); HPLC purity ) 97% (method

A).

To a suspension of NaH (2.4 mmol, 55-65% in oil) in dry

dioxane (6 mL) was added 2-methoxyethanol (158 µl, 2 mmol).

The mixture was stirred 1 h at room temperature. A solution

of 4-fluoro-N-(4-chlorophenyl)benzenesulfonamide (285 mg, 1

mmol) in dry dioxane (2 mL) was added. The resulting mixture

was heated for 24 h at 100 °C. Solvents were evaporated. NH₄-

Cl saturated solution in water (5 mL) was added and the

desired product was extracted with three portions of ethyl

acetate (3 × 5 mL). Combined organic phases were dried over

magnesium sulfate before filtering and removal of solvent. The

desired product, 4-(2-methoxyethoxy)-N-(4-chlorophenyl)ben-

zenesulfonamide was obtained as a colorless oil. This inter-

mediate was used in the next step without further purification.

MS (APCI) m/z ) 342 (M + H); HPLC purity ) 77% (method

A).

The crude N-aryl-benzenesulfonamide intermediate result-

ing from the previous step (1 mmol) was dissolved in dry

dioxane (5 mL) and was added to a suspension of NaH (1.2

mmol, 55-65% in oil) in dry dioxane (1 mL). The mixture was

stirred 1 h at room temperature. 2-Bromoacetic acid methyl

ester (133 µL, 1.4 mmol) was added dropwise. The resulting

mixture was stirred at 60 °C overnight. The solvents were

evaporated, affording methyl ({[4-(2-methoxyethoxy)phenyl]-

sulfonyl}-4-chloroanilino)acetate as a light yellow oil. This

intermediate was used in the next step without further

purification. MS (APCI) m/z ) 414 (M + H); HPLC purity 74%

(method A).

The crude carboxylic acid methyl ester resulting from the

previous step (1 mmol) was dissolved in MeOH (3.5 mL).

Hydrazine hydrate was added (0.385 mL). The reaction

mixture was stirred overnight at room temperature. Solvents

were evaporated. The crude product was dissolved in ethyl

acetate (4 mL) and was washed with water (4 mL). Aqueous

phase was extracted with ethyl acetate (3 × 2 mL) and with

DCM (2 × 2 mL). Combined organic phases were dried over

magnesium sulfate, filtered and solvents were evaporated.

N-(2-Hydrazino-2-oxoethyl)-4-(2-methoxyethoxy)-N-(4-chlo-

rophenyl)benzenesulfon-amide was isolated as a light yellow

oil. This intermediate was used in the next step without

further purification. MS (APCI) m/z ) 414 (M + H); HPLC

purity ) 86% (method A).

last condensation step, with the exceptions as shown and yields

quoted for the four final steps.

N-(4-Chlorophenyl)-4-[2-(2-methoxyethoxy)ethoxy]-N-

{2-oxo-2-[(2Z)-2-(2-oxo-1,2-dihydro-3H-indol-3-ylidene)-

hydrazino]ethyl}benzenesulfonamide (65). The title com-

pound was prepared using 2-(2-methoxy-ethoxy)-ethanol for

the aromatic substitution step and 1H-indole-2,3-dione for the

last condensation step. It was isolated by evaporation of the

solvents and purified by flash chromatography, using cyclo-

hexane/ethyl acetate mixture, 1:1 then 2:3, as eluents. A yellow

solid was obtained (447 mg, 38% over four steps). ¹H NMR

(CDCl₃, 300 MHz) δ 3.38 (s, 3H, OCH₃), 3.57 (m, 2H), 3.71 (m,

2H), 3.87 (m, 2H), 4.17 (m, 2H), 4.39 (s, 2H, NCH₂CO, major

conformer (58%)), 4.99 (s, 2H, NCH₂CO, minor conformer

(42%)), 6.88-6.97 (m, 3H, H arom.), 7.09 (m, 1H, H arom.),

7.14-7.37 (m, 5H, H arom.), 7.52 (m, 2H, H arom.), 7.70 (m,

1H, H arom.), 7.98 (br s, 1H, minor conformer (42%)), 8.28 (br

s, 1H, major conformer (58%)), 12.41 (s, 1H, minor conformer

(42%)), 13.91 (s, 1H, major conformer (58%)); MS (ESI) m/z )

587 (M + H); HPLC purity ) 95.5% (method A). Anal. (C₂₇H₂₇-

ClN₄O₇S) C, H, N.

N-(4-Chlorophenyl)-4-[2-(dimethylamino)ethoxy]-N-

{2-oxo-2-[(2Z)-2-(2-oxo-1,2-dihydro-3H-indol-3-ylidene)-

hydrazino]ethyl}benzenesulfonamide (67). The title com-

pound was prepared using 2-(dimethylamino)-ethanol for the

aromatic substitution step and 1H-indole-2,3-dione for the last

condensation step. It was isolated by evaporation of the

solvents and purified by flash chromatography, using DCM/

MeOH 20:1 mixture as eluent. A yellow oil was obtained (23

mg, 9% over four steps). ¹H NMR (CDCl₃, 300 MHz) δ 2.44 (s,

6H, N(CH₃)₂), 2.91 (m, 2H), 4.18 (m, 2H), 4.39 (s, 2H, NCH₂-

CO, major conformer (60%)), 4.99 (s, 2H, NCH₂CO, minor

conformer (40%)), 6.86-6.98 (m, 3H, H arom.), 7.07 (m, 1H,

H arom.), 7.12-7.36 (m, 5H, H arom.), 7.49 (m, 2H, H arom.),

7.69 (m, 1H, H arom.), 9.20 (br s, 1H), 12.45 (s, 1H, minor

conformer (40%)), 13.98 (s, 1H, major conformer (60%)); MS

(ESI) m/z ) 556 (M + H); m/z ) 554 (M - H); HPLC purity )

97.5% (method A); HPLC purity ) 92.3% (method B).

N-(4-Chlorophenyl)-4-[2-(4-morpholinyl)ethoxy]-N-{2-

oxo-2-[2-(2-oxo-1,2-dihydro-3H-indol-3-ylidene)hydrazino]-

ethyl}benzenesulfonamide, Hydrochloride Salt (68). The

title compound was prepared using 2-morpholin-4-yl-ethanol

for the aromatic substitution step and 1H-indole-2,3-dione for

the last condensation step. It was isolated by evaporation of

the solvents and purified by flash chromatography, using

DCM/MeOH 40:1 mixture as eluent. A yellow powder was

obtained (342 mg, 8.2% over four steps). HPLC purity ) 95%

(method A).

N-(4-Chlorophenyl)-4-[2-(4-morpholinyl)ethoxy]-N-{2-oxo-2-

[2-(2-oxo-1,2-dihydro-3H-indol-3-ylidene)hydrazino]ethyl}ben-

zenesulfonamide (342 mg, 0.572 mmol) was dissolved in DCM

(10 mL). A HCl solution in diethyl ether (1 M, 0.58 mL, 0.580

mmol) was added. Solvents were evaporated and the resulting

mass was recrystallized in MeOH, affording a yellow powder

(307 mg, 90% yield of recrystallization). M.p. 255 °C; IR (neat)

ν 1682, 1506, 1350 cm^-1; ¹H NMR (DMSO-d₆, 300 MHz) δ 3.28

(m, 2H), 3.51-3.74 (m, 4H), 3.84 (m, 2H), 4.04 (m, 2H), 4.48-

4.74 (m, 2H + 2H, NCH₂CO, major conformer (60%)), 5.12 (br

s, 2H, NCH₂CO, minor conformer (40%)), 7.03 (m, 1H, H

arom.), 7.16 (m, 1H, H arom.), 7.25 (m, 2H, H arom.), 7.35 (m,

2H, H arom.), 7.41-7.56 (m, 3H, H arom.), 7.57-7.82 (m, 3H,

H arom.), 10.91 (br s, 1H), 11.39 (s, 1H), 12.59 (br s, 1H,

CONHN, minor conformer (40%)), 13.68 (br s, 1H, CONHN,

major conformer (60%)); MS (ESI) m/z ) 598 (M + H); m/z )

596 (M - H); HPLC purity ) 100% (method A). Anal. (C₂₈H₂₉-

Cl₂N₅O₆S‚0.1H₂O) C, H, N.

Hydrazide obtained in the previous step (1 mmol) was

dissolved in EtOH/5% AcOH (3 mL). 1H-Indole-2,3-dione (118

mg, 0.8 mmol) was added. The reaction mixture was stirred

overnight at 75 °C. Solvents were evaporated and the desired

product 66 was purified by flash chromatography using a 1:1

mixture of cyclohexane and ethyl acetate. N-(4-Chlorophenyl)-

4-(2-methoxyethoxy)-N-{2-oxo-2-[(2Z)-2-(2-oxo-1,2-dihydro-3H-

indol-3-ylidene)hydrazino]ethyl}benzenesulfonamide (240 mg,

44% over four steps) was isolated as a orange solid.¹H NMR

(CDCl₃, 300 MHz) δ 3.45 (s, 3H, OCH₃), 3.76 (m, 2H), 4.16 (m,

2H), 4.38 (s, 2H, NCH₂CO, major conformer (56%)), 4.99 (s,

2H, NCH₂CO, minor conformer (44%)), 6.87-7.01 (m, 3H, H

arom.), 7.03-7.29 (m, 5H, H arom.), 7.33 (m, 1H, H arom.),

7.53 (m, 2H, H arom.), 7.61 (m, 1H, H arom.), 7.82 (br s, 1H,

minor conformer (44%)), 8.11 (br s, 1H, major conformer

(56%)), 12.42 (s, 1H, minor conformer (44%)), 13.94 (s, 1H,

major conformer (56%)); MS (APCI) m/z ) 543 (M + H); m/z

) 541 (M - H); HPLC purity ) 91.3% (method A). Anal.

(C₂₅H₂₃ClN₄O₆S) H, N; C: calcd 55.30; found 55.75.

N-(4-Chlorophenyl)-4-[3-(dimethylamino)propoxy]-N-

{2-oxo-2-[(2Z)-2-(2-oxo-1,2-dihydro-3H-indol-3-ylidene)-

hydrazino]ethyl}benzenesulfonamide, hydrochloride salt

(69). To a suspension of NaH (100 mmol, 55-65% in oil) in

dry dioxane (140 mL) was added 3-(dimethylamino)-1-propanol

(11.90 mL, 100 mmol). The mixture was stirred 1h at room

temperature. A solution of 4-fluoro-N-(4-chlorophenyl)benze-

nesulfonamide (9.53 g, 33.35 mmol, preparation as in 66, first

The following compounds were made in analogy with

compound 66, starting with 4-fluoro-N-(4-chlorophenyl)ben-

zenesulfonamide, utilizing the appropriate alcohol for the

aromatic substitution step and carbonyl compounds 27 in the

7900 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Quattropani et al.

step) in dry dioxane (35 mL) was added. The resulting mixture

was heated 24h at 100 °C. Solvents were evaporated. NH₄Cl

saturated solution in water (75 mL) was added and the desired

product was extracted with three portions of ethyl acetate (3

× 100 mL). Combined organic phases were dried over MgSO₄,

filtered and evaporated. The desired product, N-(4-chlorophe-

nyl)-4-[3-(dimethylamino) propoxy]benzenesulfonamide, was

obtained as a colorless oil. This intermediate was used in the

next step without further purification. ¹H NMR (300 MHz,

CDCl₃): δ 1.99 (m, 2H, CH₂CH₂CH₂), 2.29 (s, 6H, N(CH₃)₂),

2.50 (m, 2H, NCH₂CH₂), 4.01 (m, 2H, OCH₂CH₂), 6.84 (m, 2H,

H arom.), 7.01 (m, 2H, H arom.), 7.14 (m, 2H, H arom.), 7.65

(m, 2H, H arom.); MS (ESI) m/z ) 369 (M + H); m/z ) 367 (M

- H); HPLC purity ) 96% (method A).

N-(4-Chlorophenyl)-4-[3-(dimethylamino)propoxy]benzene-

sulfonamide (33.35 mmol) was dissolved in dry THF (500 mL).

Triphenyl phosphine (33.689 g, 128.44 mmol) was added, and

the resulting mixture was cooled to 0 °C. Diethyl azodicar-

boxylate (19.97 mL, 128.44 mmol) was added dropwise. After

15 min at room temperature, methyl glycolate in THF (100

mL) was added dropwise. The mixture was stirred 30 min. at

room temperature. Solvents were evaporated and the resulting

crude oil was dissolved in ethyl acetate (100 mL). It was

extracted with 30% citric acid solution in water (2 × 70 mL).

Combined aqueous layers were extracted with diethyl ether

(2 × 50 mL). They were then made basic with NaOH 2M until

pH 10, and extracted with EtOAc (3 × 70 mL). Combined

organic layers were dried over magnesium sulfate, filtered and

evaporated. The desired product, methyl [4-chloro({4-[3-

(dimethylamino)propoxy]phenyl}sulfonyl)anilino]acetate (12.235

g, 83% yield over two steps) was obtained as a colorless oil.

This intermediate was used in the next step without further

purification. ¹H NMR (300 MHz, CDCl₃): δ 2.03 (m, 2H,

CH₂CH₂CH₂), 2.29 (s, 6H, N(CH₃)₂), 2.49 (m, 2H, NCH₂CH₂),

3.72 (s, 3H, OCH₃), 4.09 (m, 2H, OCH₂CH₂), 4.39 (s, 2H), 6.93

(m, 2H, H arom.), 7.16 (m, 2H, H arom.), 7.28 (m, 2H, H arom.),

7.60 (m, 2H, H arom.); MS (ESI) m/z ) 441 (M + H); HPLC

purity ) 90% (method A).

of a 1 N HCl solution in diethyl ether (9.5 mL). After

evaporation of the solvents, the crude salt was recrystallized

in EtOH. The HCl salt of N-(4-chlorophenyl)-4-[3-(dimethy-

lamino)propoxy]-N-{2-oxo-2-[(2Z)-2-(2-oxo-1,2-dihydro-3H-in-

dol-3-ylidene)hydrazino]ethyl}benzenesulfonamide (1.734 g,

32% yield) was isolated as a yellow solid. M.p. 232 °C; IR (neat)

ν 3060, 1694, 1596, 1353 cm^-^{1 1}H NMR (300 MHz, DMSO-

;

d₆): δ 2.16 (m, 2H), 2.77 (s, 6H, N(CH₃)₂), 3.20 (m, 2H), 4.15

(m, 2H), 4.54 (br s, 2H, NCH₂CO, major conformer (60%)), 5.03

(br s, 2H, NCH₂CO, minor conformer (40%)), 6.96 (m, 1H, H

arom.), 7.03-7.17 (m, 3H, H arom.), 7.10 (m, 2H, H arom.),

7.33-7.74 (m, 6H, H arom.), 10.55 (br s, 1H), 11.35 (s, 1H),

12.52 (br s, 1H, CONHN, minor conformer (40%)), 13.61 (br s,

1H, CONHN, major conformer (60%)); MS (APCI) m/z ) 570

(M + H); m/z ) 568 (M - H); HPLC purity ) 98% (method A).

Anal. (C₂₇H₂₉Cl₂N₅O₅S‚0.5H₂O) C, H, N.

The following compounds were made in analogy with

compound 69, starting with 4-fluoro-N-(4-chlorophenyl)ben-

zenesulfonamide, utilizing the appropriate alcohol for the

aromatic substitution step and carbonyl compounds 27 in the

last condensation step, with the exceptions as shown and yields

quoted for the final step.

Methyl 3-({[4-Chloro({4-[3-(dimethylamino)propoxy]-

phenyl}sulfonyl)anilino]acetyl}hydrazono)-2-oxo-2,3-di-

hydro-1H-indole-7-carboxylate (74a). The title compound

was prepared using 3-(dimethylamino)-propan-1-ol for the

aromatic substitution step and 2,3-dioxo-2,3-dihydro-1H-in-

dole-7-carboxylic acid methyl ester for the final condensation

step, which was performed at 100 °C in pure AcOH. 2,3-Dioxo-

2,3-dihydro-1H-indole-7-carboxylic acid methyl ester was ob-

tained by esterification of 2,3-dioxo-2,3-dihydro-1H-indole-7-

carboxylic acid (Maybridge).³⁸The desired product was isolated

by evaporation of the solvents and purified by flash chroma-

tography, using DCM/MeOH 20:1 mixture as eluent. A yellow

solid was obtained (83 mg, 77%). ¹H NMR (DMSO-d₆, 300

MHz) δ 1.85 (m, 2H), 2.13 (s, 3H, N(CH3)2), 2.34 (m, 2H), 3.89

(s, 3H, OCH3), 4.07 (m, 2H), 4.57 (br s, 2H, major conformer

(65%)), 5.02 (m, 2H, minor conformer (35%)), 7.03-7.15 (m,

2H, H arom.), 7.17-7.33 (m, 3H, H arom.), 7.37-7.46 (m, 2H,

H arom.), 7.55 (m, 2H, H arom.), 7.81 (m, 1H, H arom.), 7.89

(m, 1H, H arom.), 11.17 (br s, 1H), 12.45 (br s, 1H, minor

conformer (35%)), 13.58 (br s, 1H, major conformer (65%)); MS

(ESI) m/z ) 628 (M + H); m/z ) 626 (M - H); HPLC purity )

92.5% (method A). Anal. (C₂₉H₃₀ClN₅O₇S‚4.2H₂O) C, H, N.

Methyl [4-chloro({4-[3-(dimethylamino)propoxy]phenyl}-

sulfonyl)anilino]acetate (12.235 g, 27.75 mmol) was dissolved

in MeOH (95 mL). Hydrazine hydrate was added (10 mL). The

reaction mixture was stirred 1 h at room temperature. Solvents

were evaporated. The crude product was dissolved in ethyl

acetate (50 mL) and extracted with 30% citric acid solution in

water (2 × 70 mL). Combined aqueous layers were extracted

with diethyl ether (2 × 50 mL). They were then made basic

with NaOH 2 M until pH 10, and extracted with EtOAc (3 ×

70 mL). Combined organic layers were dried over magnesium

sulfate, filtered and evaporated. The desired product, N-(4-

chlorophenyl)-4-[3-(dimethylamino)propoxy]-N-(2-hydrazino-

2-oxoethyl)benzenesulfonamide (8.772 g, 72% yield) was ob-

tained as a colorless solid. HPLC purity ) 87% (method A). A

fraction of this crude intermediate (842 mg) was recrystallized

3-({[4-Chloro({4-[3-(dimethylamino)propoxy]phenyl}-

sulfonyl)anilino]acetyl}hydrazono)-2-oxo-2,3-dihydro-

1H-indole-7-carboxylic Acid (77b). The title compound was

prepared using 3-(dimethylamino)-propan-1-ol for the aromatic

substitution step and 2,3-dioxo-2,3-dihydro-1H-indole-7-car-

boxylic acid (Maybridge) for the last condensation step, which

was performed at 100 °C in pure AcOH. It was isolated by

evaporation of the solvents and purified by flash chromatog-

raphy, using a gradient DCM/MeOH from 10:1 to 5:1 as eluent.

1

in EtOH, affording a colorless solid (283 mg, 34% yield). H

A yellow solid was obtained (39 mg, 35%). H NMR (DMSO-

NMR (300 MHz, CDCl₃): δ 1.79 (m, 2H, CH₂CH₂CH₂), 2.07

(s, 6H, N(CH₃)₂), 2.28 (m, 2H, NCH₂CH₂), 3.24 (s, 2H), 4.00

(m, 2H, OCH₂CH₂), 4.09 (s, 2H), 4.12 (br s, 1H), 7.01 (m, 2H,

H arom.), 7.10 (m, 2H, H arom.), 7.33 (m, 2H, H arom.), 7.45

(m, 2H, H arom.); MS (ESI) m/z ) 441 (M + H); m/z ) 439 (M

- H); HPLC purity ) 97% (method A).

N-(4-Chlorophenyl)-4-[3-(dimethylamino)propoxy]-N-(2-hy-

drazino-2-oxoethyl)benzenesulfonamide was dissolved in EtOH/

5% AcOH (40 mL). 1H-Indole-2,3-dione (2.029 g, 13.8 mmol)

was added. The reaction mixture was stirred overnight at 75

°C. Solvents were evaporated and the desired product was

purified by flash chromatography using a gradient DCM/

MeOH from 40:1 to 40:3 as eluent. Two fractions of N-(4-

chlorophenyl)-4-[3-(dimethylamino)propoxy]-N-{2-oxo-2-[(2Z)-

2-(2-oxo-1,2-dihydro-3H-indol-3-ylidene)hydrazino]ethyl}ben-

zenesulfonamide (855 mg in 92.2% purity and 5.141 g in 74%

purity (HPLC, method A) were isolated as a yellow solid (61%

yield). One fraction of the final product (5.141 g, 9.018 mmol,

74% pure) was transformed in the corresponding HCl salt, by

dissolution in DCM (120 mL) and addition of one equivalent

d₆, 300 MHz) δ 1.94 (m, 2H), 2.23 (s, 3H, N(CH3)2), 2.60 (m,

2H), 4.09 (m, 2H), 4.54 (br s, 2H, major conformer (65%)), 5.01

(m, 2H, minor conformer (35%)), 7.02-7.13 (m, 4H, H arom.),

7.28 (m, 2H, H arom.), 7.40 (m, 2H, H arom.), 7.46-7.67 (m,

3H), 7.79 (m, 1H), 11.06 (br s, 1H), 12.51 (s, 1H, minor

conformer (35%)), 13.60 (s, 1H, major conformer (65%)); MS

(ESI) m/z ) 614 (M + H); m/z ) 612 (M - H); HPLC purity )

92.0% (method A). Anal. (C₂₈H_28ClN₅O₇S‚4.3H₂O) C, H; N:

calcd 10.13; found 9.60.

2-[3-({[4-Chloro({4-[3-(dimethylamino)propoxy]phenyl}-

sulfonyl)anilino]acetyl}hydrazono)-2-oxo-2,3-dihydro-

1H-indol-1-yl]acetamide, Trifluoroacetate Salt (75). 1H-

Indole-2,3-dione (736 mg, 5 mmol) was dissolved in DMF (25

mL). NaH (7.5 mmol, 55-65% in oil) was added in one portion.

After 1 h at r.t., 2-bromoacetamide was added. After 5 h at

r.t., the reaction was quenched by the addition of water. The

desired product was extracted with EtOAc (3 × 20 mL).

Combined organic phases were dried over MgSO₄, filtered and

evaporated affording 2-(2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-

acetamide which was used without further purification. ¹H

Oxytocin Receptor Antagonists

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7901

NMR (DMSO-d₆, 300 MHz) δ 4.18 (s, 2H, N-CH₂-CO), 6.96 (m,

1H, H arom.), 7.08 (m, 1H, H arom.), 7.23 (br s, 1H, CO-NH₂),

7.51 (m, 1H, H arom.), 7.58 (m, 1H, H arom.), 7.65 (br s, 1H,

CO-NH₂); HPLC purity ) 92.6% (method A).

at 60 °C. The reaction was quenched at room temperature with

NH₄Cl saturated solution in water (5 mL). The desired product

was extracted with three portions of ethyl acetate (3 × 10 mL).

Combined organic phases were dried over magnesium sulfate

before filtering and removal of solvent. The crude product was

purified by flash chromatography, using pure ethyl acetate as

eluent. The desired product, N-(4-chlorophenyl)-4-{[2-(4-

morpholinyl)ethyl]amino}benzenesulfonamide (184 mg, 62%)

was obtained as a colorless oil. This intermediate was used in

the next step without further purification. ¹H NMR (300 MHz,

CDCl₃): δ 2.30 (m, 4H), 2.46 (m, 2H), 3.00 (m, 2H), 3.55 (m,

4H), 4.74 (br s, 1H), 6.35 (m, 2H, H arom.), 6.51 (br s, 1H),

6.84 (m, 2H, H arom.), 7.01 (m, 2H, H arom.), 7.37 (m, 2H, H

arom.); MS (ESI) m/z ) 394 (M - H); HPLC purity ) 97%

(method A).

The title compound was prepared using 3-(dimethylamino)-

propan-1-ol for the aromatic substitution step and 2-(2,3-dioxo-

2,3-dihydro-1H-indol-1-yl)acetamide for the last condensation

step, which was performed at 100 °C in pure AcOH. It was

isolated by evaporation of the solvents and purified by

preparative HPLC (reverse phase, H₂O 0.1%TFA/ MeCN

0.1%TFA gradient between 80:20 and 0:100). After lyophiliza-

tion, the TFA salt of 75 was isolated as a yellow oil. ¹H NMR

(DMSO-d₆, 300 MHz) δ 2.19 (m, 2H), 2.91 (s, 6H, N(CH₃)₂),

3.31 (m, 2H), 4.22 (m, 2H), 4.48 (br s, 2H), 4.65 (br s, 1H, major

conformer (65%)), 5.16 (br s, 1H, minor conformer (35%)),

7.12-7.84 (m, 12H, H arom.), 9.43 (br s, 1H), 12.45 (br s, 1H,

minor conformer (35%)), 13.51 (s, 1H, major conformer (65%));

MS (ESI) m/z ) 627 (M + H); m/z ) 625 (M - H); HPLC purity

) 89% (method A).

The remaining steps toward the title compound were

performed by analogy with the synthesis described for com-

pound 69, using 1H-indole-2,3-dione for the last condensation

step. Compound 70 crystallized in the reaction mixture. It was

isolated by filtration as an orange-yellow solid (220 mg, 84%

N-(4-Chlorophenyl)-4-[3-(dimethylamino)propoxy]-N-

{2-oxo-2-[(2Z)-2-(2-oxo-1,2-dihydro-3H-pyrrolo[3,2-c]pyr-

idin-3-ylidene)hydrazino]ethyl}benzenesulfonamide, Tri-

fluoroacetate Salt (76). The title compound was prepared

using 3-(dimethylamino)-propan-1-ol for the aromatic substitu-

tion and 5-azaisatin³⁷for the final condensation step. It was

isolated by evaporation of the solvents and purified by flash

chromatography, using a gradient DCM/MeOH from 10:1 to

5:1 as eluent. A light yellow powder (296 mg, 43%) was

obtained in 94% purity (HPLC, method A). It was further

purified by preparative HPLC with a gradient of H₂O/0.1%TFA

and MeCN/1%TFA as eluent, affording a beige powder (296

1

yield). H NMR (300 MHz, DMSO-d₆): δ 2.9-3.78 (m, 10H),

3.93 (m, 2H), 4.40 (br s, 2H, NCH₂CO, major conformer (65%)),

4.93 (br s, 2H, NCH₂CO, major conformer (35%)), 6.64 (m, 2H,

H arom.), 6.77 (br s, 1H, NH minor conformer (35%)), 6.90 (m,

1H, H arom.), 7.03 (m, 1H, H arom.), 7.15-7.55 (m, 8H, H

arom.), 11.38 (s, 1H, NH, major conformer (65%)), 11.22 (s,

1H, NH), 12.46 (br s, 1H, CONHN, minor conformer (35%)),

13.60 (br s, 1H, CONHN, major conformer (65%)); MS (ESI)

m/z ) 595 (M - H); HPLC purity ) 98% (method A); HPLC

purity ) 95.5% (method B).

3-{4-[(4-Chloro{2-oxo-2-[2-(2-oxo-1,2-dihydro-3H-indol-

3-ylidene)hydrazino]ethyl}anilino)sulfonyl]phenyl}-N-

[3-(dimethylamino)propyl]propanamide, Trifluoroace-

tate Salt (73). 4-Chloroaniline (957 mg, 7.5 mmol) was

dissolved in pyridine (25 mL). The resulting mixture was

cooled to 0 °C. Methyl 3-(4-chlorosulfonyl)phenylpropionate

(Lancaster, 1.314 g, 5.0 mmol) was added in portions. The

mixture was stirred between 0 °C and room-temperature

overnight. Solvents were evaporated to dryness. The crude oil

was dissolved in ethyl acetate (30 mL) and washed with 10%

HCl (2 × 15 mL) and brine (1 × 15 mL). The organic phase

was dried over magnesium sulfate before filtering and removal

of solvent. The desired product, methyl 3-{4-[(4-chloroanilino)-

sulfonyl]phenyl}propanoate (23b with R2′ ) 4-Cl, 1.458 g,

82.4%), was obtained as a colorless solid. ¹H NMR (300 MHz,

CDCl₃): δ 2.65 (t, 2H, J ) 7.0 Hz), 3.00 (t, 2H, J ) 7.0 Hz),

3.67 (s, 3H, OCH₃), 6.91 (br s, 1H, NH), 7.04 (m, 2H, H arom.),

7.22 (m, 2H, H arom.), 7.30 (m, 2H, H arom.), 7.70 (m, 2H, H

arom.); MS (ESI) m/z ) 351 (M - H); HPLC purity ) 98.5%

(method A).

A solution of sodium hydroxide (560 mg, 14.0 mmol) in water

(7.0 mL) was added to 23b (with R2′ ) 4-Cl; 1.458 g, 4.12

mmol) in 3:1 dioxane:water (30 mL). The resulting mixture

was stirred overnight at room temperature. The reaction

mixture was acidified to pH 2 with 2 N solution of HCl in

water. It was extracted with ethyl acetate (3 × 30 mL).

Combined organic phases were dried over magnesium sulfate,

filtered and evaporated. The expected product, 3-{4-[(4-

chloroanilino)sulfonyl]phenyl}propanoic acid (23g with R2′ )

4-Cl, 1.410 g, quantitative yield) was obtained as a colorless

oil. This intermediate was used in the next step without

further purification. ¹H NMR (300 MHz, DMSO-d₆): δ 2.53

(t, 2H, J ) 7.0 Hz), 2.84 (t, 2H, J ) 7.0 Hz), 7.08 (m, 2H, H

arom.), 7.28 (m, 2H, H arom.), 7.40 (m, 2H, H arom.), 7.64 (m,

2H, H arom.), 10.40 (s, 1H), 12.16 (s, 1H); MS (ESI) m/z )

340 (M + H); m/z ) 338 (M - H); HPLC purity ) 95% (method

A).

1

mg; 43%). IR (neat) ν 1718, 1674, 1651, 1487 cm^-1; H NMR

(DMSO-d₆, 300 MHz) δ 2.11 (m, 2H), 2.82 (s, 3H, N(CH₃)₂),

2.22 (m, 2H), 4.01 (br s, 2H, major conformer (65%)), 4.13 (m,

2H), 4.70 (m, 2H, minor conformer (35%)), 7.00 (m, 2H, H

arom.), 7.17-7.34 (m, 3H, H arom.), 7.43 (m, 2H, H arom.),

7.59 (m, 2H), 8.57 (m, 1H, H arom.), 9.49 (s, 1H), 11.06 (br s,

1H), 12.19 (s, 1H, major conformer (65%)), 13.38 (s, 1H, minor

conformer (35%)); MS (ESI) m/z ) 571 (M + H); m/z ) 569 (M

- H); HPLC purity ) 99.7% (method A). Anal. (C26H₂₇-

ClN₆O₅S‚2H₂O‚2C₂HF₃O₂) C, H, N.

N-(4-Chlorophenyl)-4-[3-(dimethylamino)propoxy]-N-

{2-[2-(1,3-dioxo-2,3-dihydro-4(1H)-isoquinolinylidene)hy-

drazino]-2-oxoethyl}benzenesulfonamide, Trifluoroace-

tate Salt (77). The title compound was prepared using

3-(dimethylamino)-propan-1-ol for the aromatic substitution

and 1,3,4(2H)-isoquinolinetrione^39,40for the last condensation

step, which was performed at 50 °C in H₂O/2%TFA mixture.

77 was isolated by evaporation of the solvents and purified by

preparative HPLC (reverse phase, H₂O 0.1%TFA/ MeCN

0.1%TFA gradient between 80:20 and 0:100). After lyophiliza-

tion, the TFA salt of 77 was isolated as a light yellow solid

(380 mg, 31%). IR (neat) ν 1728, 1689, 1596 cm^-^{1 1}H NMR

;

(DMSO-d₆, 300 MHz) δ 1.98 (m, 2H), 2.17 (s, 6H, N(CH₃)₂),

2.90 (m, 2H), 3.99 (m, 2H), 4.34 (br s, 2H, major conformer

(65%)), 4.80 (br s, 2H, minor conformer (35%)), 6.78 (m, 2H,

H arom.), 6.96 (m, 2H, H arom.), 7.10 (m, 2H, H arom.), 7.34-

7.74 (m, 4H, H arom.), 7.75 (m, 1H, H arom.), 7.81 (br s, 1H,

major conformer (65%)), 9.07 (s, 1H, minor conformer (35%)),

11.99 (br s, 1H),), 13.16 (br s, 1H, major conformer (65%)),

14.23 (s, 1H, minor conformer (35%)); MS (ESI) m/z ) 598 (M

+ H); m/z ) 596 (M - H); HPLC purity ) 98.1% (method A).

Anal. (C₂₈H₂₈ClN₅O₆S‚C₂HF₃O₂‚2.95H₂O) C, N; H: calcd 4.60;

found 4.04.

N-(4-Chlorophenyl)-4-{[2-(4-morpholinyl)ethyl]amino}-

N-{2-oxo-2-[2-(2-oxo-1,2-dihydro-3H-indol-3-ylidene)hydra-

zino]ethyl}benzenesulfonamide (70). N-(2-Aminoethyl)-

morpholine (293 µL, 2.25 mmol) was dissolved in THF (7.5 mL)

and was cooled to -78 °C. A 2.5 N solution of nBuLi in hexane

(1 mL, 2.5 mmol) was added dropwise. After 5 min. at -78

°C, the mixture was stirred 1h at -40 °C. 4-Fluoro-N-(4-

chlorophenyl)benzenesulfonamide (214 mg, 0.75 mmol, prepa-

ration as in 66, first step) was added as a solid. The reaction

mixture was stirred 2 h at room temperature, then overnight

The propanoic acid 23g with R2′ ) 4-Cl (2.06 mmol) was

dissolved in THF (10 mL) and cooled to -25 °C. N-Methyl

morpholine (0.57 mL, 5.15 mmol) and isobutylchloroformate

(0.29 mL, 2.27 mmol) were added successively. The resulting

mixture was stirred at -25 °C for 30 min. N,N-Dimethyl-1,3-

propanediamine (316 mg, 3.09 mmol) was added and the

mixture was allowed to gradually warm to room temperaure.

7902 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Quattropani et al.

After 16 h, solvents were removed. The crude residue was

dissolved in EtOAc and washed with water and with a 10%

solution of NaHCO₃in water. The organic layer was dried over

Na₂SO₄filtered and evaporated. The desired product, 3-{4-

[(4-chloroanilino)sulfonyl]phenyl}-N-[3-(dimethylamino)propyl]-

propanamide (816 mg, 98%), was obtained as a colorless oil.

¹H NMR (300 MHz, CDCl₃): δ 1.61 (m, 2H), 2.23 (s, 6H,

N(CH₃)₂), 2.35 (m, 2H), 2.42 (m, 2H), 2.96 (m, 2H), 3.26 (m,

2H), 7.04 (m, 2H, H arom.), 7.16 (m, 2H, H arom.), 7.24 (m,

2H, H arom.), 7.64 (m, 2H, H arom.); MS (ESI) m/z ) 424 (M

+ H); m/z ) 422 (M - H); HPLC purity ) 94.3% (method A).

The remaining steps toward the title compound were

performed by analogy with the synthesis described for com-

pound 69, using 1H-indole-2,3-dione for the last condensation

step, which was performed at 100 °C in pure AcOH. Solvents

were evaporated and the desired product 73 was purified by

preparative HPLC (reverse phase, H₂O 0.1%TFA/ MeCN

0.1%TFA gradient between 100:1 and 45:55). After lyophiliza-

tion, the TFA salt of 73 was isolated as a yellow solid (107

mg, 44% yield). ¹H NMR (300 MHz, DMSO-d₆): δ 1.68 (m, 2H),

2.42 (m, 2H), 2.73 (s, 3H, N(CH₃)₂), 2.88-2.96 (m, 4H), 3.06

(m, 2H), 4.56 (br s, 2H, NCH₂CO, major conformer (60%)), 5.05

(br s, 2H, NCH₂CO, minor conformer (40%)), 6.95 (m, 1H, H

arom.), 7.09 (m, 1H, H arom.), 7.25 (m, 2H, H arom.), 7.34-

7.68 (m, 8H), 8.00 (m, 1H),), 9.19 (br s, 1H),), 11.28 (s, 1H),

12.52 (br s, 1H, CONHN, minor conformer (40%)), 13.58 (br s,

1H, CONHN, major conformer (60%)); MS (ESI) m/z ) 625

(M + H); m/z ) 623 (M - H); HPLC purity ) 98% (method A).

Anal. (C₃₀H₃₃ClN₆O₅‚0.5H₂O‚1.7C₂HF₃O₂) C, H, N.

mg, 75%) as white solid. It was used in the next step without

further purification. MS (ESI) m/z ) 340 (M + H); m/z ) 338

(M - H).

The remaining steps of the title compound synthesis were

performed by analogy with the synthesis of compound 73,

using 1H-indole-2,3-dione for the last condensation step. The

correponding tert-butyldimethylchlorosilyl ether was first

isolated. It was dissolved in DCM/20% TFA mixture and

stirred 30 min at r.t.. The desired alcohol 71 was finally

isolated by evaporation of the solvents and purified by

preparative HPLC (reverse phase, H₂O 0.1%TFA/ MeCN

0.1%TFA 60:40). After lyophilization, the TFA salt of 71 was

isolated as a yellow solid (358 mg, 41% yield over two steps,

condensation and deprotection steps). ¹H NMR (DMSO-d₆, 300

MHz) δ 1.26 (m, 2H), 2.16 (m, 2H), 2.67 (m, 2H), 2.82 (m, 2H),

3.09 (m, 2H), 4.33 (br s, 2H, major conformer (65%)), 4.81 (m,

2H, minor conformer (35%)), 6.72 (m, 1H, H arom.), 6.87 (m,

1H, H arom.), 7.03 (m, 2H, H arom.), 7.18 (m, 5H, H arom.),

7.30 (m, 3H), 7.59 (m, 1H, H arom.), 11.06 (br s, 1H), 12.28

(br s, 1H, major conformer (65%)), 13.36 (m, 1H, minor

conformer (35%)); MS (ESI) m/z ) 527 (M + H); m/z ) 525 (M

- H); HPLC purity ) 99.53% (method A). Anal. (C₂₅H₂₃-

ClN₄O₅S‚1H₂O) C, H, N.

General Procedure for Solid-Phase Synthesis of Sul-

fanilide Derivatives of Formula 40. Fmoc-Rink amide resin

(0.84 mmol/g) was suspended in 10 mL/g of 20% piperidine in

DMF and was shaken for 30 min. The resin was filtered and

washed with DMF, DCM, DMF, DCM, MeOH and twice with

Et₂O, then dried. The disappearance of the CdO stretch in

the IR indicated a complete deprotection of the Fmoc-Rink

amide.

3-{4-[(4-Chloro{2-oxo-2-[2-(2-oxo-1,2-dihydro-3H-indol-

3-ylidene)hydrazino]ethyl}anilino)sulfonyl]phenyl}-N-

(3-hydroxypropyl)propanamide (72). The title compound

was made by analogy with compound 73, starting with 3-{4-

[(4-chloroanilino) sulfonyl]phenyl}propanoic acid (23g with R2′

) 4-Cl) and coupled with 3-[[(1,1-dimethylethyl) dimethylsilyl]-

oxy]-1-propanamine.⁴¹The final condensation step was per-

formed with 1H-indole-2,3-dione. The correponding tert-

butyldimethylchlorosilyl ether was first isolated. It was dissolved

in DCM/20% TFA mixture and stirred 30 min at r.t.. The

desired alcohol 72 was finally isolated by evaporation of the

solvents and purified by preparative HPLC (reverse phase,

H₂O 0.1%TFA/ MeCN 0.1%TFA gradient between 66:34 and

40:60). After lyophilization, the TFA salt of 72 was isolated

as a yellow solid (154 mg, 10% yield over two steps, condensa-

tion and deprotection steps). IR (neat) ν 1710, 1693, 1622, 1344

cm^-1; ¹H NMR (DMSO-d₆, 300 MHz) δ 1.72 (m, 2H, HO-CH₂-

CH₂-CH₂-Ph), 2.71 (m, 2H, HO-CH₂-CH₂-CH₂-Ph), 3.39 (m, 2H,

HO-CH₂-CH₂-CH₂-Ph), 4.54 (br s, 2H, major conformer (65%)),

5.04 (m, 2H, minor conformer (35%)), 6.95 (m, 1H, H arom.),

7.09 (m, 1H, H arom.), 7.27 (m, 2H, H arom.), 7.42 (m, 5H, H

arom.), 7.53 (m, 3H); MS (ESI) m/z ) 598 (M + H); m/z ) 596

(M - H); HPLC purity ) 94.9% (method A). Anal. (C₂₈H₂₈-

ClN₅O₆S‚0.8H₂O) C, H, N.

N-(4-Chlorophenyl)-4-(3-hydroxypropyl)-N-{2-oxo-2-[2-

(2-oxo-1,2-dihydro-3H-indol-3-ylidene) hydrazino]ethyl}-

benzenesulfonamide (71). 23b with R2′ ) 4-Cl (1.415 g, 4

mmol) was dissolved in THF (60 mL) and cooled to 0 °C. A

solution of LAH in THF (1M, 4 mmol, 4 mL) was added. The

mixture was stirred at r.t for 2 h. Solvents were evaporated.

1 N HCl (60 mL) was added and the desired product was

extracted with EtOAc (3 × 100 mL). Organic phases were dried

over MgSO₄, filtered and evaporated, affording 23e with R2′

) 4-Cl (1.30 g, quantitative yield) which was used in the next

step without further purification. MS (ESI) m/z ) 326 (M +

H); m/z ) 324 (M - H).

The Rink amine resin was suspended in DMF (10 mL/g),

Fmoc-protected amino acid 34 (3 equiv) was added, together

with diisopropylcarbodiimide (3 equiv). The suspension was

shaken overnight at room temperature. The resin was washed

with DMF, DCM, DMF, DCM, MeOH and twice with Et₂O,

then dried. The formation of this new amide bond could be

checked by negative ninhydrin test and the appearance of new

CdO stretches in IR.

Fmoc-protected amino acid loaded on Rink resin (0.84 mmol/

g) was suspended in 10 mL/g of 20% piperidine in DMF and

was shaken for 30 min. The resin was filtered and washed

with DMF, DCM, DMF, DCM, MeOH and twice with Et₂O,

then dried. The loss of the CdO stretch in IR indicated a

complete deprotection of the amino acid.

Resin amine recovered from the previous step was sus-

pended in DMF (10 mL/g). Bromoacetic acid (3 equiv) and

diisopropylcarbodiimide (3 equiv) were added and the mixture

was shaken overnight at room temperature. The resin was

washed with DMF, DCM, DMF, DCM, MeOH and twice with

Et₂O, then dried. The formation of this new amide bond could

be checked by negative ninhydrin test and the appearance of

new CdO stretches in IR.

Aniline 21 (25 equiv) was dissolved in DMSO (10 mL/g of

resin). This solution was added to the resin resulting from the

previous step and the mixture was shaken overnight at room

temperature. The resin was recovered and washed with DMF,

DCM, DMF, DCM, MeOH and twice with Et₂O, then dried.

The formation of this new amide bond was checked on a

sample of resin which was suspended in 50%TFA/DCM for 10

min at room temperature. The resulting solution was analyzed

1

by LC-MS and H NMR.

Sulfonyl chloride 22 (5 equiv) was dissolved in DCM (10

mL/g of resin) and N-methylmorpholine (5 equiv) was added.

This solution was added on the resin resulting from the

previous step. The whole mixture was shaken overnight at 60

°C. The resin was recovered and washed with DMF, DCM,

DMF, DCM, MeOH and twice with Et₂O, then dried.

A 50% TFA in DCM solution (3 × 0.5 mL) was allowed to

drip through the resin resulting from the previous step. This

procedure was repeated twice. The combined filtrate was

evaporated and analyzed by LC-MS and ¹H NMR. Through

this procedure, the desired product, 40, was isolated in >60%

purity by LC/MS.

23e with R2′ ) 4-Cl (217 mg, 0.67 mmol) was dissolved in

DMF (5 mL). Imidazole (54 mg, 0.804 mmol, 1.2 equiv) and

tert-butyldimethylsilyl chloride (111 mg, 0.737 mmol, 1.1

equiv) were added and the reaction mixture was stirred 2 h

at r.t. It was diluted with EtOAc (20 mL) and was washed

with H₂O (15 mL) and brine (15 mL). It was dried over MgSO₄,

filtered and evaporated, affording 23f with R2′ ) 4-Cl (222

Oxytocin Receptor Antagonists

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7903

Compounds 57-59 were made following this general pro-

cedure, utilizing the appropriate Fmoc-protected amino acid

34, aniline 21 and sulfonyl chloride 22.

Competition binding experiments on CHO membranes

expressing the vasopressin 1A receptor subtype were per-

formed in comparable conditions as the ones described for the

HEK293-EBNA expressing the oxytocin receptors. The beads

used were the WGA SPA beads (RPNQ0001, Amersham

Biosciences UK) at a concentration of 4 mg/mL. The radioli-

gand was the ¹²⁵I-LVA antagonist (Perkin-Elmer Life Sciences,

Boston MA), which was used at a concentration of 50 pmol

(K_Dwas determined to be 0.05 nM and B_max1 pmol/mg of

protein). Incubation time was 2 h at room temperature.

Nonspecific binding was determined in the presence of 1 µM

of vasopressin.

2-[2-({[(4-Ethoxyphenyl)sulfonyl]-4-methylanilino}-

acetyl)-1,2,3,4-tetrahydro-3-isoquinolinyl]acetamide (57a).

Starting from N-Fmoc-(1,2,3,4-tetrahydro-isoquinolin-3-yl)-

acetic acid, methyl bromoacetate, 4-toluidine and 4-ethoxy-

benzenesulfonyl chloride (preparation described above, syn-

thesis of 41), the title compound was obtained in 63.3% purity

by LC/MS. MS (ESI) m/z ) 522 (M + H); m/z ) 520 (M - H).

2-[2-({[(4-Ethoxyphenyl)sulfonyl]-4-methoxyanilino}-

acetyl)-1,2,3,4-tetrahydro-3-isoquinolinyl]acetamide (57b).

Starting from N-Fmoc-(1,2,3,4-tetrahydro-isoquinolin-3-yl)-

acetic acid, methyl bromoacetate, 4-methoxy-aniline and

4-ethoxy-benzenesulfonyl chloride (preparation described above,

synthesis of 41), the title compound was obtained in >60%

purity by LC/MS. MS (ESI) m/z ) 538 (M + H).

2-({[(4-Ethoxyphenyl)sulfonyl]-4-methoxyanilino}-

acetyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (58).

Starting from N-Fmoc-1,2,3,4-tetrahydroisoquinoline-3-car-

boxylic acid, methyl bromoacetate, 4-methoxy-aniline and

4-ethoxy-benzenesulfonyl chloride (preparation described above,

synthesis of 41), the title compound was obtained in >60%

purity by LC/MS. MS (ESI) m/z ) 524 (M + H).

2-({[(4-Ethoxyphenyl)sulfonyl]-4-methoxyanilino}-

acetyl)-1-isoindoline-carboxamide (59). Starting from

N-Fmoc-2,3-dihydro-1H-isoindole-1-carboxylic acid, methyl bro-

moacetate, 4-methoxy-aniline and 4-ethoxy-benzenesulfonyl

chloride (preparation described above, synthesis of 41), the title

compound was obtained in 84.6% purity by LC/MS. MS (ESI)

m/z ) 510 (M + H).

In Vitro Experiments. Cell Culture. CHO cell lines

expressing the vasopressin receptor subtypes were maintained

in Dulbecco’s modified Eagle’s medium supplemented with

10% de-complemented fetal calf serum, 4 mM glutamine and

500 units/mL penicillin and streptomycin, in an atmosphere

of 95% air and 5% CO₂at 37 °C. HEK293-EBNA cells,

expressing the human or rat oxytocin receptors were main-

tained in Dulbecco’s modified Eagle’s medium-F-12 (1:1),

supplemented with 10% fetal calf serum and 300 µg/mL

hygromycin B, in an atmosphere of 95% air and 5% CO₂at 37

°C.³⁵

Membrane Preparation. Membranes from CHO cell lines

expressing the vasopressin receptors were prepared according

to the protocol previously described by us.³⁵Membranes from

HEK293-EBNA cells, expressing the oxytocin receptors were

prepared as follows: Cells were detached from the culture dish

with PBS, EDTA 1 mM and washed twice with PBS without

CaCl₂and MgCl₂. Cells were homogeneized in 15 mM Tris-

HCl, 2 mM MgCl₂, 5 mM EDTA, pH 7.4, containing protease

inhibitors (protease inhibitor cocktail tablets, Roche), using a

Dounce homogeneizer, and centrifuged at 250g, 4 °C for 10

min. Supernatant was centrifuged at 40 000g, 4 °C, for 60 min

and the resulting membrane pellets were resuspended in 50

mM Tris-HCl pH 7.4, 5 mM MgCl₂, 0.1% bovine serum

albumin. Protein concentration was calculated using the DC

protein assay method (Bio-Rad). Membranes were stored at

-80 °C.

Binding Experiments. Competition binding experiments

on HEK293-EBNA membranes expressing the oxytocin recep-

tors were performed in Corning NBS 96 well plates, containing

100 µL of binding buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl₂,

0.1% bovine serum albumin), in 1% dimethyl sulfoxide. Briefly,

1 or 2 µg of membranes were mixed with 100 µg of PVT-PEI

wheat germ agglutinin-coupled type A SPA beads (RPNQ0003,

Amersham Biosciences UK) and 0.1-0.2 nM of ¹²⁵I-OVTA

antagonist (Perkin-Elmer Life Sciences, Boston MA) and

incubated at room temperature for 30 min. Optimized beads

and membranes concentrations to be used in the assay were

determined by performing dose responses. The concentration

of ¹²⁵I-OVTA antagonist used in the assay was determined by

Scatchard analysis, where the K_Dwas calculated to be 0.2 nM

and the B_maxto be 2 pmol/mg of protein. Nonspecific binding

was determined in the presence of 1 µM oxytocin.

Competition binding experiments on CHO cell membranes

expressing the V1b or the V2 vasopressin receptor subtypes

were performed as described previously.³⁵

Intracellular Ca⁺⁺Measurements. HEK293-EBNA cells

expressing the human OT receptor were seeded at 10⁵cells/

well in 96-well plates precoated with poly-^L-lysine. After 24

h, cells were loaded with 4.5 µM Fluo-4 dye in Dulbecco’s

modified Eagle’s medium F-12 without serum for 1 h at 37

°C. Antagonist compounds were added to the cells and

incubated for 30 min. Cells were washed in fluorescence

imaging plate reader (FLIPR) buffer (10 mM HEPES, 145 mM

NaCl, 5 mM KCl, 1 mM MgCl₂, and 10 mM glucose, pH 7.4),

and antagonist compounds were added again to the cells.

Intracellular calcium concentration was measured for 1min

(excitation at 488 nm emission at 510-570 nm) using a FLIPR

instrument (Molecular Probes). Cells were then stimulated

with 10 nM OT, and transient intracellular mobilization was

recorded for 4 min. Ca²⁺concentration was expressed in

percentage of maximal response elicited by OT. IC₅₀measure-

ments were calculated by analysis of the data using Excelfit

software (ID Business Solution Ltd., Guilford, Surrey, UK).

Oxidative Metabolism. Rat and human microsomes were

used to assess potential metabolic instability resulting from

phase I oxidation. Microsomes (final concentration 20 mg/mL),

0.1 M phosphate buffer pH 7.4 and compound (final concentra-

tion 40 µM, 1% DMSO) were added to the assay plate and

preincubated at 37 °C. NADPH solution (final incubation

concentration of 1 mM) was added to initiate the reaction. The

reaction was stopped by addition of 100 µL of cold acetonitrile

at the appropriate time points (t ) 0, 20 and 60 min). The

samples were centrifuged at 5000 rpm for 5 min at 4 °C to

precipitate the protein. Samples were analyzed by LC/MS-MS

and the compound disappearance as a function of time was

calculated (as % of the t ) 0 values).

Cytochrome P450 Inhibition. Five human cytochrome

P450 isoforms (CYP 3A4, 2D6, 1A2, 2C9 and 2C19) were used

to assess potential inhibition of enzymatic activity by the test

compounds. The cytochrom P450 enzymes were obtained from

baculovirus/insect cell system (BD Gentest, Woburn, MA). To

evaluate the inhibitory potential, different concentrations of

compounds in 1% DMSO were added into the incubation

mixture containing a given cytochrome P450 isoform, as well

as a specific fluorogenic substrate giving rise to a fluorescent

product during the enzymatic reaction. For all test compounds,

the % inhibition at each concentration and the IC₅₀were

calculated by using the fluorescence intensity after a defined

reaction time as the read-out (Multilabel Counter VICTOR2

- EG&G WALLAC, Turku, Finland).

In Vivo Experiments. All in vivo experiments were

performed according to the European Council Directive 86/

609/EEC and the Italian Health Ministry guidelines for the

care and use of experimental animals (decree 116/92). Each

experimental protocol was authorized by the Italian Ministry

of Health. The animals (from Charles River, Calco, Italy) were

housed in a room under the following constant environmental

conditions: temperature 22 °C ( 2, relative humidity 55% (

10, 15-20 air changes per hour (filtered on HEPA 99.99%)

and artificial light with a 12-h circadian cycle (7 a.m. to 7 p.m.).

For the entire duration of the study, rats and mice were fed

with a standard pelleted diet (4RF21 and 4RF25, respectively,

produced by Charles River Italia’s licensee Mucedola, Settimo

Milanese, Italy) and filtered water “ad libitum”. In all in vivo

7904 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24

Quattropani et al.

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glycol) 400, 20% propylene glycol, and 20% saline (NP3S),

PEG400/saline (1/1) or saline, whereas ritodrine and oxytocin

were solubililized in saline.

Oxytocin-Induced Uterine Contractions in Anesthe-

tized Nonpregnant Rats. Oxytocin-induced uterine contrac-

tions in anaesthetized nonpregnant rats were produced as

previously described by us.³⁵The contractile responses to OT

were quantified by measuring the AUC of the changes in

intraluminal uterine pressure (by Chart V4.04 for Windows

software, PowerLab AD Pty Ltd Instruments,Castle Hill,

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of the above variation values after the administration of each

dose of test compound compared to that obtained at the

corresponding time point in the group receiving the vehicle

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when possible, the relative ED₅₀value calculated (by S-Plus

2000 v. 4.6 statistical software, Mathsoft Inc., Seattle, WA).

Statistical differences between treatment groups at each time-

point were determined by using one-way ANOVA followed by

Tukey test.

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21 of pregnancy) Sprague Dawley CD (SD) BR female rats

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Supporting Information Available: Analytical data

related to the chemical purity of compounds 7, 41-44, 47-

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Internet at http://pubs.acs.org.

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JM050645F

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Discovery and development of a new class of potent, selective, orally active oxytocin receptor antagonists

DOI: 10.1021/jm050645f

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