Selection of a respiratory syncytial virus fusion inhibitor clinical candidate. 2. Discovery of a morpholinopropylaminobenzimidazole derivative (TMC353121)

J. Med. Chem. 2008, 51, 875–896

875

Selection of a Respiratory Syncytial Virus Fusion Inhibitor Clinical Candidate. 2. Discovery of

a Morpholinopropylaminobenzimidazole Derivative (TMC353121)

Jean-François Bonfanti,*^,†Christophe Meyer,^†Frédéric Doublet,^†Jérôme Fortin,^†Philippe Muller,^†Laurence Queguiner,^†

Tom Gevers,^‡Peggy Janssens,^‡Heidi Szel,^‡Rudy Willebrords,^‡Philip Timmerman,^§Koen Wuyts,^§Pieter van Remoortere,^#

Frans Janssens, Piet Wigerinck,^#and Koen Andries^‡

Medicinal Chemistry Department, Johnson & Johnson Pharmaceutical Research and DeVelopment, Campus de Maigremont, B.P. 615,

F-27106 Val de Reuil, France, Antimicrobial Research Department, ADME-Tox & Bioanalysis Department, and Medicinal Chemistry

Department, Johnson & Johnson Pharmaceutical Research and DeVelopment, Turnhoutseweg 30, B-2340 Beerse, Belgium, and Tibotec BVBA,

Generaal de Wittelaan L 11B 3, B-2800 Mechelen, Belgium

ReceiVed October 11, 2007

A preceding paper (Bonfanti et al. J. Med Chem. 2007, 50, 4572-4584) reported the optimization of

the pharmacokinetic proﬁle of substituted benzimidazoles by reducing their tissue retention. However,

the modiﬁcations that were necessary to achieve this goal also led to a signiﬁcant drop in anti-RSV

activity. This paper describes a molecular modeling study followed by a lead optimization program

that led to the recovery of the initial potent antiviral activity and the selection of TMC353121 as a

clinical candidate.

Introduction

Human respiratory syncytial virus (RSV^a) is a negative-sense

enveloped RNA virus, a member of the Paramyxoviridae family,

subfamily Pneumovirinae. Human RSV causes a spectrum of

respiratory tract diseases in people of all ages throughout the

world. In healthy adults, a relatively mild infection results in

symptoms similar to those of the common cold. Among more

susceptible populations, particularly infants under the age of

Figure 1. Chemical structures of compounds JNJ-2408068 (57) and

58.

two and immunosuppressed and elderly people, RSV infection

can induce severe bronchiolitis or pneumonia, resulting in

respectively polyclonal and monoclonal antibodies, were li-

censed to prevent RSV infection in high-risk infants.

signiﬁcant morbidity and mortality. RSV is highly contagious,

and the infection in young children can cause lung damage that

persists for years, contributing to chronic lung diseases in later

life (chronic wheezing, asthma).

Thus, there is a clear need for a new drug to prevent and

treat RSV infection. Our RSV inhibition program ﬁrst led to

the discovery of JNJ-2408068 (57) (Figure 1).⁵This derivative

demonstrated picomolar anti-RSV activity, but its long elimina-

tion half-life from several tissues (t_1/2in lung ) 153 h) created

cause for concern. Initial attempts to improve the pharmacoki-

netic proﬁle were focused on the identiﬁcation of critical

substructures responsible for the long tissue retention. We

learned that reducing the basicity of the molecule by replacing

the aminoethylpiperidine moiety successfully reduced the tissue

half-life, but the resulting compounds were signiﬁcantly less

active against RSV.¹We then initiated a molecular modeling

study in combination with a lead optimization program aiming

at recovering the original RSV inhibitory activity.

On the basis of the discovery of resistance mutations in the

fusion protein of the virus, substituted benzimidazole compounds

were assumed to inhibit the RSV fusion process.⁵More

precisely, we assumed that these inhibitors act through direct

binding to a putative site located in the core domain of the RSV

fusion protein (F protein). This hypothesis was supported by

time of addition studies demonstrating that 57 inhibits the fusion

of the virus to uninfected cells early in the infection cycle as

well as the fusion of infected cells to uninfected cells at the

end of the replication cycle.⁵Single-point mutations were

identiﬁed in the fusion protein of several compound 57-resistant

variants of RSV. We,⁵and others,⁶have described two sets of

mutations on the core and the head domains respectively.

Evidence for the presumed mechanism of action came from

There are no vaccines available to prevent RSV infection.^2,3

Only three drugs have been approved so far. The only

chemotherapeutic on the market is ribavirin (Virazole).⁴This

aerosol treatment is used in high-risk or severely ill hospitalized

infants. The route of administration, the toxicity (risk of

teratogenicity), the cost, and the highly variable efﬁcacy limit

its use. Respigam, replaced now by palivizumab (Synagys),

* Author to whom correspondence should be addressed (telephone, +33

2 32 61 74 72; fax, +33 2 32 61 72 98; e-mail, jbonfant@prdfr.jnj.com).

†

Johnson & Johnson Pharmaceutical Research and Development, Val

de Reuil.

‡

Antimicrobial Research Department, Johnson & Johnson Pharmaceuti-

cal Research and Development, Beerse.

§

ADME-Tox & Bioanalysis Department, Johnson & Johnson Pharma-

ceutical Research and Development, Beerse.

#

Tibotec BVBA.

Medicinal Chemistry Department, Johnson & Johnson Pharmaceutical

Research and Development, Beerse.

^aAbbreviations: RSV, respiratory syncytial virus; RNA, ribonucleic acid;

F protein, fusion protein; Boc, tert-butoxycarbonyl; PK, pharmacokinetic;

LC; liquid chromatography; MS, mass spectroscopy; SAR, structure–pro-

perty relationship; rt, room temperature; EDTA, ethylenediaminetetraacetic

acid; DIPE, diisopropyl ether; HOBT, 1-hydroxybenzotriazole; EDCI, 1-(3-

dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; AUCinf, area

under concentration × time curve (total systemic exposure to test

compound); CL, plasma clearance; C_max, maximum plasma concentration;

Fabs, absolute bioavailability; t_1/2, half-life; V_dz, apparent volume of

distribution during the terminal phase; po, oral; iv, intravenous.

10.1021/jm701284j CCC: $40.75

2008 American Chemical Society

Published on Web 02/07/2008

876 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4

Bonfanti et al.

Scheme 1^a

^aReagents and conditions: (a) urea, xylene, 140 °C, 12 h; (b) POCl₃, HCl, 120 °C, 6 h; (c) 160 °C, 2 h; (d) K₂CO₃, CH₃CN, reﬂux, 12 h; (e) LiAlH₄,

THF, rt, 12 h; (f) H₂, Pd/C 10%, CH₃OH, 8 bar, 40 °C 12 h; (g) NEt₃, DMF, 80 °C, 12 h; (h) MnO₂, CH₂Cl₂, rt, 12 h; (i) NaH, THF, rt, 12 h; (j)

HCl/2-propanol 5 N, 2-propanol, 60 °C, 4 h; (k) H₂, Pd/C 10%, CH₃OH/THF, 2 bar, 4 h.

mutations D486N/E/V, E487D, F488I/L, and D489Y, which

cluster on the core domain over a putative binding site described

as a potential target for small-molecule inhibitors.⁷RSV fusion

inhibitors are thought to be able to disrupt the formation of a

six-helix bundle that is essential to start the fusion between the

viral and the cellular membrane.⁸

A common feature shared by class 1 fusion proteins is a

conformational rearrangement that those proteins undergo upon

fusion⁹to adopt a stable postfusion form. An X-ray structure

of the RSV F protein core domain in the postfusion state⁷

revealed the details of the intramolecular interactions between

two heptad repeats, named HR-N and HR-C. The HR-C peptides

insert in an antiparallel way into hydrophobic grooves located

at the surface of a central HR-N coiled coil. Of particular interest

is a cavity where most of the contacts between the two partners

occur and which accommodates the side chains of two aromatic

residues F483 and F488 of the HR-C peptides. Although

disruption of protein–protein interaction by small molecules is

traditionally considered to be a difﬁcult task in drug design,

this cavity has been described as a potential target for such

small-molecule inhibitors.^8,10

The above considerations allowed a structure-based design

strategy: the most potent compound of the series (58, pEC₅₀

)

10) (Figure 1) and the lead compound (57, pEC₅₀) 9.6) were

submitted to a conformational analysis to identify their most

stable conformation. Both structures were then docked in the

putative binding site both manually and automatically. The best

docking solutions were selected and analyzed for potential areas

of chemical modiﬁcation. A subsequent lead optimization

program led to the discovery of new compounds with picomolar

anti-RSV activity and devoid of accumulation issues.

Chemistry

The common building block 10 was obtained in six steps

starting from 3,4-diaminobenzoic acid ethyl ester 1 (Scheme

Respiratory Syncytial Virus Fusion Inhibitor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 877

Scheme 2^a

^aReagents and conditions: (a) CHOH 37% in water, NaBH₃CN, CH₃CO₂H, CH₃CN, rt, 12 h; (b) SOCl₂, CH₂Cl₂, 5 °C to rt, 12 h; (c) K₂CO₃, CH₃CN,

50 °C 12 h; (d) K₂CO₃, DMF, 80 °C, 4 h; (e) MnO₂, CH₂Cl₂, rt, 12 h; (f) (3-methylbenzyl)phosphonic acid diethyl ester, NaH, THF, 5 °C to rt, 12 h; (g)

H₂, Pd/C 10%, CH₃OH/THF, 2 bar, rt, 4 h; (h) ArNH₂, BH₃CN- on solid support or NaBH₃CN, AcOH, CH₃OH, rt, 24 h; (i) bromobenzene, Mg, THF, rt,

3 h.

1). Cyclization with urea in xylene at high temperature gave

the corresponding benzimidazolone 2. POCl₃mediated chlorina-

tion in acidic medium followed by a melting reaction with

N-benzyl-4-aminopiperidine 4 afforded intermediate 5. The

introduction of the hydroxypyridine moiety was not selective

and led to the formation of two isomers, 7 and 8. The two new

analogues were separated by column chromatography over silica

gel. Reduction using LiAlH₄in THF was performed on isomer

7. The resulting alcohol analogue 9 was ﬁnally debenzylated,

affording building block 10.

12. The aldehyde group necessary for a Wittig–Horner reaction

was formed via oxidation of the methyl-alcohol group with

MnO₂. 3-Methylbenzyl phosphonate, deprotonated with NaH,

reacted on the aldehyde to afford the methylstyrene derivative

15. Part of intermediate 15 was used to reduce the double bond

with hydrogen and palladium 10% on charcoal. Acid-mediated

deprotection of the primary amino group in intermediates 15

and 17 led to the ﬁnal expected compounds 16 and 18.

The selective methylation of the piperidine moiety was

performed by reductive amination using p-formaldehyde and

sodium borohydride (Scheme 2). A two-step process, involving

SOCl₂-mediated chlorination and coupling reaction in the

presence of K₂CO₃, allowed us to introduce different thio- and

aminoaryl groups. The aldehyde building block 23 gave access

Building block 10 was used to synthesize analogues of JNJ-

2408068 bearing an extra phenethyl chain on the benzimidazole

group (Scheme 1). N-Alkylation reaction with Boc-protected

bromoethylamine 11 in the presence of NEt₃gave intermediate

878 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4

Bonfanti et al.

Scheme 3^a

^aReagents and conditions (a) 120 °C, 10 h; (b) K₂CO₃, DMF, 70 °C, 24 h; (c) HBr 48% in water, 70 °C, 12 h; (d) HCHO 37% in water, NaBH₃CN,

AcOH, CH₃CN, rt, 12 h; (e) Ni (Ra), H₂, CH₃OH, 3 bar, rt, 1 h; (f) EDCI, HOBT, CH₂Cl₂, rt, 12 h; (g) BH₃CN- on solid support, AcOH, CH₃OH, rt, 24 h.

to the styrene (24) and phenethyl (25) analogues, to different

aniline derivatives (26a, 26b, 26c), and to a shorter chain with

a secondary hydroxyl group after a Grignard reaction (27).

To continue the modulation of the spacer group between the

benzimidazole core heterocycle and the phenyl ring, we

synthesized building block 35 bearing an amino functional group

(Scheme 3).

This building block was obtained using the synthetic pathway

already described in Scheme 1, starting from the nitrochlo-

robenzimidazole 28. A carboethoxy replaced the benzyl as

protecting group for the piperidine moiety. The ﬁnal reduction

was performed in the presence of Raney nickel under a pressure

of hydrogen to yield compound 35. This new starting material

was employed in a coupling reaction to afford the amide

derivative 36 and in a reductive amination reaction to afford

the N-m-methylbenzyl targeted analogue 37.

Bromoacetic acid ethyl ester was used to introduce the

hydroxyethyl chain on the piperidine group (Scheme 4). The

synthetic route leading to the aniline derivatives remained as

described earlier in Scheme 2. Final compounds 41a and 41b

were ﬁnally obtained after reduction of the carboxylic ester into

alcohol.

In the series covering the modulation of the entire aminoet-

hylpiperidine moiety, we introduced the various chains at the

beginning of the synthesis scheme by melting with chloroben-

zimidazole 3 (Scheme 5). As before, the introduction of the

hydroxypyridine “head part” led to the formation of two isomers.

After separation by column chromatography over silica gel,

isomer 45 was smoothly oxidized and the resulting aldehyde

reacted with 3,5-dimethylaniline to yield ﬁnal compounds

48a-48e. 48f was obtained after deprotection of the diol

function in 48b.

The morpholinopropyl series was explored extensively, and

an optimized synthetic pathway was used to produce the two

requested building blocks 53 and 54 (Scheme 6). Chlorobenz-

imidazole ester 3 was ﬁrst reduced into alcohol in the presence

of LiAlH₄. This reduction prevented a possible side reaction of

the ester group with the amino moiety of the next-step reactant

and allowed us to improve signiﬁcantly the yield of the melting

reaction (from 47 to 82%). The reaction with the lutidine

derivative 6 afforded again two isomers in the same proportion

and with a yield comparable to what was achieved with the

ester starting material 42. Secondary and primary anilines were

involved in reactions with the two building blocks 53 and 54

utilizing already described nucleophilic substitution and reduc-

tive amination conditions.

Molecular Modeling

The conformational analysis of compounds 58 and 57 yielded

1244 and 735 conformers, respectively, within the energy

window of 8 kcal/mol above the global minimum. Respectively,

Respiratory Syncytial Virus Fusion Inhibitor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 879

Scheme 4^a

^aReagents and conditions: (a) NEt₃, DMF, 50 °C, 1 h; (b) MnO₂, CH₂Cl₂, rt, 12 h; (c) ArNH₂, NaBH₃CN, CH₃CN, rt, 6 h; (d) LiAlH₄, THF, 5 °C to rt,

5 h.

Scheme 5^a

^aReagents and conditions: (a) 140 °C, 3 h; (b) K₂CO₃, DMF, rt, 24 h; (c) LiAlH₄, THF, 5 °C to rt, 13 h; (d) MnO₂, CH₂Cl₂, rt, 12 h; (e) AcOH,

NaBH₃CN, CH₃CN, rt, 12 h; (f) HCl 3N, THF, rt, 6 h.

84 and 91% of the conformers were found at least twice,

meaning that the conformational space was properly sampled.

Most notably, the lowest energy conformer of both compounds

(64.4 and 57.2 kcal/mol, respectively) showed an intramolecular

880 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4

Bonfanti et al.

Scheme 6^a

^aReagents and conditions (a) LiAlH₄, THF, 5 °C, 3 h; (b) 125 °C, 4 h; (c) K₂CO₃, DMF, rt, 12 h; (d) SOCl₂, CH₂Cl₂, 5 °C to rt, 14 h; (e) MnO₂, CH₂Cl₂,

rt, 12 h; (f) K₂CO₃or Cs₂CO₃, DMF, 80 °C, 1-4 h; (g) NaBH₃CN- on solid support or NaBH₃CN, AcOH, CH₃OH, or CH₃CN, rt, 12 to 24 h.

H-bond between the planar NH group and the pyridyl nitrogen.

However, the N-H· · ·N angles (128.8 and 131.9°, respectively)

deviated signiﬁcantly from the ideal 180° geometry such that it

was not expected that this weak H-bond strongly inﬂuences the

ligand conformation upon protein binding. Both lowest energy

conformers were selected as the starting point of the docking

study.

Automated and manual docking was performed on both

compounds. It has to be stressed that docking tools perform

better on well-delineated globular cavities where directional

protein–ligand H-bonds help the algorithm to converge to a

solution. Because the shape of the putative binding site is a

hydrophobic groove and the HR-C/HR-N interaction takes place

at the surface of the HR-N coiled coil, it is not surprising that

only a few solutions were obtained with FlexX. Additional

solutions were obtained by rigid, manual docking of the lowest

energy conformer of both compounds. All complexes were

subsequently optimized using the MMFF94s force ﬁeld.

The most interesting solutions are given in Figure 2. 58

(left) binds in two (P1 and P2) of the three subpockets

forming the hydrophobic binding site. In addition to the

ligand’s isopropyl group making favorable steric and hydro-

Respiratory Syncytial Virus Fusion Inhibitor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 881

Figure 2. Automated docking solutions for compounds 58 (left) and 57 (right). Similar binding modes have been obtained; both compounds

occupy subpockets P1 and P2, thus leaving subpocket P3 unexploited. Major interactions can be observed with residues N208, Q202, and Y198 and

the salt bridge D200-K196. (Left) Best docking solution obtained for compound 58. The isopropyl group perfectly mimics the interactions made

by the side chain of HR-C residue L481. (Right) Two best docking solutions obtained for compound 57. The differences lie in the orientation of

the ﬂexible amino chain able to interact with asparagine N208 or glutamine Q202.

phobic interactions in P1, three major interactions with

surrounding residues can be observed: the ammonium sub-

stituent is performing electrostatic interactions with the side

chain of polar residue N208; the hydroxyl group located on

the pyridyl ring makes a H-bond with the salt bridge

D200-K196; and the methyl substituent at position 4 of the

benzimidazole scaffold is engaged in an alkyl-pi stacking

with the aromatic ring of Y198. 57 (right) shares a similar

binding mode with 58. However, the absence of the isopropyl

group confers a greater ﬂexibility to the amino chain, which

enables the ammonium group to span a wider conformational

space. Two docking solutions were obtained that differ only

by the region of interaction of the ammonium group either

in the vicinity of N208 or close to Q202. Neither the

consensus scoring nor the potential energy difference between

the minimized complexes allowed us to favor one solution

over the other.

Evidence in favor of our docking solutions comes from

the comparison with the F protein core domain X-ray

structure. The “natural substrate” HR-C peptide and the

Figure 3. Interaction matches between HR-C (yellow) and 58 (green).

58 in its docked orientation has been superimposed to the X-ray

docked inhibitor 58 show remarkable similarities in their

interaction scheme with the F protein (Figure 3): both

molecules have an isopropyl chain interacting in subcavity

P1; their respective hydroxyl group is located in the same

region in space and makes a H-bond with the D200-K196

salt bridge. The two molecules also have an aromatic moiety

lying in the same subpocket P2.

Another observation that adds conﬁdence to our model

comes from the resistant RSV mutants that were raised

through exposure to 57.⁵They were shown to have single-

point mutations in the F-gene that affect residues at position

486-489 of the HR-C peptide. Those residues cover sub-

pocket P3 and a part of subpocket P2 with residue D486

being in direct contact with the docked inhibitors (Figure

3). It was questioned why those mutations occur on the HR-C

peptide, whereas they would have been expected to affect

residues of the HR-N coiled coil. Consistent with a scenario

structure of the F-protein core domain. The red circles indicate a match

between equivalent groups in both substrates.

proposed recently,¹¹one explanation is the establishment of

a ternary HR-N/inhibitor/HR-C complex upon binding of the

inhibitor. Due to its presence in the hydrophobic groove, the

competitive inhibitor would prevent zipping of the HR-C/

HR-N peptides. Starting in the mutation region, the remaining

HR-C sequence of residues would unzip from the “hydro-

phobic rail”. Local stabilization would be achieved by

establishment of new favorable interactions between affected

HR-C residues and the inhibitor. Mutations clearly indicate

that residues D486, E487, F488, and D489 could be actively

involved in a ternary complex formation and might inﬂuence

inhibitor binding. As a consequence, docking studies involv-

ing only residues from the HR-N binding site and the inhibitor

882 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4

Bonfanti et al.

Table 1. In Vitro RSV Inhibitory Activities of Compounds Synthesized To Validate the Molecular Modeling Model

should be analyzed with caution and within the context of a

possible ternary complex.

dazole scaffold and should extend by three bonds along the

hydrophobic groove. An aromatic 6-membered ring was deemed

to be optimal to mimic a phenylalanine side chain. Finally,

introduction of a methyl substituent in the meta position of the

phenyl ring was meant to optimize steric and hydrophobic

interactions. To validate these results, we synthesized the

corresponding 57 derivatives 16 and 18 (Table 1). Because of

chemical feasibility considerations, the methyl substituent at

position 4 of the benzimidazole scaffold was not introduced. If

properly designed, these compounds were supposed to retain

an activity level close to that of their parent compound. This

was indeed the case with compound 18, showing nanomolar

activity against RSV. The corresponding unsaturated analogue

16 was slightly less potent, presumably as a consequence of

inherent rigidity, which could prevent the substituent to ﬁt

optimally in the hydrophobic subpocket.

Results and Discussion

We have previously outlined the importance of the

aminoethylpiperidine moiety of compound 57 regarding RSV

inhibitory activity and tissue retention propensity.¹Its

replacement led to an improvement of the pharmacokinetic

proﬁle at the expense of a large drop in antiviral activity.

This drop is consistent with the assumption that the sup-

pression of the terminal ammonium group would lead to a

large increase in binding energy (∼18 kcal/mol). Analysis

of the docking model clearly indicated that compounds 57

and 58 bind in subpockets P1 and P2, thereby leaving

subpocket P3 free. It was hypothesized that the increase in

free binding energy obtained upon removal of the aminoet-

hylpiperidine moiety might be compensated by new favorable

interactions made in subpocket P3. This pocket is known to

be a key subsite for HR-C binding, where numerous contacts

occur with residue F488. Ideally, a new substituent should

be introduced that mimics the aromatic side chain of HR-C

residue F488. If successful, this would in turn translate into

some activity recovery while preserving the enhanced phar-

macokinetic proﬁle.

Lead Optimization. The strategy was to introduce substit-

uents in position 6 of the new compounds with improved

pharmacokinetic properties,¹in an attempt to increase their in

vitro RSV inhibitory activity.

The results obtained with compounds 16 and 18 suggested

that introduction of bulky hydrophobic substituents in position

6 was allowed, and we tried to further optimize the ﬁrst

substituents. N-Methylpiperidine building blocks were the most

accessible, and we decided to perform the optimization in this

series (Table 2). As expected, the strict analogues of compounds

A modeling design showed that, to reach subpocket P3, new

substituents should be anchored at position 6 of the benzimi-

Respiratory Syncytial Virus Fusion Inhibitor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 883

Table 2. Structure–Activity Relationship in the N-Methylpiperidine

Series

substituent was less effective (compound 21c, pEC₅₀) 7.4).

Most promisingly, incorporation of an ethanol chain resulted

in a further increase in activity (compound 21b, pEC₅₀) 8.5).

Combination with the di-m-methyl substitution of the aniline

moiety afforded the most potent inhibitor (compound 21a, pEC₅₀

) 9.4). This last compound was nearly equipotent to JNJ-

2408068.

We combined the substitution of compounds 26a and 26b

with different new chains L reported in our previous paper¹

(Table 3). For the hydroxyethylpiperidine chain (compounds

41a and 41b), the additional substituent led to a 1 log increase

in pEC₅₀compare to the original compound (from pEC₅₀

)

7.7 to pEC₅₀) 8.8). No difference was observed between the

monomethyl and dimethyl substitution of the phenyl ring. A

comparable improvement in potency was obtained for the

N-methylpropylpiperazine analogue (compound 48a, pEC₅₀

)

8.4 instead of 7.6 for the original derivative). The addition of

the new R-group did not result in any improvement to the

inhibitory activity of poorly active compounds with pEC₅₀values

below 4 (compounds 48c and 48d). The same modiﬁcation led

to a disappointing outcome for the dihydroxypropyl analogue

(compound 48f, pEC₅₀) 7.5, equipotent to the original

compound). Finally, the best results were obtained for the

morpholinopropyl derivatives. A 1.7 log increase was observed

with the mono-m-methyl substituent (compound 56a, pEC₅₀

)

8.6). Incorporation of a second methyl in the meta-position

resulted in a further increase in RSV inhibitory activity

(compound 48e, pEC₅₀) 9.0).

Three molecules underwent in vivo pharmacokinetics (PK)

experiments to study tissue retention. Combination of the new

substitution with the hydroxyethylpiperidine chain proved to

have an unfavorable effect on PK proﬁle in terms of tissue

retention. Whereas the original compound gave a t_1/2in lung of

29 h, the new analogues led to much longer half-lives,

comparable for the two derivatives (67.7 h for compound 41a

and 84.9 h for compound 41b). Most promisingly, the good

trend was conﬁrmed for the propylmorpholine analogue (com-

pound 48e, t_1/2) 13.7 h).

In an attempt to further study the phenomenon of potent RSV

inhibitors with improved PK proﬁle, we tested additional

morpholinopropyl analogues (Table 4). Substantial gains in

potency were seen with the introduction of polar chains on the

aniline part of the molecules. A hydroxyethyl chain on the

nitrogen of compound 56a led to a 0.7 log increase in RSV

inhibitory activity (compound 55b, pEC₅₀) 9.3). The addition

of a second methyl group in the meta-position resulted in an

antiviral of equivalent potency (compound 55c, pEC₅₀) 9.3).

In agreement with what we saw in the previous series, the

unsubstituted analogue still showed an interesting antiviral

potency, although 1 order of magnitude lower (compound 55a,

pEC₅₀) 8.5). The hydroxyethyl chain was favorably replaced

with an amide-propyl chain (compound 55d, pEC₅₀) 9.6). We

discovered that dislocation of the alcohol chain to the ortho-

position of the phenyl ring was the optimal choice (compound

56b, pEC₅₀) 9.1). Increasing the length of the linear alkyl chain

from two to three methylene groups led to a very potent

molecule (compound 56c, pEC₅₀) 9.6). Finally, combination

with the meta-methyl substitution afforded the most potent

analogue (compound 56d, pEC₅₀) 9.9).

16 and 18 were found to be less potent (compound 24, pEC₅₀

) 6.3; compound 25, pEC₅₀) 7.7). Despite this 2 log reduction

in activity, they could be used as a basis for a further

optimization. Replacement of the methylene linked to the phenyl

group with a heteroatom led to mixed results: although the

introduction of a sulfur atom resulted in somewhat reduced

potency (compound 22, pEC₅₀) 6.6), the gain in activity for

the aniline derivative was substantial (compound 26b, pEC₅₀

) 8.3). The importance of the methyl substituent on the phenyl

ring was well demonstrated by the reduction in activity when

removed (compound 26c, pEC₅₀) 7.8). Reduction of the chain

length alongside addition of a hydroxyl group to mimic the NH

of compound 26c (compound 27, pEC₅₀) 6.8) afforded a less

potent compound. Moving the NH one carbon atom away from

the ring reduced activity (compound 37, pEC₅₀) 7.8). An even

larger decrease in potency was seen upon replacement of the

methylene with a carbonyl in this inhibitor (compound 36, pEC₅₀

) 7.2). The addition of a second methyl group in the meta-

position of the aniline moiety led to a very potent molecule

(compound 26a, pEC₅₀) 8.6). We ﬁnally investigated substitu-

tion of the aniline nitrogen. The molecule with a simple methyl

Subsequently, four new morpholinopropyl derivatives were

selected for in vivo half-life determination. We conﬁrmed that

this series of very potent RSV inhibitors was devoid of

unfavorable tissue retention (10.4 h e t_1/2e 25.1 h).

884 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4

Bonfanti et al.

Table 3. Transfer of the SAR in the Series Covering the Modulation of the Aminoethylpiperidine Moiety

Several compounds with improved PK properties were

identiﬁed for preclinical PK and safety assessment, leading to

the selection of compound 56d (TMC353121) for clinical

evaluation. 56d is further evaluated in clinical trials.

mechanism by binding to the fusion protein and, hence,

inhibiting virus-cell fusion and cell-cell fusion (syncytia

formation).

Pharmacokinetic Proﬁle of 56d in Plasma. After iv

administration, 56d was rapidly eliminated with a plasma

clearance of 5.4 L/h/kg (Table 6). The plasma concentrations

dropped quickly within the ﬁrst hour after dosing and then much

more slowly. The plasma half-life determined between 4 and

24 h (last blood sampling time point) was about 10 h. The

volume of distribution was 79 L. After oral administration of a

10 mg/kg dose, the compound was slowly absorbed and the

absolute bioavailability was moderate (about 14%). Maximum

plasma concentrations of about 47 ng/mL were observed at 4 h

postdose.

Antiviral Spectrum and Mechanism of Action of 56d.

Antiviral activity was measured against group A and group B

respiratory syncytial viruses and clinical isolates with equal

potency (Table 5). The extent of resistance and/or cross-

resistance of 56d on mutants raised against 57¹(R1LO, R2LO

in Table 5) or palizivumab (R6LO in Table 5), where all

mutations are located on the fusion protein, was assessed using

the antiviral assay. The activity proﬁle of 56d was found to be

identical to the proﬁle of JNJ-2408068. Also, the R1LO and

R2LO RSV mutants were resistant against two demonstrated

binders of the fusion protein, VP-146373¹¹and BMS4337714.⁸

Moreover, it has recently been demonstrated that JNJ-2408068

competes with VP-14637 for the same binding site.¹¹These

observations all conﬁrm that 56d interferes with the entry

56d in the Cotton Rat Model. The cotton rat model was

used to demonstrate the in vivo efﬁcacy of 56d. The cotton rat

is semipermissive for RSV infection; that is, the amount of virus

recovered from the lungs is directly proportional to the amount

Respiratory Syncytial Virus Fusion Inhibitor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 885

Table 4. Structure–Activity Relationship in the Morpholinopropyl Series

of challenge virus, suggesting that there is only one round of

replication in the cotton rat.¹²

The results of the different experiments are summarized in

Table 7.

56d was shown to be active by intravenous, oral, and aerosol

administration in the cotton rat model at doses of 10 mg/kg, 40

mg/kg, and 5 mg/mL, respectively.

The results of the studies in which 56d (10 mg/kg) was

administered intravenously at different time points before and after

RSV infection suggest that the compound has to be administered

shortly before the virus challenge (i.e., 5 min) to achieve a maximal

antiviral effect. Only minor reductions were measured when the

compound was administered 24 h before or 3 h after virus

challenge. This is due to the fact that RSV replication in the cotton

886 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4

Bonfanti et al.

Table 5. Median in Vitro Results (HeLaM Cells) for 56d

RSV EC₅₀CC₅₀

rat model results in only one replication cycle and that 56d has to

be present before viral fusion occurs at the beginning of that

replication cycle. RSV infection in humans involves several

replication cycles, and it can therefore not be excluded that 56d

will have a therapeutic effect if administered after the onset of

symptoms.

SI

subtype strain (ng/mL) pEC₅₀pEC₉₀(ng/mL) (CC₅₀/EC₅₀)

A

B

A

LO

0.07

0.03

0.04

0.02

7.03

279.91

0.04

9.9

10.3

10.1

10.5

7.9

9.6

10.0

9.8

10.1

7.5

4436

63371

147867

110900

221800

631

CI11

CI57

CI41

CI42

R1LO

R2LO

R6LO

Conclusion

6.3

10.1

<4

<6.7

16

A molecular modeling study allowed us to identify a

possible modulation to improve the binding of our RSV

inhibitors with the target protein and recover the initial

nanomolar activity. A subsequent lead optimization program

led to the discovery of new morpholinopropyl-containing

substituted benzimidazoles. These new compounds not only

were very potent RSV inhibitors but were also devoid of any

tissue retention.

Among these new antiviral compounds, 56d was selected for

clinical development. 56d showed activity against groups A and

B RSV and against a panel of clinical isolates with equal

potency. In vivo activity in the cotton rat model by different

routes of administration was demonstrated. 56d is a potent novel

RSV fusion inhibitor further evaluated in clinical trials.

110900

dissolution. An equal volume of each individual solution was added

together with an equal volume of demineralized water to make a

formulation with a ﬁnal concentration of 0.25 mg of compound/

mL. The osmolarity was measured and brought to 283 mosmol/kg

with NaCl. The ﬁnal pH of the formulation was 5.35. Before dosing,

the formulation was stored at room temperature and protected from

light. Immediately after dosage, the formulation was frozen and

stored at e-18 °C until analysis.

For the tissue retention studies, all animals were dosed by a 10

min infusion in the tail vein, to provide a ﬁnal dose of 1 mg of

compound/kg of body weight. The exact infusion speed was

calculated by taking the exact body weight of the individual animals

into account. For the bioavailability study, a dose of 2 mg/kg of

compound 56d was given intravenously in a saphenous vein, and

a 10 mg/kg dose was given orally by gastric intubation.

For the tissue retention studies, at 4, 24, 96, and 168 h after

dose administration, three individual animals per time group were

sacriﬁced by decapitation for blood collection and dissection of

lung, liver, and kidney. For the bioavailability study, the sample

time points were at 7 and 20 min and 1, 2, 4, and 8 h (after

intravenous administration) and at 30 min and 1, 2, 4, 8, and 24 h

(after oral administration). Blood was collected on EDTA K3 in

10 mL BD Vacutainer tubes. Plasma was obtained following

centrifugation at 4 °C. Individual tissue samples were dissected,

blotted on ﬁlter paper, and weighed immediately. Individual tissue

homogenates were prepared in demineralized water (1:9 w/v).

Plasma and tissue homogenate samples were stored at e-18 °C

prior to analysis.

Individual plasma and tissue samples were analyzed using a

qualiﬁed research bioanalytical method (LC-MS/MS), that is, a

bioanalytical method that takes into account all scientiﬁc features

of a method stipulated in the FDA guidelines with respect to

stability, accuracy, and precision, but not fully validated.

Chemistry. All analytically pure compounds were dried under

vacuum in a drying pistol using a Buchi Glass Oven B-580

apparatus. Melting points were determined using a Leica VMHB

apparatus and are uncorrected. TLC analyses were run on silica

gel 60 F₂₅₄plates (Merck) using a variety of solvent systems and

a ﬂuorescent indicator for visualization. Spots were visualized under

254 nm UV illumination. Column chromatography was performed

with silica gel 60 (Merck) (0.015–0.040 mm) or Kromasyl (Akzo

Nobel) (0.010 mm). Proton NMR spectra were recorded on a Bruker

Avance 300 (300 MHz) and a Bruker Avance 400 (400 MHz)

spectrometer using internal deuterium lock. Chemical shifts are

reported relative to internal DMSO (δ 2.54) in parts per million

and coupling constants (J) in hertz. Exact mass spectra (TOF) were

recorded with a Micromass LCT instrument. Elemental analyses

were performed with a Thermo Electron Corp. instrument EA 1110

or EA 1108 for C, H, and N, and the results were within (0.4% of

the theoretical values. Chemicals and solvents were purchased from

either Acros Co. or Aldrich Chemical Co. Yields refer to puriﬁed

products and are not optimized.

Experimental Section

In Vitro Antiviral Assay. Our multicycle replication assay was

developed on the basis of the HeLaM cell using a low amount of

virus as the inoculum. This ensures that compounds interfering with

any of the many molecular targets that are important for virus

replication in vitro will be detected. RSV was added to HeLaM

cells, an epithelial-like cell line, in the presence and absence of

various compound concentrations. The viability of treated and

untreated cells was assessed by the addition of Thiazolyl blue

(MTT) after an incubation period of 1 week. pEC₅₀values were

calculated from the optical density (OD) values, measured spec-

trophotometrically. Virus controls, cell controls, and cytotoxicity

controls were included in each plate. Reference compounds were

included in each test. Tests in which the virus/cell/compound

controls did not meet the objectives were not approved.

The in vitro model was validated using ribavirin and palivizumab

as controls. Both substances produced EC₅₀values in the range of

published data. Ribavirin and JNJ-2408068 were included as

positive control in each test.

Cotton Rat Model. Cotton rat experiments were carried out

using prophylactic and therapeutic drug administration schedules.

Efﬁcacy was measured at 4 days after virus challenge, at a time

when RSV pulmonary titers reach their maximum level (i.e., ∼10⁶

infectious viruses/mL of BALF) in untreated control animals. For

sample collection, lungs were ﬂushed with physiological water and

the virus concentration in these broncho-alveolar lavage ﬂuids

(BALF) was determined by plaque titration. Compounds were

administered via inhalation (INH) with a SATURN (small animal

therapeutic ultrasonic nebulizer) aerosol-generating device, intra-

venously (iv) or orally by gavage (po).

A rough calculation of the dosage per kilogram of body weight

of drug delivered to the cotton rats by aerosol (inhalation) was made

using the following formula: estimated dose ) aerosol concentration

of drug × minutes of treatment × respiratory volume per minute/

kg for cotton rats × pulmonary retention fraction.¹³Because 56d

was added to the aerosol delivery reservoir at a concentration of 5

mg/mL, it is estimated that under the conditions utilized, a 75 g

cotton rat administered a single aerosol for 60 min would have

received an estimated dose of 1.25 mg/kg.

Pharmacokinetic Determination. Male Sprague–Dawley rats

(250–300 g, Charles River) were used (three animals per time

group).

Each individual compound was dissolved in a 10% hydroxypro-

pyl-ꢀ-cyclodextrin solution at an individual concentration of 1 mg/

mL. For some compounds, HCl was added to obtain their total

2-Hydroxy-3H-benzoimidazole-5-carboxylic Acid Ethyl Ester

(Intermediate 2). A mixture of 1 (0.166 mol) and urea (0.199 mol)

in xylene (300 mL) was stirred under reﬂux for 12 h. The reaction

was cooled to room temperature. The precipitate was ﬁltered off,

rinsed with xylene and DIPE, and then dried (32 g, 93%, melting

1

point > 260 °C): H NMR (DMSO-d₆) δ 1.30 (t, 3 H, J ) 7.0

Respiratory Syncytial Virus Fusion Inhibitor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 887

Table 6. Pharmacokinetic Proﬁle in Plasma

iv

po

dose (mg/kg)

2

AUC_inf(ng·h/mL)

CL (L/h/kg)

5.4

V_dz(L/kg)

t_{1/2(4–24 h)}(h)

dose (mg/kg)

10

C_max(ng/mL)

AUC_inf(ng·h/mL)

F_abs(%)

354

79.3

10.3

47.3

246

14

Table 7. Results in Cotton Rat Model

2-propanone/CH₃CN/DIPE. The precipitate was ﬁltered off and

dried (4.9 g of intermediate 7, 25%, melting point 179 °C): ¹H

NMR (DMSO-d₆) δ 1.32 (t, 3 H, J ) 7.2 Hz), 1.57 (qd, 2 H, J )

11.1 Hz), 2.02 (d, 2 H, J ) 11.1 Hz), 2.13 (t, 2 H, J ) 11.1 Hz),

2.36 (s, 3 H), 2.81 (d, 2 H, J ) 11.1 Hz), 3.50 (s, 2 H), 3.73–3.85

(m, 1 H), 4.28 (qd, 2 H, J ) 7.2 Hz), 5.19 (s, 2 H), 7.03–7.08 (m,

2 H), 7.15 (d, 1 H, J ) 7.7 Hz), 7.20 (d, 1 H, J ) 7.7 Hz), 7.22–7.28

(m, 1 H), 7.29–7.38 (m, 4 H), 7.62 (dd, 1 H, J ) 2.0, 7.7 Hz), 7.89

(d, 1 H, J ) 2.0 Hz), 10.34 (br s, 1 H).

log reduction^a

route^b

timing^c

dose^d

0.6

1

INH

iv

po

-1 h

0.25 mg/mL

1.25 mg/mL

5 mg/mL

20 mg/mL

2 mpk

10 mpk

40 mpk

-1 h

1.5

1.6

0.4

0.2

0.4

0.3

0.5

0.9

1.2

0.5

0.2

0.4

0.8

-1 h

-5 min

-96 h

-72 h

-48 h

-24 h

-4 h

Fraction 2 was crystallized from 2-propanone/CH₃CN/DIPE. The

precipitate was ﬁltered off and dried (5.5 g of intermediate8, 28%,

1

melting point 238 °C): H NMR (DMSO-d₆) δ 1.30 (t, 3 H, J )

-1 h

-5 min

+3 h

+24 h

-2 h

7.2 Hz), 1.55 (qd, 2 H, J ) 11.1 Hz), 2.02 (d, 2 H, J ) 11.1 Hz),

2.13 (t, 2 H, J ) 11.1 Hz), 2.34 (s, 3 H), 2.80 (d, 2 H, J ) 11.1

Hz), 3.50 (s, 2 H), 3.70–3.81 (m, 1 H), 4.26 (qd, 2 H, J ) 7.2 Hz),

5.19 (s, 2 H), 6.92–7.00 (m, 1 H), 7.06 (d, 1 H, J ) 7.7 Hz), 7.15

(d, 1 H, J ) 7.7 Hz), 7.22–7.28 (m, 1 H), 7.29–7.38 (m, 5 H), 7.59

(d, 1 H, J ) 7.7 Hz), 7.75 (s, 1 H), 10.34 (br s, 1 H).

^aLog reduction in BALF of cotton rats was calculated versus control

animals. ^bAll single doses (duration inhalation treatment ) 1 h). ^cIndicates

when the compound was administered before (minus) or after (plus) virus

challenge. ^dInhalation doses are expressed in mg/mL, i.e., the concentration

of the solution in the reservoir of the nebulizer.

2-[2-(1-Benzylpiperidin-4-ylamino)-6-hydroxymethylbenzoim-

idazol-1-ylmethyl]-6-methylpyridin-3-ol (Intermediate 9). LiAlH₄

(0.009 mol) was added portionwise to a mixture of 7 (0.003 mol)

in THF (60 mL) at 5 °C under N₂ﬂow. The reaction was stirred at

5 °C for 1 h and then at room temperature for 12 h. AcOEt and

H₂O were added carefully, and the aqueous layer was saturated

with K₂CO₃(powder). The organic layer was separated, dried (over

MgSO₄), and then ﬁltered over Celite. The ﬁltrate was evaporated

until dryness (1.3 g, 97%): ¹H NMR (DMSO-d₆) δ 1.54 (qd, 2 H,

J ) 11.1 Hz), 2.04 (d, 2 H, J ) 11.1 Hz), 2.13 (t, 2 H, J ) 11.1

Hz), 2.36 (s, 3 H), 2.80 (d, 2 H, J ) 11.1 Hz), 3.50 (s, 2 H),

3.68–3.78 (m, 1 H), 4.48 (d, 2 H, J ) 4.6 Hz), 5.01 (t, 1 H, J )

4.6 Hz), 5.10 (s, 2 H), 6.78 (d, 1 H, J ) 7.2 Hz), 6.88 (d, 1 H, J

) 7.7 Hz), 7.06 (d, 1 H, J ) 7.7 Hz), 7.11 (d, 1 H, J ) 7.7 Hz),

7.15 (d, 1 H, J ) 7.7 Hz), 7.22–7.28 (m, 2 H), 7.30–7.38 (m, 4 H),

10.28 (br s, 1 H).

Hz), 4.26 (qd, 2 H, J ) 7.0 Hz), 7.02 (d, 1 H, J ) 7.7 Hz), 7.48

(s, 1 H), 7.62 (d, 1 H, J ) 7.7 Hz), 10.93 (br s, 2 H).

2-Chloro-3H-benzoimidazole-5-carboxylic Acid Ethyl Ester

(Intermediate 3). A mixture of 2(0.073 mol) in POCl₃(150 mL)

was stirred at 100 °C. Concentrated HCl (around 1.5 mL) was added

dropwise, very carefully, until dissolution of 2. Next, the mixture

was stirred at 120 °C for 6 h. The solvent was evaporated until

dryness. The residue was taken up in H₂O/ice, basiﬁed with K₂CO₃

(powder), and extracted with EtOAc + 10% CH₃OH. The organic

layer was separated, dried (over MgSO₄), and ﬁltered, and the

solvent was evaporated until dryness (13.5 g, 83%, melting point

178 °C).

2-(1-Benzylpiperidin-4-ylamino)-3H-benzoimidazole-5-car-

boxylic Acid Ethyl Ester (Intermediate 5). A mixture of 3 (0.051

mmol) and 4 (0.056 mol) was stirred at 160 °C for 2 h. The residue

was taken up in CH₂Cl₂/H₂O and basiﬁed with a 10% solution of

K₂CO₃in water. The organic layer was separated, dried (over

MgSO₄), and ﬁltered, and the solvent was evaporated until dryness.

The residue was puriﬁed by column chromatography over silica

gel (eluent, CH₂Cl₂/CH₃OH/NH₄OH 95:5:0.5). The pure fractions

2-[6-Hydroxymethyl-2-(piperidin-4-ylamino)benzoimidazol-

1-ylmethyl]-6-methylpyridin-3-ol (Intermediate 10). A mixture

of 9 (0.0028 mol) and Pd/C 10% (2.5 g) in CH₃OH (40 mL) was

hydrogenated at 40 °C for 12 h under an 8 bar pressure and then

ﬁltered over Celite. The Celite was rinsed with a solution of

CH₃OH/THF (50:50). The ﬁltrate was concentrated under reduced

pressure (1.8 g, 95%, melting point 260 °C): MS (C₂₀H₂₅N₅O₂),

m/z 368 (M + H)⁺.

(2-{4-[6-Hydroxymethyl-1-(3-hydroxy-6-methylpyridin-2-ylme-

thyl)-1H-benzoimidazol-2-ylamino]piperidin-1-yl}ethyl)carbamic

Acid tert-Butyl Ester (Intermediate 12). A mixture of 10 (0.0079

mol), 11 (0.0095 mol), and NEt₃(0.0118 mol) in DMF (60 mL)

was stirred at 80 °C for 12 h. The solvent was evaporated until

dryness. The residue was taken up in CH₂Cl₂/H₂O. The organic

layer was separated, dried (over MgSO₄), and ﬁltered, and the

solvent was evaporated. The residue (7 g) was puriﬁed by column

chromatography over silica gel (eluent, CH₂Cl₂/CH₃OH/NH₄OH

90:10:0.5). The pure fractions were collected, and the solvent was

evaporated (1.2 g, 30%): MS (C₂₇H₃₈N₆O₄), m/z 511 (M + H)⁺.

(2-{4-[6-Formyl-1-(3-hydroxy-6-methylpyridin-2-ylmethyl)-

1H-benzoimidazol-2-ylamino]piperidin-1-yl}ethyl)carbamic Acid

tert-Butyl Ester (Intermediate 13). A mixture of 12 (0.0023 mol)

and MnO₂(2.4 g) in CH₂Cl₂(80 mL) was stirred at room

temperature for 12 h and then ﬁltered over Celite. The Celite was

washed with H₂O. The ﬁltrate was concentrated under reduced

pressure. The residue (1.2 g) was puriﬁed by column chromatog-

raphy over silica gel (eluent, CH₂Cl₂/CH₃OH/NH₄OH 95:5:0.1).

The pure fractions were collected, and the solvent was evaporated

(0.8 g, 67%): MS (C₂₇H₃₆N₆O₄), m/z 509 (M + H)⁺.

1

were collected, and the solvent was evaporated (15.3 g, 79%): H

NMR (DMSO-d₆) δ 1.30 (t, 3 H, J ) 7.2 Hz), 1.52 (qd, 2 H, J )

11.1 Hz), 1.93 (d, 2 H, J ) 11.1 Hz), 2.07 (t, 2 H, J ) 11.1 Hz),

2.79 (d, 2 H, J ) 11.1 Hz), 3.48 (s, 2 H), 3.55–3.65 (m, 1 H), 4.27

(qd, 2 H, J ) 7.2 Hz), 7.04 (d, 1 H, J ) 7.7 Hz), 7.13–7.19 (m, 1

H), 7.20–7.35 (m, 5 H), 7.59 (d, 1 H, J ) 7.7 Hz), 7.72 (s, 1 H),

10.28 (s, 1 H).

2-(1-Benzylpiperidin-4-ylamino)-3-(3-hydroxy-6-methylpyri-

din-2-ylmethyl)-3H-benzoimidazole-5-carboxylic Acid Ethyl Es-

ter (Intermediate 7) and 2-(1-Benzylpiperidin-4-ylamino)-1-(3-

hydroxy-6-methylpyridin-2-ylmethyl)-1H-benzoimidazole-5-

carboxylic Acid Ethyl Ester (Intermediate 8). A mixture of 5

(0.0396 mol), 6 (0.059 mol), and K₂CO₃(0.1584 mol) in CH₃CN

(180 mL) was stirred and reﬂuxed for 12 h. The solvent was

evaporated until dryness. The residue was taken up in CH₂Cl₂. The

organic layer was washed with H₂O, dried (over MgSO₄), and

ﬁltered, and the solvent was evaporated until dryness. The residue

(20 g) was puriﬁed by column chromatography over silica gel

(eluent, toluene/2-propanol/NH₄OH 85:15:1). Two fractions were

collected, and the solvent was evaporated [5.3 g of fraction 1 (27%)

and 6.3 g of fraction 2 (32%)]. Fraction 1 was crystallized twice in

888 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4

Bonfanti et al.

(2-{4-[1-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-6-(2-m-tolyl-

vinyl)-1H-benzoimidazol-2-ylamino]piperidin-1-yl}ethyl)carbamic

Acid tert-Butyl Ester (Intermediate 15). Diethyl 3-methylben-

zylphosphonate 14 (0.0023 mol) was added at 5 °C to a mixture of

NaH in oil (0.0047 mol) in THF (20 mL) under N₂ﬂow. The

mixture was stirred at 5 °C for 30 min. A solution of 13 (0.0007

mol) in THF (10 mL) was added. The mixture was stirred at 5 °C

for 1 h and then stirred at room temperature for 12 h. H₂O was

added. The mixture was extracted with CH₂Cl₂. The organic layer

was separated, dried (over MgSO₄), and ﬁltered, and the solvent

was evaporated. The residue was taken up in 2-propanone. The

precipitate was ﬁltered off and dried (0.32 g, 68%): ¹H NMR

(DMSO-d₆) δ 1.38 (s, 9 H), 1.53 (qd, 2 H, J ) 11.1 Hz), 2.01 (d,

2 H, J ) 11.1 Hz), 2.11 (t, 1 H, J ) 11.1 Hz), 2.30–2.39 (m, 8 H),

2.83 (d, 2 H, J ) 11.1 Hz), 3.05 (qd, 2 H, J ) 6.1 Hz), 3.65–3.75

(m, 1 H), 5.17 (s, 2 H), 6.65 (t, 1 H,J ) 5.1 Hz), 6.75–6.85 (m, 1

H), 7.00 (d, 1 H, J ) 16.2 Hz), 7.02–7.09 (m, 2 H), 7.13–7.20 (m,

3 H), 7.20–7.27 (m, 2 H), 7.35 (d, 1 H, J ) 7.7 Hz), 7.39 (s, 1 H),

7.54 (s, 1 H), 10.38 (br s, 1 H).

2-[2-[1-(2-Aminoethyl)piperidin-4-ylamino]-6-(2-m-tolylvinyl)

benzoimidazol-1-ylmethyl]-6-methylpyridin-3-ol (Compound

16). A mixture of 15 (0.0001 mol) in HCl 5 N/2-propanol (0.6

mL) and 2-propanol (6 mL) was stirred at 60 °C for 4 h and then

cooled to room temperature. The precipitate was ﬁltered off, rinsed

with 2-propanol and then with DIPE, and dried (0.057 g, HCl, salt,

71%, melting point 211 °C): ¹H NMR (DMSO-d₆) δ 2.10–2.30

(m, 4 H), 2.33 (s, 3 H), 2.37 (s, 3 H), 3.13 (qd, 2 H, J ) 11.1 Hz),

3.30–3.45 (m, 4 H), 3.70–3.79 (m, 2 H), 4.15–4.27 (m, 1 H), 5.62

(s, 2 H), 7.09 (s, 1 H, J ) 7.7 Hz), 7.19–7.30 (m, 4 H), 7.35 (d, 1

H, J ) 7.7 Hz), 7.40 (s, 1 H), 7.43–7.50 (m, 3 H), 7.75 (s, 1 H),

11.33 (br s, 1 H); HRMS (ESI), calcd for C₃₀H₃₇N₆O 497.3029,

found [MH]⁺497.3017.

(qd, 2 H, J ) 11.1 Hz), 2.04 (d, 2 H, J ) 11.1 Hz), 2.12–2.30 (m,

5 H), 2.38 (s, 3 H), 2.85 (d, 2 H, J ) 11.1 Hz), 3.67–3.78 (m, 1

H), 4.48 (s, 2 H), 4.95–5.05 (m, 1 H), 5.10 (s, 2 H), 6.81 (d, 1 H,

J ) 5.0 Hz), 6.88 (d, 1 H, J ) 7.7 Hz), 7.05 (d, 1 H, J ) 7.7 Hz),

7.12 (d, 1 H, J ) 7.7 Hz), 7.16 (d, 1 H, J ) 7.7 Hz), 7.25 (s, 1 H),

10.28 (br s, 1 H).

2-[6-Chloromethyl-2-(1-methylpiperidin-4-ylamino)benzoimi-

dazol-1-ylmethyl]-6-methylpyridin-3-ol (Intermediate 20). SOCl₂

(2.1 mL) was added dropwise to a mixture of 19 (0.0018 mol) in

CH₂Cl₂(20 mL) at 5 °C. The mixture was stirred at 5 °C for 1 h

and then at room temperature for 12 h. The solvent was evaporated

until dryness (0.93 g, 100%). The crude product was used directly

in the next reaction step.

2-[6-{[(3,5-Dimethylphenyl)-(2-hydroxyethyl)amino]methyl}-

2-(1-methylpiperidin-4-ylamino)benzoimidazol-1-ylmethyl]-6-

methylpyridin-3-ol (Compound 21a). A mixture of 20 (0.0003

mol), 2-(3,5-dimethylphenylamino)ethanol (0.0005 mol), and K₂CO₃

(0.0019 mol) in DMF (30 mL) was stirred at 80 °C for 4 h and

poured into H₂O. The aqueous layer was saturated with K₂CO₃and

extracted with CH₂Cl₂/CH₃OH. The organic layer was separated,

dried (over MgSO₄), and ﬁltered, and the solvent was evaporated

until dryness. The residue (0.25 g) was puriﬁed by column

chromatography over silica gel (eluent, CH₂Cl₂/CH₃OH/NH₄OH

90:10:1). The pure fractions were collected, and the solvent was

evaporated. The residue (0.05 g, 24%) was crystallized from

2-propanone/DIPE. The precipitate was ﬁltered off and dried (0.042

1

g, 20%, melting point 176 °C): H NMR (DMSO-d₆) δ 1.53 (qd,

2 H, J ) 11.1 Hz), 1.97–2.10 (m, 4 H), 2.13 (s, 6 H), 2.20 (s, 3 H),

2.33 (s, 3 H), 2.75 (d, 2 H, J ) 11.1 Hz), 3.44 (t, 2 H, J ) 6.4 Hz),

3.55 (t, 2 H, J ) 6.4 Hz), 3.60–3.70 (m, 1 H), 4.53 (s, 3H), 5.05

(s, 2 H), 6.20 (s, 1 H), 6.33 (s, 2 H), 6.75 (br s, 1 H), 6.79 (s, 1 H,

J ) 7.7 Hz), 7.03 (d, 1 H, J ) 7.7 Hz), 7.10 (d, 1 H, J ) 7.7 Hz),

7.12–7.18 (m, 2 H), 11.30 (br s, 1 H); HRMS (ESI), calcd for

C₃₁H₄₁N₆O₂529.3291, found [MH]⁺529.3305. Anal.

(C₃₁H₄₀N₆O₂·0.4H₂O) C, H, N.

(2-{4-[1-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-6-(2-m-tolyl-

ethyl)-1H-benzoimidazol-2-ylamino]piperidin-1-yl}ethyl)carbamic

Acid tert-Butyl Ester (Intermediate 17). A mixture of 15 (0.0002

mol) and Pd/C 10% (0.03 g) in CH₃OH (5 mL) and THF (5 mL)

was hydrogenated at room temperature for 4 h under a 2 bar

pressure and then ﬁltered over Celite. The organic layer was washed

with H₂O, dried (over MgSO₄), and ﬁltered, and the solvent was

evaporated until dryness (0.15 g, 100%). The crude compound was

used directly in the next reaction step.

2-[6-{[(2-Hydroxyethyl)phenylamino]methyl}-2-(1-methylpi-

peridin-4-ylamino)-benzoimidazol-1-ylmethyl]-6-methylpyridin-

3-ol (Compound 21b). 21b was synthesized from 2-phenylami-

noethanol with the procedure described for compound 21a (28.2%,

melting point 229 °C): ¹H NMR (DMSO-d₆) δ 1.52 (qd, 2 H, J )

11.1 Hz), 1.95–2.08 (m, 4 H), 2.20 (s, 3 H), 2.33 (s, 3 H), 2.33 (s,

3 H), 2.75 (d, 2 H, J ) 11.1 Hz), 3.49 (t, 2 H, J ) 6.4 Hz), 3.58

(t, 2 H, J ) 6.4 Hz), 3.62–3.72 (m, 1 H), 4.55 (s, 2 H), 4.68 br s,

1H), 5.05 (s, 2 H), 6.53 (t, 1 H, J ) 7.7 Hz), 6.65–6.75 (m, 3 H),

6.80 (d, 1 H, J ) 7.7 Hz), 7.01–7.12 (m, 4 H), 7.13–7.18 (m, 2 H),

10.25 (br s, 1 H); HRMS (ESI), calcd for C₂₉H₃₇N₆O₂501.2978,

found [MH]⁺501.2981. Anal. (C₂₉H₃₆N₆O₂·0.3H₂O) C, H, N.

6-Methyl-2-[6-[(methylphenylamino)methyl]-2-(1-methylpip-

eridin-4-ylamino)benzoimidazol-1-ylmethyl]pyridin-3-ol (Com-

pound 21c). 21c was synthesized from methylphenylamine with

the procedure described for compound 21a (30%, melting point

189 °C): ¹H NMR (DMSO-d₆) δ 1.53 (qd, 2 H, J ) 11.1 Hz),

1.95–2.09 (m, 4 H), 2.18 (s, 3 H), 2.33 (s, 3 H), 2.73 (d, 2 H, J )

11.1 Hz), 2.97 (s, 3H), 3.58–3.70 (m, 1 H), 4.52 (s, 2H), 5.05 (s,

2 H), 6.58 (t, 1 H, J ) 7.7 Hz), 6.70–6.78 (m, 3 H), 6.80 (d, 1 H,

J ) 7.7 Hz), 7.04 (d, 1 H, J ) 7.7 Hz), 7.07–7.20 (m, 5 H), 10.30

(br s, 1 H); HRMS (ESI), calcd for C₂₈H₃₅N₆O 471.2872, found

[MH]⁺471.2875. Anal. (C₂₈H₃₄N₆O) C, H, N.

6-Methyl-2-[2-(1-methylpiperidin-4-ylamino)-6-m-tolylsulfa-

nylmethylbenzoimidazol-1-ylmethyl]pyridin-3-ol (Compound

22). A mixture of 20 (0.0018 mol), 3-methylthiophenol (0.002 mol),

and K₂CO₃(0.0077 mol) in CH₃CN (70 mL) was stirred at 50 °C

for 12 h. The solvent was evaporated under reduced pressure. The

residue was taken up in H₂O. The mixture was extracted with

CH₂Cl₂. The organic layer was separated, dried (over MgSO₄), and

ﬁltered, and the solvent was evaporated until dryness. The residue

(0.55 g) was puriﬁed by column chromatography over silica gel

(eluent, CH₂Cl₂/CH₃OH/NH₄OH 88:12:2). The pure fractions were

collected, and the solvent was evaporated. The residue (0.35 g, 39%)

was crystallized from CH₃CN/DIPE. The precipitate was ﬁltered

off and dried (0.32 g, melting point 202 °C): ¹H NMR (DMSO-d₆)

2-[2-[1-(2-Aminoethyl)piperidin-4-ylamino]-6-(2-m-tolyleth-

yl)benzoimidazol-1-ylmethyl]-6-methylpyridin-3-ol (Compound

18). A mixture of 17 (0.0002 mol) in HCl 5 N/2-propanol (1.5

mL) and 2-propanol (15 mL) was stirred at 60 °C for 4 h and then

cooled to room temperature. The precipitate was ﬁltered off, rinsed

with 2-propanol/DIPE, and dried (0.128 g, HCl salt, 76%, melting

1

point 188 °C): H NMR (DMSO-d₆) δ 2.03–2.20 (m, 4 H), 2.22

(s, 3 H), 2.33 (s, 3 H), 2.72–2.82 (m, 4 H), 2.83–2.93 (m, 4 H),

3.05–3.20 (m, 2 H), 3.30–3.45 (m, 4 H), 3.70–3.80 (m, 2 H),

4.10–4.22 (m, 1H), 5.50 (s, 2 H), 6.88–6.95 (m, 2 H), 6.99 (s, 1

H), 7.07(d, 1 H, J ) 7.7 Hz), 7.12 (d, 1 H, J ) 7.7 Hz), 7.19 (d,

1 H, J ) 7.7 Hz), 7.29 (s, 1 H), 7.32–7.40 (m, 2 H), 9.00 (br s, 1

H), 11.30 (br s, 1 H); HRMS (ESI), calcd for C₃₀H₃₉N₆O 499.3185,

found [MH]⁺499.3197.

2-[6-Hydroxymethyl-2-(1-methylpiperidin-4-ylamino)ben-

zoimidazol-1-ylmethyl]-6-methylpyridin-3-ol (Intermediate 19).

HCHO 37% in water (0.0098 mol), NaBH₃CN (0.0059 mol), and

then CH₃CO₂H (2 mL) were added at room temperature to a mixture

of 10 (0.0049 mol) in CH₃CN (50 mL). The mixture was stirred at

room temperature for 12 h. The solvent was evaporated until

dryness. The residue was taken up in EtOH (30 mL), and a 5 N

solution of HCl in 2-propanol (4 mL) was added. The mixture was

stirred at 80 °C for 8 h. The solvent was evaporated until dryness.

The residue was taken up in CH₂Cl₂/K₂CO₃10%. The organic layer

was separated, dried (over MgSO₄), and ﬁltered, and the solvent

was evaporated under reduced pressure. The residue was crystallized

from CH₃OH/2-propanone/CH₃CN. The precipitate was ﬁltered off

and dried (1.65 g, 88%). Part of this fraction (0.15 g) was

crystallized from CH₃OH/2-propanone. The precipitate was ﬁltered

off and dried (melting point 165 °C): ¹H NMR (DMSO-d₆) δ 1.56

Respiratory Syncytial Virus Fusion Inhibitor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 889

δ 1.53 (qd, 2 H, J ) 11.1 Hz), 1.95–2.09 (m, 4 H), 2.18 (s, 3 H),

2.24 (s, 3 H), 2.35 (s, 3 H), 2.75 (d, 2 H, J ) 11.1 Hz), 3.60–3.72

(m, 1 H), 4.20 (s, 1 H), 5.08 (s, 2 H), 6.80 (s, 1 H), 6.90 (d, 1 H,

J ) 7.7 Hz), 7.03–7.10 (m, 4 H), 7.17 (d, 1 H, J ) 7.7 Hz), 7.23

(d, 2 H, J ) 7.7 Hz), 7.28 (s, 1 H), 10.35 (br s, 1 H); HRMS

(ESI), calcd for C₂₈H₃₄N₅OS 488.2484, found [MH]⁺488.2469.

Anal. (C₂₈H₃₃N₅OS) C, H, N.

Hz), 6.15 (s, 1 H), 6.22 (s, 2H), 6.70–6.75 (m, 1 H), 6.95 (d, 1 H,

J ) 7.7 Hz), 7.05 (d, 1 H, J ) 7.7 Hz), 7.12 (d, 1 H, J ) 7.7 Hz),

7.17 (d, 1 H, J ) 7.7 Hz), 7.28 (s, 1H), 10.25 (br s, 1 H); HRMS

(ESI), calcd for C₂₉H₃₇N₆O 485.3029, found [MH]⁺485.3028.

6-Methyl-2-[2-(1-methylpiperidin-4-ylamino)-6-(m-tolylami-

nomethyl)benzoimidazol-1-ylmethyl]pyridin-3-ol (Compound

26b). 23 (0.0005 mol), NaBH₃CN (0.0006 mol), and then CH₃CO₂H

(0.2 mL) were added at room temperature to a mixture of

3-methylaniline (0.0006 mol) in CH₃CN (20 mL). The mixture was

stirred at room temperature for 12 h. H₂O was added. The mixture

was saturated with K₂CO₃(powder) and extracted with CH₂Cl₂/

CH₃OH. The organic layer was separated, dried (over MgSO₄), and

ﬁltered, and the solvent was evaporated until dryness. The residue

(0.3 g) was puriﬁed by column chromatography over silica gel

(eluent, CH₂Cl₂/CH₃OH/NEt₃90:10:0.1). The pure fractions were

collected, and the solvent was evaporated. The residue (0.17 g, 68%)

was crystallized from CH₃OH/2-propanone/DIPE. The precipitate

was ﬁltered off and dried (0.13 g, 52%, melting point 141 °C): ¹H

NMR (DMSO-d₆) δ 1.53 (qd, 2 H, J ) 11.1 Hz), 1.97–2.10 (m, 4

H), 2.15 (s, 3 H), 2.20 (s, 3 H), 2.33 (s, 3 H), 2.75 (d, 2 H, J )

11.1 Hz), 3.63–3.72 (m, 1 H), 4.20 (d, 2 H, J ) 5.5 Hz), 5.10 (s,

2 H), 5.94 (t, 1 H, J ) 5.5 Hz), 6.31 (d, 1 H, J ) 7.7 Hz), 6.38 (d,

1 H, J ) 7.7 Hz), 6.42 (s, 1 H), 6.77 (br s, 1H), 6.91 (t, 1 H, J )

7.7 Hz), 7.95 (d, 1 H, J ) 7.7 Hz), 7.04 (d, 1 H, J ) 7.7 Hz),

7.10–7.19 (m, 2 H), 7.29 (s, 1H), 10.30 (br s, 1 H); HRMS (ESI),

calcd for C₂₈H₃₅N₆O 471.2872, found [MH]⁺471.2898. Anal.

(C₂₈H₃₄N₆O·0.5H₂O) C, H, N.

3-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-2-(1-methylpiperi-

din-4-ylamino)-3H-benzoimidazole-5-carbaldehyde (Intermedi-

ate 23). A mixture of 19 (0.0028 mol) and MnO₂(2.5 g) in CH₂Cl₂

(40 mL) was stirred at room temperature for 12 h and then ﬁltered

over Celite. Celite was rinsed with CH₂Cl₂. The ﬁltrate was

concentrated under reduced pressure. The residue was taken up in

2-propanone. The precipitate was ﬁltered off and dried (0.75 g,

1

69%, melting point 250 °C): H NMR (DMSO-d₆) δ 1.56 (qd, 2

H, J ) 11.1 Hz), 1.95–2.08 (m, 4 H), 2.20 (s, 3 H), 2.34 (s, 3 H),

2.75 (d, 2 H, J ) 11.1 Hz), 3.72–3.82 (m, 1 H), 5.22 (s, 2 H), 7.05

(d, 1 H, J ) 7.7 Hz), 7.15 (d, 1 H, J ) 7.7 Hz), 7.18–7.28 (m, 1

H), 7.30 (d, 1 H, J ) 7.7 Hz), 7.54 (dd, 1 H, J ) 2.0, 7.7 Hz), 7.73

(d, 1 H, J ) 2.0 Hz), 9.85 (s, 1 H).

6-Methyl-2-[2-(1-methylpiperidin-4-ylamino)-6-(2-m-tolylvi-

nyl)benzoimidazol-1-ylmethyl]-pyridin-3-ol (Compound 24). 3-

Methylbenzylphosphonic acid diethyl ester 14 (0.0027 mol) was

added at 5 °C to a mixture of NaH (0.0027 mol) in THF (15 mL)

under N₂ﬂow. The mixture was stirred at 5 °C for 30 min. A

solution of 23 (0.0009 mol) in THF (5 mL) was added. The mixture

was stirred at 5 °C for 1 h and then stirred at room temperature for

12 h. H₂O was added. The mixture was extracted with CH₂Cl₂.

The organic layer was separated, dried (over MgSO₄), and ﬁltered,

and the solvent was evaporated until dryness. The residue was

puriﬁed by column chromatography over silica gel (eluent, CH₂Cl₂/

CH₃OH/NH₄OH 85:14:1). The pure fractions were collected, and

the solvent was evaporated. The residue was crystallized from

2-propanone. The precipitate was ﬁltered off and dried (0.12 g,

6-Methyl-2-[2-(1-methylpiperidin-4-ylamino)-6-phenylami-

nomethylbenzoimidazol-1-ylmethyl]pyridin-3-ol (Compound

26c). 26c was synthesized from phenylamine with the procedure

1

described for compound 26b (51.4%, melting point 206 °C): H

NMR (DMSO-d₆) δ 1.53 (qd, 2 H, J ) 11.1 Hz), 1.95–2.08 (m, 4

H), 2.19 (s, 3 H), 2.33 (s, 3 H), 2.75 (d, 2 H, J ) 11.1 Hz),

3.63–3.72 (m, 1H), 4.23 (d, 1 H, J ) 5.5 Hz), 5.09 (s, 2 H), 6.03

(t, 1 H, J ) 5.5 Hz), 6.48 (t, 1 H, J ) 7.7 Hz), 6.58 (d, 2 H, J )

7.7 Hz), 6.78 (br s, 1 H), 6.95 (d, 1 H, J ) 7.7 Hz), 6.97–7.07 (m,

3 H), 7.10–7.18 (m, 2 H), 7.30 (s, 1 H), 10.30 (br s, 1 H); HRMS

(ESI), calcd for C₂₇H₃₃N₆O 457.2716, found [MH]⁺457.2731.

Anal. (C₂₇H₃₂N₆O·0.4H₂O) C, H, N.

1

28%, melting point 244 °C): H NMR (DMSO-d₆) δ 1.54 (qd, 2

H, J ) 11.1 Hz), 1.95–2.09 (m, 4 H), 2.19 (s, 3 H), 2.33 (s, 3 H),

2.37 (s, 3 H), 2.75 (d, 2 H, J ) 11.1 Hz), 3.64–3.75 (m, 1 H), 5.17

(s, 2 H), 6.84 (s, 1 H), 6.99 (d, 1 H, J ) 16.2 Hz), 7.02–7.10 (m,

2 H), 7.13–7.30 (m, 5 H), 7.36 (d, 2 H, J ) 7.7 Hz), 7.39 (s, 1 H),

7.53 (s, 3 H), 10.35 (br s, 1 H); HRMS (ESI), calcd for C₂₉H₃₄N₅O

468.2763, found [MH]⁺468.2751. Anal. (C₂₉H₃₃N₅O·0.3H₂O) C,

H, N.

2-[6-(Hydroxyphenylmethyl)-2-(1-methylpiperidin-4-ylami-

no)benzoimidazol-1-ylmethyl]-6-methylpyridin-3-ol (Compound

27). A solution of bromobenzene (0.002 mol) in THF was added

to a mixture of magnesium (0.002 mol) in a minimum of THF under

N₂ﬂow. The mixture was stirred at a temperature above 30 °C

and then cooled to room temperature. A solution of 23 (0.0003

mol) in THF (7 mL) was added dropwise. The mixture was stirred

at room temperature for 3 h. H₂O was added. Then, CH₂Cl₂was

added. The mixture was ﬁltered over Celite. The organic layer was

separated, dried (over MgSO₄), and ﬁltered, and the solvent was

evaporated until dryness. The residue was puriﬁed by column

chromatography over silica gel (eluent, CH₂Cl₂/CH₃OH/NH₄OH

84:14:1). The pure fractions were collected, and the solvent was

evaporated. The residue (0.1 g, 55%) was crystallized from DIPE.

The precipitate was ﬁltered off and dried (0.092 g, 51%, melting

6-Methyl-2-[2-(1-methylpiperidin-4-ylamino)-6-(2-m-tolyleth-

yl)benzoimidazol-1-ylmethyl]-pyridin-3-ol (Compound 25). A

mixture of 24 (0.0001 mol) and Pd/C 10% (0.015 g) in CH₃OH (3

mL) and THF (3 mL) was hydrogenated at room temperature for

4 h under a 2 bar pressure and then ﬁltered over Celite. The Celite

was washed with H₂O. The ﬁltrate was concentrated under reduced

pressure. The residue was crystallized from CH₃OH/2-propanone.

The precipitate was ﬁltered off and dried (0.04 g, 80%, melting

1

point 246 °C): H NMR (DMSO-d₆) δ 1.53 (qd, 2 H, J ) 11.1

Hz), 1.95–2.09 (m, 4 H), 2.18 (s, 3 H), 2.27 (s, 3 H), 2.35 (s, 3 H),

2.70–2.90 (m, 6 H), 3.60–3.72 (m, 1 H), 5.09 (s, 2 H), 6.65–6.75

(m, 1 H), 6.79 (d, 2 H, J ) 5.5 Hz), 6.95–7.08 (m, 5 H), 7.12–7.22

(m, 3 H), 10.35 (br s, 1 H); HRMS (ESI), calcd for C₂₉H₃₆N₅O

470.2920, found [MH]⁺470.2911. Anal. (C₂₉H₃₅N₅O·0.3H₂O) C,

H, N.

1

point 144 °C): H NMR (DMSO-d₆) δ 1.52 (qd, 2 H, J ) 11.1

Hz), 1.95–2.09 (m, 4 H), 2.18 (s, 3 H), 2.33 (s, 3 H), 2.70–2.80

(m, 2 H), 3.60–3.70 (m, 1 H), 5.08 (s, 2 H), 5.68 (s, 1 H), 5.73 (s,

1 H), 6.75 (s, 1 H), 6.88 (d, 1 H, J ) 7.7 Hz), 7.05 (t, 2 H, J ) 7.7

Hz), 7.03–7.19 (m, 2 H), 7.25 (t, 2 H, J ) 7.7 Hz), 7.32–7.40 (m,

3 H), 10.35 (br s, 1 H); HRMS (ESI), calcd for C₂₇H₃₂N₅O₂

458.2556, found [MH]⁺458.2564. Anal. (C₂₇H₃₁N₅O₂·1H₂O) C,

H, N.

2-[6-[(3,5-Dimethylphenylamino)methyl]-2-(1-methylpiperi-

din-4-ylamino)benzoimidazol-1-ylmethyl]-6-methylpyridin-3-

ol (Compound 26a). A mixture of 23 (0.0004 mol), 3,5-dimethyl-

aniline (0.00047 mol), BH₃CN- on solid support (0.00047 mol),

and CH₃CO₂H (4 drops) in CH₃OH (8 mL) was stirred at room

temperature for 24 h. The solution was ﬁltered. The ﬁltrate was

concentrated under reduced pressure. The residue was puriﬁed by

column chromatography over silica gel (eluent, CH₂Cl₂/CH₃OH/

NH₄OH 87:12:1.5). The pure fractions were collected, and the

solvent was evaporated (0.12 g). The compound was crystallized

4-(6-Nitro-1H-benzoimidazol-2-ylamino)piperidine-1-carb-

oxylic Acid Ethyl Ester (Intermediate 30). A mixture of 28

(0.0524 mol) and 29 (0.1048 mol) was stirred at 120 °C in a Parr

pressure vessel for 10 h, then taken up into H₂O, and extracted

with AcOEt. The concentrated organic layer was puriﬁed by short

open column chromatography over silica gel (eluent, CH₂Cl₂/

CH₃OH 96:4). The product fractions were collected, and the solvent

1

in CH₃CN/DIPE (0.08 g, 42%, melting point 235 °C): H NMR

(DMSO-d₆) δ 1.47–1.60 (m, 2 H), 1.97–2.08 (m, 4 H), 2.10 (s, 6

H), 2.20 (s, 3 H), 2.33 (s, 3 H), 2.72–2.80 (m, 2 H), 3.65–3.75 (m,

1 H), 4.20 (d, 2 H, J ) 5.5 Hz), 5.10 (s, 2 H), 5.80 (t, 1 H, J ) 5.5

1

was evaporated until dryness (7.7 g, 44%): H NMR (DMSO-d₆)

δ 1.19 (t, 3H, J ) 6.9 Hz), 1.40 (qd, 2 H, J ) 11.1 Hz), 1.95 (d,

890 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4

Bonfanti et al.

2 H, J ) 11.1 Hz), 2.90–3.05 (m, 2 H), 3.80–3.90 (m, 1 H), 3.95

(d, 2 H, J ) 11.1 Hz), 4.05 (qd, 2 H, J ) 6.9 Hz), 7.24 (d, 1 H, J

) 7.7 Hz), 7.40–7.55 (m, 1 H), 7.88 (d, 1 H, J ) 7.7 Hz), 7.95 (s,

1 H), 11.25 (br s, 1 H).

were added. The mixture was stirred and reﬂuxed for 4 h. CH₃OH

was then removed under reduced pressure, and the resulting solution

extracted with CH₂Cl₂. The organic layer was separated, dried (over

MgSO₄), and ﬁltered, and the solvent was evaporated until dryness.

The residue was puriﬁed by column chromatography over silica

gel (eluent, CH₂Cl₂/CH₃OH/NH₄OH 90:10:0.5). The pure fractions

were collected, and the solvent was evaporated (0.04 g, 60%):

HRMS (ESI), calcd for C₂₈H₃₃N₆O₂485.2665, found [MH]⁺

485.2660.

4-[1-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-6-nitro-1H-ben-

zoimidazol-2-ylamino]piperidine-1-carboxylic Acid Ethyl Ester

and 4-[1-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-5-nitro-1H-

benzoimidazol-2-ylamino]piperidine-1-carboxylic Acid Ethyl

Ester (Intermediates 31 and 32). A mixture of 30 (0.0312 mol),

6 (0.0343 mol), and K₂CO₃(0.1092 mol) in DMF (100 mL) was

stirred at 70 °C for 24 h. H₂O was then added. The mixture was

extracted with CH₂Cl₂. The organic layer was separated, dried (over

MgSO₄), and ﬁltered, and the solvent was evaporated until dryness.

The residue (12.2 g) was puriﬁed by column chromatography over

silica gel (eluent, toluene/iPrOH/NH₄OH 90:10:0.5). Two fractions

were collected, and the solvent was evaporated. Four grams of

intermediate 31 (28%) were obtained: ¹H NMR (DMSO-d₆) δ 1.20

(t, 3H, J ) 6.9 Hz), 1.48 (qd, 2 H, J ) 11.1 Hz), 2.00 (d, 2 H, J

) 11.1 Hz), 2.30 (s, 3, H), 3.00–3.10 (m, 2 H), 3.94 (d, 2 H, J )

11.1 Hz), 4.05 (qd, 2 H, J ) 6.9 Hz), 5.28 (s, 2 H), 7.05 (d, 1 H,

J ) 7.7 Hz), 7.15 (d, 1 H, J ) 7.7 Hz), 7.28 (d, 1 H, J ) 7.7 Hz),

7.34 (d, 1 H, J ) 7.7 Hz), 7.94 (dd, 1 H, J ) 2.5, 7.7 Hz), 8.13 (d,

1 H, J ) 2.5 Hz), 10.32 (br s, 1 H). Intermediate 32 as obtained in

6-Methyl-2-[6-(3-methylbenzylamino)-2-(1-methylpiperidin-

4-ylamino)benzoimidazol-1-ylmethyl]pyridin-3-ol (Compound

37). A mixture of 35 (0.0005 mol), 3-methylbenzaldehyde (0.0006

mol), BH₃CN- on solid support (0.0006 mol), and acetic acid (8

drops) in CH₃OH (10 mL) was stirred at room temperature for 24 h.

The solid support was ﬁltered off and rinsed with CH₃OH. The

ﬁltrate was concentrated under reduced pressure. The residue (0.53

g) was puriﬁed by column chromatography over silica gel (eluent,

CH₂Cl₂/CH₃OH/NH₄OH 92:8:0.5 to 89:10:1). The pure fractions

were collected, and the solvent was evaporated. The residue (0.11

g) was crystallized from CH₃OH/DIPE. The precipitate was ﬁltered

off and dried (0.072 g, 28%, melting point 240 °C): ¹H NMR

(DMSO-d₆) δ 1.490 (qd, 2 H, J ) 11.1 Hz), 1.95–2.07 (m, 4 H),

2.17 (s, 3 H), 2.27 (s, 3 H), 2.35 (s, 3 H), 2.75 (d, 2 H, J ) 11.1

Hz), 3.52–3.72 (m, 1 H), 4.15 (d, 2 H, J ) 5.5 Hz), 4.95 (s, 2 H),

5.67 (t, 1 H, J ) 5.5 Hz), 6.28 (d, 1 H, J ) 7.7 Hz), 6.53 (br s, 1

H), 6.63 (s, 1 H), 6.90 (d, 1 H, J ) 7.7 Hz), 6.99–7.08 (m, 2H),

7.14–7.23 (m, 4 H), 10.33 (br s, 1 H); HRMS (ESI), calcd for

C₂₈H₃₅N₆O 471.2872, found [MH]⁺471.2853. Anal. (C₂₈H₃₄N₆O)

C, H, N.

1

a yield of 5.4 g (38%): H NMR (DMSO-d₆) δ 1.19 (t, 3 H, J )

7.1 Hz), 1.39–1.51 (m, 2 H), 1.98–2.05 (m, 2 H), 2.28 (s, 3 H),

3.00–3.10 (m, 2 H), 3.88–3.96 (m, 3 H), 4.06 (qd, 2 H, J ) 7.1

Hz), 5.28 (s, 2 H), 7.05 (d, 1 H, J ) 7.7 Hz), 7.12–7.20 (m, 3H),

7.24 (d, 1 H, J ) 7.7 Hz), 7.89 (dd, 1 H, J ) 2.0, 7.7 Hz), 8.00 (d,

1 H, J ) 2.0 Hz), 10.28 (br s, 1H).

6-Methyl-2-[6-nitro-2-(piperidin-4-ylamino)benzoimidazol-1-

ylmethyl]pyridin-3-ol (Intermediate 33). Intermediate 31 (0.0088

mol) was added portionwise to a 48% solution of HBr in water

(40 mL). The mixture was brought slowly to 70 °C and then stirred

for 12 h. The precipitate was ﬁltered, washed with CH₃CN,

and dried. The residue (4.6 g, 80%) was taken up in H₂O and

basiﬁed with K₂CO₃(powder). The precipitate was ﬁltered and then

rinsed with EtOH. The ﬁltrate was concentrated under reduced

pressure (3 g, 52%).

6-Methyl-2-[2-(1-methylpiperidin-4-ylamino)-6-nitrobenzoim-

idazol-1-ylmethyl]pyridin-3-ol (Intermediate 34). HCHO 37%

in H₂O (0.0152 mol) and then NaBH₃CN (0.0091 mol) were added

at room temperature to a mixture of 33 (0.0075 mol) in CH₃CN

(100 mL). Acetic acid (3.5 mL) was added slowly at room

temperature. The mixture was stirred at room temperature for 12 h

and poured into H₂O. The aqueous layer was saturated with K₂CO₃

(powder). The mixture was extracted with EtOAc/CH₃OH. The

organic layer was separated, dried (over MgSO₄), and ﬁltered, and

the solvent was evaporated until dryness (2.6 g, 87%).

{4-[6-Hydroxymethyl-1-(3-hydroxy-6-methylpyridin-2-yl-

methyl)-1H-benzoimidazol-2-ylamino]piperidin-1-yl}acetic Acid

Ethyl Ester (Intermediate 38). A mixture of 10 (0.0027 mol),

ethyl bromoacetate (0.0032 mol), and NEt₃(0.004 mol) in DMF

(40 mL) was stirred at 50 °C for 1 h, poured into ice–water, and

extracted three times with CH₂Cl₂. The organic layer was separated,

dried (over MgSO₄), and ﬁltered, and the solvent was evaporated

until dryness. The residue was taken up in 2-propanone/DIPE. The

precipitate was ﬁltered off, rinsed with H₂O, and dried (1 g, 82%,

1

melting point 206 °C): H NMR (DMSO-d₆) δ 1.20 (t, 3H, J )

6.9 Hz), 1.54 (qd, 2 H, J ) 11.1 Hz), 2.03 (d, 2 H, J ) 11.1 Hz),

2.28–2.40 (m, 5 H), 2.85 (d, 2 H, J ) 11.1 Hz), 3.25 (s, 2 H),

3.65–3.75 (m, 1 H), 4.09 (qd, 2 H, J ) 6.9 Hz), 4.48 (d, 1 H, J )

5.0 Hz), 5.03 (t, 1 H, J ) 5.0 Hz), 5.12 (s, 2 H), 6.80–6.85 (m, 1

H), 6.88 (d, 1 H, J ) 7.7 Hz), 7.05 (d, 1 H, J ) 7.7 Hz), 7.12 (d,

1 H, J ) 7.7 Hz), 7.15 (d, 1 H, J ) 7.7 Hz), 7.25 (s, 1 H), 10.28

(s, 1 H).

{4-[6-Formyl-1-(3-hydroxy-6-methylpyridin-2-ylmethyl)-1H-

benzoimidazol-2-ylamino]piperidin-1-yl}acetic Acid Ethyl Ester

(Intermediate 39). A mixture of 38 (0.0044 mol) and MnO₂(4 g)

in CH₂Cl₂(60 mL) was stirred at room temperature for 12 h then

ﬁltered over Celite. The Celite was rinsed with H₂O. The ﬁltrate

was concentrated under reduced pressure. The residue was taken

up in CH₃CN. The precipitate was ﬁltered off and dried (1.8 g,

2-[6-Amino-2-(1-methylpiperidin-4-ylamino)benzoimidazol-

1-ylmethyl]-6-methylpyridin-3-ol (Intermediate 35). A mixture

of 34 (0.0065 mol) and Raney nickel (2.6 g) in CH₃OH (100 mL)

was hydrogenated at room temperature for 1 h under a 3 bar

pressure and then ﬁltered over Celite. The Celite was washed with

CH₃OH. The ﬁltrate was concentrated under reduced pressure. The

residue (2.2 g) was puriﬁed by column chromatography over silica

gel (eluent, CH₂Cl₂/CH₃OH/NH₄OH 85:14:1). The pure fractions

1

91%): H NMR (DMSO-d₆) δ 1.20 (t, 3H, J ) 6.9 Hz), 1.58 (qd,

2 H, J ) 11.1 Hz), 2.00 (d, 2 H, J ) 11.1 Hz), 2.30–2.40 (m, 5 H),

2.86 (d, 2 H, J ) 11.1 Hz), 3.25 (s, 2 H), 3.75–3.85 (m, 1 H), 4.09

(qd, 2 H, J ) 6.9 Hz), 5.23 (s, 2 H), 7.05 (d, 1 H, J ) 7.7 Hz),

7.15 (d, 1 H, J ) 7.7 Hz), 7.20 (d, 1 H, J ) 7.7 Hz), 7.31 (d, 1 H,

J ) 7.7 Hz), 7.54 (d, 1 H, J ) 7.7 Hz), 7.73 (s, 1 H), 9.85 (s, 1 H),

10.32 (s, 1 H).

1

were collected, and the solvent was evaporated (0.85 g, 35%): H

NMR (DMSO-d₆) δ 1.50 (qd, 2 H, J ) 11.1 Hz), 1.97–2.10 (m, 4

H), 2.20 (s, 3 H), 2.35 (s, 3 H), 2.70–2.80 (m, 2 H), 3.55–3.65 (m,

1 H), 4.60 (br s, 2 H), 4.95 (s, 2 H), 6.25 (dd, 1 H, J ) 2.1, 7.7

Hz), 6.55 (d, 1 H, J ) 2.1 Hz), 6.60 (br s, 1 H), 6.86 (d, 1 H, J )

7.7 Hz), 7.05 (d, 1 H, J ) 7.7 Hz), 7.15 (d, 1 H, J ) 7.7 Hz),

10.20 (br s, 1 H).

N-[3-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-2-(1-methylpi-

peridin-4-ylamino)-3H-benzoimidazol-5-yl]-3-methylbenza-

mide (Compound 36). A mixture of 35 (0.000125 mol) and

3-methylbenzoic acid (0.00025 mol) in CH₂Cl₂(4 mL) was stirred

at room temperature. HOBT (0.00025 mol) and EDCI (0.00025

mol) were added. The reaction was stirred at room temperature for

12 h. The solution was concentrated under reduced pressure, and a

10% solution of NaHCO₃in water (2 mL) and CH₃OH (2 mL)

{4-[1-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-6-(m-tolylami-

nomethyl)-1H-benzoimidazol-2-ylamino]piperidin-1-yl}acetic Acid

Ethyl Ester (Intermediate 40a). CH₃CO₂H (0.2 mL) was added

at room temperature to a mixture of 39 (0.0004 mol), 3-methyl-

aniline (0.0005 mol), and NaBH₃CN (0.0005 mol) in CH₃CN (20

mL). The mixture was stirred at room temperature for 6 h.

CH₃CO₂H (0.2 mL) was added. The mixture was stirred at room

temperature for 12 h. The solvent was evaporated until dryness.

The residue was taken up in CH₂Cl₂/K₂CO₃10%. The organic layer

was separated, dried (over MgSO₄), and ﬁltered, and the solvent

Respiratory Syncytial Virus Fusion Inhibitor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 891

was evaporated until dryness (0.22 g, 100%). This product was

used directly in the next reaction step.

1.78 (qt, 2 H, J ) 7.0 Hz), 2.14 (s, 3 H), 2.20–2.40 (m, 11 H),

3.40–3.50 (m, 2 H), 4.25 (qd, 2 H, J ) 7.0 Hz), 5.18 (s, 2 H), 7.03

(d, 1 H, J ) 7.7 Hz), 7.12–7.25 (m, 3 H), 7.55–7.64 (m, 1 Hm),

7.74–7.82 (m, 1 H).

{4-[6-[(3,5-Dimethylphenylamino)methyl]-1-(3-hydroxy-6-me-

thylpyridin-2-ylmethyl)-1H-benzoimidazol-2-ylamino]piperidin-

1-yl}acetic Acid Ethyl Ester (Intermediate 40b). 40b was

synthesized from 3,5-dimethylphenylamine with the procedure

described for intermediate 40a (100%).

2-{6-Hydroxymethyl-2-[3-(4-methylpiperazin-1-yl)propylami-

no]benzoimidazol-1-ylmethyl}-6-methylpyridin-3-ol (Intermedi-

ate 45a) and 2-{5-Hydroxymethyl-2-[3-(4-methylpiperazin-1-

yl)-propylamino]benzoimidazol-1-ylmethyl}-6-methylpyridin-3-

ol (Intermediate 46a). LiAlH₄(0.009 mol) was added slowly at 5

°C to a mixture of 43a + 44a (0.0015 mol) in THF (50 mL) under

N₂ﬂow. The mixture was stirred at 5 °C for 1 h and then stirred

at room temperature for 12 h. A minimum of H₂O was added. Next,

CH₂Cl₂/CH₃OH was added. The organic layer was separated, dried

(over MgSO₄), and ﬁltered, and the solvent was evaporated until

dryness. The residue (1.2 g) was puriﬁed by column chromatog-

raphy over silica gel (eluent, CH₂Cl₂/CH₃OH/NH₄OH 80:20:2). Two

fractions were collected, and the solvent was evaporated (0.37 g

of F1 and 0.53 g of F2). F1 was crystallized from 2-propanone/

CH₃CN/DIPE. The precipitate was ﬁltered off and dried (0.34 g of

intermediate 45a, 25.2%; melting point 230 °C): ¹H NMR (DMSO-

d₆) δ 1.78 (qt, 2 H, J ) 6.4 Hz), 2.12 (s, 3 H), 2.20–2.42 (m, 13

H), 3.39 (t, 2 H, J ) 6.4 Hz), 4.46 (s, 2 H), 4.98 (br s, 1 H), 5.10

(s, 2 H), 6.79 (br s, 1 H), 6.88 (d, 1 H, J ) 7.7 Hz), 7.03 (d, 1 H,

J ) 7.7 Hz), 7.09 (d, 1 H, J ) 7.7 Hz), 7.13 (d, 1 H, J ) 7.7 Hz),

7.18 (s, 1 H), 10.12 (br s, 1 H).

2-[2-[1-(2-Hydroxyethyl)piperidin-4-ylamino]-6-(m-tolylami-

nomethyl)benzoimidazol-1-ylmethyl]-6-methylpyridin-3-ol (Com-

pound 41a). LiAlH₄(0.0008 mol) was added to a mixture of 40a

(0.0004 mol) in THF (20 mL) at 5 °C under N₂ﬂow. The mixture

was stirred at 5 °C for 1 h, then brought to room temperature, and

stirred for 4 h. A minimum of H₂O and then CH₂Cl₂were added.

The organic layer was separated, dried (over MgSO₄), and ﬁltered,

and the solvent was evaporated until dryness. The residue (0.22 g)

was puriﬁed by column chromatography over silica gel (eluent,

CH₂Cl₂/CH₃OH/NH₄OH 85:15:1). The pure fractions were col-

lected, and the solvent was evaporated. The residue (0.1 g, 50%)

was crystallized from CH₃OH/CH₃CN/DIPE. The precipitate was

ﬁltered off and dried (0.08 g, 40%, melting point 137 °C): ¹H NMR

(DMSO-d₆) δ 1.50–1.65 (m, 2 H), 2.05 (d, 2H, J ) 11.1 Hz), 2.13

(s, 3 H), 2.25–2.38 (m, 5 H), 2.45–2.60 (m, 2 H), 2.92–3.04 (m, 2

H), 3.52–3.60 (m, 2 H), 3.68–3.80 (m, 1 H), 4.20 (d, 2 H, J ) 5.2

Hz), 5.10 (s, 2 H), 5.95 (t, 1 H, J ) 5.2 Hz), 6.30 (d, 1 H, J ) 7.7

Hz), 6.37 (d, 1 H, J ) 7.7 Hz), 6.42 (s, 1 H), 6.77 (s, 1 H), 6.89

(t, 1 H, J ) 7.7 Hz), 6.95 (d, 1 H, J ) 7.7 Hz), 7.04 (d, 1 H, J )

7.7 Hz), 7.12 (d, 1 H, J ) 7.7 Hz), 7.17 (d, 1 H, J ) 7.7 Hz), 7.28

(s, 1 H), 10.30 (br s, 1 H); HRMS (ESI), calcd for C₂₉H₃₇N₆O₂

501.2978, found [MH]⁺501.2970. Anal. (C₂₉H₃₆N₆O₂·2.8H₂O) C,

H, N.

F2 was crystallized from 2-propanone/CH₃CN/DIPE. The pre-

cipitate was ﬁltered off and dried (0.5 g of intermediate 46a, 39.4%;

1

melting point 154 °C): H NMR (DMSO-d₆) δ 1.77 (qt, 2 H, J )

6.8 Hz), 2.12 (s, 3 H), 2.16–2.44 (m, 13 H), 3.39 (t, 2 H, J ) 6.8

Hz), 4.45 (s, 2 H), 4.93 (s, 1 H), 5.10 (s, 2 H), 6.80–6.85 (m, 2 H),

7.02 (d, 1 Η, J ) 7.7 Hz), 7.07–7.17 (m, 3 H), 10.09 (br s, 1 H).

2-{6-[(3,5-Dimethylphenylamino)methyl]-2-[1-(2-hydroxyeth-

yl)piperidin-4-ylamino]benzoimidazol-1-ylmethyl}-6-methylpy-

ridin-3-ol (Compound 41b). 41b was synthesized from interme-

diate 40b with the procedure described for compound 41a (0.153

3-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-2-[3-(4-methylpip-

erazin-1-yl)propylamino]-3H-benzoimidazole-5-carbaldehyde (In-

termediate 47a). A mixture of 45a (0.0006 mol) and MnO₂(0.6

g) in CH₂Cl₂(60 mL) was stirred at room temperature for 12 h

and then ﬁltered over Celite. The Celite was rinsed with H₂O. The

ﬁltrate was concentrated under reduced pressure (0.28 g, 100%).

1

g, 66%, melting point 128 °C): H NMR (DMSO-d₆) δ 1.57 (qd,

2H, J ) 11.1 Hz), 2.05 (d, 2H, J ) 11.1 Hz), 2.14 (s, 6 H), 2.23

(t, 2H, J ) 11.1 Hz), 2.37 (s, 3 H), 2.46 (t, 2H, J ) 6.1 Hz), 2.86

(d, 2H, J ) 11.1 Hz), 3.53 (t, 2H, J ) 6.1 Hz), 3.70–3.80 (m, 1

H), 4.24 (d, 2H, J ) 5.1 Hz), 5.11 (s, 2 H), 5.42 (s, 1 H), 6.19 (s,

1 H), 6.27 (s, 2 H), 6.52 (br s, 1 H), 6.95 (d, 1 H, J ) 7.7 Hz),

7.04 (d, 1 H, J ) 7.7 Hz), 7.13 (d, 1 H, J ) 7.7 Hz), 7.17 (d, 1 H,

J ) 7.7 Hz), 7.33 (s, 1 H), 10.35 (br s, 1 H); HRMS (ESI), calcd

for C₃₀H₃₉N₆O₂515.3134, found [MH]⁺515.3119.

2-{6-[(3,5-Dimethylphenylamino)methyl]-2-[3-(4-methylpip-

erazin-1-yl)propylamino]benzoimidazol-1-ylmethyl}-6-meth-

ylpyridin-3-ol (Compound 48a). Acetic acid (0.3 mL) was added

at room temperature to a mixture of 47a (0.0006 mol), 3,5-

dimethylaniline hydrochloride (0.0007 mol), and NaBH₃CN (0.0007

mol) in CH₃CN (30 mL). The mixture was stirred at room

temperature for 30 min. Acetic acid (0.3 mL) was added, and the

mixture was stirred at room temperature for 12 h. Then, the solvent

was evaporated until dryness, the residue was taken up in EtOH/

2-propanol/saturated HCl, and the mixture was stirred at 80 °C for

12 h. Next, the solvent was evaporated until dryness. The mixture

was extracted with CH₂Cl₂/K₂CO₃10%. The organic layer was

separated, dried (over MgSO₄), and ﬁltered, and the solvent was

evaporated until dryness. The residue (0.43 g) was puriﬁed by

column chromatography over silica gel (eluent, CH₂Cl₂/CH₃OH/

NH₄OH 88:12:0.5). The pure fractions were collected, and the

solvent was evaporated. The residue (0.14 g, 40%) was crystallized

from 2-propanone/DIPE. The precipitate was ﬁltered off and dried

2-[3-(4-Methylpiperazin-1-yl)propylamino]-3H-benzoimida-

zole-5-carboxylic Acid Ethyl Ester (Intermediate 42a). A mixture

of 3 (0.0222 mol) and 1-(3-aminopropyl)-4-methylpiperazine

(0.0267 mol) was stirred at 140 °C for 1 h and then taken up in

CH₂Cl₂/CH₃OH. The organic layer was washed with K₂CO₃10%,

dried (over MgSO₄), and ﬁltered, and the solvent was evaporated

until dryness. The residue was puriﬁed by column chromatography

over silica gel (eluent, CH₂Cl₂/CH₃OH/NH₄OH 88:12:1). The pure

fractions were collected, and the solvent was evaporated (1 g, 13%):

¹H NMR (DMSO-d₆) δ 1.30 (t, 3 H, J ) 6.8 Hz), 1.72 (qt, 2 H, J

) 7.0 Hz), 2.15 (s, 3 H), 2.20–2.45 (m, 10 H), 3.30 (qd, 2 H, J )

7.0 Hz), 4.25 (qd, 2 H, J ) 6.8 Hz), 6.93–7.05 (m, 1 H), 7.15 (d,

1 H,J ) 7.7 Hz), 7.57 (d, 1 H, J ) 7.7 Hz), 7.70 (s, 1 H), 11.00 (br

s, 1 H).

1

(0.107 g, 30.6%; melting point 150 °C): H NMR (DMSO-d₆) δ

3-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-2-[3-(4-methylpip-

erazin-1-yl)propylamino]-3H-benzoimidazole-5-carboxylic Acid

Ethyl Ester (Intermediate 43a) and 1-(3-Hydroxy-6-methylpy-

ridin-2-ylmethyl)-2-[3-(4-methylpiperazin-1-yl)propylamino]-

1H-benzoimidazole-5-carboxylic Acid Ethyl Ester (Intermediate

44a). A mixture of 42a (0.0055 mol), 6 (0.006 mol), and K₂CO₃

(0.0165 mol) in CH₃CN (25 mL) was stirred at room temperature

for 24 h, poured into ice–water, and extracted with CH₂Cl₂. The

organic layer was separated, dried (over MgSO₄), and ﬁltered, and

the solvent was evaporated until dryness. The residue was puriﬁed

by column chromatography over silica gel (eluent, CH₂Cl₂/CH₃OH/

NH₄OH 84:15:1). The pure fractions were collected, and the solvent

was evaporated [1.4 g of the mixture of intermediates 43a + 44a

(60:40), 54.7%]; ¹H NMR (DMSO-d₆) δ 1.31 (t, 3 H, J ) 6.8 Hz),

1.78 (qt, 2 H, J ) 6.4 Hz), 2.08 (s, 6 H), 2.12 (s, 3 H), 2.20–2.40

(m, 13 H), 3.39 (t, 2 H, J ) 6.4 Hz), 4.17 (d, 2 H, J ) 6.4 Hz),

5.10 (s, 2 H), 5.82 (t, 1 H, J ) 5.1 Hz), 6.14 (s, 1 H), 6.20 (s, 2 H),

6.93 (d, 1 H, J ) 7.7 Hz), 7.02 (d, 1 H, J ) 7.7 Hz), 7.10 (d, 1 H,

J ) 7.7 Hz), 7.14 (d, 1H, J ) 7.7 Hz), 7.20 (s, 1 H), 10.20 (brs, 2

H); HRMS (ESI), calcd for C₃₁H₄₂N₇O 528.3451, found [MH]⁺

528.3434. Anal. (C₃₁H₄₁N₇O·0.45H₂O) C, H, N.

2-[(2,2-Dimethyl-[1,3]dioxolan-4-ylmethyl)amino]-3H-ben-

zoimidazole-5-carboxylic Acid Ethyl Ester (Intermediate 42b).

42b was synthesized from (2,2-dimethyl-[1,3]dioxolan-4-yl)meth-

ylamine with the procedure described for compound 42a (43.7%):

¹H NMR (DMSO-d₆) δ 1.25 (s, 3 H), 1.32 (t, 3 H, J ) 6.8 Hz),

1.37 (s, 3 H), 3.37–3.50 (m, 2 H), 3.72 (t, 1 H, J ) 7.7 Hz), 4.02

892 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4

Bonfanti et al.

(t, 1 H, J ) 7.7 Hz), 4.20–4.30 (m, 3 H), 6.90–7.00 (m, 1 H),

7.10–7.20 (m, 1 H), 7.55–7.65 (m, 1 H), 7.72 (s, 1 H), 10.90 (br s,

1H).

(qd, 2 H, J ) 7.0 Hz), 4.25 (qd, 2 H, J ) 6.8 Hz), 7.01 (t, 1 H, J

) 7.0 Hz), 7.15 (d, 1 H, J ) 7.7 Hz), 7.57 (dd, 1 H, J ) 2.0, 7.7

Hz), 7.70 (d, 1 H, J ) 2.0 Hz), 11.05 (br s, 1 H).

2-[(2,2-Dimethyl-[1,3]dioxolan-4-ylmethyl)amino]-3-(3-hydroxy-

6-methylpyridin-2-ylmethyl)-3H-benzoimidazole-5-carboxylic Acid

Ethyl Ester (43b) and 2-[(2,2-Dimethyl-[1,3]dioxolan-4-yl-

methyl)amino]-1-(3-hydroxy-6-methylpyridin-2-ylmethyl)-1H-

benzoimidazole-5-carboxylic Acid Ethyl Ester (44b). 43b and

44b were synthesized from 42b with the procedure described for

compounds 43a + 44a (40:60 mixture, 78.9%): ¹H NMR (DMSO-

d₆) δ 1.27 (s, 3 H), 1.31 (t, 3 H, J ) 6.8 Hz), 1.37 (s, 3 H),

2.30–2.35 (m, 3 H), 3.50–3.60 (m, 2 H), 3.75 (t, 1 H, J ) 7.7 Hz),

4.03 (t, 1 H, J ) 7.7 Hz), 4.25 (qd, 3 H, J ) 6.8 Hz), 4.35 (qt, 1

H, J ) 5.7 Hz), 5.15–5.25 (m, 2 H), 7.05 (d, 1 H, J ) 7.7 Hz),

7.12–7.35 (m, 3 H), 7.56–7.65 (m, 1 H), 7.75 (s, 1 H), 10.23 (br s,

1 H).

2-Butylamino-3-(3-hydroxy-6-methylpyridin-2-ylmethyl)-3H-

benzoimidazole-5-carboxylic Acid Ethyl Ester (43c) and 2-Bu-

tylamino-1-(3-hydroxy-6-methylpyridin-2-ylmethyl)-1H-ben-

zoimidazole-5-carboxylic Acid Ethyl Ester (44c). 43c and 44c

were synthesized from 42c with the procedure described for 43a

1

+ 44a (16.4% of 43c and 23.7% of 44c): H NMR (DMSO-d₆) δ

0.93 (t, 3 H, J ) 7.7 Hz), 1.30 (t, 3 H, J ) 6.8 Hz), 1.45 (sx, 2 H,

J ) 7.7 Hz), 1.62 (qt, 2 H, J ) 7.7 Hz), 2.30 (s, 3 H), 3.41 (qd, 2

H, J ) 5.1 Hz), 4.25 (qd, 2 H, J ) 6.8 Hz), 5.17 (s, 2 H), 6.94 (t,

1H, J ) 5.1 Hz), 7.04 (d, 1 H, J ) 7.7 Hz), 7.15 (d, 1 H, J ) 7.7

Hz), 7.25 (d, 1 H, J ) 7.7 Hz), 7.55 (d, 1 H, J ) 7.7 Hz), 7.74 (s,

1 H), 10.24 (br s, 1 H)).

2-(2-Butylamino-6-hydroxymethylbenzoimidazol-1-ylmethyl)-

6-methylpyridin-3-ol (45c). 45c was synthesized from 43c with

the procedure described for 45a (100%).

2-Butylamino-3-(3-hydroxy-6-methylpyridin-2-ylmethyl)-3H-

benzoimidazole-5-carbaldehyde (47c). 47c was synthesized from

45c with the procedure described for 47a (94%).

2-{2-[(2,2-Dimethyl-[1,3]dioxolan-4-ylmethyl)amino]-6-hy-

droxymethylbenzoimidazol-1-ylmethyl}-6-methylpyridin-3-ol (45b)

and 2-{2-[(2,2-Dimethyl-[1,3]dioxolan-4-ylmethyl)amino]-5-hy-

droxymethylbenzoimidazol-1-ylmethyl}-6-methylpyridin-3-

ol (46b). 45b and 46b were synthesized from the mixture 43b +

44b with the procedure described for 45a and 46a. 22% of 45b:

¹H NMR (DMSO-d₆) δ 1.28 (s, 3 H), 1.38 (s, 3 H), 2.34 (s, 3 H),

3.45–3.55 (m, 2 H), 3.75 (t, 1 H, J ) 7.7 Hz), 4.04 (t, 1 H, J ) 7.7

Hz), 4.35 (qt, 1 H, J ) 6.4 Hz), 4.47 (d, 2 H, J ) 5.1 Hz), 5.0 (t,

1 H, J ) 6.4 Hz), 5.05–5.20 (m, 2 H), 6.89 (d, 1 H, J ) 7.7 Hz),

6.95–7.07 (m, 2 H), 7.13 (d, 1 H, J ) 7.7 Hz), 7.15 (d, 1 H, J )

2-{2-Butylamino-6-[(3,5-dimethylphenylamino)methyl]ben-

zoimidazol-1-ylmethyl}-6-methylpyridin-3-ol (48c). 48c was syn-

thesized from 47c with the procedure described for 48a (14.5%,

1

melting point 181 °C): H NMR (DMSO-d₆) δ 0.92 (t, 2 H, J )

7.2 Hz), 1.39 (sx, 2 H, J ) 7.5 Hz), 1.61 (qt, 2 H, J ) 7.5 Hz),

2.08 (s, 6 H), 2.30 (s, 3 H), 3.35–3.45 (m, 2 H), 4.17 (d, 2 H, J )

5.1 Hz), 5.10 (s, 2 H), 5.82 (t, 1 H, J ) 5.1 Hz), 6.13 (s, 1 H), 6.20

(s, 2 H), 6.70 (br s, 1 H), 6.93 (d, 1 H, J ) 7.7 Hz), 7.02 (d, 1 H,

J ) 7.7 Hz), 7.09 (d, 1 H, J ) 7.7 Hz), 7.14 (d, 1H, J ) 7.7 Hz),

7.23 (s, 1 H), 10.25 (br s, 2 H); HRMS (ESI), calcd for C₂₇H₃₄N₅O

444.2763, found [MH]⁺444.2760. Anal. (C₂₇H₃₃N₅O) C, H, N.

2-Phenethylamino-3H-benzoimidazole-5-carboxylic Acid Eth-

yl Ester (42d). 42d was synthesized from phenethylamine with

the procedure described for 42a (48.4%, melting point 153 °C):

¹H NMR (DMSO-d₆) δ 1.30 (t, 3 H, J ) 6.8 Hz), 2.90 (t, 2 H, J

) 7.1 Hz), 3.55 (qd, 2 H, J ) 7.1 Hz), 4.27 (qd, 2 H, J ) 6.8 Hz),

7.00–7.13 (m, 1 H), 7.15–7.25 (m, 2 H), 7.25–7.35 (m, 4 H), 7.58

(d, 1 H, J ) 7.7 Hz), 7.73 (s, 1H), 11.00 (br s, 1H).

1

7.7 Hz), 7.23 (s, 1 H), 10.22 (br s, 1 H). 6.2% of 46b: H NMR

(DMSO-d₆) δ 1.28 (s, 3 H), 1.37 (s, 3 H), 2.34 (s, 3 H), 3.45–3.65

(m, 2 H), 3.75 (t, 1 H, J ) 7.7 Hz), 4.04 (t, 1 H, J ) 7.7 Hz),

4.30–4.40 (m, 1 H), 4.45 (d, 2 H, J ) 5.1 Hz), 4.95 (t, 1 H, J )

5.1 Hz), 5.05–5.20 (m, 2 H), 6.85 (d, 1 H, J ) 7.7 Hz), 6.95–7.02

(m, 1 H), 7.03 (d, 1 H, J ) 7.7 Hz), 7.10–7.18 (m, 3 H), 10.20 (br

s, 1 H).

2-[(2,2-Dimethyl-[1,3]dioxolan-4-ylmethyl)amino]-3-(3-hydroxy-

6-methylpyridin-2-ylmethyl)-3H-benzoimidazole-5-carbalde-

hyde (47b). 47b was synthesized from 45b with the procedure

described for 47a (90%).

2-{2-[(2,2-Dimethyl-[1,3]dioxolan-4-ylmethyl)amino]-6-[(3,5-

dimethylphenylamino)methyl]benzoimidazol-1-ylmethyl}-6-me-

thylpyridin-3-ol (48b). 48b was synthesized from 47b with the

procedure described for 48a (45.5%, melting point 212 °C): ¹H

NMR (DMSO-d₆) δ 1.28 (s, 3 H), 1.37 (s, 3 H), 2.08 (s, 6H), 2.32

(s, 3 H), 3.42–3.58 (m, 2 H), 3.74 (t, 1 H, J ) 7.3 Hz), 4.02 (t, 1

H, J ) 7.3 Hz), 4.18 (d, 2 H, J ) 5.6 Hz), 4.36 (qt, 1 H, J ) 7.3

Hz), 5.11 (dd, 2 H, J ) 3.6, 11 Hz), 5.84 (t, 1 H, J ) 5.6 Hz), 6.14

(s, 1 H), 6.20 (s, 2 H), 6.95 (d, 1 H, J ) 8.1 Hz), 6.97–7.05 (m, 2

H), 7.13 (t, 1 H, J ) 8.1 Hz), 7.24 (s, 1 H), 10.21 (br s, 1 H).

3-[6-[(3,5-Dimethylphenylamino)methyl]-1-(3-hydroxy-6-me-

thylpyridin-2-ylmethyl)-1H-benzoimidazol-2-ylamino]propane-

1,2-diol (48f). A mixture of 48b (0.0004 mol) in a 3 N solution of

HCl in water (20 mL) and THF (20 mL) was stirred at room

temperature for 6 h, basiﬁed with K₂CO₃(powder), and extracted

with CH₂Cl₂. The organic layer was separated, dried (over MgSO₄),

and ﬁltered, and the solvent was evaporated until dryness. The

residue (0.25 g) was puriﬁed by column chromatography over silica

gel (eluent, CH₂Cl₂/CH₃OH/NH₄OH 92:8:0.5). The pure fractions

were collected, and the solvent was evaporated. The residue (0.17

g, 92%) was crystallized from CH₃CN/DIPE. The precipitate was

3-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-2-phenethylamino-

3H-benzoimidazole-5-carboxylic Acid Ethyl Ester (43d) and

1-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-2-phenethylamino-

1H-benzoimidazole-5-carboxylic Acid Ethyl Ester (44d). 43d and

44d were synthesized from 42d with the procedure described for

43a + 44a. 19.8% of 43d: ¹H NMR (DMSO-d₆) δ 1.32 (t, 3 H, J

) 6.8 Hz), 2.25 (s, 3 H), 2.95 (t, 2 H, J ) 7.1 Hz), 3.65 (t, 2 H,

J ) 7.1 Hz), 4.25 (qd, 2 H, J ) 6.8 Hz), 5.18 (s, 2 H), 7.03 (d, 1

H, J ) 7.7 Hz), 7.13 (d, 1 H, J ) 7.7 Hz), 7.18–7.33 (m, 7 H),

7.64 (dd, 1 H, J ) 2.0, 7.7 Hz), 7.81 (d, 1 H, J ) 2.0 Hz), 10.25

1

(br s, 1H). 49.5% of 44d: H NMR (DMSO-d₆) δ 1.32 (t, 3 H, J

) 6.8 Hz), 2.24 (s, 3 H), 2.95 (t, 2 H, J ) 7.1 Hz), 3.64 (t, 2 H,

J ) 7.1 Hz), 4.27 (qd, 2 H, J ) 6.8 Hz), 5.18 (s, 2 H), 7.03 (d, 1

H, J ) 7.7 Hz), 7.10–7.17 (m, 2 H), 7.18–7.32 (m, 6 H), 7.58 (dd,

1 H, J ) 2.0, 7.7 Hz), 7.78 (d, 1 H, J ) 2.0 Hz), 10.25 (br s, 1H).

2-(6-Hydroxymethyl-2-phenethylaminobenzoimidazol-1-yl-

methyl)-6-methylpyridin-3-ol (45d). 45d was synthesized from

43d with the procedure described for 45a (70%, melting point 185

1

°C): H NMR (DMSO-d₆) δ 2.25 (s, 3 H), 2.96 (t, 2 H, J ) 7.1

Hz), 3.61 (t, 2 H, J ) 7.1 Hz), 4.47 (s, 2 H), 4.99 (br s, 1H), 5.10

(s, 2 H), 6.74–7.4 (m, 11H), 10.23 (br s, 1 H).

1

ﬁltered off and dried (0.127 g, 69%, melting point 128 °C): H

NMR (DMSO-d₆) δ 2.08 (s, 6 H), 2.32 (s, 3 H), 3.30–3.42 (m, 3

H), 3.47–3.56 (m, 1 H), 3.70 (qt, 1 H, J ) 4.6 Hz), 4.18 (d, 2 H,

J ) 5.1 Hz), 5.05–5.18 (m, 4 H), 5.84 (t, 1 H, J ) 5.1 Hz), 6.15

(s, 1 H), 6.20 (s, 2 H), 6.92–7.04 (m, 3 H), 7.10 (d, 1 H, J ) 7.7

Hz), 7.13 (d, 1 H, J ) 8.2 Hz), 7.25 (s, 1 H), 10.20 (brs, 2 H);

HRMS (ESI), calcd for C₂₆H₃₂N₅O₃462.2505, found [MH]⁺

462.2513. Anal. (C₂₆H₃₁N₅O₃·0.35H₂O) C, H, N.

3-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-2-phenethylamino-

3H-benzoimidazole-5-carbaldehyde (47d). 47d was synthesized

from 45d with the procedure described for 47a (100%).

2-{6-[(3,5-Dimethylphenylamino)methyl]-2-phenethylami-

nobenzoimidazol-1-ylmethyl}-6-methylpyridin-3-ol (48d). 48d

was synthesized from 47d with the procedure described for 48a

1

(75.4%, melting point 190 °C): H NMR (DMSO-d₆) δ 2.08 (s, 6

2-Butylamino-3H-benzoimidazole-5-carboxylic Acid Ethyl

Ester (42c). 42c was synthesized from butylamine with the

H), 2.25 (s, 3 H), 2.95 (t, 2 H, J ) 7.2 Hz), 3.45–3.55 (m, 2 H),

4.18 (d, 2 H, J ) 5.1 Hz), 5.10 (s, 2 H), 5.82 (t, 1 H, J ) 5.1 Hz),

6.14 (s, 1 H), 6.20 (s, 2 H), 6.88 (br s, 1 H), 6.94 (d, 1 H, J ) 7.7

Hz), 7.00 (d, 1 H, J ) 7.7 Hz), 7.13 (d, 2 H, J ) 7.7 Hz), 7.18–7.24

1

procedure described for 42a (26%): H NMR (DMSO-d₆) δ 0.92

(t, 3 H, J ) 7.7 Hz), 1.27–1.40 (m, 5 H), 1.50–1.60 (m, 2 H), 3.29

Respiratory Syncytial Virus Fusion Inhibitor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 893

(m, 2 H), 7.25–7.33 (m, 4 H), 10.20 (br s, 2 H); HRMS (ESI),

calcd for C₃₁H₃₄N₅O 492.2763, found [MH]⁺492.2788. Anal.

(C₃₁H₃₃N₅O) C, H, N.

taken up in CH₂Cl₂/CH₃OH. The organic layer was washed with a

10% solution of K₂CO₃in water, dried (over MgSO₄), and ﬁltered

and the solvent was evaporated until dryness. The residue (37 g)

was puriﬁed by column chromatography over silica gel (eluent,

CH₂Cl₂/CH₃OH/NH₄OH 90:10:0.5). The pure fractions were col-

2-(3-Morpholin-4-yl-propylamino)-3H-benzoimidazole-5-car-

boxylic Acid Ethyl Ester (42e). 42e was synthesized from

3-morpholin-4-yl-propylamine with the procedure described for 42a

(47%): ¹H NMR (DMSO-d₆) δ 1.30 (t, 3 H, J ) 7.1 Hz), 1.62 (qt,

2 H, J ) 6.9 Hz), 2.30–2.40 (m, 6 H), 3.30–3.38 (m, 2 H), 3.57 (t,

4 H, J ) 4.0 Hz), 4.25 (qd, 2 H, J ) 7.1 Hz), 7.00–7.08 (m, 1 H),

7.15 (d, 1 H, J ) 7.7 Hz), 7.53–7.63 (m, 1 H), 7.70 (s, 1 H), 11.10

(s, 1 H).

3-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-2-(3-morpholin-4-

ylpropylamino)-3H-benzoimidazole-5-carboxylic Acid Ethyl Es-

ter (43e) and 1-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-2-(3-

morpholin-4-ylpropylamino)-1H-benzoimidazole-5-carboxylic

Acid Ethyl Ester (44e). 43e and 44e were synthesized from 42e

with the procedure described for 43a + 44a. 20%, melting point

184 °C for 43e: ¹H NMR (DMSO-d₆) δ 1.31 (t, 3 H, J ) 7.4 Hz),

1.79 (qt, 2 H, J ) 6.6 Hz), 2.26–2.42 (m, 9 H), 3.45 (t, 2 H, J )

6.6 Hz), 3.55 (t, 4 H, J ) 5.6 Hz), 4.25 (qd, 2 H, J ) 7.4 Hz), 5.18

(s, 2 H), 7.05 (d, 1 H, J ) 7.7 Hz), 7.10–7.18 (m, 2 H), 7.19 (d, 1

H, J ) 7.7 Hz), 7.62 (d, 1 H, J ) 7.7 Hz), 7.80 (s, 1 H), 10.25 (br

s, 1 H). 34%, melting point 176 °C for 44e: ¹H NMR (DMSO-d₆)

δ 1.30 (t, 3 H, J ) 7.4 Hz), 1.79 (qt, 2 H, J ) 6.6 Hz), 2.30 (s, 3

H), 2.31–2.40 (m, 6 H), 3.45 (t, 2 H, J ) 6.6 Hz), 3.55 (t, 4 H, J

) 5.6 Hz), 4.25 (qd, 2 H, J ) 7.4 Hz), 5.18 (s, 2 H), 6.92–7.02

(m, 1 H), 7.05 (d, 1 H, J ) 7.7 Hz), 7.15 (d, 1 H, J ) 7.7 Hz),

7.23 (d, 1 H, J ) 7.7 Hz), 7.55 (d, 1 H, J ) 7.7 Hz), 7.75 (s, 1 H),

10.25 (br s, 1 H).

1

lected, and the solvent was evaporated (16.5 g, 82%): H NMR

(DMSO-d₆) δ 1.72 (qt, 2 H, J ) 6.4 Hz), 2.28–2.42 (m, 6 H), 3.29

(qd, 2 H, J ) 6.4 Hz), 3.57 (t, 4 H, J ) 5.1 Hz), 4.46 (s, 2 H), 5.07

(br s, 1 H), 6.54 (t, 1 H, J ) 6.4 Hz), 6.81 (d, 1 H, J ) 7.7 Hz),

7.04 (d, 1 H, J ) 7.7 Hz), 7.08 (s, 1 H), 10.71 (br s, 1 H).

2-[6-Hydroxymethyl-2-(3-morpholin-4-ylpropylamino)ben-

zoimidazol-1-ylmethyl]-6-methylpyridin-3-ol (51) and 2-[5-Hy-

droxymethyl-2-(3-morpholin-4-ylpropylamino)benzoimidazol-

1-ylmethyl]-6-methylpyridin-3-ol (52). A mixture of 50 (0.0396

mol), 6 (0.0475 mol), and K₂CO₃(0.1188 mol) in dimethylforma-

mide (110 mL) was stirred at room temperature for 12 h. The

reaction was poured into ice/water. The aqueous layer was saturated

with K₂CO₃(powder) and extracted with a solution of CH₂Cl₂/

CH₃OH (95:5). The residue was puriﬁed by chromatography over

silica gel (eluent, CH₂Cl₂/CH₃OH/NH₄OH 90:10:1). The pure

fractions were collected, and the solvent was evaporated. 5.4 g of

51 (33%, melting point 192 °C): ¹H NMR (DMSO-d₆) δ 1.79 (qt,

2 H, J ) 6.4 Hz), 2.31 (s, 3 H), 2.33–2.45 (m, 6 H), 3.41 (t, 2 H,

J ) 6.4 Hz), 3.54 (t, 4 H, J ) 5.1 Hz), 4.47 (s, 2 H), 4.99 (br s, 1

H), 5.11 (s, 2 H), 6.80 (br s, 1 H), 6.88 (d, 1 H, J ) 7.7 Hz), 7.03

(d, 1 H, J ) 7.7 Hz), 7.09 (d, 1 H, J ) 7.7 Hz), 7.15 (d, 1 H, J )

7.7 Hz), 7.19 (s, 1 H), 10.26 (br s, 1 H). 5 g of 52 (31%, melting

point 134 °C): ¹H NMR (DMSO-d₆) δ 1.79 (qt, 2 H, J ) 6.4 Hz),

2.31 (s, 3 H), 2.33–2.43 (m, 6 H), 3.41 (t, 2 H, J ) 6.4 Hz), 3.56

(t, 4 H, J ) 5.1 Hz), 4.45 (s, 2 H), 4.94 (br s, 1 H), 5.11 (s, 2 H),

6.76 (br s, 1 H), 6.82 (d, 1 H, J ) 7.7 Hz), 7.02 (d, 1 H, J ) 7.7

Hz), 7.06–7.17 (m, 3 H), 10.26 (br s, 1 H).

2-[6-Hydroxymethyl-2-(3-morpholin-4-ylpropylamino)benzo-

imidazol-1-ylmethyl]-6-methyl-pyridin-3-ol (45e). 45e was syn-

thesized from 43e with the procedure described for 45a (83%,

1

2-[6-Chloromethyl-2-(3-morpholin-4-ylpropylamino)benzoim-

idazol-1-ylmethyl]-6-methylpyridin-3-ol (53). SOCl₂(0.81 mL)

was added dropwise to a mixture of 51 (0.0006 mol) in CH₂Cl₂

(10 mL) at 5 °C. The mixture was stirred at 5 °C for 2 h, then

brought to room temperature, and stirred for 12 h. The solvent was

evaporated until dryness (0.42 g, 100%, 4 HCl). The crude

compound was used directly in the next reaction step.

3-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-2-(3-morpholin-4-

ylpropylamino)-3H-benzoimidazole-5-carbaldehyde (54). 54 is

the same compound as 47e.

melting point 192 °C): H NMR (DMSO-d₆) δ 1.79 (qt, 2 H, J )

6.4 Hz), 2.31 (s, 3 H), 2.33–2.45 (m, 6 H), 3.41 (t, 2 H, J ) 6.4

Hz), 3.54 (t, 4 H, J ) 5.1 Hz), 4.47 (s, 2 H), 4.99 (br s, 1 H), 5.11

(s, 2 H), 6.80 (br s, 1 H), 6.88 (d, 1 H, J ) 7.7 Hz), 7.03 (d, 1 H,

J ) 7.7 Hz), 7.09 (d, 1 H, J ) 7.7 Hz), 7.15 (d, 1 H, J ) 7.7 Hz),

7.19 (s, 1 H), 10.26 (br s, 1 H).

3-(3-Hydroxy-6-methylpyridin-2-ylmethyl)-2-(3-morpholin-4-

ylpropylamino)-3H-benzoimidazole-5-carbaldehyde (47e). 47e

was synthesized from 45e with the procedure described for 47a

1

(90%, melting point 206 °C): H NMR (DMSO-d₆) δ 1.80 (qt, 2

H, J ) 6.6 Hz), 2.30 (s, 3 H), 2.31–2.40 (m, 6 H), 3.48 (t, 2 H, J

) 6.6 Hz), 3.55 (t, 4 H, J ) 5.6 Hz), 5.22 (s, 2 H), 7.03 (d, 1 H,

J ) 7.7 Hz), 7.14 (d, 1 H, J ) 7.7 Hz), 7.22–7.32 (m, 2 H), 7.53

(d, 1 H, J ) 7.7 Hz), 7.65 (s, 1 H), 9.81 (s, 1 H).

2-[6-{[(2-Hydroxyethyl)phenylamino]methyl}-2-(3-morpholin-

4-ylpropylamino)benzoimidazol-1-ylmethyl]-6-methylpyridin-3-

ol (55a). A mixture of 53 (0.0009 mol), 2-(phenylamino)ethanol

(0.0004 mol), and K₂CO₃(0.0019 mol) in DMF (30 mL) was stirred

at 80 °C for 1 h. H₂O was added, and the mixture was extracted

with CH₂Cl₂. The organic layer was separated, dried (over MgSO₄),

and ﬁltered, and the solvent was evaporated. The residue (0.16 g)

was crystallized from CH₃CN/DIPE. The precipitate was ﬁltered

off, rinsed with DIPE, and dried (0.051 g, 25%, melting point 190

°C): ¹H NMR (DMSO-d₆) δ 1.78 (qt, 2 H, J ) 7.4 Hz), 2.32 (s, 3

H), 2.33–2.42 (m, 6 H), 3.40 (t, 2 H, J ) 6.4 Hz), 3.47 (t, 2 H, J

) 6.4 Hz), 3.53–3.63 (m, 6 H), 4.55 (s, 2 H), 5.05 (s, 2 H), 6.53

(t, 1 H, J ) 7.7 Hz), 6.68 (d, 2 H, J ) 7.7 Hz), 6.78 (d, 1 H, J )

7.7 Hz), 6.98–7.15 (m, 6 H), 10.20 (br s, 1 H); HRMS (ESI), calcd

for C₃₀H₃₉N₆O₃531.3084, found [MH]⁺531.3088. Anal. (C₃₀H₃₈-

N₆O₃·0.5H₂O) C, H, N.

2-[6-[(3,5-Dimethylphenylamino)methyl]-2-(3-morpholin-4-yl-

propylamino)benzoimidazol-1-ylmethyl]-6-methylpyridin-3-ol

(48e). 48e was synthesized from 47e with the procedure described

for 48a (48%, melting point 199 °C): ¹H NMR (DMSO-d₆) δ 1.78

(qt, 2 H, J ) 7.4 Hz), 2.08 (s, 6 H), 2.30 (s, 3 H), 2.31–2.42 (m,

6 H), 3.40 (t, 2 H, J ) 6.4 Hz), 3.55 (t, 4 H, J ) 4.0 Hz), 4.18 (t,

2 H, J ) 5.1 Hz), 5.10 (s, 2 H), 5.82 (t, 1 H, J ) 5.1 Hz), 6.14 (s,

1 H), 6.20 (s, 2 H), 6.77 (br s, 1 H), 6.93 (d, 1 H, J ) 7.7 Hz),

7.02 (d, 1 H, J ) 7.7 Hz), 7.09 (d, 1 H, J ) 7.7 Hz), 7.14 (d, 1 H,

J ) 7.7 Hz), 7.20 (s, 1 H), 10.24 (br s, 1 H); HRMS (ESI), calcd

for C₃₀H₃₉N₆O₂515.3134, found [MH]⁺515.3144. Anal.

(C₃₀H₃₈N₆O₂) C, H, N.

(2-Chloro-3H-benzoimidazol-5-yl)methanol (49). LiAlH₄(0.146

mol) was added portionwise to a solution of tetrahydrofuran (200

mL) at 5 °C under N₂ﬂow. A solution of 3 (0.073 mol) in

tetrahydrofuran (200 mL) was then added dropwise. The mixture

was stirred at 5 °C for 3 h. A minimum of H₂O was then added,

followed by a solution of CH₂Cl₂/CH₃OH (90:10). The resulting

mixture was dried (over MgSO₄) and ﬁltered, and the solvent was

evaporated until dryness (12.6 g, 95%, melting point 179 °C): H

NMR (DMSO-d₆) δ 4.58 (s, 2 H), 5.20 (br s, 1 H), 7.18 (d, 1 H,

J ) 7.7 Hz), 7.40–7.49 (m, 2 H), 13.14 (br s, 1 H).

2-[6-{[(2-Hydroxyethyl)-m-tolylamino]methyl}-2-(3-morpho-

lin-4-ylpropylamino)benzoimidazol-1-ylmethyl]-6-methylpyri-

din-3-ol (55b). A mixture of 53 (0.0005 mol), 2-[(3-methylphe-

nyl)amino]ethanol (0.0006 mol), and K₂CO₃(0.0025 mol) in DMF

(30 mL) was stirred at 80 °C for 4 h and then poured into H₂O.

The aqueous layer was saturated with K₂CO₃(powder) and extracted

with CH₂Cl₂. The organic layer was separated, dried (over MgSO₄),

and ﬁltered, and the solvent was evaporated. The residue (0.7 g)

was puriﬁed by column chromatography over silica gel (eluent,

CH₂Cl₂/CH₃OH/NH₄OH 93:7:0.5). The pure fractions were col-

lected, and the solvent was evaporated. The residue (0.12 g) was

crystallized from 2-propanone/DIPE. The precipitate was ﬁltered

off, rinsed with DIPE, and dried (0.1 g, 36%, melting point 205

1

[2-(3-Morpholin-4-ylpropylamino)-3H-benzoimidazol-5-yl]-

methanol (50). A mixture of 49 (0.069 mol) and 3-morpholin-4-

ylpropylamine (0.207 mol) was stirred at 125 °C for 4 h and then

894 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4

Bonfanti et al.

°C): ¹H NMR (DMSO-d₆) δ 1.78 (qt, 2 H, J ) 7.4 Hz), 2.32 (s, 3

H), 2.33–2.42 (m, 6 H), 3.40 (t, 2 H, J ) 6.4 Hz), 3.47 (t, 2 H, J

) 6.4 Hz), 3.53–3.63 (m, 6 H), 4.55 (s, 2 H), 5.05 (s, 2 H), 6.53

(t, 1 H, J ) 7.7 Hz), 6.68 (d, 2 H, J ) 7.7 Hz), 6.78 (d, 1 H, J )

7.7 Hz), 6.98–7.15 (m, 6 H), 10.20 (br s, 1 H); HRMS (ESI), calcd

for C₃₁H₄₁N₆O₃545.3240, found [MH]⁺545.3254. Anal.

(C₃₁H₄₀N₆O₃·0.3H₂O) C, H, N.

2-[6-{[(3,5-Dimethylphenyl)-(2-hydroxyethyl)amino]methyl}-

2-(3-morpholin-4-ylpropylamino)benzoimidazol-1-ylmethyl]-6-

methylpyridin-3-ol (55c). A mixture of 3,5-dimethylaniline (0.04

mol), 2-bromoethanol (0.033 mol), and K₂CO₃(0.033 mol) in

CH₃CN (50 mL) was stirred at 80 °C for 12 h. The reaction was

cooled to room temperature, and the solvent was evaporated. The

residue was taken up in CH₂Cl₂/CH₃OH (95:5) and washed with a

saturated solution of K₂CO₃in water. The organic layer was

separated, dried (over MgSO₄), and ﬁltered, and the solvent was

evaporated until dryness. The residue was puriﬁed by column

chromatography over silica gel (eluent, CH₂Cl₂/CH₃OH/ NH₄OH

98:2:0.1). The pure fractions were collected, and the solvent was

evaporated [1.9 g of 2-(3,5-dimethylphenylamino)ethanol (29%)]:

¹H NMR (DMSO-d₆) δ 2.12 (s, 6 H), 3.05 (qd, 2 H, J ) 6.1 Hz),

3.51 (qd, 2 H, J ) 6.1 Hz), 4.63 (t, 1 H, J ) 6.1 Hz), 5.25 (t, 1 H,

J ) 6.1 Hz), 6.12–6.23 (m, 3 H).

2 H), 5.06 (s, 2 H), 6.20 (s, 1 H), 6.32 (s, 2 H), 6.73 (br s, 1H),

6.77 (d, 1 H, J ) 7.7 Hz), 6.83 (s, 1 H), 7.01 (d, 1 H, J ) 7.7 Hz),

7.05–7.15 (m, 3 H), 7.33 (s, 1 H), 10.23 (br s, 1 H); HRMS (ESI),

calcd for C₃₄H₄₆N₇O₃600.3662, found [MH]⁺600.3704. Anal.

(C₃₄H₄₅N₇O₃) C, H, N.

6-Methyl-2-[2-(3-morpholin-4-ylpropylamino)-6-(m-tolylami-

nomethyl)benzoimidazol-1-ylmethyl]pyridin-3-ol (56a). Acetic

acid (0.2 mL) was added at room temperature to a mixture of 54

(0.0004 mol), 3-methylaniline (0.0005 mol), and NaBH₃CN (0.0005

mol) in CH₃CN (25 mL). The mixture was stirred at room

temperature for 30 min. Acetic acid (0.2 mL) was added, and the

mixture was stirred at room temperature for 12 h. The solvent was

evaporated until dryness. The residue was taken up in CH₂Cl₂/

K₂CO₃10%. The organic layer was separated, dried (over MgSO₄),

and ﬁltered, and the solvent was evaporated until dryness. The

residue (0.22 g) was puriﬁed by column chromatography over silica

gel (eluent, CH₂Cl₂/CH₃OH/NH₄OH 94:6:0.5). The pure fractions

were collected, and the solvent was evaporated. The residue (0.16

g, 65%) was crystallized from 2-propanone/DIPE. The precipitate

was ﬁltered off and dried (0.114 g, 46.5%, melting point 195 °C):

¹H NMR (DMSO-d₆) δ 1.78 (qt, 2 H, J ) 7.4 Hz), 2.12 (s, 3 H),

2.30 (s, 3 H), 2.31–2.40 (m, 6 H), 3.40 (t, 2 H, J ) 6.4 Hz), 3.55

(t, 4 H, J ) 4.0 Hz), 4.18 (t, 2 H, J ) 5.1 Hz), 5.10 (s, 2 H), 5.94

(t, 1 H, J ) 5.1 Hz), 6.30 (d, 1 H, J ) 7.7 Hz), 6.37 (d, 1 H, J )

7.7 Hz), 6.40 (s, 1 H), 6.77 (br s, 1 H), 6.88 (t, 1 H, J ) 7.7 Hz),

6.94 (d, 1 H, J ) 7.7 Hz), 7.03 (d, 1 H, J ) 7.7 Hz), 7.10 (d, 1 H,

J ) 7.7 Hz), 7.14 (d, 1 H, J ) 7.7 Hz), 7.20 (s, 1 H), 10.24 (br s,

1 H); HRMS (ESI), calcd for C₂₉H₃₇N₆O₂501.2978, found [MH]⁺

501.2991. Anal. (C₂₉H₃₆N₆O₂) C, H, N.

2-[6-{[2-(2-Hydroxyethyl)phenylamino]methyl}-2-(3-morpho-

lin-4-ylpropylamino)benzoimidazol-1-ylmethyl]-6-methylpyri-

din-3-ol (56b). A mixture of 54 (0.0002 mol), 2-(2-aminophe-

nyl)ethanol (0.0002 mol), and BH₃CN- (polymer support) (0.0003

mol) in CH₃OH (10 mL) and acetic acid (0.1 mL) was stirred at

room temperature for 16 h and then ﬁltered. The ﬁltrate was

concentrated under reduced pressure. The residue was taken up in

K₂CO₃10%, saturated with K₂CO₃(powder), and extracted with

CH₂Cl₂/CH₃OH. The organic layer was separated, dried (over

MgSO₄), and ﬁltered, and the solvent was evaporated until dryness.

The residue was crystallized from 2-propanone/DIPE. The precipi-

tate was ﬁltered off and dried (0.12 g, 93%, melting point 140 °C):

¹H NMR (DMSO-d₆) δ 1.78 (qt, 2 H, J ) 7.4 Hz), 2.30 (s, 3 H),

2.32–2.40 (m, 6 H), 2.67 (t, 2 H, J ) 6.4 Hz), 3.40 (t, 2 H, J ) 6.4

Hz), 3.55 (t, 2 H, J ) 3.9 Hz), 3.63 (t, 2 H, J ) 6.4 Hz), 4.25 (d,

2 H, J ) 5.1 Hz), 5.10 (s, 2 H), 5.57 (t, 1 H, J ) 5.1 Hz), 6.40–6.50

(m, 2 H), 6.77 (br s, 1 H), 6.88 (t, 1 H, J ) 7.7 Hz), 6.90–6.98 (m,

2 H), 7.03 (d, 1 H, J ) 7.7 Hz), 7.09 (d, 1 H, J ) 7.7 Hz), 7.15 (d,

1 H, J ) 7.7 Hz), 7.24 (s, 1 H), 10.20 (br s, 1 H); HRMS (ESI),

calcd for C₃₀H₃₉N₆O₃531.3084, found [MH]⁺531.3097. Anal.

(C₃₀H₃₈N₆O₃·1.35H₂O) C, H, N.

A mixture of 53 (0.000695 mol), 2-(3,5-dimethylphenylamino)

ethanol (0.0009 mol), and K₂CO₃(0.0035 mol) in dimethylforma-

mide (40 mL) was stirred at 80 °C for 4 h. H₂O was added. The

solution was saturated with K₂CO₃(powder) and extracted with

CH₂Cl₂/CH₃OH (95:5). The organic layer was separated, dried (over

MgSO₄), and ﬁltered, and the solvent was evaporated. The residue

(0.5 g) was puriﬁed by column chromatography over silica gel

(eluent, CH₂Cl₂/CH₃OH/NH₄OH 93:7:0.5). The pure fractions were

collected, and the solvent was evaporated (0.120 g, 31%). The

residue was crystallized from CH₃CN/DIPE. The precipitate was

ﬁltered off, rinsed with DIPE, and dried (0.1 g, 26%, melting point

1

180 °C): H NMR (DMSO-d₆) δ 1.79 (qt, 2 H, J ) 7.4 Hz), 2.13

(s, 6 H), 2.30 (s, 3 H), 2.32–2.41 (m, 6 H), 3.40 (qd, 4 H, J ) 6.8

Hz), 3.51–3.66 (m, 6 H), 4.52 (s, 2 H), 4.65 (br s, 1 H), 5.07 (s, 2

H), 6.20 (s, 1 H), 6.32 (s, 2 H), 6.74 (br s, 1H), 6.78 (d, 1 H, J )

7.7 Hz), 7.01 (t, 1 H, J ) 7.7 Hz), 7.05–7.15 (m, 3 H), 10.20 (br

s, 1 H); HRMS (ESI), calcd for C₃₂H₄₃N₆O₃559.3397, found [MH]⁺

559.3395. Anal. (C₃₂H₄₂N₆O₃) C, H, N.

4-{(3,5-Dimethylphenyl)-[3-(3-hydroxy-6-methylpyridin-2-yl-

methyl)-2-(3-morpholin-4-ylpropylamino)-3H-benzoimidazol-5-

ylmethyl]amino}butyramide (55d). 3-(3,5-Dimethylphenylami-

no)butyric acid ethyl ester was prepared analogously to 2-(3,5-

dimethylphenylamino)ethanol starting from 3,5-dimethylaniline and

3-bromopropionic acid ethyl ester: ¹H NMR (DMSO-d₆) δ 1.18 (t,

3 H, J ) 7.1 Hz), 1.75 (qt, 2 H, J ) 6.9 Hz), 2.13 (s, 6 H), 2.38

(t, 2 H, J ) 6.9 Hz), 2.98 (qd, 2 H, J ) 6.9 Hz), 4.05 (qd, 2 H, J

) 7.1 Hz), 5.40 (t, 1 H, J ) 6.9 Hz), 6.15 (s, 3 H).

3-(3,5-Dimethylphenylamino)butyric acid ethyl ester (0.0026

mol) in a 7 N solution of NH₃in CH₃OH was stirred at 80 °C in

a sealed vessel. The reaction was cooled to room temperature, and

the solvent was evaporated until dryness [0.5 g of 3-(3,5-

2-[6-{[2-(3-Hydroxypropyl)phenylamino]methyl}-2-(3-mor-

pholin-4-ylpropylamino)benzoimidazol-1-ylmethyl]-6-methylpy-

ridin-3-ol (56c). A mixture of 54 (0.0004 mol), 3-(2-aminophe-

nyl)propan-1-ol (0.0005 mol), and BH₃CN- (polymer support)

(0.0007 mol) in CH₃OH (20 mL) and acetic acid (0.2 mL) was

stirred at room temperature for 24 h. The polymer was ﬁltered off.

The ﬁltrate was concentrated under reduced pressure. The residue

was taken up in CH₂Cl₂. The organic layer was washed with K₂CO₃

10%, dried (over MgSO₄), and ﬁltered, and the solvent was

evaporated until dryness. The residue was crystallized from CH₃OH/

DIPE. The precipitate was ﬁltered off and dried (0.14 g, 66%,

1

dimethylphenylamino)butyramide (100%)]: H NMR (DMSO-d₆)

δ 1.72 (qt, 2 H, J ) 7.1 Hz), 2.10–2.18 (m, 8 H), 2.95 (qd, 2H, J

) 7.1 Hz), 5.35 (t, 1 H, J ) 7.1 Hz), 6.15 (s, 3 H), 6.74 (s, 1 H),

7.25 (s, 1 H).

A mixture of 53 (0.0125 mol), 3-(3,5-dimethylphenylamino)bu-

tyramide (0.0145 mol), and Cs₂CO₃(0.0605 mol) in DMF (300

mL) was stirred at 80 °C for 4 h, poured into ice–water, and

extracted with CH₂Cl₂. The organic layer was separated, dried (over

MgSO₄), and ﬁltered, and the solvent was evaporated. The residue

(11.3 g) was puriﬁed by column chromatography over silica gel

(eluent, CH₂Cl₂/CH₃OH/NH₄OH 93:7:0.5). The pure fractions were

collected, and the solvent was evaporated (2.6 g, 35%). This fraction

was crystallized from 2-propanone/CH₃OH/DIPE. The precipitate

was ﬁltered off and dried (2.17 g, 29%, melting point 170 °C): ¹H

NMR (DMSO-d₆) δ 1.70–1.85 (m, 4 H), 2.04–2.12 (m, 2 H), 2.13

(s, 6 H), 2.30 (s, 3 H), 2.31–2.41 (m, 6 H), 3.30 (t, 2 H, J ) 6.8

Hz), 3.40 (t, 2 H, J ) 6.8 Hz), 3.55 (t, 4 H, J ) 3.8 Hz), 4.47 (s,

1

melting point 178 °C): H NMR (DMSO-d₆) δ 1.72 (qt, 2 H, J )

7.4 Hz), 1.79 (qt, 2 H, J ) 7.4 Hz), 2.30 (s, 3 H), 2.30–2.40 (m,

6 H), 2.50–2.58 (m, 2 H), 3.40 (t, 2 H, J ) 6.4 Hz), 3.47 (t, 2 H,

J ) 6.4 Hz), 3.55 (t, 4 H, J ) 3.9 Hz), 4.30 (d, 2 H, J ) 5.1 Hz),

5.10 (s, 2 H), 5.48 (t, 1 H, J ) 5.1 Hz), 6.40–6.50 (m, 2 H), 6.80

(br s, 1 H), 6.85 (t, 1 H, J ) 7.7 Hz), 6.90–7.00 (m, 2 H), 7.04 (d,

1 H, J ) 7.7 Hz), 7.09 (d, 1 H, J ) 7.7 Hz), 7.15 (d, 1 H, J ) 7.7

Hz), 7.24 (s, 1 H), 10.20 (br s, 1 H); HRMS (ESI), calcd for

C₃₁H₄₁N₆O₃545.3240, found [MH]⁺545.3230. Anal. (C₃₁H₄₀N₆O₃)

C, H, N.

Respiratory Syncytial Virus Fusion Inhibitor

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 895

2-[6-{[2-(3-Hydroxypropyl)-5-methylphenylamino]methyl}-2-

(3-morpholin-4-ylpropylamino)benzoimidazol-1-ylmethyl]-6-me-

thylpyridin-3-ol (56d). A mixture of 1-iodo-4-methyl-2-nitroben-

zene (0.019 mol), acrylic acid ethyl ester (0.0238 mol), NEt₃(0.019

mol), and Pd(OAc)₂in CH₃CN was stirred at 100 °C for 2.5 h and

then cooled to room temperature. The mixture was ﬁltered through

a pad of Celite. The Celite was rinsed with CH₂Cl₂. The ﬁltrate

was concentrated under reduced pressure. The residue was taken

up in DIPE with a few drops of acetone. The precipitate was ﬁltered

off and dried [4.47 g of 3-(4-methyl-2-nitrophenyl)acrylic acid ethyl

ester, 100%, melting point 67 °C]: ¹H NMR (DMSO-d₆) δ 1.28 (t,

3 H, J ) 7.1 Hz), 2.45 (s, 3 H), 4.22 (qd, 2 H, J ) 7.1 Hz), 6.62

(d, 1 H, J ) 16.2 Hz), 7.60 (d, 1H, J ) 7.7 Hz), 7.85–7.93 (m,

3H).

Dibal-H 20% in toluene (0.0255 mol) was added at -35 °C to

a mixture of 3-(4-methyl-2-nitrophenyl)acrylic acid ethyl ester

(0.0085 mol) in THF (80 mL) under N₂ﬂow. The mixture was

stirred at -35 °C for 15 min. H₂O (20 mL) was added dropwise at

-35 °C under N₂ﬂow. The mixture was half-evaporated, and

CH₂Cl₂was added. The mixture was ﬁltered over Celite. The Celite

was washed with CH₂Cl₂. The ﬁltrate was washed with H₂O. The

organic layer was separated, dried (MgSO₄), and ﬁltered, and the

solvent was evaporated [2 g of 3-(4-methyl-2-nitrophenyl)prop-2-

en-1-ol, 100%]: ¹H NMR (DMSO-d₆) δ 2.40 (s, 3 H), 4.15 (t, 2 H,

J ) 4.9 Hz), 5.03 (t, 1 H, J ) 4.9 Hz), 6.47 (dt, 1 H, J ) 4.3, 16.2

Hz), 6.80 (d, 1 H, J ) 16.2 Hz), 7.50 (d, 1 H, J ) 7.7 Hz), 7.70

(d, 1H, J ) 7.7 Hz), 7.75 (s, 1 H).

Figure 4. Molcad representation of the binding site used for molecular

docking. The molecular lipophilic potential has been mapped on the

Connolly surface (brown, lipophilic regions; blue, polar regions). The

binding site encompasses subcavities P1, P2, and P3 that accommodate

the hydrophobic side chains of the three HR-C residues L481, F483,

and F488, respectively (yellow tube, HR-C backbone; white-capped

sticks, the three residue side chains).

A mixture of 3-(4-methyl-2-nitrophenyl)prop-2-en-1-ol (0.0085

mol) and Raney nickel (1.6 g) in CH₃OH (30 mL) was hydrogenated

at room temperature for 2 h under a 3 bar pressure and then ﬁltered

over Celite. The Celite was rinsed with CH₃OH. The ﬁltrate was

concentrated under reduced pressure [1.7 g of 3-(2-amino-4-

allowed for the minimization step with the Powell method and the

MMFF94S force ﬁeld and charge set. A dielectric constant of 80

with a distance-dependent attenuation function was used to simulate

solvent effects. All calculations were performed on a Silicon

Graphics O2 workstation [400 MHz MIPS R12000 (IP35) proces-

sor].

Docking. The initial protein coordinates were retrieved from the

Protein Data Bank (code 1g2c) and processed to yield a model

appropriate for molecular docking. According to the indications

given by the X-ray structure authors,⁷the inhibitor-binding region

was identiﬁed on a hydrophobic groove located on the HR-N coiled

coil structure. The C-terminal end of the groove features a cavity

where the majority of contacts occur between six cavity-lining

HR-N residues and two aromatic HR-C residues, F483 and F488.

To deﬁne the binding site required for docking, we extended this

region by including one adjacent cavity in which the hydrophobic

side chain of the HR-C residue L481 is deeply buried. The three

binding site subcavities have been denoted P1, P2, and P3 as shown

in Figure 4.

Compounds 58 and 57 were docked both manually and auto-

matically within the putative binding site. We used FlexX, as

implemented in the SYBYL 6.7.2 package, for automatic docking

with the genetic algorithm parameters set to their default value.

Consensus scoring was applied and used in the decision-making

process. All protein–ligand complexes were optimized using the

MMFF94s force ﬁeld. Within the binding site region all residues

and ligand atoms were allowed to move. Residues outside this

region were kept rigid during the calculation.

1

methylphenyl)propan-1-ol, 86%, melting point 65 °C]: H NMR

(DMSO-d₆) δ 1.62 (qt, 2 H, J ) 7.1 Hz), 2.12 (s, 3 H), 2.40 (t, 2

H, J ) 7.1 Hz), 3.40 (qd, 2 H, J ) 6.0 Hz), 4.45 (t, 1 H, J ) 5.1

Hz), 4.70 (s, 2 H), 6.30 (d, 1 H, J ) 7.7 Hz), 6.42 (s, 1 H), 6.77

(d, 1 H, J ) 7.7 Hz).

Acetic acid (0.2 mL) was added at room temperature to a mixture

of 54 (0.0004 mol), 3-(2-amino-4-methylphenyl)propan-1-ol (0.0005

mol) and BH₃CN- on solid support (0.0007 mol) in CH₃OH (20

mL). The mixture was stirred at room temperature for 12 h. The

solid support was ﬁltered off and rinsed with CH₃OH, and the ﬁltrate

was concentrated. The residue was taken up in a 10% solution of

K₂CO₃in water and extracted with CH₂Cl₂/CH₃OH (95:5). The

organic layer was separated, dried (over MgSO₄), and ﬁltered, and

the solvent was evaporated until dryness. The residue was puriﬁed

by column chromatography over silica gel (eluent, CH₂Cl₂/CH₃OH/

NH₄OH 92:8:1). The pure fractions were collected, and the solvent

was evaporated. The residue was crystallized from 2-propanone/

DIPE. The precipitate was ﬁltered off and dried (0.223 g, 82%,

1

melting point 208 °C): H NMR (DMSO-d₆) δ 1.69 (qt, 2 H, J )

7.4 Hz), 1.79 (qt, 2 H, J ) 7.4 Hz), 2.05 (s, 3 H), 2.30 (s, 3 H),

2.32–2.52 (m, 8 H), 3.40 (t, 2 H, J ) 6.4 Hz), 3.45 (t, 2 H, J ) 6.4

Hz), 3.54 (t, 4 H, J ) 3.9 Hz), 4.28 (d, 2 H, J ) 5.1 Hz), 5.10 (s,

2 H), 5.32 (t, 1 H, J ) 5.1 Hz), 6.25–6.31 (m, 2 H), 6.80 (d, 1 H,

J ) 7.7 Hz), 6.81 (br s, 1 H), 6.95 (d, 1 H, J ) 7.7 Hz), 7.02 (d,

1 H, J ) 7.7 Hz), 7.10 (d, 1 H, J ) 7.7 Hz), 7.13 (d, 1 H, J ) 7.7

Hz), 7.24 (s, 1 H), 10.10 (br s, 1 H); HRMS (ESI), calcd for

C₃₂H₄₃N₆O₃559.3397, found [MH]⁺559.3403. Anal. (C₃₂H₄₂N₆O₃)

C, H, N.

Acknowledgement. We gratefully acknowledge Line Harmi-

er for analysis and Cyril Henry for puriﬁcation of compounds.

We are also grateful to Luc Geeraert for proofreading the

manuscript.

Conformational Analysis. The initial structures of compounds

58 and 57 (Figure 1) were constructed using the sketcher of

SYBYL¹⁴and further optimized using the MMFF94S¹⁵force ﬁeld

and charge set to yield a suitable starting point for the searching

algorithm.

Supporting Information Available: Elemental analyses of the

target compounds. This material is available free of charge via the

Internet at http://pubs.acs.org/.

The Random Search tool available in SYBYL was used to

perform a random torsional search of each compound. All rotatable

bonds were searched except those of the terminal groups. The

Random Search parameters were set to default values except for

the following: max cycle 20000, E cutoff 8 kcal/mol, and chirality

check turned on (for 58 only). A maximum of 1200 iterations was

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JM701284J

Article Doi

Selection of a respiratory syncytial virus fusion inhibitor clinical candidate. 2. Discovery of a morpholinopropylaminobenzimidazole derivative (TMC353121)

DOI: 10.1021/jm701284j

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