1,4-Benzodiazepines as inhibitors of respiratory syncytial virus. The identification of a clinical candidate

Article abstract of DOI:10.1021/jm060747l

Respiratory syncytial virus (RSV) is the cause of one-fifth of all lower respiratory tract infections worldwide and is increasingly being recognized as representing a serious threat to patient groups with poorly functioning or immature immune systems. Rac

Full text of DOI:10.1021/jm060747l

J. Med. Chem. 2007, 50, 1685-1692
1685
1,4-Benzodiazepines as Inhibitors of Respiratory Syncytial Virus. The Identification of a
Clinical Candidate
Elisa A. Henderson,* Dagmar G. Alber, Robert C. Baxter, Sian K. Bithell, Joanna Budworth, Malcolm C. Carter, Ann Chubb,
G. Stuart Cockerill, Verity C. L. Dowdell, Ian J. Fraser, Robert A. Harris, Sally J. Keegan, Richard D. Kelsey,
James A. Lumley, Jeremy N. Stables, Natasha Weerasekera, Lara J. Wilson, and Kenneth L. Powell
Arrow Therapeutics, Britannia House, 7 Trinity Street, London, SE1 1DA, United Kingdom
ReceiVed June 23, 2006
Respiratory syncytial virus (RSV) is the cause of one-fifth of all lower respiratory tract infections worldwide
and is increasingly being recognized as representing a serious threat to patient groups with poorly functioning
or immature immune systems. Racemic 1,4-benzodiazepines show potent anti-RSV activity in vitro. Anti-
RSV evaluation of 3-position R- and S-benzodiazepine enantiomers and subsequent optimization of this
series resulted in selection of a clinical candidate. Antiviral activity was found to reside mainly in the
S-enantiomer, and the R-enantiomers were consistently less active against RSV. Analogues of 1,4-(S)-
benzodiazepine were synthesized as part of the lead optimization program at Arrow and tested in the XTT
assay. From this exercise, (S)-1-(2-fluorophenyl)-3-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]-diazepin-
3-yl)-urea, 17b (RSV-604) was identified as a clinical candidate, exhibiting potent anti-RSV activity in the
XTT assay, which was confirmed in secondary assays. Compound 17b also possessed a good pharmacokinetic
profile and has now progressed into the clinic.
Introduction
analyzed using chiral HPLC, and both were found to have
enantiomeric excesses greater than 99.9%.
The preceding paper in this series1 described the lead
optimization program based on racemic benzodiazepines identi-
fied as inhibitors of RSV in an XTTa based cell screen. This
activity was confirmed in secondary assays (plaque and ELISA),
and the compounds exhibited good pharmacokinetic (PK)
properties when dosed in the rat.
Synthesis and evaluation of 3R- and 3S-enantiomers of lead
compounds showed that S-enantiomers were consistently more
active against RSV than R-enantiomers. Subsequent lead
optimization of these enantiomers led to the identification of
17b (RSV-604), (S)-1-(2-fluorophenyl)-3-(2-oxo-5-phenyl-2,3-
dihydro-1H-benzo[e][1,4]-diazepin-3-yl)-urea, as a candidate
compound for clinical evaluation.
Enantiomerically pure amide and urea derivatives were
prepared as shown in Schemes 2 and 3.6 Several specific
carboxylic acids were required as part of the lead optimization
program. 2-Methoxy-4-methylsulfanylbenzoic acid was oxidized
with oxone to produce 4-methanesulfonyl-2-methoxybenzoic
acid,7 which was used in the synthesis of compound 18.
Cyclization of ethenesulfonyl ethane onto 2-amino-5-chloroben-
zoic acid afforded the intermediate acid8 required for the
synthesis of compound 19. Microwave heating of 2,4-difluo-
robenzoic acid with piperidine resulted in the formation of
4-fluoro-2-piperidin-1-ylbenzoic acid,9 which was used in the
synthesis of compound 22. Functionalization at the N-1 position
was carried out as in Scheme 4.10 Compound 17b was
deprotonated with sodium hydride and treated with methyl
bromoacetate to yield intermediate 11. Deprotection of the ester
with lithium hydroxide11 followed by coupling with a variety
of amines in the presence of HBTU yielded the desired products
of the general structure of 13.
Chemistry
The unsubstituted, racemic benzodiazepine (1a) was synthe-
sized as described in the previous paper.1 Initial attempts to
separate the isomers via crystallization with chiral salts2 were
unsuccessful, so a synthetic route was developed involving the
synthesis of diastereomeric analogues coupled to S-phenyl-
alanine3 (Scheme 1).
The racemic amine 1a was coupled to N-Boc-S-phenylalanine
using HBTU,4 followed by deprotection with hydrogen chloride
in ethyl acetate to yield amines 3b and 3c. At this stage, repeated
crystallizations using methanol and ethyl acetate yielded dias-
tereomerically pure quantities of each diastereomeric amine.
Amines 3b and 3c were independently treated with isothio-
cyanatobenzene to yield the thioureas, 4b and 4c, which yielded
the enantiomeric amines 1b and 1c upon Edman degradation.5
The enantiomeric purity of intermediates 1b and 1c was
Structure Confirmation. To obtain preliminary identification
of the isomers, compounds analogous to 3b and 3c were
synthesized using N-Boc-D-phenylalanine instead of N-Boc-L-
phenylalanine, so that comparison with a published crystal
structure could be used to assign stereochemistry. The isomers
were separated and N1-methylated, and 1H NMR in CDCl3 was
performed. This data was compared to previously published
structural analyses on the N1-methylated derivative of compound
3b12 and, as the diastereoisomers showed differing signals, it
was possible to assign the stereochemistry of these analogues.
Both isomers were deprotected to give compounds 1b and 1c.
These samples were compared by chiral HPLC to samples of
1b and 1c derived from the route using N-Boc-L-phenylalanine,
which resulted in compound 3b being identified as the S-isomer.
The most pronounced difference in the spectra is the signal due
to the C3 proton; in the S-isomer, this is quoted as 5.55 ppm,
and we found that in the R-isomer it lay at 5.57 ppm (Figure
* To whom correspondence should be addressed. Tel.: +44 207 015
1033. Fax: +44 207 015 1020. E-mail: ehenderson@arrowt.co.uk.
a Abbreviations: XTT, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxanilide disodium salt; HBTU, O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate; HEp-2, human respi-
ratory epithelial cells; F-protein, fusion protein; N-protein, nucleocapsid
protein; P-protein, phosphoprotein.
10.1021/jm060747l CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/07/2007

1686 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Henderson et al.
Scheme 1a
Scheme 4a
a Reagents and conditions: (a) NaH, methyl bromoacetate, DMF; (b)
LiOH, THF, H2O; (c) R2-NH, HBTU, Et3N, DMF.
a Reagents and conditions: (a) N-Boc-L-Phe, HBTU, Et3N, CH2Cl2; (b)
HCl, EtOAc; (c) PhNCS, CH2Cl2; (d) TFA.
Scheme 2a
a Reagents and conditions: (a) R-COOH, HBTU, Et3N.
Scheme 3a
Figure 1. 1H NMR of diastereomers derived from intermediates 3b
and 3c.
a Reagents and conditions: (a) R-NCO, Et3N.
Figure 2. Structure for X-ray crystallography.
1). The NMR sample of 3b was spiked with a small quantity
of 3c to confirm the differences in the spectra.
To unequivocally confirm this assignment, a crystal structure
determination was obtained. To obtain satisfactory resolution,
it was necessary to synthesize a derivative with at least one
heavy heteroatom incorporated into the structure to ensure
scattering of the X-rays.13 Compound 14 (Figure 2) was
synthesized from intermediate 1b, and 4-bromo-2-chlorophenyl
isocyanate and crystals of suitable quality for X-ray crystal

1,4-Benzodiazepines as Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1687
Table 1. Activity Against RSV in XTT Assay of Racemates and
Separate Enantiomers
Figure 3. Structure determination by X-ray crystallography.
structure determination were produced. The X-ray crystal
structure (Figure 3) confirmed that compound 14 had the
expected S-stereochemistry at the C-3 position of the benzodi-
azepine ring system (shown as C-11 in Figure 3).
Computational conformational analysis utilizing the MMFF94s
forcefield14 identified the low-energy conformations for R- and
S-isomers of compound 14. In each case, the pendent phenyl
amide was maintained in the equatorial position by an alternate
“boat”-shaped conformation of the seven-membered heterocyclic
ring. The boat-shaped conformation of the benzodiazepine seen
here is consistent with previously published structural reports.15
A clear energetic preference of over 10 kcal mol-1 was seen
for the R- or S-equatorial over the axial isomer.
this series.1 A series of compounds that showed a range of
activities in addition to structural differences in the side chain
and linker were chosen to be synthesized as chirally pure
enantiomers to quickly examine any enantiomeric preference
and trends. The results of three of these enantiomeric pairs are
presented in Table 1. It was observed that in all three cases the
relative potency across the chiral and achiral forms remained
the same, but the S-isomer showed higher potency than the
racemate, and the R-isomer was consistently less potent.
With this initial indication of enantiospecific anti-RSV activity
and trends in place, the strategy within the chiral series was to
optimize utilizing the SARs gleaned from the racemic optimiza-
tion.1 Particular attention was paid to substituents that would
potentially modify physical properties and maintain or improve
the PK profile of the series. Therefore, 5-phenyl-1,3-dihydrobenzo-
[e][1,4]diazepin-2-one was used as the core structure for this
optimization.
From the SAR in the racemic series it was known that
electron-rich side chain substituents were favorable for greater
potency against RSV. However, some of these electron-rich
substituents exhibited poor PK profiles so further analogues were
synthesized with substituents that could potentially block
metabolically vulnerable sites.16
The strategy was that by using side chains containing sulfones
and amines (Table 2), as well as increasing potency, the PK
profile of the compounds could be improved by increasing
solubility and lowering CLogP17 while maintaining the electron-
rich status of the aromatic ring.18
Biology
XTT Assay. All compounds were tested against the RSS
strain of RSV in the XTT (2,3-bis-(2-methoxy-4-nitro-5-
sulfophenyl)-2H-tetrazolium-5-carboxanilide disodium salt) as-
say. Because this metabolic assay cannot distinguish between
virally induced cell death and cell death due to toxic effects of
compound, the XTT assay was simultaneously run in the
absence of virus to allow a preliminary toxicity assessment of
the compounds.
HEp-2 cells were infected with RSV in the presence of
compound and incubated for 6 days at 37 °C. Residual cell
viability was then measured by metabolism of the XTT substrate
to a colored product, and 50% inhibitory concentrations (IC50)
were determined. Toxicity was measured as TD50 and defined
as the concentration of compound required for 50% cell death
in the absence of virus.
Plaque Assay. Monolayer cultures of HEp-2 cells were
infected with the RSS strain of RSV. Following adsorption for
2 h, compounds were added in medium containing 0.6% agarose.
After a 5-day incubation at 37 °C, the monolayers were fixed
and the overlay was removed. Plaques were visualized by
staining with methylene blue.
ELISA Assay. HEp-2 cells were infected with the RSS strain
of RSV and exposed to test compound for 3 days at 37 °C.
Monoclonal antibodies to the RSV F-, N-, and P-proteins were
used and detected with an enzyme-linked secondary antibody.
Subsequent conversion of substrate to a colored product was
measured by optical density, and the IC50 was determined.
Replacing the nitro-substituent of compound 15b with another
electron-withdrawing group, methyl sulfone 18, resulted in an
equipotent compound (Table 2) with none of the potential
toxicity issues that are associated with the metabolic reduction
of aromatic nitro groups.19
From CLogP calculations it was predicted that compounds
containing a thiomorpholine S,S-dioxide substituent, for example
19 and 20 (CLogPs of 2.53 and 1.96, respectively) could prove
Results and Discussion
The structure-activity relationship (SAR) of racemic ben-
zodiazepines is described in detail in the preceding paper in

1688 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Table 2. Activity Against RSV with Varying Substituents at C-3
Henderson et al.
Table 3. Activity against RSV with Varying Substituents at N-1
It was concluded that the benzodiazepine core with no
substitution at the N-1 position was optimum for activity against
RSV, and three of the most potent compounds (15b, 17b, and
21) were chosen to proceed to secondary assays (ELISA and
plaque) and PK profiling, Table 4.
All three compounds showed excellent potency against RSV
in the primary and secondary assays with IC50 values in the
range of 0.5-1.5 µM, and the calculated logP of the compounds
were within an acceptable range. The initial PKs showed that
compound 15b reached a relatively high Cmax compared to the
other two analogues. However, the presence of the aromatic
nitro substituent and the associated developability issues19
precluded further interest in this compound. The PK profile of
the other two compounds showed that 17b had higher exposure
and also a higher solubility in water than compound 21.
Compound 17b, (S)-1-(2-fluorophenyl)-3-(2-oxo-5-phenyl-2,3-
dihydro-1H-benzo[e][1,4]-diazepin-3-yl)-urea, was, therefore,
chosen to proceed into further studies.
As part of the preclinical and clinical candidate selection
process, an early assessment of the ADME characteristics of
17b was undertaken. In a focused selection of in vivo and in
vitro experiments, the suitability for progression into safety
assessment studies and, ultimately, clinical evaluation was
determined.
When a high throughput equilibrium dialysis approach with
LC-MS/MS detection was used, 17b showed high binding to
serum proteins; fu ) 0.05 (95% protein bound), with equiva-
lence observed in rat dog and human sera.
The susceptibility of 17b for oxidative metabolism was
assessed in vitro using rat and human microsomes. Turnover
of compound was slow for both species, with the apparent
intrinsic clearance (disregarding fraction unbound in the mi-
crosomal protein incubations) of 23.5 ( 2.8 and 4.5 ( 2.4 µL/
min/mg of rat and human microsomal protein, respectively.
Early in vivo PK characterization of 17b was undertaken in
the rat using both oral and intravenous routes of administration,
with the parameters obtained detailed in Table 5.
to be more water soluble than compound 15b (CLogP of 3.13).
Potential metabolically vulnerable sites were blocked with
halogens to produce active analogues with fluorine, 20, being
preferred over chlorine, 19.
A range of other potentially solubilizing substituents on the
aromatic ring gave varying results. A morpholine group on either
a phenyl ring, 21, or a pyridyl ring, 23, showed potent activity
against RSV, but when the phenyl ring was substituted with
piperidine, 22, a significant decrease in potency was observed.
It has been demonstrated in benzodiazepines targeted as
CCK-B antagonists that N-1 substitution on the benzodiazepine
core can enhance activity.10 In the previous paper in this series,1
it was shown that N-1 methylation reduced potency against RSV.
To further investigate this observation, a number of compounds
were synthesized with N-1 amide substituents that were
analogous to those published as CCK-B antagonists.
The unsubstituted compound, 17b, was a potent RSV
inhibitor, but all other substitutions at this position resulted in
a significant reduction of activity against RSV, Table 3. The
only exception was when the substituent was pyridinylamide,
26, but this was still significantly less active than the unsub-
stituted analogue, 17b.
The plasma clearance (CLp) was shown to be low in the rat
at 7.2 mL/min/kg, which supported the findings of the microso-

1,4-Benzodiazepines as Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1689
Table 4. Biological Evaluation of Lead Compounds
XTT
(µM)
ELISA
(µM)
plaque
(µM)
aqueous
solubility
pH 7
plasma levelsa
cmpd
15b
IC50
0.9
TD50
IC50
1.0
TD50
IC50
0.5
TD50
CLogP
3.13
(µg/mL)
Cmax (ng/mL)
>50
>50
>50
-
C
max 6172
max 6 h
max 1023
max 4 h
max 204
max 6 h
max 2197
max 8 h
T
C
17b
RSV-604
21
0.9
1.4
0.6
>50
>50
>50
1.4
1.5
0.6
>50
>50
>50
0.7
0.8
0.5
>50
>50
>50
3.02
2.67
1.88
4.1
0.9
22.0
T
C
T
C
18
A-315
T
a 20 mg/kg dosed orally as a suspension in 5% PEG400 with 1% CMC.
Table 5. Pharmacokinetic Parameters of 17b Following Intravenous and Oral Administration to Rat (n ) 3)
dose
AUC0-∞
Cmax
CLp
Vss
t1/2
route
(mg/kg of body wt)
(µg‚hr/mL)
(µg/mL)
(mL/min/kg)
(L/kg)
(hours)
PO
IV
5
5
10.9 (( 1.5)
11.7 (( 1.4)
0.91 (( 0.10)
3.3 (( 0.48)
NDa
7.2 (( 0.87)
NDa
2.2 (( 0.12)
2.6 (( 0.2)
3.8 (( 0.9)
a ND ) not determinable following oral administration.
mal turnover screen. Compound 17b exhibited a high volume
of distribution (Vss), which at 2.2 L/kg was much greater than
total body water. This finding implied that compound 17b was
unlikely to be restricted to the systemic circulation and could
have significant tissue distribution.
These preclinical data aided the selection of 17b for pre-
clinical study and its move into the clinical arena, where it has
successfully progressed through phase I and into phase II clinical
development. PK data thus far obtained from these studies
correlates well with the preclinical findings reported from these
early ADME characterizations.
No cellular toxicity was observed in the three antiviral in
vitro assays for either of the enantiomers or the racemate of
compound 17.
The racemate (17a) and the S-isomer (17b) have been
screened against a wide range of receptors in a CEREP screen.21
The racemate, 17a, was shown to hit CCKa receptors at low
levels as well as CCKb and the benzodiazepine receptor. (S)-
1-(2-Fluorophenyl)-3-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e]-
[1,4]-diazepin-3-yl)-urea, 17b, showed a cleaner profile than
the racemate when tested in the CEREP screen. It showed
marginal activity at CCKa and CCKb receptors, and there was
no effect observed on the benzodiazepine receptor.
Resistant mutant studies on compounds 15b and 17b showed
amino acid changes in the same region of the N-protein as the
original lead compound in this series,1 indicating the same mode
of action.
This observation was further supported following oral PK
studies in colostrum deprived calves where nasal mucosa
showed significant levels of parent compound at trough.20 These
data helped support the decision to progress 17b into clinical
development, as the findings in calves suggested the compound
was capable of reaching the target site of RSV infection at
concentrations that exceed the bovine IC50 (123 ng/mL) at 24
h post-dose. These data provided confidence that efficacious
target tissue concentrations could readily be achieved using an
appropriate dosing regimen, thus providing an opportunity to
demonstrate in vivo potency assuming these observations
extrapolate to man.
The oral dose was given in a lipid-based formulation to
enhance bioavailability (F) and maximize exposure and circulat-
ing plasma levels of 17b. The compound showed good absorp-
tion, with F > 90% at 5 mg/kg. When 17b was administered
as a suspension in 1% carboxymethylcellulose (CMC), bio-
availability was reduced to <40% at an equivalent dose but
dose proportionality in this formulation was demonstrated from
2 to 20 mg/kg. The apparent high bioavailability in the
solubilized form correlated well with the low clearance and the
in vitro experimentally determined apparent permeability coef-
ficient (Papp) using a MDCK cell line of 13 × 10-6 cm/sec,
indicating that 17b had moderate to high passive permeability
characteristics.
A subsequent program at Arrow Therapeutics identified (S)-
4-methanesulfonyl-2-methoxy-N-(2-oxo-5-phenyl-2,3-dihydro-
1H-benzo[e][1,4]diazepin-3-yl)-benzamide, 18 (A-315), as a
back-up molecule to 17b. Compound 18 showed potent anti-
RSV activity across the primary and secondary assays, with a
different PK profile and improved aqueous solubility over 17b.
Conclusion
Although 17b has low plasma clearance, it has been shown
to be a substrate for cytochrome P450 (CYP450) metabolism
using the Bactosome recombinant isoform expression system.
CYP450 3A4 was established to be the major contributor to
the compound’s metabolism, with both 2C19 and 2D6 also
implicated in metabolic turnover in vitro. To further assess the
potential for 17b to undergo drug interaction when dosed in
combination with other CYP450 metabolized therapeutics, CYP
inhibition and induction were assessed. Compound 17b was
found to be a moderate inducer of CYP3A4 and CYP1A in a
concentration-dependent manner but was demonstrated to have
no inhibitory effect on the metabolism of specific probe
substrates for CYPs 3A4, 1A2, 2D6, 2C9, or 2C19.
Separation of the R- and S-enantiomer of intermediates 3b
and 3c followed by synthesis of a variety of analogues resulted
in the discovery that the RSV activity resides mainly in the
S-enantiomer of these benzodiazepine derivatives. Optimization
of this series produced compounds with comparable activity,
with a range of physicochemical properties, and with increased
solubility. Substitution at the N-1 position was found to greatly
reduce RSV activity. Compound 17b, (S)-1-(2-Fluorophenyl)-
3-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]-diazepin-3-yl)-
urea, was selected as a clinical candidate, as it showed potent
anti-RSV activity across a range of in vitro assays as well as a
good PK profile. It was subsequently tested in CEREP screens
and shown to have a clean in vitro toxicity profile. This

1690 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Henderson et al.
was added and stirring was continued for 10 min. The colorless
precipitate was collected by filtration and partitioned between
dichloromethane (25 mL) and water (25 mL). The organic phase
was dried by filtration through a hydrophobic frit and evaporated,
and the residue was purified on a silica gel SPE cartridge. Elution
with ethyl acetate/petrol 1:1 gave the title compound.
General Procedure 5. A solution of {3-[3-(2-fluorophenyl)-
ureido]-2-oxo-5-phenyl-2,3-dihydrobenzo [e][1,4]diazepin-1-yl} ace-
tic acid, 12 (40 mg), in dry N,N-dimethylformamide (1.5 mL) was
treated with O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (37 mg), triethylamine (0.018 mL), and amine
(1 equiv) and stirred at ambient temperature under a nitrogen
atmosphere for 2 h. Water (10 mL) was added and the product
was collected by filtration.
compound has now successfully finished Phase I and entered
into Phase II clinical trials. Further work allowed us to identify
18, (S)-4-methanesulfonyl-2-methoxy-N-(2-oxo-5-phenyl-2,3-
dihydro-1H-benzo[e][1,4]diazepin-3-yl)-benzamide, as a back
up compound to 17b. This compound possesses potent anti-
RSV activity across the primary and secondary assays as well
as improved solubility and a different PK profile.
Experimental Section
Chemistry. 1H NMR spectra were recorded on a Bruker Avance
spectrometer at 250 MHz and chemical shifts are reported in ppm
relative to DMSO-d6 or CDCl3. Elemental analyses were performed
by G. A. Maxwell at University College London. Thin-layer
chromatography was performed on precoated silica gel F-254 plastic
plates (0.2 mm, Macherey-Nagel) and was visualized with UV light.
LC/MS conditions: Samples were run on a Micromass ZMD, using
electrospray with simultaneous positive-negative ion detection.
Column, YMC-PACK FL-ODS AQ, 50 × 4.6 mm I.D. S-5 µm;
gradient, 95:5 to 5:95 v/v H2O/CH3CN + 0.05% formic acid over
4.0 min, hold 3 min, return to 95:5 v/v H2O/CH3CN + 0.05%
formic acid over 0.2 min and hold at 95:5 v/v H2O/CH3CN + 0.05%
formic acid over 3 min; detection, PDA 250-340 nm; flow rate,
1.5 mL/min.
Chiral high-performance liquid chromatography (HPLC) was
carried out on a HP1090 chromatograph equipped with a UV
detector (λ ) 280 nm and 254 nm). Enantiomeric excess of final
compounds was measured using a 250 × 4 mm Chiralpak AD
column with an isocratic mobile phase of 50:50 propan-2-ol/hexane.
All temperatures are in °C. Solid-phase extraction (SPE) chro-
matography was carried out using Jones chromatography (Si)
cartridges under 15 mmHg vacuum with stepped gradient elution.
Temperature-controlled microwave heating was carried out using
a CEM Discover single-mode synthesizer. All the organic phases
were dried over MgSO4 or by passing through a hydrophobic,
single-fritted column (Isolute SPE accessories). All chemicals were
purchased from commercial suppliers and used directly without
further purification.
General Procedure 1. A solution of amine (1 equiv), acid (1
equiv), triethylamine (0.07 mL), and O-benzotriazol-1-yl-N,N,N′,N′-
tetramethyluronium hexafluorophosphate (121 mg) in dry tetrahy-
drofuran (3 mL) was stirred at ambient temperature for 18 h under
a nitrogen atmosphere. The mixture was then partitioned between
potassium carbonate solution (15 mL) and dichloromethane (15
mL). The organic phase was passed through a hydrophobic frit and
evaporated. The residue was purified on a silica gel SPE cartridge,
eluting with dichloromethane followed by dichloromethane/
ethanol: 0.880 ammonia 400:8:1 and then 200:8:1, to yield the
title compound.
General Procedure 2. A solution of amine (1 equiv) in
tetrahydrofuran (2 mL) containing triethylamine (0.085 mL) was
treated with acid chloride (1 equiv) and stirred at ambient
temperature under a nitrogen atmosphere for 18 h. The mixture
was partitioned between water (10 mL) and dichloromethane (10
mL). The organic extract was evaporated, and the residue was
purified on a silica gel SPE cartridge, eluting with dichloromethane
followed by dichloromethane/ethanol: 0.880 ammonia 400:8:1 and
then 200:8:1, to yield the title compound.
(S,S)- and (R,S)-[1-(2-Oxo-5-phenyl-2,3-dihydro-1H-benzo-
[e][1,4]diazepin-3-ylcarbamoyl)-2-phenylethyl] Carbamic Acid
tert-Butyl Ester, 2. A solution of 3-amino-5-phenyl-1,3-dihydro-
benzo[e][1,4]diazepin-2-one, 1 (34.9 g), (S)-2-tert-butoxycarbonyl-
amino-3-phenylpropionic acid (55.3 g), triethylamine (100 mL),
and O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluo-
rophosphate (116 g) in dichloromethane (1 L) was stirred at ambient
temperature for 18 h under a nitrogen atmosphere. The solvent was
evaporated, and the residue was partitioned between 10% citric acid
solution and ethyl acetate. The organic phase was further washed
with 2 M sodium hydroxide, water, and brine before being dried
(MgSO4). The organic phase was evaporated, giving an oil that
was used crude in the following step. LC/MS ES+ ) 498. 1H NMR
(DMSO-d6) δ 1.29 (s, 9H), 2.72-2.84 (m, 1H), 3.05-3.18 (m, 1H),
4.32-4.44 (m, 1H), 5.20-5.25 (m, 1H), 6.97-7.05 (m, 1H), 7.16-
7.68 (m, 14H), 9.17-9.21 (d, 1H, J ) 7.0 Hz), 10.90 (s, 1H).
(S,S)-2-Amino-N-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e]-
[1,4]diazepin-3-yl)-2-phenylpropionamide, 3b, and (R,S)-2-
Amino-N-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-
3-yl)-2-phenylpropionamide, 3c. [1-(2-Oxo-5-phenyl-2,3-dihydro-
1H-benzo[e][1,4]diazepin-3-ylcarbamoyl)-phenylethyl] carbamic
acid tert-butyl ester, 2, was added in a single portion to a cooled
(-10 °C) solution of HCl (34 g) in ethyl acetate (1 L). The reaction
was stirred at this temperature for 1 h, before being warmed to 20
°C and stirred for a further 2 h. The reaction was cooled to 0 °C,
and water (300 mL) was added at a rate that maintained a
temperature below 10 °C. The aqueous layer was washed with ethyl
acetate (2 × 150 mL), and the aqueous layer was returned to the
reaction flask. The reaction was again cooled to 0 °C, and
concentrated aqueous ammonia was added at a rate that maintained
the temperature below 5 °C until pH 9.0 had been achieved. The
reaction was washed with ethyl acetate (5 × 150 mL), the combined
organic extracts were washed with brine (100 mL) and dried with
magnesium sulfate, and the solvent was evaporated, producing a
yellow oil. The yellow oil was stirred rapidly with a 5% solution
of methanol in ethyl acetate until a thick white precipitate formed.
The precipitate was filtered, and the mother liquor was evaporated.
The residual gum was again stirred with 5% methanol in ethyl
acetate until a thick precipitate had formed. This sequence was
repeated several times. On each occasion, the precipitate was
analyzed to assess the diastereomeric excess by TLC (SiO2, DCM/
EtOH/NH3, 200:8:1). Pure, or mostly pure, batches of each
diastereomer were kept aside, and mixtures were returned to the
precipitation procedure at the evaporation stage after first dissolving
in a mixture of 5% methanol in dichloromethane. The combined
batches that contained pure or mainly pure 3b (S,S)-diastereomer
(Rf ) 0.25, higher spot) were stirred as a slurry in 5% methanol in
ethyl acetate for 10 min and filtered to produce 3b (S,S)-
diastereomer (>99% d.e.), a pure sample as a white powder (26.1
General Procedure 3. A mixture of amine (1 equiv) and
isocyanate (1 equiv) in dry tetrahydrofuran (4 mL) was treated with
triethylamine (1 equiv) and stirred at ambient temperature for 18
h. The mixture was partitioned between water (10 mL) and
dichloromethane (10 mL). The organic extract was dried by
filtration through a hydrophobic frit and concentrated in vacuo. The
residue was triturated with petroleum ether to yield the final
compound.
General Procedure 4. (S)-3-Amino-5-phenyl-1,3-dihydro-benzo-
[e][1,4]diazepin-2-one, 1b (100 mg), O-benzotriazol-1-yl-N,N,N′,N′-
tetramethyluronium hexafluorophosphate (150 mg), triethylamine
(0.083 mL), and acid (1 equiv) in dry N,N-dimethylformamide (1
mL) was stirred at ambient temperature for 1 h. Water (10 mL)
1
g, 47% over two steps). LC/MS ES+ ) 399. H NMR (CDCl3) δ
1.45 (br s, 2H), 2.81 (dd, 1H, J ) 13.9 Hz, 10.1 Hz), 3.33 (dd, 1H,
J ) 13.9 Hz, 3.8 Hz), 3.72 (dd, 1H, J ) 9.5 Hz, 3.8 Hz), 5.55 (d,
1H, J ) 8.2 Hz), 7.53-7.12 (m, 14H), 8.52 (s, 1H), 8.88 (d, 1H,
J ) 8.8 Hz).
Compound 3c (R,S)-diastereomer (Rf ) 0.22, lower spot) was
isolated by recrystallization (as described above) as a white solid
(15.4 g, 28% over two steps). LC/MS ES+ ) 399. 1H NMR (CDCl3)

1,4-Benzodiazepines as Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1691
δ 1.48 (br s, 2H), 2.76 (dd, 1H, J ) 13.3 Hz, 10.1 Hz), 3.40 (dd,
1H, J ) 13.3 Hz, 3.8 Hz), 3.79 (dd, 1H, J ) 10.1 Hz, 3.8 Hz),
5.57 (d, 1H, J ) 8.2 Hz), 7.17-7.56 (m, 14H), 8.91 (d, 1H, J )
8.2 Hz), 9.02 (s, 1H).
(s, 1H). Anal. (C23H18N4O5‚0.36H2O) C, H, N. HPLC: retention
time, 10.21 min (e.e. >99%).
(R)-2-Methoxy-4-nitro-N-(2-oxo-5-phenyl-2,3-dihydro-1H-
benzo[e][1,4]-diazepin-3-yl-benzamide, 15c. This material was
prepared as described in General Procedure 1, using (R)-3-amino-
5-phenyl-1,3-dihydrobenzo[e][1,4]diazepin-2-one, 1c (40 mg), and
2-methoxy-4-nitrobenzoic acid (47 mg). The title compound was
(S,S)-N-(2-Oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-
3-yl)-3-phenyl-2-(3-phenylthioureido)propionamide, 4b. A solu-
tion of (S,S)-2-amino-N-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e]-
[1,4]-diazepin-3-yl)-2-phenylpropionamide,3b(27.1g),indichloromethane
(500 mL) was treated with isothiocyanatobenzene (14.7 g), and the
mixture was stirred at ambient temperature for 18 h. The solvent
and excess reagent were removed by evaporation, and the residue
was redissolved in dichloromethane and then precipitated with
petrol, giving a colorless solid that was collected by filtration (36.1
1
isolated as a white solid (57 mg, 83%). LC/MS ES+ ) 431. H
NMR (DMSO-d6) δ 4.13 (s, 3H), 5.45 (d, 1H, J ) 7.0 Hz), 7.27-
7.70 (m, 9H), 7.98-8.09 (m, 3H), 9.67 (d, 1H, J ) 7.6 Hz), 11.01
(s, 1H). Anal. (C23H18N4O5‚0.15H2O) C, H, N. HPLC: retention
time, 22.82 min (e.e. >99%).
Cyclohexanecarboxylic Acid (2-Oxo-5-phenyl-2,3-dihydro-
1H-benzo[e][1,4]diazepin-3-yl-amide, 16a. This material was
prepared as described in General Procedure 2, using 3-amino-5-
phenyl-1,3-dihydrobenzo[e][1,4]diazepin-2-one, 1a (100 mg), and
cyclohexanecarbonyl chloride (0.053 mL). The title compound was
isolated as a colorless solid (57 mg, 40%). LC/MS ES+ ) 362. 1H
NMR (DMSO-d6) δ 1.10-1.43 (m, 5H), 1.60-1.82 (m, 5H), 2.44
(m, 1H), 5.22 (d, 1H, J ) 7.6 Hz), 7.20-7.67 (m, 9H), 8.81 (d,
1H, J ) 7.6 Hz), 10.75 (s, 1H). Anal. (C22H23N3O2‚0.50H2O) C,
H, N. HPLC: retention time, 4.45 min (53.3%), 5.61 min (46.7%).
(S)-Cyclohexanecarboxylic Acid (2-Oxo-5-phenyl-2,3-dihy-
dro-1H-benzo[e][1,4]diazepin-3-yl-amide, 16b. This material was
prepared as described in General Procedure 2, using (S)-3-amino-
5-phenyl-1,3-dihydrobenzo[e][1,4]diazepin-2-one, 1b (40 mg), and
cyclohexanecarbonyl chloride (0.021 mL). The title compound was
1
g, 99%). LC/MS ES- ) 532. H NMR (CDCl3) δ 3.25-3.30 (m,
2H), 5.35-5.41 (m, 2H), 6.66 (d, 1H, J ) 7.6 Hz), 6.94-7.50 (m,
19H), 7.83 (s, 1H), 8.61 (br s, 1H).
(R,S)-N-(2-Oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-
3-yl)-3-phenyl-2-(3-phenylthioureido)propionamide, 4c. This
compound was prepared as described for compound 4b using (R,S)-
2-amino-N-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-
3-yl)-2-phenylpropionamide, 3c (15 g). The title compound was
1
isolated as a white solid (18.7 g, 93%). LC/MS ES+ ) 534. H
NMR (CDCl3) δ 3.20-3.52 (m, 2H), 5.41 (d, 1H, J ) 7.6 Hz),
5.62 (m, 1H), 6.84-7.53 (m, 20H), 7.80 (m, 1H), 8.10 (m, 1H),
9.21 (br s, 1H).
(S)-3-Amino-5-phenyl-1,3-dihydrobenzo[e][1,4]diazepin-2-
one, 1b. (S,S)-N-(2-Oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]-
diazepin-3-yl)-2-phenyl-2-(3-phenylthioureido)propionamide, 4b
(24 g), was heated to 50 °C and treated with trifluoroacetic acid
(64 mL). The mixture was stirred rapidly for 40 min and evaporated
to dryness, giving a yellow oil. This material was purified by silica
gel chromatography. Elution with dichloromethane/methanol/acetic
acid/water, 90:10:1:1, gave the acetate salt of the amine as a pale
yellow foam (13.1 g, 94%). LC/MS ES+ ) 252. 1H NMR (CDCl3)
δ 2.17 (s, 3H), 4.68 (br s, 1H), 6.98-7.47 (m, 9H), 9.56 (br s,
1H), 10.68 (br s, 1H). HPLC: retention time, 6.16 min (e.e. >99%).
The free base of 1b was isolated as follows: 0.5 g of the acetate
salt of 1b was dissolved in dichloromethane (1 mL) and basified
by the addition of 0.880 ammonia (1 mL), giving a colorless
precipitate that was collected by filtration and dried (380 mg, 94%).
(R)-3-Amino-5-phenyl-1,3-dihydrobenzo[e][1,4]diazepin-2-
one, 1c. A solution of (R,S)-N-(2-oxo-5-phenyl-2,3-dihydro-1H-
benzo[e][1,4]diazepin-3-yl)-2-phenyl-2-(3-phenylthioureido)propi-
onamide, 4c (5 g), in dichloromethane (35 mL) was treated with
trifluoroacetic acid (3.5 mL) and stirred at ambient temperature
for 6 h. The reaction was quenched with ice water (60 mL), and
the organic extract was washed with water (40 mL). The combined
aqueous extracts were basified to pH 9 with 10% aq potassium
carbonate, and the precipitate was collected by filtration to yield
1
isolated as a white solid (28 mg, 49%). LC/MS ES+ ) 362. H
NMR (DMSO-d6) δ 1.20-1.43 (m, 5H), 1.60-1.82 (m, 5H), 2.47
(m, 1H), 5.29 (d, 1H, J ) 8.2 Hz), 7.25-7.72 (m, 9H), 8.93 (d,
1H, J ) 8.2 Hz), 10.85 (s, 1H). Anal. (C22H23N3O2) C, H, N.
HPLC: retention time, 4.45 min (e.e. >99%).
(R)-Cyclohexanecarboxylic Acid (2-Oxo-5-phenyl-2,3-dihy-
dro-1H-benzo[e][1,4]diazepin-3-yl-amide, 16c. This material was
prepared as described in General Procedure 2, using (R)-3-amino-
5-phenyl-1,3-dihydrobenzo[e][1,4]diazepin-2-one, 1c (100 mg), and
cyclohexanecarbonyl chloride (0.053 mL). The title compound was
1
isolated as a white solid (42 mg, 29%). LC/MS ES+ ) 362. H
NMR (DMSO-d6) δ 1.16-1.40 (m, 5H), 1.60-1.81 (m, 5H), 2.43
(m, 1H), 5.21 (d, 1H, J ) 7.5 Hz), 7.21-7.67 (m, 9H), 8.86 (d,
1H, J ) 7.5 Hz), 10.78 (s, 1H). Anal. (C22H23N3O2) C, H, N.
HPLC: retention time, 5.62 min (e.e. >99%).
1-(2-Fluorophenyl)-3-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo-
[e][1,4]-diazepin-3-yl)-urea, 17a. This material was prepared as
described in General Procedure 3, using 3-amino-5-phenyl-1,3-
dihydrobenzo[e][1,4]diazepin-2-one, 1a (30 mg), and 2-fluoroiso-
cyanatobenzene (0.010 mL). The title compound was isolated as a
beige solid (29 mg, 62%). LC/MS ES+ ) 389. 1H NMR (DMSO-
d6) δ 5.21 (d, 1H, J ) 8.2 Hz), 6.90-7.70 (m, 12H), 8.07 (m, 2H),
8.93 (s, 1H), 10.94 (s, 1H). Anal. (C22H17FN4O2‚0.40H2O) C, H,
N. HPLC: retention time, 7.52 min (49.4%), 8.65 min (50.6%).
(S)-1-(2-Fluorophenyl)-3-(2-oxo-5-phenyl-2,3-dihydro-1H-
benzo[e][1,4]-diazepin-3-yl)-urea, 17b. This material was prepared
as described in General Procedure 3, using (S)-3-amino-5-phenyl-
1,3-dihydrobenzo[e][1,4]diazepin-2-one, 1b (30 mg), and 2-fluor-
oisocyanatobenzene (0.010 mL). The title compound was isolated
the title compound as a white solid (1.3 g, 55%). LC/MS ES+
252. H NMR (DMSO-d6) δ 2.58 (br s, 2H), 4.24 (s, 1H), 7.15-
7.29 (m, 3H), 7.43-7.62 (m, 6H), 10.69 (br s, 1H). HPLC:
retention time, 4.40 min (e.e. >98%).
)
1
2-Methoxy-4-nitro-N-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo-
[e][1,4]-diazepin-3-yl)benzamide, 15a. This material was prepared
as described in General Procedure 1, using 3-amino-5-phenyl-1,3-
dihydrobenzo[e][1,4]diazepin-2-one, 1a (40 mg), and 2-methoxy-
4-nitrobenzoic acid (47 mg). The title compound was isolated as a
colorless solid (51 mg, 74%). LC/MS ES+ ) 431. 1H NMR
(DMSO-d6) δ 4.09 (s, 3H), 5.69 (d, 1H, J ) 7.0 Hz), 7.08-7.49
(m, 9H), 7.80-7.86 (m, 2H), 8.27 (s, 1H), 8.31 (s, 1H), 9.52 (d,
1H, J ) 7.1 Hz). Anal. (C23H18N4O5‚0.48H2O) C, H, N. HPLC:
retention time, 10.39 min (49.9%), 22.92 min (50.1%).
(S)-2-Methoxy-4-nitro-N-(2-oxo-5-phenyl-2,3-dihydro-1H-
benzo[e][1,4]-diazepin-3-yl)benzamide, 15b. This material was
prepared as described in General Procedure 1, using (S)-3-amino-
5-phenyl-1,3-dihydrobenzo[e][1,4]diazepin-2-one, 1b (40 mg), and
2-methoxy-4-nitrobenzoic acid (47 mg). The title compound was
isolated as a colorless solid (37 mg, 54%). LC/MS ES+ ) 431. 1H
NMR (DMSO-d6) δ 4.13 (s, 3H), 5.44 (d, 1H, J ) 7.0 Hz), 7.29-
7.70 (m, 9H), 7.97-8.10 (m, 3H), 9.63 (d, 1H, J ) 7.0 Hz), 11.05
1
as a white solid (34 mg, 73%). LC/MS ES+ ) 389. H NMR
(DMSO-d6) δ 5.24 (d, 1H, J ) 7.5 Hz), 6.90-7.75 (m, 12H), 8.11-
8.17 (m, 2H), 8.95 (d, 1H, J ) 2.5 Hz), 10.95 (s, 1H). Anal. (C22H17-
FN4O2) C, H, N. HPLC: retention time, 8.64 min (e.e. >99%).
(R)-1-(2-Fluorophenyl)-3-(2-oxo-5-phenyl-2,3-dihydro-1H-
benzo[e][1,4]-diazepin-3-yl)-urea, 17c. This material was prepared
as described in General Procedure 3, using (R)-3-amino-5-phenyl-
1,3-dihydrobenzo[e][1,4]diazepin-2-one, 1c (30 mg), and 2-fluor-
oisocyanatobenzene (0.010 mL). The title compound was isolated
1
as a white solid (32 mg, 69%). LC/MS ES+ ) 389. H NMR
(DMSO-d6) δ 5.20 (d, 1H, J ) 8.2 Hz), 6.90-7.68 (m, 12H), 8.05-
8.11 (m, 2H), 8.92 (s, 1H), 10.93 (s, 1H). Anal. (C22H17FN4O2;
0.37 H2O) C, H, N. HPLC: retention time, 7.52 min (e.e. >99%).
(S)-4-Methanesulfonyl-2-methoxy-N-(2-oxo-5-phenyl-2,3-di-
hydro-1H-benzo[e][1,4]diazepin-3-yl)-benzamide, 18. This mate-

1692 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Henderson et al.
(2) Reider, P. J.; Davis, P.; Hughes, D. L.; Grabowski, E. J. J.
Crystallization-induced asymmetric transformation: Stereospecific
synthesis of a potent peripheral CCK antagonist. J. Org. Chem. 1987,
52, 955-957.
(3) Rittle, K. E.; Evans, B. E.; Bock, M. G.; DiPardo, R. M.; Whitter,
W. L.; Homnick, C. F.; Veber, D. F.; Freidinger, R. M. A new amine
resolution method and its application to 3-aminobenzodiazepines.
Tetrahedron Lett. 1987, 28, 5, 521-522.
(4) Dourtoglou, V.; Gross, B. O-Benzotriazolyl-N,N,N′,N′-tetramethyl-
uronium hexafluorophosphate as coupling reagent for the synthesis
of peptides of biological interest. Synthesis 1984, 572-574.
(5) Edman, P. Method for determination of the amino acid sequence in
peptides. Acta Chem. Scand. 1950, 4, 283-293.
(6) Bock, M. G.; DiPardo, R. M.; Evans, B. E.; Rittle, K. E.; Whitter,
W. L.; Garsky, V. M.; Gilbert, K. F.; Leighton, J. L.; Carson, K. L.;
et al. Development of 1,4-benzodiazepine cholecystokinin type B
antagonists. J. Med. Chem. 1993, 36, 4276-4292.
(7) Webb, K. S. A mild, inexpensive and practical oxidation of sulfides.
Tetrahedron Lett. 1994, 35, 3457-3460.
(8) Chen, J. J.; Lu, C. V.; Brockman, R. N. Catalyzed double Michael
addition of anilines to vinyl sulfone. Tetrahedron Lett. 2003, 44,
3459-3462.
(9) Leost, F.; Chantegrel, B.; Deshayes, C. Tandem Wolff rearrangement-
“tert-amino effect” sequence: Synthesis of 2-oxoindolinium enolate
derivatives. Tetrahedron 1997, 53, 7557-7576.
(10) Semple, G.; Ryder, H.; Rooker, D. P.; Batt, A. R.; Kendrick, D. A.;
Szelke, M.; Ohta, M.; Satoh, M.; Nishida, A.; Akuzawa, S.; Miyata,
K. (3R)-N-(1-(tert-Butylcarbonylmethyl)-2,3-dihydro-2-oxo-5-(2-py-
ridyl)-1H-1,4-benzodiazepin-3-yl)-N′-(3-(methylamino)phenyl)urea
(YF476): A potent and orally active gastrin/CCK-B antagonist. J.
Med. Chem. 1997, 40, 331-341.
(11) Corey, E. J.; Szekely, I.; Shiner, C. S. Synthesis of 6,9R-oxido-11R,-
15R-dihydroxyprosta-(E)5, (E)13-dienoic acid, an isomer of PGI2
(vane’s PGX). Tetrahedron Lett. 1977, 18, 3529-3532.
(12) Bock, M. G.; DiPardo, R. M.; Evans, B. E.; Rittle, K. E.; Veber, D.
F.; Freidinger, R. M.; Hirshfield, J.; Springer, J. P. Synthesis and
resolution of 3-amino-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-
2-ones. J. Org. Chem. 1987, 52, 3232-3239.
(13) Engel, D. W.; Zechmeister, K.; Hoppe, W. Experience in the
determination of absolute configuration of light atom compounds by
X-ray anomalous scattering. Tetrahedron Lett. 1972, 13, 1323-1326.
(14) Halgren, T. A. MMFF VI. MMFF94s option for energy minimization
studies. J. Comput. Chem. 1999, 20, 720-729.
(15) Visnjevac, A.; Kojic-Prodic, B. Ethyl 7-chloro-2,3-dihydro-2-oxo-
5-phenyl-1H-1,4-benzodiazepine-3-carboxylate. Acta Crystallogr.
2001, E57, o356-o357.
(16) Bathelt, C. M.; Ridder, L.; Mulholland, A. J.; Harvey, J. N. Aromatic
hydroxylation by cytochrome P450: Model calculations of mecha-
nism and substituent effects. J. Am. Chem. Soc. 2003, 125, 15004-
15005.
rial was prepared as described as in General Procedure 2, using
(S)-3-amino-5-phenyl-1,3-dihydro-benzo[e][1,4]diazepin-2-one, 1b
(100 mg), and 4-methanesulfonyl-2-methoxybenzoic acid, 8 (46
mg). The title compound was isolated as a colorless solid (55 mg,
1
30%). LC/MS ES+ ) 464. H NMR (DMSO-d6) δ 3.33 (s, 3H),
4.13 (s, 3H), 5.44 (d, 1H, J ) 7.5 Hz), 7.33-7.71 (m, 11H), 8.10
(d, 1H, J ) 10.0 Hz), 9.61 (d, 1H, J ) 7.5 Hz), 11.06 (s, 1H).
Anal. (C24H21N3O5S‚1.19H2O) C, H, N. HPLC: retention time, 8.28
min (e.e. >99%).
(S)-2-Chloro-4-morpholin-4-yl-N-(2-oxo-5-phenyl-2,3-dihydro-
1H-benzo[e][1,4]diazepin-3-yl)-benzamide, 21. This material was
prepared as described in General Procedure 4, using 2-chloro-4-
morpholin-4-yl benzoic acid (86 mg). The title compound was
isolated as a colorless solid (112 mg, 59%). LC/MS ES+ ) 475.
1H NMR (DMSO-d6) δ 3.21 (m, 4H), 3.70 (t, 4H, J ) 4.7 Hz),
5.36 (d, 1H, J ) 8.2 Hz), 6.90-6.97 (m, 2H), 7.21-7.66 (m, 10H),
9.21 (d, 1H, J ) 8.2 Hz), 10.86 (s, 1H). Anal. (C26H23ClN4O3‚
1.0H2O) C, H, N. HPLC: retention time, 14.08 min (e.e. >99%).
(S)-{3-[3-(2-Fluorophenyl)ureido]-2-oxo-5-phenyl-2,3-dihydro-
benzo[e][1,4]diazepin-1-yl}-acetic Acid Methyl Ester, 11. A
solution of (S)-1-(2-fluorophenyl)-3-(2-oxo-5-phenyl-2,3-dihydro-
1H-benzo[e][1,4]diazepin-3-yl)-urea, 17b (500 mg), in dry N,N-
dimethylformamide (15 mL) was treated with 60% sodium hydride
in mineral oil and stirred at ambient temperature for 30 min under
a nitrogen atmosphere. Methyl bromoacetate (0.13 mL) was added,
and the solution was stirred for 3 h. Water (70 mL) was added,
and the crude product was collected by filtration. Purification on a
silica gel SPE, eluting with dichloromethane followed by dichlo-
romethane/ethanol/0.880 ammonia 200:8:1, yielded the title product
as a pale yellow solid (385 mg, 65%). LC/MS ES+ ) 461. 1H NMR
(CDCl3) δ 3.62 (s, 3H), 4.63 (ABq, 2H, J ) 17.3 Hz), 5.62 (d, 1H,
J ) 8.3 Hz), 6.75 (d, 1H, J ) 8.2 Hz), 6.89-7.12 (m, 4H), 7.22-
7.70 (m, 9H), 7.99 (t, 1H, J ) 8.2 Hz).
(S)-{3-[3-(2-Fluorophenyl)-ureido]-2-oxo-5-phenyl-2,3-dihy-
dro-benzo[e][1,4]diazepin-1-yl} Acetic Acid, 12. A solution of
(S)-{3-[3-(2-fluorophenyl)ureido-2-oxo-5-phenyl-2,3-dihydro-benzo-
[e][1,4]diazepin-1-yl} acetic acid methyl ester, 11 (50 mg), in
tetrahydrofuran (1 mL) and water (3 mL) was treated with lithium
hydroxide (50 mg) and stirred at ambient temperature for 1 h. The
solution was cooled to 0 °C and acidified to pH 2, and the
precipitate was collected by filtration to yield the title compound
as a pale yellow solid (41 mg, 85%). LC/MS ES+ ) 447. 1H NMR
(DMSO-d6) δ 4.66 (ABq, 2H, J ) 19.0 Hz, 17.7 Hz), 5.33 (d, 1H,
J ) 8.9 Hz), 6.90-7.73 (m, 12H), 8.00-8.11 (m, 2H), 8.94 (s,
1H), 13.14 (br s, 1H).
(17) CLOGP, version 3.63; Daylight Chemical Information Systems:
Santa Fe, NM, 1997.
Acknowledgment. We thank Christopher Otton for chiral
analysis of all final compounds. CHN analyses were kindly
performed by Gillian Maxwell at University College London.
We are grateful to David J. Williams at Imperial College who
determined the X-ray crystal structure of compound 14.
(18) (a) Hennequin, L. F.; Stokes, E. S. E.; Thomas, A. P.; Johnstone,
C.; Ple´, P. A.; Ogilvie, D. A.; Dukes, M.; Wedge, S. R.; Kendrew,
J.; Curwen, J. O. Novel 4-anilinoquinazolines with C-7 basic side
chains: Design and structure-activity relationship of a series of
potent, orally active, VEGF receptor tyrosine kinase inhibitors. J.
Med. Chem. 2002, 45, 1300-1312. (b) Delaney, J. S. Predicting
aqueous solubility from structure. Drug DiscoVery Today 2005, 10,
289-295.
(19) Sternson, L. A.; Gammans, R. E. Effect of aromatic nitro compounds
on oxidative metabolism by cytochrome P-450 dependent enzymes.
J. Med. Chem. 1976, 19, 174-177.
(20) Gershwin, L. J.; Schelegle, E. S.; Gunther, R. A.; Anderson, M. L.;
Woolums, A. R.; Larochelle, D. R.; Boyle, G. A.; Friebertshauser,
K. E.; Singer, R. S. A bovine model of vaccine enhanced respiratory
syncytial virus pathophysiology. Vaccine 1998, 16, 1225-1236.
(21) CEREP screen: see http://www.cerep.fr for details of screen and
protocols.
Supporting Information Available: Experimental procedures
for the synthesis of intermediates and characterization of final
products, including X-ray crystal structure determination. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Carter, M. C.; Alber, D. G.; Baxter, R. C.; Bithell, S. K.; Budworth,
J.; Chubb, A.; Cockerill, G. S.; Dowdell, V. C. L.; Henderson, E.
A.; Keegan, S. J.; Kelsey, R. D.; Lockyer, M. J.; Stables, J. N.;
Wilson, L. J.; Powell, K. L. 1,4-Benzodiazepines as inhibitors of
respiratory syncytial virus. J. Med. Chem. 2006, 49, 2311-2319.
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