From hit to lead. Combining two complementary methods for focused library design. Application to μ opiate ligands

Article abstract of DOI:10.1021/jm010877o

Compound 1 obtained by random screening and displaying a micromolar activity on the μ opiate receptor was chosen as a starting point for optimization. Two complementary concepts of similarity were used for the design of analogues and compared. These are based, respectively, on a computer-aided comparison of pharmacophoric patterns and on topological similarity. The structure-activity relationships are discussed in light of both similarity concepts. Compound 40, an N-methyl-3-(4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decyl)acetamide derivative, designed by combining the structure-activity relationships enlightened by each method, has a subnanomolar affinity for μ (h) receptor (IC₅₀=0.9 nM). It is a promising lead, allowing the design of a new series of analogues substituted at the N-3 of the spirocycle moiety.

Full text of DOI:10.1021/jm010877o

3378
J . Med. Chem. 2001, 44, 3378-3390
F r om Hit to Lea d . Com bin in g Tw o Com p lem en ta r y Meth od s for F ocu sed
Libr a r y Design . Ap p lica tion to µ Op ia te Liga n d s
Re´becca Poulain,*,† Dragos Horvath,†,‡ Be´atrice Bonnet,†, Christian Eckhoff,§ Be´atrice Chapelain,|
Marie-Christine Bodinier,§ and Benoˆıt De´prez†,
Department of Chemistry, CEREP, 1 rue du Pr. Calmette, F-59000 Lille, France, Department of Pharmaceutical Development,
CEREP Inc., 15318 NE 95th Street, Redmond, Washington 98052, and Department of Pharmacology, CEREP,
Celle l’Evescault, BP1, F-86600 Le Bois L’Eveˆque, France
Received March 14, 2001
Compound 1 obtained by random screening and displaying a micromolar activity on the µ opiate
receptor was chosen as a starting point for optimization. Two complementary concepts of
similarity were used for the design of analogues and compared. These are based, respectively,
on a computer-aided comparison of pharmacophoric patterns and on topological similarity. The
structure-activity relationships are discussed in light of both similarity concepts. Compound
40, an N-methyl-3-(4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decyl)acetamide derivative, designed
by combining the structure-activity relationships enlightened by each method, has a
subnanomolar affinity for µ (h) receptor (IC50 ) 0.9 nM). It is a promising lead, allowing the
design of a new series of analogues substituted at the N-3 of the spirocycle moiety.
Ch a r t 1
In tr od u ction
In recent years, parallel synthesis1 and high-through-
put screening (HTS),2 relying in various extents on
molecular modeling for library management,3 descrip-
tion,4 similarity evaluation,5 and quantitative structure-
activity relationships (SARs),6 have led to a dramatic
increase in throughput in the primary stages of drug
discovery.
We report here a generic hit-to-lead strategy, relying
on the systematic generation and evaluation of ana-
logues around the parent hit compound. This strategy
makes concerted uses of two different analoging meth-
ods to cover efficiently the structural space around the
hits. The first method is a classical exploratory analog-
ing based on similar topology. The second method relies
on (1) algorithms for rapid generation of 3D molecular
fingerprints7 based on multiconformational models of
candidate compounds and (2) similarity8 evaluation
tools for the comparison of these 3D fingerprints. It is
therefore called the similar pharmacophoric pattern
search.
To validate experimentally our hit-to-lead strategy,
we have applied it to the well-known µ receptor. Our
goal was not primarily to discover new µ ligands but to
exemplify our approach in the case of a universally
recognized model. To mimic a real situation of drug
discovery, a diverse library, of 10560 compounds, fitting
the Lipinski rules,9 obtained by parallel synthesis, was
screened at 10 µM on the µ opiate receptor (rat cortex)
for binding ability. The hit rate obtained at 10 µM for
an inhibition threshold of 50% was 1.7%. Hits were then
resynthesized, purified, and tested for affinity against
the µ receptor (human) and the two other opiate
subtypes (δ, κ). Out of these primary hits, compound 1
(Chart 1) was selected as a starting point for optimiza-
tion.
Analogues of 1 were designed either by pharmaco-
phoric pattern similarity searching against a virtual
library of potentially synthesizable combinatorial prod-
ucts or by exploring various changes of the pharma-
cophoric pattern, while keeping the topology. Automated
parallel synthesis was used for the rapid synthesis of
the different analogues. A total of 294 analogues of 1
were synthesized on the 5 µmol scale and screened on
the µ (h) receptor at 10 µM. Eventually, 30 compounds
were selected for resynthesis and purification on a larger
scale, to allow both complete purity and identity as-
sessment and biological evaluation. After this first step,
six new analogues displaying combinations of the best
structural features evidenced by each analogue design
method were synthesized and tested.
* To whom correspondence should be addressed. Current address:
UMR8525, Faculte´ des Sciences Pharmaceutiques et Biologiques,
Chimie Organique, 3 rue du Pr. Laguesse, BP 83, F-59006 Lille Cedex,
France. Phone: 33.3.20.96.47.07. Fax: 33.3.20.96.47.09. E-mail:
rpoulain@phare.univ-lille2.fr.
† Department of Chemistry.
Design of th e An a logu es
‡ Current address: CEREP, 128 rue Danton, BP50601, F-92506
Rueil-Malmaison, France.
To select and synthesize the analogues, two indepen-
dent methods of analogue design have been used,
allowing us to investigate in a systematic way the
structural space around 1.
§ Department of Pharmaceutical Development.
| Department of Pharmacology.
Current address: Devgen NV, Technologiepark 9, B-9052 Ghent,
Zwijnaarde, Belgium.
10.1021/jm010877o CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/12/2001

Combining Two Methods for Focused Library Design
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3379
Sch em e 1a
(a ) Meth od A consists of exploring the distribution
of the pharmacophoric10 elements of the active com-
pound while keeping a similar molecular topology. This
method uses modifications of structures such as chain
lengthening, substitutions, ring opening or closures, and
simplifications to “mutate”, “enrich”, or “simplify” the
distribution of the pharmacophoric groups, also referred
to as the pharmacophoric pattern, of the hit. At least
one obvious retrosynthetic route of wide chemical scope
was available for compound 1 since it was primarily
issued from parallel synthesis. Therefore, most of the
analogues were generated as combinations of com-
mercially available or purposely synthesized topologi-
cally similar but pharmacophorically different analogues
of the original building blocks used for the synthesis of
1.
a
Reagents and Conditions: (a) (i) K2CO3, DMF, 70 °C, overnight;
(ii) EtOH, Pd-C (10%), NH4+HCOO-, 70 °C, overnight; (iii) AcOEt,
DIPEA, 0 °C, triphosgene; (iv) TFA/CH2Cl2 (50/50).
Sch em e 2a
(b) Meth od B aims at retrieving analogues that
display a similar pharmacophoric pattern, out of a
virtual library of all feasible combinations of available
building blocks (more than 7000 diversity reagents, in
our case). This method does not explicitly take molecular
topology into account. Each member of the virtual
library was described by an autocorrelogram referred
to as an FBPA (fuzzy bipolar pharmacophore autocor-
relogram).7 The 252-dimensional FBPAs are the molec-
ular descriptors at the basis of the similarity metrics
used in this work.8 The FBPA of a molecule M, hereafter
denoted as ΨM(a,b,∆), monitors the numbers of atom
pairs within each of the 252 ) 21 × 12 categories that
can be defined in terms of the 21 combinations of six
pharmacophoric features a, b ∈ {hydrophobicity, aro-
maticity, hydrogen bond acceptor and donor, cation,
anion} times 12 considered distance ranges ∆ (Å) ∈ {3-
4, 4-5, ..., 14-15}. The associated fuzzy-logic-based
dissimilarity score ∆FBP(m,M) between a candidate
molecule m and the reference M relies on partial
dissimilarity scores δa,b(m,M) calculated with respect to
the atom pair distributions matching every pharma-
cophore feature pair a, b:
a
Reagents and Conditions: (a) (i) K2CO3, DMF, 70 °C, overnight;
(ii) Fe, HCl (concentrated), EtOH, reflux, 3.5 h; (iii) triphosgene,
AcOEt, rt, overnight; (iv) Pd-C, HCOOH, MeOH, reflux, over-
night; (b) (i) K2CO3, DMF, 70 °C, overnight; (ii) Fe, HCl (concen-
trated), EtOH, reflux, 3.5 h; (iii) oxalic acid, HCl (3 N), reflux,
overnight; (iv) Pd-C, HCOOH, MeOH, reflux, overnight.
sented biological activity classes within a most diverse
subset selected by the dissimilarity metric from a
collection of reference ligands of various receptors.
Candidates selected from a virtual compound collection
and displaying the lowest dissimilarity scores when
their FBPAs were compared to that of the reference
compound (here, hit 1) were then, in a second step,
superimposed on the reference and reranked according
to the more accurate ComPharm similarity score.8
2(Ψm X ΨM)a,b
δ
a,b(m,M) ) 1 -
(ΨM X ΨM)a,b + (Ψm X Ψm)a,b
Ch em istr y
where
(a ) Libr a r y of Exp lor a tor y An a logu es (Meth od
A, Sim ila r Top ology). Compound 1 is a carbamate.
Various analogues of the amine moiety were purchased
or prepared (Schemes 1 and 2) to determine the effect
of ring opening of the piperidine, substitution of one of
the hydrogen atoms of the benzimidazolinyl group, and
homologation of the 2-oxo-1-benzimidazolinyl group by
insertion of another carbonyl moiety. Different alcohols,
amines, and carboxylic acids were coupled, using vari-
ous synthetic methods (Scheme 3), to these amines to
generate carbamates, ureas, and amides, respectively.
The presence of a basic nitrogen is known to be critical
for binding to opiate receptors as well as to many other
G-protein-coupled receptors (GPCRs) displaying a con-
served aspartate residue in the potential binding cleft.11
Moreover, this feature has a major impact on the
physical-chemical behavior. We thus investigated the
synthesis of N-(3-phenylpropyl)piperidinyl derivatives
as analogues of 1. Alkylation procedures from aldehyde
or halide, which can readily generate a broad structural
Nbin Nbin
(Ψm X ΨM)a,b
)
Ψ (a,b,∆ ) Ψ (a,b,∆ ) ×
∑ ∑
∆1)1∆2)1
m
1
M
2
exp[-R(∆1 - ∆2)2]
The global dissimilarity score ∆FBP(m,M) is defined
as a weighted average of the pharmacophore pair partial
scores, with weighing factors wk associated with each
pharmacophore feature:
Nf Nf
w w δa,b(m,M)
∑∑
a
b
∆FBP(m,M) ) a)1b)a
Nf Nf
w w
∑∑
a
b
a)1b)a
The weighing factors and the fuzziness parameter R
were once calibrated to maximize the number of repre-

3380 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Poulain et al.
Sch em e 3a
Sch em e 4a
a
Reagents and conditions: (a) alcohol in DMF, N,N′-carbonyl-
diimidazole (CDI) in THF, 86-98%; (b) carboxylic acid in DMF,
CDI in THF, 87-90%; (c) amine in DMF, CDI in THF, 90-92%;
(d) aldehyde, NaHB(OAc)3, CH2Cl2, 81-88%; (e) halide, DIPEA,
DMF, 95-99%.
diversity, are the obvious synthetic way to obtain these
compounds. However, in our hands, aliphatic aldehydes
were not stable in the synthesis conditions, and 3-phe-
nylpropyl halides are poorly reactive. The carbon chain
between the basic nitrogen and the phenyl group was
thus shortened to use benzyl aldehydes and halides
(Scheme 3). These N-benzylpiperidinyl analogues are
related to the 4-(2-oxo-1-benzimidazolinyl)piperidine
derivatives already described as displaying in vivo
analgesic properties.12
a
Reagents and Conditions: (a) (i) DMF, reflux, 1 h; (ii) HCl (3
N), 100 °C, 2 h; (b) (i) THF, CDI, MeNH2·HCl, TEA; (ii) THF, HCl
(1 N), Et2O, H2SO4, 65 °C, overnight; (c) (i) LiI, pyridine, reflux,
overnight; (ii) ClCOOEt, DIPEA, THF, 0 °C, 1.5 h; (iii) NaBH4,
H2O, 0 °C, 2 h.
Sch em e 5a
(b) Libr a r y of An a logu es w ith Sim ila r P h a r m a -
cop h or ic P a t t er n s (Met h od B). These compounds
could be sorted into two series. The first series consisted
of analogues with a topology completely different from
that of 1. Examples are presented in Chart 2 (com-
pounds 6-9). Intermediates 10-12 had to be prepared
as shown in Scheme 4 to synthesize these analogues.
The second and best fitting series consisted of analogues
derived from 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one
or its substituted derivative 11.
Analogues displaying a spirocyclic moiety and re-
trieved by method B could be easily synthesized as
described in Scheme 5. Some analogues displaying a
good similarity score were derived from alcohol 12,
which had been previously synthesized from the corre-
sponding methyl ester.
(c) An a lytica l Con tr ol. Each of the 294 analogues
synthesized by parallel synthesis, at an “HTS grade”,
was controlled for purity and identity using LC/MS. In
each case, the purity exceeded 80% and the mass
spectrum of the major peak was consistent with the
expected structure. Further pharmacological character-
ization was undertaken on fully purified and controlled
samples (“SAR grade”).
a
Reagents and conditions: (a) alcohol in DMF, CDI in THF,
85-95%; (b) carboxylic acid in DMF, CDI in THF, 90-94%; (c)
amine in DMF, CDI in THF, 90-96%.
Scr een in g
The libraries were tested at 10 µM for binding at the
µ human receptor. Crude products displaying a percent-
age of inhibition above 50% were selected for prelimi-
nary IC50 determination prior to resynthesis and puri-
fication. Eventually, 30 compounds were selected for
resynthesis and purification on a larger scale to allow
biological characterization on µ (h), κ (h), and δ (h).
Resu lts a n d Discu ssion
IC50 is defined as the concentration inhibiting 50% of
specific binding of 0.5 nM DAMGO, U69593, and DP-
DPE to µ, κ, and δ receptors, respectively. Binding
Ch a r t 2

Combining Two Methods for Focused Library Design
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3381
Ta ble 1. Binding Affinities (IC50) on the Opiate Receptors of Exploratory Analogues 13-27 of 1 Retrieved by Method A (Similar
Topology)
IC50 (nM)a,b
compd
no.
solc
(µM)
R1
R2
R3
R4
n
p
µ (h)
κ (h)
δ (h)
>10000
log Dd
1
13
14
15
16
17
18
NO2
Cl
Cl
Cl
NO2
Cl
-CH2CH2-
-CH2CH2-
H
H
H
H
H
H
H
CH3
1
1
1
0
1
1
1
1
1
1
1
1
2
1
1050 (1020-1090)
147 (142-152)
2480 (1160-3800)
>10000
>10000
2590 (2340-2840)
1235 (1184-1292)
>10000
<10
<10
<10
<10
f
<10
<10
f
284 (265-303)
1740 (1429-2051)
>10000
2530 (2507-2554)
>10000
4.2
e
3.2
f
1.1
3.4
H
H
H
>10000
-CH2CHd
-CH2CH2-
-CH2CH2-
6540 (6290-6790)
1060 (983-1137)
797 (770-825)
>10000
>10000
Cl
5640 (3960-7320)
IC50 (nM)a,b
κ (h)
compd
no.
solc
(µ M)
X
µ (h)
δ (h)
log Dd
19
20
CH2
-CHdCH-
>10000
960 (959-961)
1320 (1316-1324)
4030 (3247-4812)
>10000
>10000
<10
57.4
2.4
3
IC50 (nM)a,b
κ (h)
1.7 (1.6-1.8)
compd
no.
solc
(µM)
R1
R2
R3
R4
R5
R6
p
µ (h)
δ (h)
37.4 (36.9-37.9)
log Dd
21
22
23
24
25
26
27
Cl
Cl
NO2
NO2
Cl
-OCH2O-
-OCH2O-
-OCH2O-
-OCH2O-
-OCH2O-
-OCH2O-
-CH2CH2-
H
H
H
H
H
H
CH3
H
1
1
1
1
2
1
1
1.2 (1.1-1.2)
235 (231-240)
1.5 (1.4-1.6)
89.6
185.7
<10
3.0
1.8
2.4
2.9
1.3
2.6
2.5
H
507 (401-612) 3330 (295-03710)
37 (32.4-41.8) 264 (248-279)
-CH2CH2-
-CH2CHd
-CH2CH2-
-CH2CH2-
-CH2CH2-
19.3 (17.2-21.3) 311 (286-336) 1040 (1010-1070) <10
6.3 (4.4-8.3)
2.4 (2.2-2.5)
16 (14.7-17.2)
32 (30-34)
2.9 (2.7-3.0)
620 (589-651)
117 (101-133)
46.4
<10
195.3
Cl
Cl
OCH3
H
270 (270-270) 1410 (304-2510)
a
IC50 values were obtained from an experiment performed in duplicate from 11 concentrations. Both values are given in parentheses.
The radioligands used were [3H]DAMGO (µ (h)), [3H]U69593 (κ (h)), and [3H]DPDPE (δ (h)). c The solubility value is determined in PBS,
b
pH 7.4. Partition coefficient between PBS, pH 7.4, and octanol. e Determination could not be made since the compound was not detected
d
by MS or UV in the buffer phase (log D likely >4). f Not determined.
affinities (IC50 values, nM) to the µ, κ, and δ receptors
of the selected analogues of 1 are reported in Tables
1-3.
13 (IC50(µ) ) 147 nM) and 21 (IC50(µ) ) 1.2 nM). In the
meantime, solubility increases and log D decreases.
Simultaneous removal of a methylene group and ring
opening led to a complete loss of activity (compound 15).
A change in the structural constraint, thanks to a double
bond, in the piperidine ring of 1 or 23 led to a 10-fold
decrease in activity (16 or 24, respectively).
Quinoxalinedione derivatives 17 and 25 were found
to be 5-20 times less active than the corresponding
benzimidazolones. These analogues displayed the lowest
log D of the series.
(a ) Exp lor a tor y An a logu es (Meth od A, Sim ila r
Top ology). As one might expect,11 introduction of a
basic nitrogen, conjugated with shortening of the chain,
enhances binding affinity by 2 or 3 orders of magnitude
(1 vs 23), while decreasing subtype selectivity. Isosteric
replacement of the carbamate in 1 by a cinnamoyl
moiety (compound 20) has poor incidence on the activity
(except on κ (h)) but increases solubility. The carbamate
link seems not favorable for aqueous solubility.
The open ring analogues 14 and 22 (IC50(µ) ) 2480
nM and IC50(µ) ) 235 nM, respectively) are 10-100 times
less active than the corresponding piperidine compounds
Affinities of analogues bearing a methyl group at
position 6 on the benzimidazolone moiety decrease (1
and 21 vs 18 and 26) by only a factor of 2. Interestingly,
analogue 16 displays a better affinity for κ (h) than for

3382 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Poulain et al.
Ta ble 2. Binding Affinities (IC50) on the Opiate Receptors of Spirocycle Analogues 28-38 of 1 Retrieved by Method B (Similar
Pharmacophoric Pattern)
IC50 (nM)a,b
compd
no.
solc
R1
R2
R3
R4
R5
X
µ (h)
CH >10000
CH2CONHCH3 CH 179 (173-184)
κ (h)
5070 (5000-5140 ) >10000
333 (286-380) 1765 (1600-1930)
δ (h)
(µM) log Dd
28
29
30
31
32
33
34
35
H
NO2
Cl
Cl
H
-OCH2O-
-OCH2O-
-OCH2O-
-OCH2O-
-thiadiazole-
H
H
H
H
<10
36.9
3.1
3.1
2.9
3.7
3.4
e
H
H
H
H
H
H
H
H
H
H
H
CH 1730 (1700-1759 ) 2145 (1710-2580) >10000
CH 2285 (2090-2480) 2235 (2080-2390) >10000
<10
<10
<10
<10
<10
105
H
H
CH >10000
CH >10000
1480 (1140-1820) >10000
1810 (1510-2100) >10000
-OC6H5
-CH2CH2-
H
H
H
CH 1470 (1160-1780) 617 (532-702)
>10000
e
2.5
H
N
>10000
3550 (2350-4750) >10000
IC50 (nM)a,b
compd
solc
no.
R1
R2
R3
X
µ (h)
κ (h)
δ (h)
(µM)
log Dd
36
37
38
-OC H2O-
-OC H2O-
H
NO2
NO2
-CH2NH-
-CHdCH-
-CHdCH-
>10000
>10000
>10000
2280 (1610-2950)
>10000
>10000
>10000
>10000
>10000
28.8
24.3
34.8
2.8
3.1
3.0
OCH3
OCH3
a-e See footnotes of Table 1.
Ta ble 3. Binding Affinities (IC50) on the Opiate Receptors of Analogues 6-9 of 1 Retrieved by Method B (Similar Pharmacophoric
Pattern)
IC50 (nM)a,b
compd
no.
solc
(µM)
µ (h)
κ (h)
δ (h)
log Dd
6
7
8
9
4955 (2740-7170)
2285 (2090-2480)
>10000
7240 (2980-11500)
>10000
1010 (1000-1020)
>10000
>10000
>10000
>10000
<10
<10
<10
46.5
4.2
e
3
2890 (1170-4610)
7130 (3160-11100)
3.4
a-e See footnotes of Table 1.
µ (h). Analogue 26, differing from 21 only by a methyl
group, is however much less water soluble.
Interestingly, relative subtle modification of structure
(13, 1, and 21 vs 17, 18, and 26) can lead to significant
changes in solubility properties.
A chlorine atom in the ortho-position of the benzyl
ring improves both binding and solubility in comparison
with the nitro group (13 and 21 vs 1 and 23), but
selectivity between µ (h) and κ (h) decreases. Suppres-
sion of the methylenedioxy group and replacement by
a single methoxy substituant (27 vs 21) results in a 10-
fold decrease of affinity for µ (h) but a better solubility.
The two key features that thus should be taken into
consideration for the optimization of compound 1 are
the presence of the basic nitrogen, to reach nanomolar
activities, and the replacement of the NO2 group by a
chlorine atom, to increase solubility.
compound 28 shows no detectable activity, while re-
placement of the nitro group by a chlorine atom in 30
allows activities similar to that of 1. ortho-Substitution
on the benzyl ring allows micromolar activity on µ (h).
Interestingly, compound 34, which bears a 2-indan
moiety instead of an ortho-subtituted benzyl ring, also
displays micromolar activity on µ (h) (IC50(µ) ) 1470 nM),
and submicromolar activity on κ (h) (IC50(κ) ) 617 nM).
In this carbamate series, the only features that increase
solubility are the presence of a pyridine ring (35) or the
presence of an N-methylacetamide group on position 3
of the spirodecanone (29). Interestingly, the latter
substitution was also found to be, in our series, the most
beneficial feature for binding. The isosteric replacement
of the carbamate link by an amide (compounds 37 and
38) or a urea (compound 36) did improve solubility, but
had no effect on activity.
(b) An a logu es w ith Sim ila r P h a r m a cop h or ic
P a tter n s (Meth od B). Among others, of micromolar
activities, method B produced several analogues where
the piperidinylbenzimidazolone is replaced by a phenyl-
triazaspirodecanone moiety (28-38). In that series,
Besides the spirodecanone template, method B identi-
fied several other pharmacophorically similar backbones
(6-9, Table 3) that actually had micromolar IC50 values.
However, none of these structures were found in a
consistent family, rendering the elaboration of the

Combining Two Methods for Focused Library Design
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3383
Ta ble 4. Binding Affinities (IC50) on the Opiate Receptors of Analogues 39-44 of 1 Resulting from Combining Methods A and B
IC50 (nM)a,b
compd
no.
solc
(µM)
R1 R2
R3
X
R4
µ (h)
κ (h)
δ (h)
log Dd
39
40
41
42
43
44
Cl
Cl
H
H
H
-OCH2O-
-OCH2O-
-OCH2O-
-thiadiazole- CH2
-OC 6H5 CH2
-OCH2O-
CH2
CH2
CH2
H
2.0 (1.5-2.4)
5.7 (5.7-5.8)
2.1 (2.0-2.3)
138 (109-167)
11.4 (11.1-11.7)
1190 (997-1370)
20.1
208. 9
183. 2
3.0
2.9
2.2
2.6
2.9
0.7
CH2CONHCH3 0.9 (0.9-1.0)
H
H
H
16.7 (15.6-17.8) 56.1 (50.1-62.1)
24.9 (24.3-25.4) 104 (86-121)
83.5 (83.4-83.7) 101 (96-106)
4120 (3980-4250) 197
1840 (1770-1900) <10
H
Cl
CH2OCO CH2COOH
735 (687-784)
6570 (6490-6650) >10000
98.1
a-d
See footnotes of Table 1.
Ta ble 5. Binding Affinities (IC50) on the Opiate Receptors of Some Reference Ligands
IC50 (nM)a,b
entry
name
µ (h)
κ (h)
δ (h)
1
2
3
4
5
6
7
8
9
morphine sulfate
codeine monohydrate
nalbuphine
buprenorphine hydrochloride
naloxone
pethidine
loperamide hydrochloride
fentanyl
1.0 (0.8-1.2)
520 (507-534)
1.0 (0.8-1.2)
1.7 (1.1-2.3)
2.0 (1.9-2.0)
315 (301-329)
1.5 (1.1-1.8)
1.3 (1.3-1.4)
4.1 (3.8-4.4)
217 (191-234)
c
83 (72-94)
1.2 (1.1-1.3)
1.5 (1.2-1.8)
2370 (1920-2810)
155 (126-184)
1580 (991-2180)
512 (511-513)
242 (237-247)
>10000
353 (349-358)
0.9 (0.5-1.3)
151 (149-153)
>10000
123 (116-130)
431 (362-500)
1090 (977-1200)
methadone
a,b
See footnotes of Table 1. c Could not be determined.
primary SAR difficult. Therefore, at the end of that first
optimization cycle, we selected the spirodecanone frame-
work as a basis for a second round of synthesis.
displayed a submicromolar activity on µ (h). The influ-
ence of various substituents at this position on the
spirocyclic moiety could be the next point to investigate
to optimize selectivity and ADME properties for ex-
ample. Indeed, the methyl group could be easily replaced
to give other amides.
Applying the key features evidenced by method A on
the spirodecanone framework, we designed five more
analogues (39-43, Table 4). The syntheses were per-
formed using benzyl aldehydes or benzyl chlorides for
N-alkylation, except for compound 42, which was syn-
thesized from alcohol 12 via the corresponding mesylate.
As in the benzimidazolone series, introduction of the
basic nitrogen allowed nanomolar activities to be reached
and increased aqueous solubility (while log D was
distributed between 2.5 and 3). Large substituents on
the benzyl ring, such as a phenoxy group in 43, decrease
activity by a factor of 40 and have a dramatic impact
on solubility. Interestingly, benzothiadiazole seems to
be a good bioisoster of the methylenedioxyphenyl group,
since analogue 42 has activities, solubility, and log D
similar to those of analogue 41. In the case of opiate
receptors, benzothiadiazole could be considered as a
potential substitute for the methylenedioxyphenyl group
that, notwithstanding its occurrence in a number of
natural or synthetic therapeutic compounds, is known
to potentially inhibit cytochrome P450 and saturate
metabolic pathways. Analogue 40 bears a chlorine atom
on the benzyl ring and is substituted by an N-methyl-
acetamide group on the spiro moiety. It is the most
active compound of the series, and it has the best
solubility. It is more than 3 orders of magnitude more
active than 1, being within the range of the best
reported nonpeptidic ligands (entries 1-9, Table 5).
Interestingly, the N-3 substitution on the spirocyclic
moiety is quite tolerant since compound 44, having no
basic nitrogen but even bearing a carboxylate group,
Con clu sion
Hit optimization of compound 1 relied on two different
analogue design strategies: a computer-aided approach
based on a quantitative estimation of the similarity of
pharmacophoric patterns in the “structural space”, and
an open-ended exploratory approach consisting of vol-
untary changes of the molecular pharmacophores while
preserving the overall “skeleton” (topology) of the com-
pounds.
The exploratory approach may introduce dramatic
changes in the pharmacophoric patterns of the mol-
ecules, with radical effects on the activity. It evidenced
here again the beneficial introduction of a basic nitrogen
to reach nanomolar activities, on the opiate receptors.
The analogues of 1 with similar pharmacophoric pat-
terns included some representatives in which the benz-
imidazolone moiety has been replaced by a topologically
different triazaspirodecanone ring system. Though simi-
lar activities for derivatives of these two moieties had
already been reported in the serotoninergic and dopam-
inergic series,13,14 substitution by a N-methylacetamide
at N-3 of the spirocyclic moiety has, to our knowledge,
never been reported. In our hands, the spirodecanone
moiety has proven to be pharmacophorically equivalent
to the benzimidazolone, with respect to the opiate
receptors, having moreover the advantage of accom-
modating at the N-3 position a variety of substituents

3384 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Poulain et al.
Gen er a l P r oced u r e A for Ca r ba m a tes (Com p ou n d s 1,
13-18, 28-35, a n d 44). A 42 µL (10.3 µmol, 2.1 equiv) sample
of a solution (0.247 M, in THF) of N,N′-carbonyldiimidazole
was added at room temperature to 50 µL (5 µmol, 1 equiv) of
a solution (0.1 M, in DMF) of alcohol. After the resulting
solution was stirred at room temperature for 2 h, 50 µL (5
µmol, 1 equiv) of a solution (0.1 M, in DMF) of amine was
added. The reaction mixture was stirred overnight, evaporated
under reduced pressure, and, except for 1, purified using
preparative HPLC. For 1, the residue was dissolved in AcOEt
and purified by acidobasic extractions. The organic layer was
dried over MgSO4 and filtered before evaporation.
ranging from hydrogen to carboxylate or N-methylcar-
boxamide. The latter result could be exploited to opti-
mize other properties of the compound such as selec-
tivity or ADME profile, without affecting the binding
of the molecule to the µ opiate receptor.
In conclusion, our pharmacophore-oriented analogue
design (method B) suggests completely novel molecular
skeletons compatible with a primary pharmacophore
assumption, and exploratory modifications (method A)
can then be applied to refine the assumption. The two
methods can therefore be regarded as complementary
and should be used in parallel for the elucidation of
structure-activity relationships. In the present case,
the combination of information provided by each ana-
logue series has allowed us to design chimeric com-
pounds that (1) were more active than the best of the
two series, (2) had improved physical-chemical proper-
ties, and (3) bear a readily modifiable substituent,
opening a promising way to a second step of optimiza-
tion.
Gen er a l P r oced u r e B for Am id es (Com p ou n d s 8, 19,
20, 37, a n d 38). A 42 µL (10.3 µmol, 2.1 equiv) sample of a
solution (0.247 M, in THF) of N,N′-carbonyldiimidazole was
added at room temperature to 9 µL (5 µmol, 1 equiv) of a
solution (0.56 M, in DMF) of acid. After the resulting solution
was stirred at room temperature for 2 h, 50 µL (5 µmol, 1
equiv) of a solution (0.1 M, in DMF) of amine was added. The
reaction mixture was stirred for 2 h and then evaporated under
reduced pressure. The residue was purified by preparative
HPLC.
Gen er a l P r oced u r e C for Ur ea s (Com p ou n d s 9 a n d
36). A 42 µL (10.3 µmol, 2.1 equiv) sample of a solution (0.247
M, in THF) of N,N′-carbonyldiimidazole was added at room
temperature to 50 µL (5 µmol, 1 equiv) of a solution (0.1 M, in
DMF) of the first amine. After the resulting solution was
stirred at room temperature for several minutes, 50 µL (5 µmol,
1 equiv) of a solution (0.1 M, in DMF) of the second amine
was added. The reaction mixture was stirred overnight and
then evaporated under reduced pressure. The residue was
purified by preparative HPLC.
Further biological evaluation of the analogues ob-
tained in these first steps of optimization is now
considered. In particular, the use of pharmacological
profiling on a large panel of receptors is currently being
investigated to study the structure-profile relation-
ships.
Exp er im en ta l Section
Ch em istr y. Gen er a l In for m a tion . All commercial re-
agents and solvents were used without further purification.
NMR spectra were recorded on either a Bruker DRX-300 or a
Bruker DMX-600 spectrometer. Chemical shifts are in parts
per million (ppm). The assignments were made using one-
dimensional (1D) 1H and 13C spectra and two-dimensional (2D)
HMQC spectra. Mass spectra were determined using a Micro-
mass Platform instrument equipped with an APCI interface.
SAR-grade compounds were purified by extraction or prepara-
tive HPLC using a Shimadzu LC-10 preparative chromatog-
raphy system equipped with LC-8A pumps, an FRC-10A
fraction collector, an SPD-10A detector, and a Vydac C18 5
µm particle size column, dimensions 250 × 25.4 mm. A
gradient starting from 100% H2O and reaching 100% H2O/
80% CH3CN within 18 min at a flow rate of 50 mL/min was
used. Organic layers obtained after extraction of aqueous
solutions were dried over MgSO4 and filtered before evapora-
tion in vacuo. Purity was determined by two different HPLC
systems. In the first system, analyses were performed using
a C18 TSK-GEL Super ODS 3 µm particle size column,
dimensions 50 × 4.6 mm. A gradient starting from 100% H2O/
0.05% TFA and reaching 100% H2O/80% CH3CN/0.0425% TFA
Gen er a l P r oced u r e D for Red u ctive Am in a tion (Com -
p ou n d s 23 a n d 24). A mixture of amine (1.5 mmol, 1 equiv),
aldehyde (1.5 mmol, 1 equiv), and NaHB(OAc)3 (3.75 mmol,
2.5 equiv) in CH2Cl2 was stirred overnight at room tempera-
ture and then evaporated under reduced pressure. The residue
obtained was triturated in an aqueous solution of Na2CO3,
washed with H2O (2 × 10 mL), filtered, and washed with Et2O/
pentane (50/50).
Gen er a l P r oced u r e E for N-Alk yla tion s (Com p ou n d s
21, 22, 25-27, 39-41, a n d 43). A 52 µL (0.3 mmol, 1 equiv)
sample of DIPEA and 600 µL of a solution of halide (0.5 M, in
DMF) (0.3 mmol, 1 equiv) were added to 3 mL of a solution
(0.1 M, in DMF) of amine (0.3 mmol, 1 equiv). The reaction
mixture was heated overnight at 70 °C and then evaporated
under reduced pressure. The residue obtained was purified by
preparative HPLC as described above, except for 22 and 40,
which were purified with HPLC solvents containing 0.5%
trifluoroacetic acid.
P r oced u r e F for th e P r ep a r a tion of Com p ou n d s 6 a n d
7. A 25 µL (5 µmol, 1 equiv) sample of a solution (0.2 M, THF)
of sulfochloride was added at room temperature to 50 µL (5
µmol, 1 equiv) of a solution (0.1 M, DMF) of N-Boc-piperazine.
After being stirred for 2 h at room temperature, the reaction
mixture was evaporated under reduced pressure, and the
residue obtained was dissolved in 100 µL of a solution of TFA/
DCM (50/50). The solution was stirred for 1/2 h and then
evaporated under reduced pressure. A 42 µL (10.3 µmol, 2.1
equiv) sample of a solution (0.247 M, in THF) of N,N′-
carbonyldiimidazole was added at room temperature to 9 µL
(5 µmol, 1 equiv) of a solution (0.56 M, in DMF) of acid. After
the resulting solution was stirred at room temperature for 2
h, 50 µL (5 µmol, 1 equiv) of a solution (0.1 M, in DMF) of the
deprotected amine was added. The reaction mixture was
stirred overnight and then evaporated under reduced pressure.
The residue was purified by preparative HPLC.
within 5 min at a flow rate of 2.75 mL/min was used (tR
)
retention time). Chromatograms were recorded on an LC/MS
instrument using a Micromass Platform mass analyzer coupled
to an HP 1100 LC system with a diode array (200-400 nm)
detector or using a Micromass ZMD system coupled with a
Gilson 307 diode array detector and LC pumps 306. In the
second system, analyses were performed on an Adsorbosphere
CN 3U from Alltech 3 µm particle size column, dimensions 50
× 4.6 mm, using a Shimadzu instrument equipped with LC-
10AS pumps and an SPD-10A dual wavelength (215, 254 nm)
detector. A gradient starting from 100% H2O/0.05% TFA and
reaching 100% H2O/80% CH3CN/0.0425% TFA within 25 min
at a flow-rate of 2.75 mL/min was used (tR′ ) retention time).
Purification yields were not optimized. Melting points were
measured on a Bu¨chi B-450 apparatus and are uncorrected.
All the reactions on the 5 µmol scale were performed in 96-
well polypropylene plates, Beckmann reference 26 7006. When
more material was needed (SAR grade compounds), the
reactions were performed 30 or 40 times in the same conditions
and reaction products pooled before purification.
4-(2-Oxoben zim idazol-1-yl)piper idin e-1-car boxylic Acid
(6-Nitr oben zo[1,3]d ioxol-5-yl)m eth yl Ester (1) (P r oce-
d u r e A). HPLC: tR ) 2.39 min. 1H NMR (DMSO-d6, 600
MHz): δ 1.71 (dd, J ) 9.9 Hz, J ′ ) 2.1 Hz, 2H), 2.22 (l s, 2H),
3.05-3.07 (m, 2H), 4.15 (d, J ) 13.6 Hz, 2H), 4.38-4.39 (m,

Combining Two Methods for Focused Library Design
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3385
1H), 5.36 (s, 2H), 6.26 (s, 2H), 6.97-6.99 (m, 3H), 7.17 (s, 1H),
7.20-7.22 (m, 1H), 7.71 (s, 1H), 10.85 (s, 1H, NHCO). MS
(APCI+): m/z 441 [M + H]+.
mixture was evaporated under reduced pressure to afford an
oil which solidified upon triturating with Et2O/pentane. The
hydrochloride salt was obtained as a beige powder (1.4 g, 25%).
1
HPLC: tR ) 1.48 min. H NMR (DMSO-d6, 300 MHz): δ 1.77
1-(2-Am in oeth yl)-1,3-d ih yd r oben zim id a zol-2-on e Tr i-
flu or oa ceta te (2). A mixture of 1-fluoro-2-nitrobenzene (1.72
mL, 16.3 mmol), tert-butyl-3-(aminoethyl)carbamate (2.61 mL,
16.3 mmol), and potassium carbonate (3.61 g, 26.1 mmol, 1.6
equiv) in DMF (42 mL) was heated at 70 °C overnight. The
reaction mixture was evaporated under reduced pressure.
After precipitation of mineral salts with CH2Cl2, evaporation
of the filtrate afforded 5.2 g of an orange oil. The oil (5.2 g)
was dissolved in EtOH (105 mL). Pd-C (10%) (1.96 g, 1.8
mmol) and ammonium formate (4.61 g, 72 mmol, 4 equiv) were
then added to the solution. The suspension was heated at 70
°C overnight, filtered on Celite, and rinsed with additional
EtOH (2 × 50 mL). Evaporation of the filtrate afforded an oil
(2.75 g, 10.95 mmol) which was then dissolved in AcOEt (140
mL) with DIPEA (3.82 mL, 21.8 mmol, 2 equiv). The solution
was cooled at 0 °C before addition of triphosgene (1.7 g, 3.61
mmol, 0.33 equiv). After being stirred for 10 min at 0 °C and
overnight at room temperature, the reaction mixture was
washed with water (3 × 50 mL). The organic layer was dried
(MgSO4), filtered, and evaporated under reduced pressure to
yield an oil. Flash chromatography on silica gel using a solvent
system of CH2Cl2/MeOH (97/3) afforded an oil. Recrystalliza-
tion from CH3CN afforded 1.1 g of purified residue. The Boc
protection was then removed by dissolving the protected amine
(1.1 g, 2.99 mmol) in CH2Cl2 (7.485 mL), TFA (7.485 mL), and
H2O (0.748 mL) at room temperature. After being stirred for
2 h, the reaction mixture was evaporated under reduced
pressure. The resulting oil was triturated with Et2O to afford
(d, J ) 12 Hz, 2H), 2.29 (s, 3H), 2.57-2.69 (m, 2H), 2.98-3.10
(m, 2H), 3.36 (d, J ) 12 Hz, 2H), 4.48 (m, 1H), 6.76 (d, J )
7.84 Hz, 1H), 6.83 (d, J ) 7.84 Hz, 1H), 7.33 (s, 1H), 9.15 (m,
2H, H2N+(piperidine)), 10.76 (s, 1H, NHCO). MS (APCI+): m/z
232 [M + H of free base]+.
1-P ip er id in -4-yl-1,4-d ih yd r oqu in oxa lin e-2,3-d ion e Hy-
d r och lor id e (5). A mixture of 2-nitrofluorobenzene (7.46 g,
52.9 mmol), 4-amino-1-benzylpiperidine (10.07 g, 52.9 mmol),
and K2CO3 (11.54 g, 83.5 mmol) in DMF (100 mL) was heated
at 70 °C overnight, and evaporated under reduced pressure.
The red residue was dissolved in EtOH (300 mL). A mixture
of this solution with Fe (17.4 g, 331 mmol) and concentrated
HCl (12 N) (26 mL) was refluxed for 2 h. After cooling, the
mixture was evaporated under reduced pressure. The residue
was dissolved in HCl (3 N) (130 mL) with oxalic acid (9.36 g,
104 mmol). The mixture was refluxed overnight, cooled,
neutralized with a 1 M aqueous solution of Na2CO3, and
extracted with AcOEt. The organic layer was washed with H2O
(2 × 30 mL), dried (MgSO4), filtered, and evaporated under
reduced pressure. The benzyl protection was removed as
described for 4 to yield the hydrochloride salt (2.34 g, 8.04
mmol, 15.2%). HPLC: tR ) 1.48 min. 1H NMR (DMSO-d6, 300
MHz): δ 1.82 (d, J ) 12.9 Hz, 2H), 2.86 (dq, J ) 3.6 Hz, J ′ )
13.2 Hz, 2H), 3.12 (q, J ) 13.4 Hz, 2H), 3.34 (d, J ) 12 Hz,
2H), 4.82 (m, 1H), 7.13-7.18 (m, 3H), 7.76-7.78 (m, 1H), 9.36
(m, 2H, H2N+(piperidine)), 12.0 (s, 1H, NHCO). MS (APCI+):
m/z 232 [M + H of free base]+.
4-Meth oxy-5-[4-(2-p h en yla m in oben zoyl)p ip er a zin e-1-
su lfon yl]th iop h en e- 3-ca r boxylic Acid Meth yl Ester (6)
(P r oced u r e F ). HPLC: tR ) 2.82 min and tR′ ) 14.75 min.
1H NMR (DMSO-d6, 300 MHz): δ 3.00-3.10 (m, 4H), 3.40-
3.60 (m, 4H), 3.89 (s, 3H), 3.94 (s, 3H), 6.83 (t, J ) 7.3 Hz,
1H), 6.91 (d, J ) 7.8 Hz, 2H), 7.06 (t, J ) 7.3 Hz, 1H), 7.15 (t,
J ) 7.7 Hz, 2H), 7.26 (d, J ) 8.1 Hz, 1H), 7.31 (dd, J ) 1.3 Hz,
J ′ ) 7.6 Hz, 1H), 7.40 (dt, J ) 1.4 Hz, J ′ ) 8.3 Hz, 1H), 7.82
(s, 1H, PhNHPh), 8.72 (s, 1H). 13C NMR (DMSO-d6, 300 MHz)
δ 41.0, 46.2, 52.8, 63.8, 117.4, 120.1, 120.6, 121.8, 123.8, 126.8,
129.5, 129.7, 131.0, 140.0, 140.5, 144.1, 158.5, 161.4, 168.5.
MS (APCI+): m/z 516 [M + H]+.
0.946 mg of the white trifluoroacetate salt (20%). HPLC: tR
)
1.18 min. 1H NMR (DMSO-d6, 600 MHz): δ 3.14-3.16 (m, 2H),
4.06 (t, J ) 6.2 Hz, 2H), 7.04-7.07 (m, 3H), 7.19 (dd, J ) 5.8
Hz, J ′ ) 1.8 Hz, 1H), 7.90 (br s, 3H, NH3+), 10.98 (s, 1H,
NHCO). MS (APCI+): m/z 178 [M + H of free base]+.
1-(2-Am in op r op yl)-1,3-d ih yd r oben zim id a zol-2-on e Tr i-
flu or oa ceta te (3). The title compound was obtained from tert-
butyl-3-(aminopropyl)carbamate, as described for 2. Mp: 208-
209 °C. HPLC: tR ) 1.40 min. 1H NMR (DMSO-d6, 300 MHz):
δ 1.93-1.95 (m, 2H), 2.80-2.85 (m, 2H), 3.88 (t, J ) 6.6 Hz,
2H), 7.01-7.05 (m, 3H), 7.19 (d, J ) 5.5 Hz, 1H), 7.75 (br s,
3H, NH3+), 10.95 (s, 1H, NHCO). MS (APCI+): m/z 192 [M +
H of free base]+.
(5-Ch lor o-1H-in dol-2-yl)[4-(2,4-din itr oben zen esu lfon yl)-
p ip er a zin -1-yl]m eth a n on e (7) (P r oced u r e F ). HPLC: tR
) 2.88 min and tR′ ) 15.27 min. MS (APCI+): m/z 495 [M +
H]+. Mixture of two conformers (68/32). (Conformer 1) 1H NMR
(DMSO-d6, 300 MHz): δ 3.45-3.48 (m, 4H), 3.93-3.98 (m, 4H),
6.87 (dd, J ) 2.0 Hz, J ′ ) 0.6 Hz, 1H), 7.27 (dd, J ) 2.1 Hz, J ′
) 8.8 Hz, 1H), 7.50 (d, J ) 8.8 Hz, 1H), 7.74 (d, J ) 2.1 Hz,
1H), 8.37 (d, J ) 8.7 Hz, 1H), 8.68 (dd, J ) 2.3 Hz, J ′ ) 8.7
Hz, 1H), 9.11 (d, J ) 2.3 Hz, 1H), 11.87 (d, J ) 1.4 Hz, 1H,
NHindole). 13C NMR (DMSO-d6, 300 MHz): δ 45.8, 46.4, 50.4,
104.6, 114.4, 120.8, 121.1, 124.2, 125.0, 127.7, 128.4, 131.5,
133, 134.7, 135.1, 148.5, 150.9, 162.5. (Conformer 2) 1H NMR
(DMSO-d6, 300 MHz): δ 3.45-3.48 (m, 4H), 3.93-3.98 (m, 4H),
6.95 (dd, J ) 2.0 Hz, J ′ ) 0.6 Hz, 1H), 7.29 (dd, J ) 2.1 Hz, J ′
) 8.8 Hz, 1H), 7.51 (d, J ) 8.8 Hz, 1H), 7.76 (d, J ) 2.1 Hz,
1H), 8.39 (d, J ) 8.7 Hz, 1H), 8.72 (dd, J ) 2.3 Hz, J ′ ) 8.7
Hz, 1H), 9.11 (d, J ) 2.3 Hz, 1H), 11.93 (d, J ) 1.4 Hz, 1H,
NHindole). 13C NMR (DMSO-d6, 300 MHz): δ 45.8, 46.4, 50.4,
104.6, 114.4, 121.7, 123.9, 124.2, 125.0, 128.4, 129.0, 131.5,
134.7, 135.1, 148.9, 150.9, 162.5.
(6-Meth yl-1-p ip er id in -4-yl)-1,3-d ih yd r oben zim id a zol-
2-on e Hyd r och lor id e (4). A mixture of 3-fluoro-4-nitrotolu-
ene (3.26 g, 21 mmol, 1 equiv), 4-amino-1-benzylpiperidine (4
g, 21 mmol, 1 equiv), and K2CO3 (4.65 g 33.63 mmol, 1.6 equiv)
in DMF (42 mL) was heated at 70 °C overnight, and evapo-
rated under reduced pressure. The red residue was dissolved
in CH2Cl2 and washed with H2O (2 × 30 mL). The organic layer
was then evaporated under reduced pressure to afford 7.64 g
of a red oil. This residue was dissolved in EtOH (150 mL). A
mixture of this solution with Fe (7 g, 6 equiv) and concentrated
HCl (8.6 mL, 5 equiv) was refluxed for 3.5 h. After cooling,
the mixture was filtered on Celite and rinsed with DMF. The
filtrate was evaporated under reduced pressure, dissolved in
H2O/AcOEt, and neutralized with Na2CO3. The organic layer
was washed twice with water, dried (MgSO4), and evaporated
under reduced pressure. A mixture of the obtained oil (4.8 g,
16.2 mmol, 1 equiv) in AcOEt (65 mL) with DIPEA (4.61 g,
35,73 mmol, 2.2 equiv) was cooled to 0 °C, and 1.78 g (6 mmol,
0.37 equiv) of triphosgene was added. The mixture was allowed
to warm to room temperature and stirred overnight. The
solvent was removed under reduced pressure to yield an oil.
AcOEt (25 mL) and H2O (25 mL) were added. The organic layer
was evaporated. Recrystallization from CH3CN (100 mL)
provided 2 g (6.2 mmol) of the amine as a brown powder. A
solution of the intermediate benzyl-protected amine in MeOH
(39 mL) with HCOOH (1.69 mL, 44.8 mmol, 7.2 equiv) and
870 mg of Pd-C (10%) was heated at 60 °C and stirred for 4
h. Evaporation under reduced pressure of the mixture gave a
black oil. A solution of this oil in HCl (1 N) (30 mL) and AcOEt
(5 mL) was stirred for 30 min at room temperature. The
4-(7-Nit r ob en zo[1,2,5]oxa d ia zol-4-yl)p ip er a zin e H y-
d r och lor id e (10). A mixture of 4-chloro-7-nitrobenzo[1,2,5]-
oxadiazole (4.5 g, 22.5 mmol, 1 equiv), N-(tert-butyloxycarbonyl)-
4-piperidine (5.04 g, 27.1 mmol, 1.2 equiv), and DMF (49.6 mL)
was refluxed for 1 h. The reaction mixture was then evaporated
under reduced pressure. The residue was then washed with
H2O (3 × 30 mL) and filtered to afford 7.315 g (20.9 mmol) of
a yellow powder. A mixture of this product, EtOH (20 mL),
and HCl (3 N) (40 mL) was refluxed for 2 h. The mixture was
evaporated under reduced pressure, and the residue was

3386 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Poulain et al.
recrystallized from Et2O/pentane (50/50) to afford 5.75 g of the
8.07 (d, J ) 9.0 Hz, 1H). MS (APCI+): m/z 167 [M + H]+, 208
[M + MeCN]+.
purple hydrochloride salt (73.1%). Mp: 297-299 °C. HPLC:
1
tR ) 2.04 min. H NMR (D2O-Na2CO3, 600 MHz): δ 2.78 (m,
4-(2-Oxo-2,3-d ih yd r ob en zim id a zol-1-yl)p ip er id in e-1-
ca r boxylic Acid (6-Ch lor oben zo[1,3]d ioxol-5-yl)m eth yl
Ester (13) (P r oced u r e A). HPLC: tR ) 2.45 min and tR′ )
4H), 3.38 (m, 2H), 6.26 (d, J ) 9.6 Hz, 1H), 8.6 (d, J ) 9.6 Hz,
1H). MS (APCI+): m/z 250 [M + H of free base]+.
1
14.1 min. H NMR (DMSO-d6, 300 MHz): δ 1.65 (l d, J ) 12
2-(5-Br om o-1H-in d ol-3-yl)-1-[4-(7-n itr oben zo[1,2,5]oxa -
d ia zol-4-yl)p ip er a zin -1-yl]eth a n on e (8) was obtained from
Hz, 2H), 2.17 (ddd, J ) 12 Hz, J ′ ) 4.3 Hz, 2H), 2.83-2.90 (l
s, 2H), 4.09 (l d, J ) 12 Hz, 2H), 4.32 (m, 1H), 5.02 (s, 2H),
6.05 (s, 2H), 6.91-6.93 (m, 3H), 7.04 (s, 1H), 7.09 (s, 1H), 7.10-
7.15 (m, 1H), 10.8 (s, 1H, NHCO). 13C NMR (DMSO-d6, 300
MHz): δ 29.4, 41.2, 50.5, 64.8, 103.0, 109.4, 109.8, 110.6, 110.8,
121.2, 121.5, 125.7, 128.0, 129.1, 130.0, 147.4, 148.9, 154.5,
155.0. MS (APCI+): m/z 430 [M + H]+ and 386 [M - 44]+.
1
10 according to procedure B. HPLC: tR ) 2.54 min. H NMR
(DMSO-d6, 300 MHz): δ 3.70-3.73 (m, 2H), 3.79 (s, 2H), 3.81-
3.84 (m, 2H), 4.00-4.10 (m, 4H), 6.54 (d, J ) 9.2 Hz, 1H), 7.11
(d, J ) 8.6 Hz, J ′ ) 1.9 Hz, 1H), 7.26 (d, J ) 8.5 Hz, 1H), 7.29
(s, 1H), 7.71 (d, J ) 1.8 Hz, 1H), 8.46 (d, J ) 9.2 Hz, 1H),
11.09 (s, 1H, NHindole). 13C NMR (DMSO-d6, 300 MHz): δ 31.8,
42.0, 45.5, 50.1, 50.4, 104.7, 115, 122.8, 125, 127.1, 137.8. MS
(APCI+): m/z 485 [M + H]+.
[3-(2-Oxo-2,3-dih ydr oben zim idazol-1-yl)pr opyl]car bam -
ic Acid (6-Ch lor oben zo[1,3]d ioxol-5-yl)m eth yl Ester (14),
Obta in ed fr om 3 (P r oced u r e A). HPLC: tR ) 2.42 min and
4-[5-(2,4-Dich lor op h en yl)-2H-n itr oim id a zol-1-yl)eth yl]-
a m id e (9) (P r oced u r e C). HPLC: tR ) 2.66 min and tR′ )
13.77 min. 1H NMR (DMSO-d6, 300 MHz): δ 1.44-1.56 (m,
2H), 1.93-1.97 (m, 2H), 2.48 (s, 3H), 2.80-2.97 (m, 3H), 3.47
(dt, J ) 5.6 Hz, J ′ ) 5.7 Hz, 2H), 3.98-4.02 (m, 2H), 4.41 (t,
J ) 5.7 Hz, 2H), 6.64 (s, 1H), 6.84 (t, J ) 5.4 Hz, 1H, NHCON),
7.54 (dd, J ) 8.4 Hz, J ′ ) 1.8 Hz, 1H), 7.74 (d, J ) 1.7 Hz,
1H), 8.40 (d, J ) 8.4 Hz, 1H), 8.11 (s, 1H), 13.0 (s, 1H,
NHpyrazole). 13C NMR (DMSO-d6, 300 MHz): δ 14.5, 31.9, 33.6,
39.9, 43.9, 46.6, 103.0, 128.1, 130.2, 132.0, 132.3, 132.9, 133.8,
139.2, 148.3, 152.2, 157.6. MS (APCI+): m/z 493 [M + H]+.
N-Met h yl-2-(4-oxo-1-p h en yl-1,3,8-t r ia za sp ir o[4,5]d ec-
3-yl)a ceta m id e Hyd r och lor id e (11). A 3.9 g (10 mmol)
sample of 3-[4-oxo-1-phenyl-8-(tert-butyloxycarbonyl)-1,3,8-
triazaspiro[4,5]decyl]acetic acid was dissolved in a solution of
N,N′-carbonyldiimidazole (0.247 M, in THF) (80 mL). After the
reaction mixture was stirred at room temperature for 2 h, 1.35
g of methylamine hydrochloride salt (20 mmol, 2 equiv) and 1
g of triethylamine were added. After being stirred overnight,
the reaction mixture was evaporated under reduced pressure.
The crude product was dissolved in CH2Cl2 and washed with
acidic water (pH 2, 3 × 50 mL). The organic layer was
evaporated under reduced pressure. HPLC: tR ) 2.44 min. MS
(APCI+) m/z 303 ([M + H]+ - Boc). A mixture of the residue,
THF (75 mL), HCl (1 N, in Et2O) (20 mL, 20 mmol, 2 equiv),
and H2SO4 (560 µL) was stirred overnight at 65 °C. The
suspension was filtered and washed with AcOEt (30 mL). The
solid was then dissolved in H2O (50 mL), and Na2CO3 was
added. The free base was extracted with CH2Cl2 (3 × 30 mL),
and the organic layer was evaporated under reduced pressure
to afford 600 mg (19.8%) of a white powder. HPLC: tR ) 1.47
min. 1H NMR (DMSO-d6, 300 MHz): δ 1.63 (d, J ) 14 Hz,
2H), 2.53-2.60 (m, 2H), 2.59 (d, J H-NH ) 4.5 Hz, 3H), 3.02
(dd, J ) 13 Hz, J ′ ) 4.4 Hz, 2H), 3.22-3.25 (m, 2H), 3.95 (s,
2H), 4.66 (s, 2H), 6.71-6.83 (m, 2H), 6.90 (d, J ) 8.2 Hz, 2H),
7.22 (t, J ) 7.4 Hz, 2H), 8.00-8.05 (m, 1H, NHCO). MS
(APCI+): m/z 303 [M + H of free base]+.
1
tR′ ) 13.40 min. H NMR (DMSO-d6, 300 MHz): δ 1.69-1.75
(m, 2H), 2.98 (dt, J ) 6.2 Hz, J ′ ) 6.7 Hz, 2H), 3.74 (t, J ) 7.0
Hz, 2H), 4.92 (s, 2H), 6.03 (s, 2H), 6.93-6.96 (m, 3H), 6.99 (s,
1H), 7.31 (m, 2H), 7.31 (t, J ) 5.5 Hz, 1H, NHCOO), 10.80 (s,
1H, NHCON). 13C NMR (DMSO-d6, 300 MHz): δ 29.5, 39.0,
39.3, 63.9, 103.4, 109.4, 110.8, 111.2, 110.3, 122.2. MS (AP-
CI+): m/z 404 [M + H]+ and 360 [M - 44]+.
[3-(2-Oxo-2,3-d ih yd r oben zim id a zol-1-yl)eth yl]ca r ba m -
ic Acid (6-Ch lor oben zo[1,3]d ioxol-5-yl)m eth yl Ester (15),
Obta in ed fr om 2 (P r oced u r e A). HPLC: tR ) 2.46 min and
tR′ ) 13.58 min. 1H NMR (DMSO-d6, 300 MHz): δ 3.21 (q,
J H-NH ) 5.9 Hz, J ) 6.1 Hz, 2H), 2.78 (t, J ) 6.1 Hz, 2H), 4.88
(s, 2H), 6.04 (s, 2H), 6.90-6.92 (m, 4H), 6.99 (m, 1H), 7.06 (s,
1H), 7.39 (t, J H-NH ) 5.9 Hz, 1H, NHCOO), 10.7 (s, 1H,
NHCON). 13C NMR (DMSO-d6, (300 MHz): δ 40.5, 41.3, 63.6,
102.9, 108.2, 110.4, 110.5, 121.2, 121.5. MS (APCI+): m/z 392
[M + H]+.
4-(2-Oxo-2,3-d ih yd r oben zim id a ol-1-yl)-3,6-d ih yd r o-2H-
p yr id in e-1-ca r b oxylic Acid (6-Nit r oben zo[1,3]d ioxol-5-
yl)m eth yl ester (16) (P r oced u r e A). HPLC: tR ) 2.43 min.
1H NMR (DMSO-d6, 300 MHz): δ 2.45 (s, 2H), 3.64 (l s, 2H),
4.12 (l d, 2H), 5.33 (s, 2H), 5.90 (s, 1H), 6.60 (s, 2H), 6.93-
6.98 (m, 4H), 7.03 (m, 1H), 7.16 (s, 1H), 7.68 (s,1H), 10.94 (s,
1H, NHCO). 13C NMR (DMSO-d6, 300 MHz): δ 27.3, 41.1, 43.3,
64.6, 104.6, 105.1, 108.9, 109.6, 110.0, 122.1, 122.8. MS
(APCI+): m/z 439 [M + H]+.
4-(2,3-Dioxo-3,4-dih ydr o-2H-qu in oxalin -1-yl)piper idin e-
1-ca r boxylic Acid (6-ch lor oben zo[1,3]d ioxol-5-yl)m eth yl
Ester (17), Obta in ed fr om 5 a n d 6-Ch lor op ip er on yl
Alcoh ol (P r oced u r e A). HPLC: tR ) 2.40 min and tR′ ) 3.41
1
min. H NMR (DMSO-d6, 300 MHz): δ 1.63 (l d, J ) 11.2 Hz,
2H), 2.48-2.56 (ddd, J ) 12.7 Hz, J ′ ) 4.2 Hz, 2H), 2.83-2.90
(l s, 2H), 4.09 (l d, J ) 10 Hz, 2H), 4.68 (m, 1H), 5.05 (s, 2H),
6.09 (s, 2H), 7.04 (s, 1H), 7.09 (s, 1H), 7.15-7.18 (m, 3H), 7.63
(m, 1H),10.7 (s, 1H, NHCO). 13C NMR (DMSO-d6, 300 MHz):
δ 27.6, 44.1, 55.3, 64.8, 103.0, 110.8, 110.9, 115.8, 116.9, 123.9,
124.5, 125.8, 127.0, 127.6, 128.0, 147.4, 148.9, 154.9, 156.3.
MS (APCI+): m/z 458 [M + H]+ and 414 [M - 44]+.
Ben zo[1,2,5]th ia d ia zol-5-ylm eth a n ol (12). A mixture of
benzo[1,2,5]thiadiazole-5-carboxylic acid methyl ester (3 g,
15.44 mmol) in pyridine (51 mL) and LiI (14.46 g, 108.1 mmol)
was refluxed overnight. The solvent was removed under
reduced pressure, and the resulting oil was dissolved in H2O
(25 mL). Addition of HCl (3 N) at 0 °C allowed the carboxylic
acid intermediate to precipitate. The solid was washed with
H2O (2 × 20 mL) and acetonitrile (2 × 20 mL) and dissolved
in THF (65 mL) and DIPEA (2.9 mL, 16.64 mmol). The mixture
was cooled to 0 °C, and ClCOOEt (1.54 mL, 16 mmol) was
added. After the resulting mixture was stirred for 1.5 h at 0
°C, a solution of NaBH4 (1.36 g, 36.06 mmol) in H2O (8.5 mL)
was added dropwise. The mixture was stirred for 2 h at room
temperature and evaporated under reduced pressure. The
orange residue was dissolved in AcOEt (40 mL) and washed
with a saturated solution of Na2CO3 (2 × 30 mL) and brine (1
× 30 mL), separated, dried (MgSO4), and evaporated under
reduced pressure to yield 1.45 g (63%) of a yellow powder.
4-(6-Meth yl-2-oxo-2,3-d ih yd r oben zim id a zol-1-yl)p ip e-
r id in e-1-ca r boxylic Acid (6-Ch lor oben zo[1,3]d ioxol-5-yl)-
m eth yl Ester (18), Obta in ed fr om 4 (P r oced u r e A).
1
HPLC: tR ) 2.67 min and tR′ ) 14.74 min. H NMR (DMSO-
d6, 300 MHz): δ 1.63 (l d, J ) 12 Hz, 2H), 2.18 (ddd, J ) 12.7
Hz, J ′ ) 4.2 Hz, 2H), 2.26 (s, 3H), 2.83-2.90 (l s, 2H), 4.09 (l
d, J ) 11.7 Hz, 2H), 4.28 (m, 1H), 5.01 (s, 2H), 6.05 (s, 2H),
6.73 (dd, J ) 7.9 Hz, J ′ ) 0.6 Hz, 1H), 6.80 (d, J ) 7.9 Hz,
1H), 6.98 (l s, 1H), 7.04 (s, 1H), 7.09 (s, 1H), 10.7 (s, 1H,
NHCO). 13C NMR (DMSO-d6, 300 MHz): δ 21.15, 28.6, 43.5,
49.6, 64.2, 103.0, 108.6, 109.1, 109.8, 110.2, 121.3. MS (AP-
CI+): m/z 445 [M + H]+ and 400 [M - 44]+.
1-{1-[2-(6-Nitr oben zo[1,3]d ioxol-5-yl)a cetyl]p ip er id in -
4-yl}-1,3-d ih yd r oben zim id a zol-2-on e (19) (P r oced u r e B).
HPLC: tR ) 2.17 min and tR′ ) 13.02 min. MS (APCI+): m/z
442 [M + H]+. Mixture of two conformers (52/48). (Conformer
1) 1H NMR (DMSO-d6, 300 MHz): δ 1.67 (l d, J ) 12 Hz, 1H),
2.03-2.11 (m, 1H), 2.68 (t, J ) 12 Hz, 1H), 3.82 (s, 3H), 3.85
(s, 3H), 4.07 (d, J ) 16 Hz), 4.10-4.18 (m, 1H), 4.20 (d, J ) 16
1
Mp: 54.8-55.5 °C. HPLC: tR ) 2.23 min. H NMR (DMSO-
d6, 300 MHz): δ 4.74 (d, J ) 5.8 Hz, 2H), 5.57 (t, J ) 5.7 Hz,
1H, OH), 7.69 (dd, J ) 1.4 Hz, J ′ ) 9.2 Hz, 1H), 7.98 (s, 1H),

Combining Two Methods for Focused Library Design
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3387
Hz, 1H), 6.92-6.95 (m, 3H), 7.06 (s, 1H), 7.24-7.27 (m, 1H),
7.65 (s, 1H), 10.83 (s, 1H, NHCON). 13C NMR (DMSO-d6, 300
MHz): δ 30.0, 39.7, 42.7, 51.2, 57.2, 57.7, 109.1, 110.2, 110.4,
117.0, 122.0. (Conformer 2) 1H NMR (DMSO-d6, 300 MHz): δ
1.76 (l d, J ) 12 Hz, 1H), 2.36-2.45 (m, 1H), 3.21-3.28 (m,
1H), 3.82 (s, 3H), 3.85 (s, 3H), 4.07 (d, J ) 16 Hz, 1H), 4.20 (d,
J ) 16 Hz, 1H), 4.41-4.49 (m, 2H), 6.92-6.95 (m, 3H), 7.06
(s, 1H), 7.24-7.27 (m, 1H), 7.65 (s, 1H), 10.83 (s, 1H, NHCON).
13C NMR (DMSO-d6, 300 MHz): δ 30.6, 39.7, 45.8, 51.2, 57.2,
57.7, 109.1, 110.2, 110.4, 117.0, 122.0.
1H), 4.69-4.80 (m, 1H), 6.12 (s, 2H), 7.12-7.30 (m, 5H), 7.59
(s, 1H), 11.98 (s, 1H, NHCO). MS (APCI+): m/z 415 [M + H]+.
1-{1-[(6-Ch lor oben zo[1,3]d ioxol-5-yl)m eth yl]p ip er id in -
4-yl}-6-m eth yl-1,3-d ih yd r oben zoim id a zol-2-on e (26), Ob-
ta in ed fr om 4 (P r oced u r e E). HPLC: tR ) 2.34 min. 1H
NMR (DMSO-d6, 300 MHz): δ 1.58 (d, J ) 10.4 Hz, 2H), 2.13
(t, J ) 11.2 Hz, 2H), 2.28-2.38 (m, 2H), 2.91 (d, J ) 10.2 Hz,
2H), 3.28 (s, 3H), 3.48 (s, 2H), 4.04-4.12 (m, 1H), 6.03 (s, 2H),
6.72 (d, J ) 7.9 Hz, 1H), 6.78 (d, J ) 7.9 Hz, 1H), 7.01 (s, 1H),
7.02 (s, 1H), 7.05 (s, 1H), 10.65 (s, 1H, NHCO). 13C NMR
(DMSO-d6, 300 MHz): δ 22.0, 29.5, 50.9, 53.5, 59.1, 102.7,
109.3, 110.0, 110.2, 110.9, 121.8, 125.5, 126.9, 129.9, 130.3,
140.4, 154.7. MS (APCI+): m/z 400 [M + H]+.
1-[1-(4-Meth oxyben zyl)p ip er id in -4-yl]-1,3-d ih yd r oben -
zoim id a zol-2-on e (27) (P r oced u r e E). HPLC: tR ) 2.10 min.
1H NMR (DMSO-d6, 300 MHz): δ 1.58 (d, J ) 11.2 Hz, 2H),
2.03 (t, J ) 9.6 Hz, 2H), 2.22-2.35 (m, 2H), 2.88 (d, J ) 11.2
Hz, 2H), 3.45 (s, 2H), 3.69 (s, 3H), 4.05-4.13 (m, 1H), 6.85 (d,
2H), 6.91-6.96 (m, 3H), 7.15-7.20 (m, 1H), 7.21 (d, 2H), 10.75
(s, 1H, NHCO). 13C NMR (DMSO-d6, 300 MHz): δ 29.5, 51.0,
53.3, 55.8, 62.1, 109.5, 109.6, 114.4, 121.2, 121.3, 125.6, 129.1,
130.0, 130.9, 154.5, 159.1. MS (APCI+): m/z 338 [M + H]+.
4-Oxo-1-p h en yl-1,3,8-tr ia za sp ir o[4.5]d eca n e-8-ca r box-
ylic Acid (6-Nitr oben zo[1,3]d ioxol-5-yl)m eth yl Ester (28)
(P r oced u r e A). HPLC: tR ) 2.61 min and tR′ ) 13.87 min.
1H NMR (DMSO-d6, 300 MHz): δ 1.62 (d, J gem ) 14.2 Hz, 2H),
2.35 (m, 2H), 3.46 (m, 2H), 3.91 (dd, 2H), 4.56 (s, 2H), 5.31 (l
d, 2H), 6.21 (s, 2H), 6.68 (d, J ) 8.25 Hz, 2H), 6.72 (t, J ) 7.3
Hz, 1H), 7.13 (s, 1H), 7.16 (dd, J ) 7.4 Hz, J ) 8.6 Hz, 2H),
7.67 (s, 1H), 8.77 (s, 1H, NHCO). 13C NMR (DMSO-d6, 300
MHz): δ 28.8, 59.1, 64.8, 104.6, 106.5, 109.2, 115.1, 130.3. MS
(APCI+): m/z 455 [M + H]+.
1-{1-[3-(4,5-Dim eth oxy-2-n itr op h en yl)a cr yloyl]p ip er i-
d in -4-yl}-1,3-d ih yd r oben zim id a zol-2-on e (20) (P r oced u r e
B). HPLC: tR ) 2.36 min and tR′ ) 13.84 min. MS (APCI+):
m/z 453 [M + H]+. Mixture of two conformers (66/33). (Con-
former 1) 1H NMR (DMSO-d6, 300 MHz): δ 1.75-1.78 (m, 2H),
2.29 (l d, J ) 9.6 Hz, 1H), 2.77 (t, J ) 12 Hz, 1H), 2.92 (t, J )
12 Hz, 1H), 3.25 (t, J ) 6.8 Hz, 1H), 3.85 (s, 3H), 3.94 (s, 3H),
6.97-6.99 (m, 3H), 7.23-7.26 (m, 1H), 7.33 (d, J ) 15 Hz, 1H),
7.42 (s, 1H), 7.63 (s, 1H), 7.86 (d, J ) 15 Hz, 1H), 10.87 (s,
1H, NHCON). 13C NMR (DMSO-d6, 300 MHz): δ 29.8, 42.7,
45.8, 57.2, 57.7, 109.1, 110.2, 110.4, 112.0, 122.0, 123.5, 137.0.
(Conformer 2) 1H NMR (DMSO-d6, 300 MHz): δ 1.75-1.78
(m, 2H), 2.30 (l d, J ) 9.6 Hz, 1H), 3.85 (s, 3H), 3.94 (s, 3H),
3.97 (d, J ) 12 Hz, 1H), 4.40-4.47 (m, 1H), 4.64 (d, J ) 13
Hz, 1H), 6.97-6.99 (m, 3H), 7.23-7.26 (m, 1H), 7.42 (s, 1H),
7.63 (s, 1H), 8.19 (d, J ) 2 Hz, 1H), 8.55 (d, J ) 1 Hz, 1H),
10.83 (s, 1H, NHCON). 13C NMR (DMSO-d6, 300 MHz): δ 30.6,
45.8, 48.0, 57.2, 57.7, 109.1, 110.2, 110.4, 112.0, 122.0, 137.0,
144.8.
1-{1-[(6-Ch lor oben zo[1,3]d ioxol-5-yl)m eth yl]p ip er id in -
4-yl}-1,3-d ih yd r oben zim id a zol-2-on e (21) (P r oced u r e E).
1
HPLC: tR ) 2.19 min. H NMR (DMSO-d6, 300 MHz): δ 1.06
3-(Met h ylca r b a m oylm et h yl)-4-oxo-1-p h en yl-1,3,8-t r i-
a za sp ir o[4.5]d eca n e-8-ca r boxylic Acid (6-Ch lor oben zo-
[1,3]d ioxol-5-yl)m eth yl Ester (29), Obta in ed fr om 11
(P r oced u r e A). HPLC: tR ) 2.81 min and tR′ ) 14.39 min.
1H NMR (DMSO-d6, 300 MHz): δ 1.64 (d, J ) 13.6 Hz, 2H),
(d, J ) 9.4 Hz, 2H), 2.13 (t, J ) 11.2 Hz, 2H), 2.26-2.38 (m,
2H), 2.90 (d, J ) 11.2 Hz, 2H), 3.48 (s, 2H), 4.07-4.17 (m,
1H), 6.03 (s, 2H), 6.91-6.96 (m, 3H), 7.01 (s, 1H), 7.05 (s, 1H),
7.19-7.20 (m, 1H), 10.79 (s, 1H, NHCO). 13C NMR (DMSO-
d6, 300 MHz): δ 29.5, 50.9, 53.5, 59.0, 102.7, 109.6, 110.2,
110.8, 121.2, 121.3, 125.5, 129.1, 130.0, 130.1, 147.5, 147.8,
154.5. MS (APCI+): m/z 387 [M + H]+.
2.32 (ddd, J ) 13.6 Hz, J ′ ) 5.43 Hz, 2H), 2.57 (d, J NH-H
)
4.53, 3H), 3.41-3.45 (l s, 2H), 3.88 (l s, 2H), 3.93 (s, 2H), 4.65
(s, 2H), 5.03 (l d, J ) 34.4 Hz, 2H), 6.05 (s, 2H), 6.66 (d, J )
8.10 Hz, 2H), 6.74 (t, J ) 7.3 Hz, 1H), 7.05 (s, 1H), 7.09 (s,
1H), 7.16 (t, J ) 7.2 Hz, 2H), 7.99 (m, 1H, CH3NHCO). 13C
NMR (DMSO-d6, 300 MHz): δ 26.8, 30.0, 41.8, 44.5, 65.5, 65.8,
103.7, 111.3, 111.8, 116.1, 119.8, 130.9. MS (APCI+): m/z 515
[M + H]+ and 471 [M - 44]+.
1-{3-[[(6-Ch lor ob en zo[1,3]d ioxol-5-yl)m et h yl]a m in o]-
p r op yl}-1,3-d ih yd r oben zim id a zol-2-on e Tr iflu or oa ceta te
(22), Obta in ed fr om 3 (P r oced u r e E). HPLC: tR ) 2.12 min.
1H NMR (DMSO-d6, 300 MHz): δ 1.92-2.02 (m, 2H), 2.95-
2.98 (m, 2H), 3.84 (t, J ) 6.7 Hz, 2H), 4.09-4.12 (m, 2H), 6.07
(s, 2H), 6.94-6.99 (m, 3H), 7.11-7.14 (m, 3H), 8.85 (s, 2H,
NH2+), 10.91 (s, 1H, NHCO). 13C NMR (DMSO-d6, 300 MHz):
δ 25.7, 37.9, 45.4, 48.0, 103.3, 108.6, 109.8, 110.6, 111.9, 121.4,
121.9, 123.2, 125.6, 126.9, 130.8, 147.6, 149.7, 155.2. MS
(APCI+): m/z 360 [M + H of free base]+.
1-{1-[(6-Nit r ob en zo[1,3]d ioxol-5-yl)m et h yl]p ip er id in -
4-yl}-1,3-d ih yd r oben zim id a zol-2-on e (23) (P r oced u r e D).
HPLC: tR ) 1.76 min. 1H NMR (CD3COOD, 300 MHz): δ 2.09
(d, J ) 12.3 Hz, 2H), 2.80-2.97 (m, 2H), 3.44 (t, 2H), 3.88 (d,
J ) 11.7 Hz, 2H), 4.68 (s, 2H), 4.69-4.81 (m, 1H), 6.21 (s, 2H),
7.08-7.12 (m, 2H), 7.17-7.20 (m, 1H), 7.28 (s, 1H), 7.34-7.36
(m, 1H), 7.68 (s, 1H). 13C NMR (CD3COOD, 300 MHz): δ 26.0,
48.1, 52.9, 57.7, 104.7, 106.4, 110.0, 110.8, 113.2, 121.1, 122.1,
122.5, 128.1, 128.7, 144.3, 150.2, 153.0, 155.6. MS (APCI+):
m/z 397 [M + H]+.
4-Oxo-1-p h en yl-1,3,8-tr ia za sp ir o[4.5]d eca n e-8-ca r box-
ylic Acid (6-Ch lor oben zo[1,3]d ioxol-5-yl)m eth yl Ester
(30) (P r oced u r e A). HPLC: tR ) 2.74 min and tR′ ) 14.59
1
min. H NMR (DMSO-d6, 300 MHz): δ 1.59 (d, J ) 13.7 Hz,
2H), 2.32 (ddd, J ) 13.6 Hz, J ′ ) 5.5 Hz, 2H), 3.42-3.49 (m,
2H), 3.87-3.91 (m, 2H), 4.55 (s, 2H), 4.97 (s, 1H), 5.09 (s, 1H),
6.04 (s, 2H), 6.64 (d, J ) 8.0 Hz, 2H), 6.72 (t, J ) 6.7 Hz, 1H),
7.04 (s, 1H), 7.09 (s, 1H), 7.14 (t, J ) 8.4 Hz, 2H), 8.76 (m, 1H,
NHCO). 13C NMR (DMSO-d6, 300 MHz): δ 29.6, 41.9, 60.4,
65.8, 103.7, 111.5, 111.8, 115.7, 119.4, 130.7. MS (APCI+): m/z
445 [M + H]+.
4-Oxo-1-p h en yl-1,3,8-tr ia za sp ir o[4.5]d eca n e-8-ca r box-
ylic Acid Ben zo[1,3]d ioxol-5-ylm eth yl Ester (31) (P r oce-
1
d u r e A). HPLC: tR ) 2.66 min and tR′ ) 13.97 min. H NMR
(DMSO-d6, 300 MHz): δ 1.59 (d, J ) 13.8 Hz, 2H), 2.30 (ddd,
J ) 5.1 Hz, J ′ ) 13.2 Hz, 2H), 3.46 (l s, 2H), 3.90 (dd, J ) 4.1
Hz, J ′ ) 12.7 Hz, 2H), 4.55 (s, 2H), 4.98 (ld, J ) 7.5 Hz), 5.96
(s, 2H), 6.63 (d, J ) 8.0 Hz, 2H), 6.71 (t, J ) 7.3 Hz, 1H), 6.8-
6.85 (m, 2H), 6.91 (d, J ) 0.6 Hz, 1H), 7.13 (t, J ) 7.4 Hz,
2H), 8.75 (s, 1H, NHCO). 13C NMR (DMSO-d6, 300 MHz): δ
29.3, 41.6, 59.7, 59.7, 67.6, 102.6, 109.6, 110.0, 115.9, 119.4,
123.1, 130.8. MS (APCI+): m/z 410 [M + H]+ and 366 [M -
44]+.
1-{1-[(6-Nitr oben zo[1,3]d ioxol-5-yl)m eth yl]-1,2,3,6-tet-
r ah ydr opyr idin -4-yl}-1,3-dih ydr oben zim idazol-2-on e (24)
1
(P r oced u r e D). HPLC: tR ) 1.73 min. H NMR (CD3COOD,
300 MHz): δ 3.09 (s, 2H), 3.90 (t, J ) 11.4 Hz, 2H), 4.31 (s,
2H), 4.88 (s, 2H), 6.1 (s, 1H), 6.30 (s, 2H), 7.18-7.22 (m, 2H),
7.25-7.36 (m, 2H), 7.42 (s, 1H), 7.77 (s, 1H). 13C NMR (CD3-
COOD, 300 MHz): δ 23.7, 49.3, 49.8, 56.3, 103.2, 106.1, 109.4,
110.3, 112.8, 119.3, 120.6, 122.0, 122.7, 127.8, 129.3, 130.4,
144.0, 149.7, 152.6, 154.5. MS (APCI+): m/z 395 [M + H]+.
4-Oxo-1-p h en yl-1,3,8-tr ia za sp ir o[4.5]d eca n e-8-ca r box-
ylic Acid Ben zo[1,2,5]th ia d ia zol-5-ylm eth yl Ester (32),
Obta in ed fr om 12 (P r oced u r e A). HPLC: tR ) 2.64 min
and tR′ ) 12.07 min. 1H NMR (DMSO-d6, 300 MHz): δ 1.63
(d, J ) 13.6 Hz, 2H), 2.34 (ddd, J ) 13.6 Hz, J ′ ) 5.5 Hz, 2H),
3.5 (l s, 2H), 3.97 (l s, 2H), 4.55 (s, 2H), 5.31 (l d, J ) 9.6 Hz,
1-{1-[(6-Ch lor oben zo[1,3]d ioxol-5-yl)m eth yl]p ip er id in -
4-yl}-1,4-d ih yd r oq u in oxa lin e-2,3-d ion e (25), Obt a in ed
fr om 5 (P r oced u r e E). HPLC: tR ) 2.09 min. 1H NMR
(DMSO-d6, 300 MHz): δ 1.85-1.88 (m, 2H), 2.50-2.58 (m, 1H),
2.85-3.01 (m, 2H), 3.23-3.57 (m, 1H), 3.44 (s, 2H), 4.28 (s,

3388 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Poulain et al.
2H), 6.64 (d, J ) 8.0 Hz, 2H), 6.67 (t, J ) 7.3 Hz, 1H), 7.07 (t,
J ) 7.6 Hz, 2H), 7.7 (dd, J ) 9.0 Hz, J ′ ) 1.4 Hz, 1H), 8.02 (s,
1H), 8.07 (d, J ) 9.0 Hz, 1H), 8.77 (s, 1H, NHCO). 13C NMR
(DMSO-d6, 300 MHz): δ 29.8, 42.0, 60.2, 67.3, 115.8, 119.5,
120.4, 122.8, 130.6, 131.4. MS (APCI+): m/z 424 [M + H]+.
467 [M + H]+. Mixture of two conformers (50/50). (Conformer
1
1) H NMR (DMSO-d6, 300 MHz): δ 1.60 (m, 2H), 2.40-2.45
(m, 2H), 3.30-3.35 (m, 1H), 3.84 (s, 3H), 3.93 (s, 3H), 4.24 (d,
2H, J ) 11.1 Hz), 4.58 (s, 2H), 6.68 (d, J ) 8.1 Hz, 2H), 6.71
(t, J ) 7.4 Hz, 1H), 7.17 (t, J ) 7.5 Hz, 2H), 7.27 (d, J ) 15.2
Hz, 1H), 7.36 (s, 1H), 7.58 (s, 1H), 7.82 (d, J ) 15.2 Hz, 1H),
8.79 (s, 1H, NHCO). 13C NMR (DMSO-d6, 300 MHz): δ 29.8,
40.4, 57.6, 58.3, 60.2, 109.4, 112.1, 116.0, 119.8, 123.9, 130.8,
4-Oxo-1-p h en yl-1,3,8-tr ia za sp ir o[4.5]d eca n e-8-ca r box-
ylic Acid 3-P h en oxyben zyl Ester (33) (P r oced u r e A).
1
HPLC: tR ) 3.38 min. H NMR (DMSO-d6, 300 MHz): δ 1.59
1
138.1. (Conformer 2) H NMR (DMSO-d6, 300 MHz): δ 1.60
(d, J ) 14 Hz, 2H), 2.24-2.35 (m, 2H), 3.4-3.6 (l s, 2H), 3.90
(dd, J ) 12.3 Hz, J ′ ) 3.6 Hz, 2H), 4.55 (s, 2H), 5.08 (l d, J )
10.8 Hz, 2H), 6.64 (d, J ) 8.4 Hz, 2H), 6.70 (t, J ) 7.3 Hz,
1H), 6.91 (m, 3H), 6.96 (d, J ) 0.9 Hz, 1H), 7.06-7.12 (m, 2H),
7.13 (t, J ) 8.3 Hz, 2H), 7.30-7.37 (m, 3H), 8.76 (s, 1H,
CONH). 13C NMR (DMSO-d6, 300 MHz): δ 29.8, 42.1, 60.5,
67.3, 115.7, 118.4, 119.0, 119.4, 120.5, 123.5, 125.3, 130.5,
132.7. MS (APCI+): m/z 458 [M + H]+.
(m, 2H), 2.40-2.45 (m, 2H), 3.75-3.80 (m, 1H), 3.84 (s, 3H),
3.93 (s, 3H), 4.37 (d, 2H, J ) 11.1 Hz), 4.58 (s, 2H), 6.68 (d, J
) 8.1 Hz, 2H), 6.71 (t, J ) 7.4 Hz, 1H), 7.17 (t, J ) 7.5 Hz,
2H), 7.27 (d, J ) 15.2 Hz, 1H), 7.36 (s, 1H), 7.58 (s, 1H), 7.82
(d, J ) 15.2 Hz, 1H), 8.79 (s, 1H, NHCO). 13C NMR (DMSO-
d6, 300 MHz): δ 31.1, 43.5, 57.6, 58.3, 60.2, 109.4, 112.1, 116.0,
119.8, 123.9, 130.8, 138.1.
4-Oxo-1-p h en yl-1,3,8-tr ia za sp ir o[4.5]d eca n e-8-Ca r box-
8-[(6-Ch lor ob en zo[1,3]d ioxol-5-yl)m et h yl]-1-p h en yl-
1,3,8-t r ia za sp ir o[4.5]d eca n -4-on e (39) (P r oced u r e E ).
HPLC: tR ) 2.26 min. H NMR (DMSO-d6, 300 MHz): δ 1.56
ylic Acid In d a n -1-yl Ester (34) (P r oced u r e A). HPLC: tR
1
1
) 3.09 min. H NMR (DMSO-d6, 300 MHz): δ 1.57 (l s, 2H),
1.96-2.02 (m, 1H), 2.34-2.40 (l s, 2H), 2.44-2.47 (m, 1H),
2.81-2.86 (m, 1H), 2.92-2.97 (m, 1H), 3.41-3.45 (m, 2H),
3.85-3.87 (m, 2H), 4.54 (s, 2H), 6.04 (ls, 1H), 6.65 (d, J ) 8.1
Hz, 2H), 6.72 (t, J ) 7.2 Hz, 1H), 7.15-7.19 (m, 3H), 7.26 (d,
J ) 3.8 Hz, 2H), 7.35 (d, J ) 7.3 Hz, 1H), 8.75 (s, 1H, NHCO).
13C NMR (DMSO-d6, 300 MHz): δ 29.5, 30.9, 41.8, 60.5, 80,
115.7, 119.7, 126.5, 128.2, 130.4, 130.8. MS (APCI+): m/z 392
[M + H]+.
(d, J ) 13 Hz, 2H), 2.48-2.56 (m, 2H), 2.72-2.88 (m, 4H), 3.51
(s, 2H), 4.56 (s, 2H), 6.06 (s, 2H), 6.75 (t, J ) 7.3 Hz, 1H), 6.86
(d, J ) 8.2 Hz, 2H), 7.04 (s, 1H), 7.07 (s, 1H), 7.24 (t, J ) 7.3
Hz, 2H), 8.64 (s, 1H, NHCO). 13C NMR (DMSO-d6, 300 MHz):
δ 28.8, 49.6, 58.5, 58.8, 59.1, 102.3, 109.8, 110.4, 114.7, 118.1,
125.1, 129.5, 143.7, 147.0, 176.6. MS (APCI+): m/z 400 [M +
H]+.
2-{8-[(6-Ch lor ob en zo[1,3]d ioxol-5-yl)m et h yl]-4-oxo-1-
p h e n yl-1,3,8-t r ia za sp ir o[4.5]d e c-3-yl}-N-m e t h yla ce t a -
m id e Tr iflu or oa ceta te (40), Obta in ed fr om 11 (P r oce-
d u r e E). HPLC: tR ) 2.26 min. 1H NMR (DMSO-d6, 300
MHz): δ 1.90 (d, J ) 14.4 Hz, 2H), 2.57 (d, J NH-H ) 4.5 Hz,
3H), 2.69-2.80 (m, 2H), 3.46 (d, J ) 10.0 Hz, 2H), 3.64-3.71
(m, 2H), 3.95 (s, 2H), 4.36 (s, 2H), 4.65 (s, 2H), 6.12 (s, 2H),
6.79 (t, J ) 7.2 Hz, 1H), 6.88 (d, J ) 8.4 Hz, 2H), 7.22 (t, J )
7.7 Hz, 2H), 7.23 (s, 1H), 7.26 (s, 1H), 8.03 (d, J NH-H ) 4.5 Hz,
1H, NHCO). 13C NMR (DMSO-d6, 300 MHz): δ 26.4, 27.2, 43.9,
49.3, 57.4, 58.4, 64.7, 103.6, 110.9, 113.3, 115.5, 119.6, 125.6,
130.0, 143.2, 143.2, 150.4, 167.7, 173.6. MS (APCI+): m/z 471
[M + H of free base]+.
4-Oxo-1-p h en yl-1,3,8-tr ia za sp ir o[4.5]d eca n e-8-ca r box-
ylic Acid P yr id in -3-ylm eth yl Ester (35) (P r oced u r e A).
1
HPLC: tR ) 1.87 min. H NMR (DMSO-d6, 300 MHz): δ 1.60
(d, J ) 13.8 Hz, 2H), 2.31 (ddd, J ) 13.5 Hz, J ′ ) 5.5 Hz, 2H),
3.48 (l s, 2H), 3.92 (dd, J ) 13.1 Hz, J ′ ) 4.9 Hz, 2H), 4.55 (s,
2H), 5.13 (l d, J ) 9.9 Hz, 2H), 6.63 (d, J ) 8.0 Hz, 2H), 6.72
(t, J ) 7.3 Hz, 1H), 7.15 (t, J ) 7.4 Hz, 2H), 7.37 (ddd, J ) 4.9
Hz, J ′ ) 7.9 Hz, J ′′ ) 0.7 Hz, 1H), 7.77 (dt, J ) 7.9 Hz, J ′ )
1.8 Hz, 1H), 8.51 (dd, J ) 4.8 Hz, J ) 1.6 Hz, 1H), 8.58 (d, J
) 1.7 Hz, 1H), 8.76 (s, 1H, NHCO). 13C NMR (DMSO-d6, 300
MHz): δ 29.8, 41.7, 60.2, 65.7, 115.7, 119.5, 125.1, 130.8, 137.4,
150.5, 150.7. MS (APCI+): m/z 367 [M + H]+.
4-Oxo-1-p h en yl-1,3,8-tr ia za sp ir o[4.5]d eca n e-8-ca r box-
ylic Acid (Ben zo[1,3]d ioxol-5-ylm eth yl)a m id e (36) (P r o-
ced u r e C). HPLC: tR ) 2.23 min and tR′ ) 12.29 min. 1H NMR
(DMSO-d6, 300 MHz): δ 1.49 (d, J ) 13.5 Hz, 2H), 2.35 (ddd,
J ) 5.2 Hz, J ′ ) 13.5 Hz, 2H), 3.37 (dt, J ) 2.8 Hz, J ′ ) 12.9
Hz, 2H), 4.25 (dd, J ) 3.3 Hz, J ′ ) 12.3 Hz, 2H), 4.15 (d, J NH-H
) 5.7 Hz, 2H), 4.54 (s, 1H), 5.93 (s, 2H), 6.59 (d, J ) 8.1 Hz,
2H), 6.68 (t, J ) 7.3 Hz, 1H), 6.72 (dd, J ) 7.8 Hz, J ′ ) 1.6
Hz, 1H), 6.78 (s, 1H), 6.81 (dd, J ) 1.5 Hz, J ′ ) 4.8 Hz, 1H),
7.08 (t, J ) 7.5 Hz, 2H), 7.12 (t, J ) 5.8 Hz, 1H, OCONH),
8.71 (s, 1H, NHCO). 13C NMR (DMSO-d6, 300 MHz): δ 29.2,
42.1, 43.4, 60.2, 102.3, 109.4, 109.6, 115.4, 119.2, 121.8, 130.8.
MS (APCI+): m/z 409 [M + H]+.
8-(Be n zo[1,3]d ioxol-5-ylm e t h yl)-1-p h e n yl-1,3,8-t r i-
a za sp ir o[4.5]d eca n -4-on e (41) (P r oced u r e E). HPLC: tR
) 2.19 min. 1H NMR (DMSO-d6, 300 MHz): δ 1.51 (d, J )
13.4 Hz, 2H), 2.42-2.52 (m, 2H), 2.65-2.68 (d, J ) 7.8 Hz,
4H), 3.38 (s, 2H), 4.52 (s, 2H), 5.94 (s, 2H), 6.68-6.75 (m, 2H),
6.71-6.82 (m, 3H), 6.87 (d, J ) 1.4 Hz, 1H), 7.21 (t, J ) 7.4
Hz, 2H), 8.59 (s, 1H, NHCO). 13C NMR (DMSO-d6, 300 MHz):
δ 29.3, 49.9, 59.0, 59.5, 62.7, 101.6, 108.7, 109.8, 115.0, 118.4,
122.7, 129.9, 133.0, 144.2, 146.9, 148.0, 177.1. MS (APCI+):
m/z 366 [M + H]+.
8-(Ben zo[1,2,5]t h ia d ia zol-5-ylm et h yl)-1-p h en yl-1,3,8-
tr ia za sp ir o[4.5]d eca n -4-on e Tr iflu or oa ceta te (42). A 500
µL sample of a solution (1.2 M, in DCM) of MeSO2Cl (2 equiv)
was added dropwise to 188 µL of a solution (1.6 M, in DCM)
of benzo[1,2,5]thiadiazol-5-ylmethanol (12) (1 equiv) and DI-
PEA (1 equiv) at 0 °C. The reaction mixture was allowed to
warm to room temperature and stirred for 30 min. The reaction
mixture was washed with water (2 × 1 mL), HCl (10%) (2 ×
1 mL), and a saturated solution of NaHCO3 (2 × 1 mL). The
organic layer was evaporated under reduced pressure. The
residue obtained was dissolved in 143 µL of THF. A 150 µL
sample of a solution (2 M, in THF) of 1-phenyl-1,3,8-triazaspiro-
[4.5]decan-4-one was added to this solution. The reaction
mixture was stirred for 1 h at room temperature and evapo-
rated under reduced pressure. The residue was purified by
preparative HPLC with H2O/TFA (99.95/0.05) and H2O/CH3-
CN/TFA (19.96/80/0.04), to give 11.6 mg of the trifluoroacetate
salt (7.8%). HPLC: tR ) 2.16 min. 1H NMR (DMSO-d6, 300
MHz): δ 1.91 (d, J ) 14.5 Hz, 2H), 2.72-2.81 (m, 2H), 3.51
(d, J ) 10.8 Hz, 2H), 3.73-3.84 (m, 2H), 4.61 (s, 2H), 4.64 (s,
2H), 6.80 (t, J ) 7.3 Hz, 1H), 6.90 (d, J ) 8.1 Hz, 2H), 7.20 (t,
J ) 8.3 Hz, 2H), 7.90 (dd, J ) 9.0 Hz, J ′ ) 1.5 Hz, 1H), 8.20
(d, J ) 9.0 Hz, 1H), 8.41 (s, 1H), 9.04 (s, 1H, NHCO). 13C NMR
(DMSO-d6, 300 MHz): δ 27.0, 49.6, 57.4, 59.7, 115.5, 119.3,
8-[3-(6-Nitr oben zo[1,3]d ioxol-5-yl)a cr yloyl]-1-p h en yl-
1,3,8-t r ia za sp ir o[4.5]d eca n -4-on e (37) (P r oced u r e B).
HPLC: tR ) 2.67 min and tR′ ) 12.44 min. MS (APCI+): m/z
451 [M + H]+. Mixture of two conformers (50/50). (Conformer
1) 1H NMR (DMSO-d6, 300 MHz): δ 1.68 (d, J ) 14.5 Hz, 2H),
2.30-2.40 (m, 2H), 3.29-3.39 (m, 1H), 4.24 (d, J ) 11.1 Hz,
2H), 4.57 (s, 2H), 6.22 (s, 2H), 6.67 (d, J ) 7.9 Hz, 2H), 6.70 (t,
J ) 7.3 Hz, 1H), 7.16 (t, J ) 7.5 Hz, 2H), 7.27 (d, J ) 15.1 Hz,
1H), 7.60 (s, 1H), 7.61 (s, 1H), 7.71 (d, J ) 15.1 Hz, 1H), 8.79
(s, 1H, NHCO). 13C NMR (DMSO-d6, 300 MHz): δ 29.8, 40.3,
60.2, 105.2, 106.4, 108.7, 115.9, 119.6, 123.9, 130.9, 137.4.
1
(Conformer 2) H NMR (DMSO-d6, 300 MHz): δ 1.68 (d, J )
14.5 Hz, 2H), 2.30-2.40 (m, 2H), 3.75 (t, 1H), 4.37 (d, J ) 11.1
Hz, 2H), 4.57 (s, 2H), 6.22 (s, 2H), 6.67 (d, J ) 7.9 Hz, 2H),
6.70 (t, J ) 7.3 Hz, 1H), 7.16 (t, J ) 7.5 Hz, 2H), 7.27 (d, J )
15.1 Hz, 1H), 7.60 (s, 1H), 7.61 (s, 1H), 7.71 (d, J ) 15.1 Hz,
1H), 8.79 (s, 1H, NHCO). 13C NMR (DMSO-d6, 300 MHz): δ
30.1, 42.4, 60.2, 105.2, 106.4, 108.7, 115.9, 119.6, 123.9, 130.9,
137.4.
8-[3-(4,5-Dim eth oxy-2-n itr op h en yl)a cr yloyl]-1-p h en yl-
1,3,8-t r ia za sp ir o[4.5]d eca n -4-on e (38) (P r oced u r e B).
HPLC: tR ) 2.67 min and tR′ ) 12.86 min. MS (APCI+): m/z

Combining Two Methods for Focused Library Design
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3389
122.5, 125.8, 130.7, 132.7, 133.0, 143.6, 154.8, 175.6. MS
Ack n ow led gm en t. We thank F. Revah for thought-
ful discussions. We are grateful to C. J arousseau for the
HTS experiments and P. George, D. Hamelin, and F.
Dezitter for their skillful help.
(APCI+): m/z 366 [M + H of free base]+.
8-(3-P h en oxyb en zyl)-1-p h en yl-1,3,8-t r ia za sp ir o[4.5]-
1
d eca n -4-on e (43) (P r oced u r e E). HPLC: tR ) 2.61 min. H
NMR (DMSO-d6, 300 MHz): δ 1.56 (d, J ) 13.2 Hz, 2H), 2.45-
2.50 (m, 2H), 2.71-2.73 (d, J ) 7.8 Hz, 4H), 3.55 (s, 2H), 4.56
(s, 2H), 6.76 (t, J ) 7.3 Hz, 1H), 6.82 (d, J ) 8.2 Hz, 2H), 6.88-
6.92 (m, 1H), 7.01-7.04 (m, 2H), 7.08-7.17 (m, 2H), 7.21 (T,
J ) 7.5 Hz, 2H), 7.35-7.48 (m, 4H), 8.65 (s, 1H, NHCO). 13C
NMR (DMSO-d6, 300 MHz): δ 29.1, 50.0, 58.8, 59.5, 62.3,
115.0, 117.8, 119.1, 119.5, 119.7, 124.4, 125.5, 128.8, 130.7,
130.9, 144.1, 157.4, 157.7, 177.0. MS (APCI+): m/z 414 [M +
H]+.
Su p p or tin g In for m a tion Ava ila ble: NMR spectra, mass
spectra, and HPLC chromatograms for all the compounds
described in the Experimental Section. This material is
available free of charge via the Internet at http://pubs.acs.org.
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and Future Trends in High Throughput Screening for Drug
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(3) Cerius2.Align, Molecular Simulations Inc., San Diego, CA.
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3-(Ca r b oxym et h yl)-4-oxo-1-p h en yl-1,3,8-t r ia za sp ir o-
[4.5]d eca n e-8-ca r boxylic Acid (6-Ch lor oben zo[1,3]d ioxol-
5-yl)m eth yl Ester (44). A suspension of 3-[4-oxo-1-phenyl-
8-(tert-butyloxycarbonyl)-1,3,8-triazaspiro[4.5]decyl]acetic acid
(3.9 g, 10 mmol), THF (30 mL), HCl (1 N, in Et2O) (20 mL, 20
mmol, 2 equiv), and H2SO4 (560 µL) was stirred overnight at
65 °C. The reaction mixture was then evaporated under
reduced pressure. The residue was triturated with AcOEt (50
mL) and Et2O (2 × 50 mL) to afford 3.87 g (100%) of a gray
powder of 3-(4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decyl)acetic
acid as its sulfuric acid salt. The product was used without
1
further purification. HPLC: tR ) 1.45 min. H NMR (DMSO-
d6, 300 MHz): δ 1.77 (d, J ) 14.3 Hz, 2H), 2.71 (dt, J ) 4.8
Hz, J ′ ) 14.2 Hz, 2H), 3.29-3.31 (m, 2H), 3.47-3.55 (m, 2H),
4.10 (s, 2H), 4.70 (s, 2H), 6.81 (t, J ) 7.3 Hz, 1H), 6.95 (d, J )
8.2 Hz, 2H), 7.25 (t, J ) 7.6 Hz, 2H), 8.75 (br s, 2H, NH2+).
MS (APCI+): m/z 290 [M + H of free base]+. The title
compound was then prepared from this intermediate according
1
to procedure A. HPLC: tR ) 2.82 min and tR′ ) 14.04 min. H
NMR (DMSO-d6, 300 MHz): δ 1.60 (d, J ) 13.6 Hz, 2H), 2.32
(m, 2H), 3.35 (l s, 2H), 3.90 (dd, 2H), 4.07 (s, 2H), 4.66 (s, 2H),
5.03 (l d, J ) 18 Hz, 2H), 6.04 (s, 2H), 6.67 (d, J ) 8.15 Hz,
2H), 6.76 (t, J ) 7.3 Hz, 1H), 7.05 (s, 1H), 7.09 (s, 1H), 7.17 (t,
J ) 7.6 Hz, 2H), 9.04 (s, 1H, COOH). 13C NMR (DMSO-d6,
300 MHz): δ 30, 41.8, 43.6, 65.0, 65.7, 104.1, 111.3, 111.7,
116.2, 120, 130.7. MS (APCI+): m/z 502 [M + H]+ and 458
[M - 44]+.
(5) Willett, P.; Barnard, J . M.; Downs, G. M. Chemical Similarity
Searching. J . Chem. Inf. Comput. Sci. 1998, 38, 983-996.
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B., Boyd, D. B., Eds.; Wiley: New York, 1997; Vol. 11, pp 127-
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Molecu la r Mod elin g. All computations were performed on
desktop Silicon Graphics workstations using a proprietary
software integrated into Cerius2 environment thanks to the
software developer kit (SDK) from Molecular Simulations Inc.
(San Diego, CA).
Recep tor Bin d in g Assa ys. The affinities for the different
receptors were determined using conventional in vitro receptor
binding methodology. All incubations of the radioligand with
membranes, and the studied compounds or the nonspecific
product, were terminated by adding cold buffer followed by
rapid filtration using a cell harvester from Packard. The bound
radioactivity was counted using a Topcount from Packard,
thanks to a scintillation liquid (Formula 989 or Microscint from
Packard). Reference compounds were assayed in duplicate,
using at least eight concentrations. All IC50 calculations were
done using nonlinear regression. Primary screening of the
10560 compounds was done on membranes from rat cerebral
cortex.15 Compounds were tested at 10 µM in duplicate against
[3H]DAMGO (1 nM). Naloxone (1 µM) was used to determine
nonspecific binding. Screening of the analogues (1 µM in
duplicate) and determination of IC50 values (eight concentra-
tions in duplicate) were done on human recombinant receptors,
expressed in mammalian cells.16 The radioligand and nonspe-
cific ligand were, respectively, [3H]DAMGO (0.5 nM) and
naloxone (10 µM) for µ (h), [3H]U69593 (0.5 nM) and U50488
(10 µM) for κ (h), and [3H]DPDPE (0.5 nM) and naltrexone
(10 µM) for δ (h).
(7) Horvath, D. High Throughput Conformational Sampling and
Fuzzy Similarity Metrics: A Novel Approach to Similarity
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Role in the Drug Discovery Laboratory. In Combinatorial
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Applications; Ghose, A. K., Vishwadhan, V. N., Eds.; Marcel
Dekker: New York, in press.
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Nova Science Publishers: New York, 2001; pp 395-439.
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ity and permeability in drug discovery and development settings.
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a sense
differing from the definition given by IUPAC (Wermuth, C.-G.;
Ganellin, C. R.; Lindberg, P.; Mitscher, L. A. Glossary of terms
used in Medicinal Chemistry (IUPAC recommendations 1997).
Annu. Rep. Med. Chem. 1998, 33, 385-395). Pharmacophore
refers here to the ensemble of all steric and electronic features
of a given molecule, which could potentially ensure interactions
with a biological target structure.
Solu bilit y Det er m in a t ion . A 10 mM solution of the
compound in DMSO was diluted to 0.2 mM in PBS (pH 7.4)
or to 0.2 mM in methanol. The solubility was obtained by
comparison of the HPLC peaks areas.17
log D Deter m in a tion . log D is the partition coefficient of
the compounds between 1-octanol and PBS (pH 7.4). It was
evaluated by LC/MS.18
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