Meta-substituted benzofused macrocyclic lactams as zinc metalloprotease inhibitors

Article abstract of DOI:10.1021/jm960583g

The design, synthesis, and biochemical profile of meta-substituted benzofused macrocyclic lactams are described. The meta-substituted benzofused macrocyclic lactams were designed to have a degree of flexibility allowing the amide bond to occupy two comple

Full text of DOI:10.1021/jm960583g

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506
J . Med. Chem. 1997, 40, 506-514
Meta -Su bstitu ted Ben zofu sed Ma cr ocyclic La cta m s a s Zin c Meta llop r otea se
In h ibitor s
Gary M. Ksander,* Reynalda de J esus, Andrew Yuan, Raj D. Ghai, Colin McMartin,† and Regine Bohacek‡
Research Department, Pharmaceuticals Division, CIBA-GEIGY Corporation, 556 Morris Avenue, Summit, New J ersey 07901
Received August 8, 1996X
The design, synthesis, and biochemical profile of meta-substituted benzofused macrocyclic
lactams are described. The meta-substituted benzofused macrocyclic lactams were designed
to have a degree of flexibility allowing the amide bond to occupy two completely different
conformations while maintaining sufficient rigidity to allow for strong interaction between
enzyme and inhibitor. Using TFIT, a novel molecular superimposition program, it was shown
that the meta analogs could be readily superimposed onto our ACE inhibitor template whereas
no low-energy superimpositions of the ortho-substituted macrocycles could be found. The
macrocycles were prepared by tethering aldehyde 1 derived from S-glutamic acid or S-aspartic
acid to a meta-substituted phosphonium bromide 2. Homologation to a monocarboxylic acid
methyl ester malonate followed by deprotection and cyclization gave the macrocyclic frame.
Further manipulation gave the desired compounds. Unlike the ortho-substituted benzofused
macrocyclic lactams described in the previous paper which are selective NEP inhibitors, the
meta-substituted compounds are dual inhibitors of both NEP and ACE. The most potent
member of this new series, compound 16a , inhibited both enzymes with an IC50 ) 8 nM in
NEP and 4 nM in ACE.
In tr od u ction
here. A zinc atom was connected to the zinc-ligating
group of each inhibitor using geometrical parameters
obtained from X-ray crystal diffraction data both of
small molecules3 and enzyme/inhibitor complexes. A
new computer program, TFIND,4a,b was then used to
superimpose the four inhibitors so that chemically
equivalent atoms of one molecule occupied the same
volume as the corresponding atoms of the other mol-
ecules. This program combines a thorough search of
low-energy conformations of each molecule with simul-
taneous superimposition by adding a superimposition
force field to the potential function. In this way, the
internal energy of the molecule and its superimposition
energy are cominimized. For more details, see the
Experimental Section. TFIND found three different
good superimpositions of low-energy conformations of
these four molecules.
Three-dimensional templates for ACE inhibitors have
previously been developed by other researchers using
a variety of different methods.3,5,6 All of these templates
as well as the superimpositions found by TFIND agree
as to the conformations the inhibitors adopt between
the P1′ side chain and the C-terminal carboxylic acid.
However, there is little agreement concerning the bio-
active conformation of the remaining portions of the
inhibitors.
To develop a hypothesis concerning the conformation
adopted by ACE inhibitors between the catalytic zinc
and the P1′ carbonyl, we turned to structural data.
Examination of the crystal structures of known zinc
metallo exo- and endo-protease/inhibitor complexes
revealed strong conformational similarities of this re-
gion for all of the inhibitors.
Although the crystal structure for ACE has not been
determined, similarity in conserved sequence motifs and
mutagenesis experiments suggest that the region of the
two active sites of ACE close to the zinc may resemble
that found in other zinc metalloproteases. ACE displays
the characteristic HEXXH7 motif found in thermolysin,8
NEP,9,10 and the metzincins.11,12 From the crystal
structures of thermolysin13 and members of four sub-
There has been considerable interest over the past
few years in dual angiotensin converting enzyme (ACE)
and neutral endopeptidase (NEP) inhibitors. Simulta-
neous blockade of angiotensin II production and in-
creasing atrial naturetic peptide levels should have a
beneficial effect in the treatment of hypertension and
other cardiovascular and renal diseases. A number of
investigators have studied the structural similarities of
the active site of these two enzymes and the incorpora-
tion of both NEP and ACE inhibitory activities within
a single chemical entity.1a-1g The previous paper
describes the use of an active site model to predict NEP
activity based on the crystal structures of the bacterial
zinc metalloprotease thermolysin. The macrocyclic lac-
tams described in the previous paper were either
inactive or very weak inhibitors of ACE. However, they
exhibited potent NEP and/or thermolysin inhibitory
activity. Using the previously described model of NEP
and a template compiled from existing ACE inhibitors,
we set out to investigate if modifications could be made
that would give these compounds the flexibility to bind
to both NEP and ACE while maintaining a degree of
rigidity that allows for a strong interaction between
enzyme and inhibitor. In this paper we describe a
simple structural change to the previously described
macrocycles which allows these selective NEP inhibitors
to inhibit both enzymes, NEP and ACE.
Molecu la r Mod elin g
Con str u ction of a n ACE In h ibitor Com p osite
Tem p la te. An ACE inhibitor template had previously
been constructed using four potent conformationally
restricted inhibitors. (See compounds A-D shown in
Figure 2.2) The procedure will be briefly summarized
* Author to whom correspondence should be addressed.
† Current address: Thistlesoft, Lindsley Dr., Suite 302, Morris
Township, NJ 07960.
‡ Current address: Ariad Pharmaceutical, Inc., 26 Landsdowne St.,
Cambridge, MA 02139.
X Abstract published in Advance ACS Abstracts, J anuary 15, 1997.
S0022-2623(96)00583-3 CCC: $14.00 © 1997 American Chemical Society

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Meta-Substituted Benzofused Macrocyclic Lactams
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 507
families of the metzincins,14-24 it is known that the two
histidines bind to the catalytic zinc. The glutamic acid
adopts the same relative position in all of these enzymes
and has been proposed to be essential for catalysis.13
Mutagenesis experiments suggest that these residues
have the same function in NEP.25,26 A second conserved
sequence, NEXIXD, is shared by ACE, thermolysin, and
NEP. Again, mutagenesis experiments with ACE27 and
NEP28 indicate that, as in thermolysin, the glutamic
acid is the third zinc binding residue.
We, therefore, made the assumption that the regions
close to the zinc in ACE are likely to be similar to those
found in other zinc metalloproteases and that when
inhibitors bind to ACE, they will also adopt the confor-
mation near zinc found in the crystal structures of other
metalloprotease inhibitor complexes. Only one of the
templates generated by the TFIND program displayed
this geometry and was, therefore, proposed as a phar-
macophore for ACE. This ACE inhibitor template has
been extensively tested and was able to distinguish
between active and inactive ACE and dual ACE/NEP
inhibitors from a number of different laboratories.2
The first set of macrocycles described in this work has
a phenyl ring in the P1′ position. To provide more
information concerning the allowed volume in the S1′
subsite of ACE, we added an additional potent, confor-
mationally restricted inhibitor, E,29 to the template.
Figure 3 shows the resulting ACE inhibitor template
used in the present work.
Once the template was constructed, the TFIT program
was used to determine if low-energy conformations could
be found for each macrocycle that would allow chemi-
cally similar atoms of the macrocycle to be superimposed
onto the corresponding atoms of the template. Like
TFIND, the TFIT program also uses the superimposi-
tion force field. However, when using TFIT, the tem-
plate is held rigid and only the test molecule is subjected
to conformational searching and cominimization of
internal and superimposition energy. (Additional de-
tails are given in the Experimental Section.)
Besides the superimposed structures, the output of
the TFIT program reports two parameters: the ligand
strain energy which approximates the cost in energy
required for a test compound to adopt the superimposed
conformation and the superimposition energy which is
a measure of how closely atoms of the test compound
can fit on chemically similar atoms of the template.
The starting structures for the template fitting were
either obtained directly from the NEP studies described
in the preceding paper or constructed using the proce-
dures also described in that paper. Next, using the
MACROMODEL30 molecular modeling program, a zinc
atom was added to the sulfur of each macrocycle with a
zinc-sulfur bond length of 2.3 Å and a zinc-sulfur-
carbon angle of 100°. These parameters are based on
small molecule crystal structures of relevant compounds
from Cambridge Structural Database given by Hausin
and Codding.3 Then each macrocycle was subjected to
template fitting with 1000 search cycles. To provide an
additional measure of how well each structure fit onto
the template, the volume of the template plus the
superimposed macrocycle was determined using MAC-
ROMODEL.
standard Wittig conditions gave the olefin 3 in good
yield (Scheme 1). Metal halogen exchange, using BuLi
with aryl bromide 3, followed by DMF condensation
gave the benzaldehyde derivative 4. Homologation with
the mixed benzyl methyl malonate ester followed by
hydrogenation in one step reduced the two double bonds
and unmasked one of the carboxylic acid groups to give
compound 6. Cyclization with EDCI to the macrocyclic
lactam 8, after deprotection of the amino alcohol, was
accomplished in low yield under dilute reaction condi-
tions. Oxidation of the macrocyclic alcohol 8 with
sodium periodate/ruthenium trichloride followed by
saponification with sodium hydroxide gave the diacid
10. The exocyclic double bond was introduced by
reaction of the carboxylic acid/amide with paraformal-
dehyde and piperidine followed by decarboxylative
elimination. Addition of thioacetic acid to the enone 11
and reesterification with diazomethane gave a mixture
of two diastereomers 13a ,b. These diastereomers could
be readily separated by flash chromatography. The
diastereomers were easily distinguishable in the NMR
by the position of the NH and the NHCHCO2CH3
methine protons. However, the relative stereochemistry
(cis and trans) could not be assigned by NMR studies
alone, due to the flexibility of the macrocyclic ring.
The relative and thus the absolute stereochemical
assignment is based on the synthetic procedures (start-
ing from L-glutamic acid) and the X-ray of 13b, Figure
1. The X-ray of the 14-membered ring macrocycle, 13b,
clearly shows a trans relationship between the carboxy
and methylenethiolacetyl groups. Since the synthetic
sequence begins with L-glutamic acid, 13b can be
assigned the R(methylene thiol acetyl),S(carboxy) con-
figuration. Therefore, 13a should be the S,S diastere-
omer. The stereochemistry of the 13-membered ring
diastereomers 15a ,b were assigned by comparing the
chemical shift of the NHCHCO2CH3 methine proton in
the 1HNMR and polarity relationship of the S,S dia-
stereomers 13a and 15a to the R,S diastereomers 13b
and 15b on silica gel plates. The S,S diastereomers
were more polar on silica gel thin layer plates (EtOAc/
Hex, 1:4), displayed a downfield shift in the proton NMR
for the NH amide protons (5.87 and 5.60 ppm vs 5.65
and 5.40 ppm, respectively) and an upfield shift for the
NHCHCO2CH3 methine proton (4.28 and 4.02 ppm vs
4.47 and 4.50 ppm, respectively). This relationship
between polarity and proton NMR shifts from the
assigned diastereomers was also consistent within the
ortho-substituted macrocyclic lactam series outlined in
the preceding paper. Therefore the stereochemical
assignments were made on the basis of chemical syn-
thesis, X-ray analysis, Rf (silica gel), and proton NMR
shifts. In addition the stereochemical assignment is
also supported by the corresponding chemical shifts
determined for the 10-membered ring macrocycle,1c the
cysteine-containing macrocycle,1n and the ortho-substi-
tuted benzofused macrocycles described in the previous
paper.
Simultaneous saponification of the S-acetyl and meth-
yl ester in 13a ,b gave the desired dual inhibitors 14a
and 14b. Compounds 16a and 16b were prepared in
an analogous fashion.
Resu lts a n d Discu ssion
Ch em istr y
Condensation of aldehyde 1 derived from L-glutamic
acid with the aryl phosphonium bromide 2 under
The ortho-substituted benzofused macrocycles (11-,
12-, and 13-membered rings) are potent inhibitors of

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508 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Ksander et al.
Sch em e 1a
a
(a) t-BuOK; (b) BuLi, DMF; (c) benzyl methyl malonate, piperidine, benzoic acid; (d) H2/Pd-C; (e) HCl; (f) EDCl, HOBT; (g) NaIO4,
RuCl3; (h) NaOH; (i) paraformaldehyde, piperidine; (j) thiolacetic acid; (k) diazomethane, separate diastereomers; (l) LiOH.
F igu r e 1. X-ray crystal structure showing absolute configu-
ration of compound 13b.
F igu r e 2. Structures used to generate ACE template.
NEP (Table 1). However they lack significant ACE
inhibitory activity. Therefore, these compounds were
matched to an ACE molecular model template fitting
program to determine if steric fit, hydrogen bonding,
or thiol zinc chelation might play a role in the inactivity
of these compounds.
On the basis of the structural data from five potent
ACE inhibitors a template was constructed to define an
active site that would encompass these large molecules.
The macrocycles considered here possess considerable
flexibility. However, when the thiol, amide, and car-
boxylic acid moeities are selectively fixed in positions

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Meta-Substituted Benzofused Macrocyclic Lactams
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 509
F igu r e 3. Molecular model of the ACE template.
F igu r e 4. Thirteen-membered ring P1′(S,S) ortho-substituted
benzofused macrocyclic lactam 19a superimposed on the ACE
template. Does not fit well on the template which is consistent
with experimental results.
Ta ble 1. In Vitro NEP, ACE, and Thermolysin Inhibition of
P1′ Ortho-Substituted Benzofused Macrocyclic Lactams
Ta ble 2. In Vitro NEP, ACE, and Thermolysin Inhibition of
P2′ Ortho-Substituted Benzofused Macrocyclic Lactams
IC50 (nM)
compd
ring size
stereochem
NEP
ACE
TL
68
2300
600
4000
1800
41000
17a
17b
18a
18b
19a
19b
11
11
12
12
13
13
S
R
S
R
S
R
0.9
>10000
>10000
8000
7
3
4
27
224
IC50 (nM)
2000
compd ring size
stereochem
NEP
99
67
136
900
ACE
TL
>10000
20
12
13
13
14
14
RS,SR (trans)
SS,RR (cis)
RS,SR (trans)
SS,RR (cis)
6000 49000
725 40000
470 16000
>10000
21a
21b
22a
22b
NT
NT
required by the template, the conformation of the carbon
backbone becomes less flexible. In some cases a par-
ticular high-energy conformation can adopt positions
required for good binding to ACE.
RS,SR (trans) >10000 >10000 60000
molecule is observed with a very low strain energy. At
this time, an explanation defining the limitations of the
template model/superimposition is worth noting. In this
superimposition, three methylene atoms protrude into
a space directly behind the template, Figure 4, so that
the rest of the macrocycle can match the corresponding
template atoms. Without the actual structure of the
enzyme/inhibitor complex, it is impossible to accurately
identify those regions of the binding site inaccessible
to the inhibitor. We assume that if a good overlap
occurs between a test molecule and the atoms of the
template and if the test molecule is in a low-energy
conformation, then good enzyme-inhibitor interactions
and high binding affinities should be observed. If,
however, atoms of the test molecule lie outside the
template in an unmapped area, then the binding
interactions between the test molecule and the enzyme
cannot be accurately predicted. To explain the poor
ACE inhibition of 19a , however, we must assume that
the protruding methylene atoms are positioned in an
area forbidden to ligand atoms.
Six P1′ ortho-substituted benzofuzed macrocycles
varying in ring size from 11 to 13 bonds were compared
to an ACE template using the molecular superposition
program, TFIT. This program is designed to identify
low-energy conformations of the macrocycle which al-
lows the best superimposition of chemically equivalent
atoms of the macrocycle onto those of the ACE inhibitor
template. The program was biased to retain only those
conformations of the macrocycles in which the zinc, the
amide carbonyl, and the carboxylic acid lie close to the
corresponding atoms of the template. For example, the
superimposition of the 13-membered macrocycle 19a
onto the ACE template generated by the program is
shown in Figure 4. The energy requirements necessary
to obtain the best fit are summarized in Table 4.
None of the P1′ ortho-substituted macrocycles fit well
onto the template. This finding is in agreement with
the weak ACE inhibitory activity obtained for these
compounds. For the four diastereomers of the 11- and
12-membered rings (17, 18), the poor activity can be
explained by the high strain energy imposed upon the
molecule to acquire the superimposition. However, for
19a the (S,S)-13-membered ring diastereomer, a rea-
sonable superimposition of the functional face of the
The in vitro data for macrocycles with a P21 ortho-
substituted benzofused ring are tabulated in Table 2.
Visual inspection of the template modeling with a P21
benzofused macrocycle, 21a (Figure 5), resulted in good
overlap along the functional side of the template and

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510 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Ksander et al.
F igu r e 5. Thirteen-membered ring P2′(racemic cis) ortho-
substituted benzofusedmacrocyclic lactam 21a superimposed
on the ACE template.
F igu r e 7. Thirteen-membered ring P1′(S,S) meta-substituted
benzofused macrocyclic lactam 16a superimposed on the ACE
template. Fits well on the template which is consistent with
experimental results.
Ta ble 3. In Vitro NEP, ACE, and Thermolysin Inhibition of
P1′ Meta-Substituted Benzofused Macrocyclic Lactams
F igu r e 6. Compound 23.
good containment within the volume defined by the
lipophilic backbone except for the aromatic ring at the
P21 pocket.
The ACE inhibitory activity of 21a is a modest IC50
) 725 nM. Interestingly, we searched our database for
compounds that might occupy the same spatial arrange-
ment seen by the protruding aromatic ring at the P21
site. We found a captopril analog 23, Figure 6, which
models similarly as 21a and inhibits ACE with an IC50
value of 500 nM. One might conclude that occupation
of this space is not absolutely forbidden but definitely
hinders the binding to the enzyme.
IC50 (nM)
compd
ring size
stereochem
NEP
ACE
TL
14a
14b
16a
16b
14
14
13
13
S
R
S
4
445
8
175
118
4
34000
>10000
48000
Returning to the modeling of 19a , we rationalized that
the lack of ACE activity was due to poor overlap along
the backbone of the molecule, i.e., occupation of lipo-
philic sites above and below the plane of the template.
Conversely, another way to model the same compound
is to constrain the backbone and examine the confor-
mational effect on the functional face of the molecule.
A very important structural feature of dual ACE/NEP
inhibitory activity is the proper orientation of the amide
bond. Since the relative dipole direction of the amide
carbonyls for good inhibitors with the active site of ACE
and NEP are opposite, some degree of flexibility within
the molecule is crucial for dual activity. For the ortho-
substituted benzofused macrocycles, steric repulsion
between the methylene side chain and the amide
carbonyl prevents the amide bond from occupying a
favorable conformation required for good ACE interac-
tion. Elimination of the steric interaction between the
amide bond and the methylene backbone could be
accomplished by replacing the “cis” double bond con-
strained within the aromatic ring/macrocycle with a
“trans” double bond. Preparation of the meta substi-
R
1000
1400
>10000
the protruding methylene chain. In addition to excel-
lent overlap of the backbone and the functional face of
the molecule with the template, a low strain energy is
calculated for this conformation. Therefore, good bind-
ing to the enzyme would be anticipated. Experimen-
tally, 16a inhibits ACE with IC50 ) 4 nM (Table 3).
The (R,S) diastereomer (16b) when tested as an
inhibitor of ACE was found to be considerably less active
than the (S,S) diastereomer (IC50 ) 1.4 µM). The
results from template fitting shows a small directional
change for the carboxyl group and a poor overlap of the
methylene sulfhydryl moiety, as well as some twisting
out of plane of the methylene backbone chain. These
changes from optimal overlap should result in decreased
enzyme interactions, as observed.
The 14-membered ring macrocycles in the meta
substituted series 14a and 14b ((SS) and (RS) diaster-
eomers) showed moderate ACE inhibition of 187 and
118 nM, respectively. To accommodate the larger ring
size, a less than optimal template overlap was observed
due to twisting of the aromatic ring out of the template
volume. Both structures are predicted to interact with
ACE to a lesser degree than 16a based on template
overlay as observed.
1
tuted P1 benzo derivatives would satisfy this goal.
The overlap of the meta-substituted benzofused mac-
rocycle 16a on the ACE template is shown in Figure 7.
In contrast to 19a , the structural overlay of 16a clearly
shows the absence, in the assumed forbidden space, of

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Meta-Substituted Benzofused Macrocyclic Lactams
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 511
Ta ble 4. Energy and Volume Calculations of Host Fit to ACE Template
superimposition
energy (kJ /mol)b
strain energyc
(kJ /mol)
volume
compd
16a
16b
14a
14b
17a
17b
18a
ACE IC50 (nM)
(A3)d
visual description
4
1400
175
118
-337
-334
-338
-339
-334
-332
-335
5
2
9
13
31
17
39
458
457
467
464
449
449
462
excellent match
carbon adjacent to sulfur does not match the template
extends beyond template on top
extends beyond template on top
extends beyond template in back
extends beyond template in back
alkane chain extends beyond template near the
carboxyl group
>10000
>10000
8000
18b
2000
-335
-333
-339
-350
-317
-331
19
4
12
26
15
26
455
463
465
474
480
488
good match
19a
>10000
extends beyond template in back
extends beyond template in back
extends beyond template in back
poor match
extends beyond template in back; sulfur and adjacent
carbon do not superimpose
19b
>10000
20 (S,S)
20 (S,R)
20 (R,S)
a
a
a
20 (R,R)
21 (S,S)
21 (S,R)
21 (R,S)
21 (R,R)
a
a
a
a
a
-337
-347
-336
-342
-329
46
15
39
25
43
476
493
491
494
494
carboxylate and amide carbonyl do not superimpose
good match
carboxylic acid does not match template
good match
sulfur, adjacent methylene, and carboxylic acid do not
match template
amide carbonyl does not superimpose
no conformation found with phenyl ring on the
carboxylic acid side of the template
extends slightly beyond template in back
carboxylic acid does not match template
22 (S,S)
22 (S,R)
a
-353
22
491
22 (R,S)
22 (R,R)
a
a
-364
-345
23
25
489
499
a
b
See Table 2 for IC50 values. A measure of the match between the host and the template. c The difference between the energy of the
superimposed conformation and the global minimum internal energy of the host in a vacuum. The volume Å3 is the sum of the template
d
(433.8) and the non-superimposible volume of the host.
The goal of this research project was to convert our
existing NEP inhibitors into dual ACE/NEP inhibitors.
As described earlier, we used molecular modeling tools
to rationalize why the ortho-substituted macrocycles
were potent NEP inhibitors, but devoid of good ACE
inhibitory activity. A minor structural change produced
the meta-substituted benzofused macrocycles (13- and
14-membered rings) which resulted in dual ACE/NEP
activity. The most potent dual inhibitor was 16a , IC50
) 8 nM for NEP and 4 nM for ACE.
The NEP activities of compounds 14a ,b and 16a ,b are
shown in Table 3. However, using the molecular
modeling program based on thermolysin described in
the previous paper, the prediction of NEP inhibitory
activity for the meta-substituted derivatives 14a and
16a should be minimal. However potent NEP inhibi-
tion, IC50 ) 8 nM and IC50 ) 4 nM, was observed with
compounds 14a and 16a , respectively. As predicted the
activity against thermolysin was poor (48 and 34 µM,
respectively). As pointed out earlier, compounds with
F igu r e 8. Thirteen-membered ring P1′(S,S) meta-substituted
benzofused macrocyclic lactam 16a in the active site of
different size substituents in the P1′ pocket have dra-
matic differences in binding affinity to thermolysin and
NEP. For example, occupation of the P1′ pocket of NEP
by a biphenyl group significantly enhances binding
potency in the dicarboxylic acid31a,b and aminophospho-
nic acid series32 as compared to the unsubstituted
phenyl derivatives. Therefore the depth of the P1′
pocket is clearly different between NEP and thermol-
ysin. However, less is known about the width of the
NEP P1′pocket. Examining the area surrounding the
P1′ site in the thermolysin model of either 14a or 16a
(Figure 8) shows the meta substituents protruding from
the cavity, unlike the ortho-derived benzofused macro-
cycle 17a , shown in the preceding paper. The NEP
binding results of 14a and 16a could be explained if one
assumes NEP is deeper and wider than thermolysin in
this region of the enzyme.
thermolysin. Arrows indicate atoms penetrating outside the
accessible surface, indicating a poor fit of the macrocycle in
the active site.
omers, with IC50 values of 445 and 1000 nM, respec-
tively. Excluding the effect at the P1′ pocket, visual
inspection of 14b and 16b in thermolysin show poor
interaction of the functional face of the molecules in the
thermolysin model. Therefore weaker interactions would
be expected.
In summary, the requirements for both ACE and NEP
inhibitory activity in the same molecule are good
hydrophobic interactions, zinc chelation, carboxylate
and hydrogen binding, and flexibility of the amide bond
which can occupy two different low-energy conforma-
tions. Although conformational restrictions are usually
a desirable property, in this case, a constrained amide
bond would not allow proper alignment for beneficial
hydrogen bonding to occur with both enzymes. This can
be appreciated visually in Figures 7 and 8. The ACE
The (R,S) diastereomers, 13- and 14-membered ring
macrocycles, 14b and 16b, are considerably weaker as
NEP inhibitors than the corresponding (S,S) diastere-

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512 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Ksander et al.
A suspension of 10% Pd/C (0.75 g) and diester 5 (6.1 g, 10.9
mmol) in 150 mL of ethyl acetate was hydrogenated at 50 psi
for 17 h. The mixture was recharged with hydrogen and
shaken for an additional 6 h. The reaction was concentrated
and filtered through a pad of Celite. Solvent was removed to
give 5.1 g (93%) of 6. The product was used as is in the next
step: 1H NMR (CDCl3) δ 7.17 (t, 1H), 7.0 (s, 1H), 6.98 (d, 2H)
3.86 (m, 1H), 3.70 (s, 3H), 3.70 (m, 2H), 3.20 (d, 2H), 2.55 (t,
2H), 1.53 (s, 3H), 1.50 (s, 3H), 1.40 (s, 9H), 1.3-1.6 (m, 10H).
[[3-(7(S)-Am in o-8-h yd r oxyoct yl)p h en yl]m et h yl]-1,3-
p r op a n ed ioic Acid 1-Meth yl Ester (7). A solution of 6 (5.0
g, 10.2 mmol) in 30 mL of methylene chloride at 0 °C was
purged with dry HCl gas for 5 min. The ice bath was removed,
HCl gas was bubbled into the solution for an additional 5 min,
and the solution was stirred at room temperature overnight.
The solvent was removed, and the residue was used as is in
the next step.
(6S )-6-(H yd r oxym e t h yl)-4-oxo-5-a za b icyclo[11.3.1]-
h ep ta d eca -1(17),13,15-tr ien e-3-ca r boxylic Acid Meth yl
Ester (8). To a solution of EDCI (3.96 g, 20.4 mmol) and
HOBT (2.12 g, 15.7 mmol) in 5 L of methylene chloride was
slowly added 7 (3.9 g, 11.1 mmol) in 35 mL of DMF, triethy-
lamine (7.4 g, 10.2 mmol), and 500 mL of methylene chloride.
The solution was mixed with a mechanical stirrer overnight.
The solvents were removed under reduced pressure and taken
up in ethyl acetate and sodium bicarbonate solution. The
organic layer was separated. The aqueous layer was re-
extracted with ethyl acetate, and the organics were combined,
washed with 1 N HCl and brine, dried (MgSO4), filtered, and
evaporated to dryness. The residue was chromatographed on
silica gel, eluting with ethyl acetate to give 1.3 g (35%) of 8 as
a mixture of diastereomers: 1H NMR (CDCl3) δ 7.2-6.9 (m,
4H), 6.78 (d, 0.3H), 5.4 (d, 0.7H), 3.95 (m, 1H), 3,75 (s, 0.3H),
3.72 (s, 0.7H), 3.7-3.1 (m, 5H), 2.6 (m, 1H), 2.1 (m, 1H), 1.7
(m, 1H), 1.4-1.0 (m, 9H).
(6S)-4-Oxo-5-a za bicyclo[11.3.1]h ep ta d eca -1(17),13,15-
tr ien e-3,6-d ica r boxylic Acid 3-Meth yl Ester (9). The
macrocyclic lactam alcohol 8 (1.3 g, 3.9 mmol) in 50 mL of
acetonitrile and 50 mL of water was oxidized with sodium
periodate (3.34 g, 15.6 mmol) and ruthenium trichloride
hydrate (0.019 g, 0.086 mmol). The mixture was stirred for
30 min at room temperature and left standing for 2 h. The
reaction mixture was diluted with methylene chloride, and the
organic layer was washed with water, dried (MgSO4), filtered,
and evaporated to dryness to give 1.13 g of 9 as a crude
product. NMR shows (DMSO-d6) amide protons at δ 8.25 (d,
0.3H), 6.2 (d, 0.7H) and two methyl ester protons at 3.75 (s,
0.3H), 3.70 (s, 0.7H). The compound was used without further
purification in the next step.
(6S)-4-Oxo-5-a za bicyclo[11.3.1]h ep ta d eca -1(17),13,15-
tr ien e-3,6-d ica r boxylic Acid (10). To the ester acid 9 (1.1g,
3.2 mmol) in 10 mL of THF was added 8.5 mL of 1 N NaOH
(2.6 equiv). The reaction mixture was stirred overnight and
diluted with ether. The aqueous layer was acidified and re-
extracted with ethyl acetate, dried (MgSO4), filtered, and
concentrated to dryness to give 1.0 g of diacid 10. 1H NMR
shows two amide protons (DMSO-d6) δ at 8.27 (d, 0.7H), 8.19
(d, 0.3H). The diacid was used without further purification
in the next step.
template shows the direction of the amide NH pointing
into the ring with the carbonyl moiety exocyclic to the
macrocycle cavity. In contrast, the NH of the NEP
template is rotated in the opposite direction. A dual
inhibitor must be flexible enough to meet both of these
requirements. This series of compounds benefit from
the rigid nature of the macrocycle structure. However,
these compounds demonstrate a balance between rigid-
ity and flexibility enabling the molecules to meet both
of the requirements necessary for dual ACE and NEP
inhibition.
We have shown, using molecular modeling tools, how
inhibitors bind to two similar enzymes, ACE and NEP.
We rationalized how small structural changes would
allow selective NEP inhibitors to adopt new conforma-
tions allowing dual inhibition to occur. These com-
pounds will not be drugs due to their inherent synthetic
difficulties of macrolactonization; however, they do
represent a good example of how medicinal chemistry
and molecular modeling were used to design, refine, and
prioritize synthetic targets.
Exp er im en ta l Section
General procedures. 1H NMR spectra were recorded on
Varian XL 400 MHz, Varian VR 300 MHz, and/or Bruker AC
250 MHz spectrometers with tetramethylsilane as internal
standard. Infrared spectra were recorded on a Nicolet 5SXFT
spectrometer. Optical rotations were measured with a Perkin-
Elmer Model 241 polarimeter. Melting points were taken on
a Thomas-Hoover melting point apparatus and are uncor-
rected.
(4S)-4-[6-(3-Br om op h en yl)-3-h exen yl]-3-t-BOC-2,2-d im -
eth yloxa zolid in e (3). To a mixture of phosphonium salt 2
(37g, 63 mmol) and aldehyde 1 (8.6g, 33.4 mmol) in 500 mL of
methylene chloride was added 63 mmol of 1.0 M potassium
tert-butoxide in THF at 0 °C. Disappearance of starting
material was monitored by TLC (2.5 h). To the reaction
mixture was added silica gel. The reaction mixture was
evaporated to dryness and flash chromatographed on silica gel
eluting with ether/hexane (1:4) to give 9.3 g (64%) of 3 as a
colorless oil.
3-[6-[4(S)-(3-t-BOC-2,2-dimethyloxazolidinyl)]-3-hexenyl]-
ben za ld eh yd e (4). The aryl bromide 3 (9.3 g, 21.2 mmol) in
200 mL of THF was cooled to -78 °C. A solution of 2.5 M
nBuLi (15.3 mL, 38.3 mmol) was added dropwise. After
completion of the nBuLi addition, DMF was added and the
mixture was warmed to 0 °C over a 1 h period. The reaction
solution was poured into ice water and extracted with ether
two times. The ethereal layer was washed with water (2×)
and brine (2×), dried over MgSO4, filtered, and evaporated to
dryness. The residue was flash chromatographed on silica gel,
eluting with ether/hexane (1:3), affording 5.16 g (63%) of 4 as
a colorless oil: 1H NMR (CDCl3) δ 9.97 (s, 1H), 7.7 (s, 2H),
7.43 (m, 2H), 5.40 (m, 2H), 3.85 (m, 1H), 3.69 (d, 2H), 2.7 (t,
2H), 2.34 (m, 2H), 1.9 (m, 2H), 1.7 (m, 2H), 1.56 (s, 3H), 1.50
(s, 3H), 1.40 (s, 9H).
(6S)-3-Meth ylen e-4-oxo-5-a za bicyclo[11.3.1]h ep ta d eca -
1(17),13,15-tr ien e-3-ca r boxylic Acid (11). The diacid 10
(1.0 g, 3.1 mmol) was refluxed for 3 h with paraformaldehyde
(0.135 g, 4.5 mmol) and piperidine (0.059 mL, 0.60 mmol) in 5
mL of pyridine. The mixture was cooled, concentrated, and
taken up in an ethyl acetate/1 N HCl solution. The organic
layer was washed with HCl and brine, dried (MgSO4), and
concentrated to give 0.9 g (96%) of the exo-methylene product
11: 1H NMR (CDCl3) δ 7.23 (d, 1H), 7.19 (d, 1H), 7.06 (d, 1H),
7.0 (d, 1H), 6.08 (d, 1H), 5.9 (s, 1H), 5.55 (s, 1H), 4.57 (m, 1H),
3.93 and 3.52 (dd, 2H), 2.64 (m, 2H) 1.8-1.0 (m, 10H).
(6S)-3-[(Acetylth io)m eth yl]-4-oxo-5-a za bicyclo[11.3.1]-
h ep ta d eca -1(17),13,15-tr ien e-6-ca r boxylic Acid (12). The
exo-methylene macrocyclic lactam 11 (0.9 g, 3.0 mmol) was
stirred in thioacetic acid (10 mL) for 2 days, concentrated to
give 12, and used as is in the next step.
2-[[3-[6-[4(S)-(3-t-BOC-2,2-dim eth yloxazolidin yl)]-3-h ex-
en yl]p h en yl]m eth ylen e]-1,3-p r op a n ed ioic Acid 1-Meth yl
3-Ben zyl Ester (5). A mixture of benzyl methyl malonate
(2.79 mL, 14.6 mmol), piperidine (0.99 mL, 10.0 mmol), benzoic
acid (0.93 g, 7.6 mmol), and the aldehyde 4 (5.16 g, 13.3 mmol)
in 200 mL of toluene was refluxed with water removal using
a Dean-Stark condenser for 6 h. The mixture was cooled to
room temperature, stirred overnight, and diluted with ether,
and the organic layer was washed with 1 N HCl (2×), NaHCO3
(2×), and brine (2×), dried (MgSO4), and evaporated to
dryness. The residue was chromatographed on silica gel
eluting with ether/hexane (1:3) to give 6.15 g (82%) of 5 as a
yellow oil: 1H NMR (CDCl3) δ 7.75 (d, 1H), 7.4-7.1 (m, 8H),
5.32 (m, 2H), 5.29 (s, 2H), 3.84 (s, 3H), 3.8 (m, 2H), 3.7 (m,
1H), 2.65 (t, 1H), 2.08 (t, 1H), 2.3 (m, 2H), 1.95 (m, 2H), 1.6
(m, 2H), 1.55 (two s, 3H), 1.45 (two s, 9H), 1.25 (s, 3H).
2-[[3-[6-[4(S)-(3-t-BOC-2,2-d im eth yloxa zolid in yl)]h exy-
l]p h en yl]m eth yl]-1,3-p r op a n oic Acid 1-Meth yl Ester (6).
3-[(Ac e t y lt h io )m e t h y l]-4-oxo -5-a za b ic y c lo [11.3.1]-
h ep ta d eca -1(17),13,15-tr ien e-6-ca r boxylic Acid Meth yl

+
+
Meta-Substituted Benzofused Macrocyclic Lactams
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 513
Ester [13a (3S,6S) Dia ster eoisom er , 13b (3R,6S) Dia ste-
r eoisom er ]. To the acid 12 (crude product, 3.0 mmol theo-
retical) in 10 mL of methanol at 0 °C was added diazomethane
in ether. The solution was stirred at room temperature for
30 min, concentrated, and chromatographed on silica gel,
eluting with ethyl acetate/hexane (1:4) to give 346 mg of the
(S,S) isomer 13a and 300 mg of the (R,S) isomer 13b. The
(R,S) diastereomer 13b was crystallized from methylene
chloride-hexane-ether to obtain X-ray quality crystals. 13a :
1H NMR (CDCl3) δ 7.2 (m, 2H), 7.0 (m, 2H), 5.86 (d, 1H), 4.30
(m, 1H), 3.7 (s, 3H), 3.3-2.5 (multiplets, 7H), 2.37 (s, 3H),
their conformations in torsion space. Various conformations
of the cyclic parts of the molecules are also generated.
TFIT is used to evaluate superimposition of molecules on a
proposed template or pharmacophore. The template is fixed
and a single test molecule is fitted to it using superimposition
plus internal energy. TFIT explores the fit of different
conformations of cyclic molecules by generating a set of low-
energy conformations (Monte Carlo search with internal
energy minimization) and then fitting all the conformations
using torsional, rotational, and transitional degrees of freedom.
The output of TFIT contains a set of conformers which fit
the template. The ligand strain energy and the superposition
energy are reported for each conformation. The ligand strain
energy is the difference between the energy of the superim-
posed conformation and the global minimum internal energy
of the compound in vacuum. The superposition energy is a
measure of the match between the test compound and the
template. This energy is computed using the superposition
potential: when an atom of the ligand approaches an atom of
the template of similar type, a negative potential energy is
experienced. The superposition energy reaches a minimum
value when two atoms of the same type are superimposed. This
term is a useful measure for comparing the degree of super-
imposition that test compounds with closely similar structures
can achieve.
1
1.70-1.0 (m, 10H). 13b: [R]D ) +56.9 (c ) 10.1 CH2Cl2); H
NMR (CDCl3) δ 7.15 (m, 2H), 7.0 (m, 2H), 5.71 (d, 1H), 4.48
(m, 1H), 3.17 (s, 3H), 3.1 (d, 2H), 2.84 (m, 2H), 2.57 (m, 2H),
2.37 (s, 3H), 1.7-1.0 (m, 10H).
Com p ou n d s (3R,6S)-3-(m er ca p tom eth yl)-4-oxo-5-a za -
b icyclo[10.3.1]h exa d eca -1(16),12,14-t r ien e-6-ca r b oxylic
a cid (15a ) a n d (3S,6S)-3-(m er ca p t om et h yl)-4-oxo-5-
a za bicyclo[10.3.1]h exa d eca -1(16),12,14-tr ien e-6-ca r boxy-
lic a cid (15b), the 13-membered ring S-acetyl derivatives,
were prepared analogous to 13a and 13b, starting from the
aldehyde 1b derived from S-aspartic acid.
15a : 1H NMR (CDCl3) δ 7.25 (m, 1H), 7.0 (m, 3H), 5.6 (d,
1H), 4.02 (m, 1H), 3.65 (s, 3H), 3.2 (m, 3H), 2.7 (m, 2H), 2.55
(s, 3H), 1.7 (m, 4H), 1.3 (m, 2H), 0.68 (m, 2H). 15b: 1H NMR
(CDCl3) δ 7.2 (m, 1H), 7.0 (m, 3H), 5.40 (d, 1H), 4.5 (m, 1H),
3.66 (s, 3H), 3.2-3.4 (m, 7H), 2.33 (s, 3H), 1.87 (m, 2H), 1.35
(m, 6H).
Ack n ow led gm en t. We thank the CIBA analytical
staff in Summit, NJ , for collecting the analytical data.
We also thank Ms. C. Berry, Ms.Y. Sakane, Mr. M. Beil,
and Ms. T. Gerlock for assistance in in vitro measure-
ments of NEP and TLN and the ANP assay. The TFIT
and TFIND programs are available from Colin McMar-
tin.
3-(Mer ca p t om et h yl)-4-oxo-5-a za b icyclo[11.3.1]h ep t a -
d eca -1(17),13,15-tr ien e-6-ca r boxylic Acid [14a (3S,6S)
Dia ster eom er , 14b (3R,6S) Dia ster eom er ]. The thioac-
etate methyl ester 13a (200 mg, 0.51 mmol) in 15 mL of THF
and 4 mL of water was deoxygenated by purging the solution
with nitrogen. To this solution was added lithium hydroxide
(85 mg, 2.0 mmol) and stirring was continued for 4 h. The
reaction was quenched with 1 N HCl, and the mixture was
extracted with ethyl acetate, dried (MgSO4), filtered, and
concentrated to give 172 mg (98%) of 14a as a colorless solid
melting at 186-191 °C: 1H NMR (DMSO-d6) δ 12.3 (br, 1H),
8.02 (d, 1H), 7.12 (m, 1H), 6.96 (m, 2H), 3.7 (m, 1H), 3.0-2.5
(m, 9H), 1.6 (m, 4H), 1.1 (m, 6H). Anal. (C18H25NO3S) C, H,
N. 14b prepared from 13b: 1H NMR (DMSO-d6) δ 12.5 (br,
1H), 8.12 (d, 1H), 7.16 (t, 1H), 6.96 (t, 2H), 4.20 (m, 1H), 2.8-
2.3 (m, 9H), 1.8-0.8 (m, 10H). Anal. (C18H25NO3S) C, H, N.
Prepared similarly from 15a and 15b respectively are
(3R,6S)-3-[(a cetylth io)m eth yl]-4-oxo-5-a za bicyclo[10.3.1]-
h exa d eca -1(16),12,14-tr ien e-6-ca r boxylic a cid m eth yl es-
Refer en ces
(1) (a) Robl, J . A.; Cimarusti, M. P.; Simpkins, L. M.; Brown, B.;
Ryono, D. E.; Bird, J . E.; Asaad, M. M.; Schaeffer, T. R.;
Trippodo, N. C. Dual Metalloprotease Inhibitors. 6. Incorporation
of Bicyclic and Substituted Monocyclic Azepinones as Dipeptide
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dopeptidase Inhibitors J . Med. Chem. 1996, 39, 494-502. (b)
De Lombaert, S.; Tan, J .; Stamford, L.; Sakane, Y.; Berry, C.;
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2715-2720. (c) MacPherson, L. J .; Baybert, E. K.; Capparelli,
M. P.; Bohacek, R. S.; Clarke, F. H.; Ghai, R. D.; Sakane, Y.;
Berry, C. J .; Peppard, J . V.; Trapani, A. Design and Synthesis
of an Orally Active Macrocyclic Neutral Endopeptidase 24.11
Inhibitor. J . Med. Chem. 1993, 36, 3821-3828. (d) Bhagwat, S.
S.; Fink, C. A.; Gude, C.; Chan, K.; Qiao, Y.; Sakane, Y.; Berry,
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Angiotensin Converting Enzyme and Neutral Endopeptidase
24.11. Bioorg. Med. Chem. Lett. 1994, 4, 2673-2676. (e) Turcaud,
S.; Gonzalez, W.; Michel, J .; Roques, B. P.; Fournie-Zaluski, C.
Diastereoselective Synthesis of Mixanpril, an Orally Active Dual
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Slusarchyk, W. A.; Robl, J . A.; Taunk, P. C.; Asaad, M. M.; Bird,
J . E.; DiMarco, J .; Pan, Y. Dual Metalloprotease Inhibitors. V.
Utilization of Bicyclic Azepinonethiazolidines and Azepinonetet-
rahydrothiazines in Constrained Peptidomimetics of Mercap-
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(g) Das, J .; Robl, J . A.; Reid, J . A.; Sun, C. Q.; Misra, R. N.;
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zazepinone Based Mercaptoacetyls. Bioorg. Med. Chem. Lett.
1994, 4, 1795-1800. (j) Robl, J . A.; Simpkins, L. M.; Stevenson,
J .; Kelly, Y. F.; Sun, C. Q.; Murugesan, N.; Barrish, J . C.; Asaad,
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1
ter (16a ): H NMR (MeOD4) δ 7.2 (m, 1H), 7.0 (m, 3H), 3.56
(m, 1H), 2.9 (t, 2H), 2.7-2.5 (m, 4H), 2.0-1.2 (m, 6H), 0.9 (m,
2H). Anal. (C17H23NO3S) C, H, N.
(3S,6S)-Met h yl 3-[(a cet ylt h io)m et h yl]-4-oxo-5-a za b i-
cyclo[10.3.1]h exadeca-1(16),12,14-tr ien e-6-car boxylic acid
(16b): 1H NMR (MeOD4) δ 8.0 (d, 1H), 7.2 (m, 1H), 7.0 (m,
3H), 4.3 (m, 1H), 2.9-2.5 (m, 6H), 2.0-0.8 (m, 8H). Anal.
(C17H23NO3S) C, H, N.
TF IND a n d TF IT. Template making and template fitting
were performed using a program called QXP (quick explore).4a,b
This program explores conformations by making Monte Carlo
perturbations in torsion space followed by rapid Cartesian
minimization. Atoms which are part of a cyclic system receive
Cartesian perturbations at the Monte Carlo step. The program
has application scripts for template making (TFIND) and for
template fitting (TFIT).
For both template applications the program uses an ex-
tended force field potential which includes a superposition
energy. When the superposition energy is switched on, atoms
that are close to each other in space and of similar chemical
type but belong to different molecules receive an attractive
potential which forces them to superimpose. Atoms are typed
according to hydrophobicity, hydrogen bonding character, and
formal charge. The internal energies of the aligned molecules
are computed with the intermolecular electrostatic and van
der Waals forces switched off. These searches result in
structures of low internal strain energy which are aligned to
optimize the match between atoms.
For template finding, several molecules can be submitted.
These molecules are allowed to translate, rotate, and alter

+
+
514 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Ksander et al.
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of human neutrophil collagenase inhibited by
a substrate
analogue reveals the essentials for catalysis and specificity.
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