Potent and selective non-peptidic inhibitors of endothelin-converting enzyme-1 with sustained duration of action

Article abstract of DOI:10.1021/jm990507o

Potent and selective non-peptidic inhibitors of human endothelin- converting enzyme-1 (ECE-1) have been designed as potential modulators of endothelin (ET-1) production in vivo. Because of its unique structural characteristics and long duration of action

Full text of DOI:10.1021/jm990507o

488
J . Med. Chem. 2000, 43, 488-504
P oten t a n d Selective Non -P ep tid ic In h ibitor s of En d oth elin -Con ver tin g
En zym e-1 w ith Su sta in ed Du r a tion of Action
Ste´phane De Lombaert,* Louis Blanchard, Lisa B. Stamford, J enny Tan, Eli M. Wallace, Yoshitaka Satoh,
J ohn Fitt, Denton Hoyer, David Simonsbergen, J ohn Moliterni, Nicholas Marcopoulos, Paula Savage,
Mary Chou, Angelo J . Trapani, and Arco Y. J eng
Metabolic and Cardiovascular Diseases, Novartis Institute for Biomedical Research, Summit, New J ersey 07901
Received October 7, 1999
Potent and selective non-peptidic inhibitors of human endothelin-converting enzyme-1 (ECE-
1) have been designed as potential modulators of endothelin (ET-1) production in vivo. Because
of its unique structural characteristics and long duration of action in vivo, the dual ECE-1 and
neutral endopeptidase 24.11 (NEP) inhibitor, CGS 26303, was selected as an attractive lead
for further optimization of potency and selectivity. Replacement of the P1′ biphenyl substituent
of CGS 26303 by a conformationally restricted 3-dibenzofuranyl group led to more potent and
more selective ECE-1 inhibitors, such as the tetrazole 27. The remarkable effect of this P1′
modification allowed for the first time phosphonomethylcarboxylic acids, such as 29, to display
both potent (IC50 ) 22 nM) and selective (104-fold vs NEP) ECE-1 inhibition. Chemoenzymatic
syntheses of the new R-amino acid (S)-3-dibenzofuran-3-ylalanine intermediate were developed,
and improved procedures to generate substituted R-aminoalkylphosphonic acids were devised
to support the production of various analogues. Although additional gains in intrinsic ECE-1
inhibitory potency could occasionally be achieved by addition of a P1 side chain, these compounds
(e.g. 43a ) showed poor functional activity in vivo in the big ET-1 pressor test. Phosphonoalkyl
dipeptides featuring 3-dibenzofuranyl groups in both the P1′ and P2′ positions were also very
potent ECE-1 inhibitors, albeit lacking the desired selectivity against NEP. Functionally, 27
and 29 were the two most efficacious compounds from this study, producing sustained inhibition
of ECE-1 activity in rats, as measured by their ability to block the hypertensive effects induced
by big ET-1. This profile was similar to that of a potent ETA/ETB dual receptor antagonist, SB
209670. Due to their favorable in vitro and in vivo profiles, 27 (CGS 34043) and 29 (CGS 35066)
constitute new pharmacological tools useful in assessing the role of ECE-1 in pathological
conditions.
In tr od u ction
sented a more challenging task.8 Endothelin-converting
enzyme-1 (ECE-1) is a zinc metalloprotease catalyzing
the posttranslational conversion of big ET-1 to ET-1 in
a rate-limiting fashion9 and, therefore, constitutes a
prime therapeutic target for the regulation of ET-1
production in vivo.10 It is ubiquitously distributed in the
human vascular endothelium, where both big ET-1 and
mature ET-1 have been observed.11 In addition, ECE-1
activity has been detected in brain tissues. Until now,
three isoforms, ECE-1a, ECE-1b, and ECE-1c, differing
only in their N-terminal regions as a result of alterna-
tive splicing of a single gene, have been identified in
human tissues.11-13 Recent supporting evidence for the
significance of ECE-1 in ET-1 biosynthesis includes the
observation that disruption of the ECE-1 gene in mice
results in term embryos exhibiting phenotypes similar
to those lacking the ET-1 or ETA receptor genes.14
Although ECE-1 emerges as a critical player in the
biosynthesis of ET-1, it still remains possible that other
enzymes could partially contribute to the proteolytic
cleavage of big ET-1 under physiological or pathological
conditions, or even under chronic ECE-1 inhibition in
vivo. Selective inhibitors of ECE-1 with good in vivo
efficacy would represent novel pharmacological tools to
address this question. Until recently, the most potent
ECE-1 inhibitors reported have also demonstrated
inhibitory activity against other zinc metalloproteases,
Since its initial characterization as an exceedingly
potent vasoconstrictive peptide, endothelin-1 (ET-1) has
been the target of an intense research effort aimed at
elucidating its potential significance in health and
diseases.1 From these studies, ET-1 has emerged as an
important regulatory element during fetal development,
while its overproduction, resulting in abnormally high
plasma or tissue levels, has been implicated in a
heterogeneous list of diseases encompassing systemic
and pulmonary hypertension, cerebral vasospasm and
stroke, asthma, cardiac and renal failure, atheroscle-
rosis, preeclampsia, benign prostatic hyperplasia, and
carcinogenesis.2-5 Among several conceptual therapeu-
tic approaches to block the biological effects of ET-1, the
antagonism of its G-protein-coupled receptors, ETA and/
or ETB, is currently the most advanced. Indeed, several
potent peptidic and non-peptidic ET-1 receptor antago-
nists, such as SB 209670, have been described,6 and
several members of this class of compounds are pres-
ently undergoing clinical trials.7
By contrast, the discovery of potent and selective
inhibitors of ET-1 biosynthesis has apparently repre-
* Address for correspondence: Neurogen Corp., 35 Northeast In-
dustrial Rd., Branford, CT 06405. Tel: 203-315-3009. Fax: 203-483-
7027. E-mail: sdelombaert@nrgn.com.
10.1021/jm990507o CCC: $19.00 © 2000 American Chemical Society
Published on Web 01/14/2000

Non-Peptidic Inhibitors of ECE-1
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 489
Ch a r t 1
in particular neutral endopeptidase 24.11 (NEP), an
enzyme involved in the degradation of several vaso-
active peptides including atrial natriuretic peptide
(ANP).8 From an efficacy point of view, we and oth-
ers15,16 have favored the rationale that dual ECE/NEP
inhibitors could exhibit a superior therapeutic profile
in the treatment of various cardiovascular and renal
disorders, by depressing the effects of ET-1 while
concomitantly potentiating those of ANP. In parallel,
we have also searched for agents able to selectively
inhibit ECE-1 because inhibition of NEP may not
always be necessary or even desirable under some
pathological conditions such as asthma.17 Several at-
tempts to obtain selective ECE-1 inhibitors by system-
atic structural modification of the dipeptidic metallo-
protease inhibitor phosphoramidon18,19 or by screening
compound and natural product libraries have been
reported.8,20-22 Although moderate success has been
reported occasionally, the identification of potent and
selective ECE-1 inhibitors that possess sustained func-
tional activity in vivo still remains a challenge. Conse-
quently, the in vivo characterization of ECE inhibitors
remains scarce.23,24
Discovered several years ago, CGS 26303 (Chart 1)
is a structurally unique non-peptidic dual ECE/NEP
inhibitor characterized by a long duration of action in
vivo.25 As such, this compound produced therapeutic
benefits in preclinical models of hypertension25 and cere-
bral vasospasm following subarachnoid hemorrhage,26-28
a condition linked to a local overproduction of ET-1.
Although CGS 26303 represented an attractive lead
toward the rational design of more potent and, possibly,
more selective ECE inhibitors, preliminary structure-
activity relationship investigations indicated that even
minor structural alterations of the core template gener-
ally resulted in a dramatic loss of ECE inhibitory
activity. One successful approach, however, consisted
in adding a lipophilic P1 side chain, as in CGS 31447,
thereby increasing the in vitro potency significantly.29
However, this compound, and other related analogues,
retained potent NEP inhibitory activity.30 More recently,
our search for surrogates of the biphenyl group has led
to the discovery of 2-fluorophenylacetylene derivatives
(e.g. 1) which showed enhanced inhibitory potency and
selectivity (defined as the ratio of IC50 NEP to IC50 ECE)
relative to CGS 26303, without the need for a P1
substituent.31 Di- and tripeptide derivatives (e.g. 2a and
2b) displayed an even superior profile in vitro, although
they departed from our major focus of designing truly
non-peptidic inhibitors. When administered intrave-
nously to anesthetized rats, 2a and 2b inhibited by 51%
and 64%, respectively, the pressor response induced by
big ET-1 for at least 15 min.31 Unfortunately, possibly
because of their peptidic nature, these highly selective
ECE inhibitors failed to maintain their inhibitory effects
when big ET-1 was administered 90 min post-dosing.
At this time point, the levels of pressor response
inhibition by 2a and 2b had been reduced to 12% and
13%, respectively (results not shown). In addition, we
noted that, despite their higher ECE-1 inhibitory po-
tency and selectivity in vitro, these two compounds were
less effective in vivo than the intrinsically weaker ECE
inhibitor, phosphoramidon.
Remarkably, despite weak potency and poor ECE
selectivity, the non-peptidic inhibitors CGS 26303 and
its 2-fluoroacetylene tetrazole analogue 1 were charac-
terized by a strong and sustained functional inhibition
in vivo (50% and 83% inhibition of the pressor response
at 90 min, respectively). Since 1 was a more potent and
more selective ECE inhibitor than CGS 26303, we aimed
for further structural optimization of the P1′ substituent.
Toward that goal, and considering the narrow structural
tolerance in that region of the inhibitors, we focused on
altering the conformation of the distal phenyl ring at
the P1′ position as a possible means to enhance the
potency and selectivity of these non-peptidic inhibitors
toward ECE. In this article, we describe a new series of

490 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
De Lombaert et al.
Sch em e 1a
a
(a) H2N(CH2)2CN, EDC; (b) (CF3SO)2O, NaN3; (c) H2NNH2; (d) HCl; (e) Ph2CdNH; (f) NaHMDS, RBr; (g) NaHCO3; (h)
(EtO)2P(O)CH2OSO2CF3, EtN(iPr)2; (i) DBU; (j) TMSBr; (k) NaOH then CHP-20.
ECE-1 inhibitors featuring coplanar biphenyl surrogates
at the P1′ position.
method 1, Heck coupling between 3-iododibenzofuran
and methyl 2-acetamidoacrylate afforded the R,â-
unsaturated R-amido ester 12 which was hydrogenated
to yield the racemic ester 13. Incubation of 13 with
alcalase led to the enantiospecific hydrolysis of the S
enantiomer, giving the carboxylic acid 14. The unreacted
R enantiomer 13(R) was recycled by a sequence of
epimerization/resolution to provide an additional amount
of acid 14. On larger scale, this recycling protocol could
be repeated several times. The latter compound was
eventually converted in four straightforward steps to
the BOC-protected R-amino acid 18 (Scheme 2).
Method 2 relies on a biaryl formation via ortho
palladation35 of the readily available diaryl ether 1936
and provided a more direct access to the acid intermedi-
ate 14. Besides the desired product 20, a small amount
(14%) of regioisomer 21 was also formed when the
reaction was carried out in acetic acid. The reaction
proceeded faster in trifluoroacetic acid (3 h at reflux),
but a larger amount of undesired isomer 21 was formed
under these conditions. Although the latter compound
could not be easily separated from 20, a small amount
was eventually obtained for characterization purposes
by successive triturations in ether. It was also deter-
mined that 21 was not a substrate to alcalase. There-
fore, subsequent enzymatic digestion of the mixture of
ethyl esters 20 and 21 provided both an efficient
separation and a resolution method, affording the key
carboxylic acid intermediate 14 in high chemical and
enantiomeric purity (Scheme 3). Recycling of the enan-
Ch em istr y
The synthesis of 11a (X ) CH2), 11b (X ) NCH3), and
11c (X ) CdO), the fluorene, 9-methylcarbazole, and
fluorenone analogues of CGS 26303, respectively, ex-
ploits a modification of the alkylation protocol of tetra-
zole benzophenone imines reported previously (Scheme
1).32
In the current variation, the 2-cyanoethyl group was
chosen as the N1-protecting group of the tetrazole
instead of the originally prescribed 4-methoxybenzyl,
because the conditions for its removal were milder and
chemically more compatible. When the alkylation step
was carried out at -78 °C, the loss of the 2-cyanoethyl
group by â-elimination was minimized, allowing the
reaction to proceed with moderate yields. After acid
hydrolysis of the imines 7a -c, the target aminophos-
phonic acids were elaborated by alkylation of the
corresponding amines 8a -c with dimethylphosphono-
methyl triflate, as described previously.31,33 The final
compounds were usually converted to their trisodium
salts 11a -c to maximize water solubility. When neces-
sary, these salts were purified by chromatography on a
CHP-20P resin34 (see Experimental Section).
The novel R-amino acid, (S)-3-dibenzofuran-3-ylala-
nine (15), was selected as a common building block for
the preparation of various derivatives and was obtained
by two alternative methods (Schemes 2 and 3). In

Non-Peptidic Inhibitors of ECE-1
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 491
Sch em e 2a
a
(a) Methyl 2-acetamidoacrylate, Pd(OAc)2, nBu4NCl, DMF; (b) Pd-C, H2; (c) alcalase, NaHCO3, MeCN; (d) HCl; (e) HCl, MeOH; (f)
BOC2O; (g) LiOH, MeOH; (h) DBU.
Sch em e 3a
a
(a) Pd(OAc)2, AcOH; (b) alcalase, NaHCO3, MeCN; (c) DBU or EtOH, EtONa(cat.).
tiomer 20(R) was performed as described above, but the
number of cycles was limited by the accumulation of
regioisomer 21 which hindered the crystallization of 14.
For the synthesis of 27, the elaboration of the tetra-
zole ring and the introduction of the phosphonomethyl
group followed procedures specifically developed earlier
for the preparation of CGS 26303 (Scheme 4).37 Par-
ticularly noteworthy is the use of the diphenyl phos-
phonate 26 (CGS 34753) which serves both as a chemi-
cal precusor and as a potential orally bioavailable
prodrug of 27.
Starting from the R-amino ester 16, a similar syn-
thetic approach was used to prepare the corresponding
N-phosphonomethyl R-amino acid analogue 29. In this
case, an exhaustive acid hydrolysis of the diphenyl
phosphonate intermediate 28 afforded 29 directly
(Scheme 5). The high enantiopurity (>96%) of this
compound was confirmed by NMR analysis.38

492 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
De Lombaert et al.
Sch em e 4a
a
(a) H2N(CH2)2CN, BOP; (b) TMSN3, DIAD, PPh3; (c) HCOOH; (d) HCHO(aq), NaHCO3; (e) PO(OPh)2; (f) DBU; (g) NaHCO3.
Sch em e 5a
a
(a) HCHO(aq), NaHCO3; (b) PO(OPh)2; (c) HCl(aq), 100 °C.
C-Terminal amide derivatives (30, 31) and dipeptides
(32-36) (Table 2) were obtained readily according to the
methods already described for the biphenyl and pheny-
lacetylene analogues.31,39,40
(Scheme 6). Compounds 37a -v were generally obtained
as mixtures of two diastereoisomers (A and B), which
could often be separated by chromatography on SiO2.
If the resolution was poor, the mixtures were then
converted to the corresponding acids 38a -v and sepa-
rated on Lichroprep DIOL columns. However, because
the saponification of 37a -v often resulted in some
hydrolysis of the dimethyl phosphonate, the mixture of
products, containing mainly 38a -v, was typically used
directly in the final deprotection step with HBr or
TMSBr. The phosphonic acids 39a -v were usually
converted to their sodium salts 40a -v to allow their
solubilization in water (Table 3). When necessary, these
salts could be purified by chromatography on a CHP-
20P resin34 (see Experimental Section). For our purpose,
the lack of a stereoselectivity of the R-aminophospho-
nate synthesis was advantageous since the absolute
configuration required for the P1 side chain was not
Introduction of various substituents at the P1 position
required a convergent access to R-substituted R-amino-
phosphonic acids. Since the published procedures af-
forded low yields of the expected products,30 a synthetic
optimization effort was devoted to address this issue.
We found that the desired substituted R-aminophos-
phonates could be conveniently obtained using a Lewis
acid-promoted addition of trimethyl phosphite to a
preformed imine intermediate41,42 generated from the
condensation of (S)-3-dibenzofuran-3-ylalanine methyl
ester hydrochloride (16) with an aldehyde. The use of
BF3‚Et2O turned out to be particularly advantageous
in this case, readily affording dimethyl R-aminophos-
phonates in satisfactory yields under mild conditions

Non-Peptidic Inhibitors of ECE-1
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 493
Sch em e 6a
a
(a) RCHO (see Table 3 for definitions of R), NEt3, Na2SO4; (b) BF3‚OEt2, P(OMe)3; (c) NaOH; (d) HBr or TMSBr.
Ta ble 1. In Vitro ECE and NEP Inhibitory Potencies of
Sch em e 7a
Phosphonomethyl Aminoalkyl Tetrazoles
a
ECE selectivity is defined as the ratio IC50 NEP/IC50 ECE.
decreased (about 225-fold). Since forcing coplanarity of
the aryl rings not only was tolerated but even appeared
to increase the ECE selectivity and, to a lesser extent,
the ECE inhibition, we have concentrated on incorpo-
rating structural attributes that could potentially en-
hance the inhibitory activity of the phosphonic acids.
Although the 9-methylcarbazole 11b displayed much
weaker activities than CGS 26303, the fluorenone 11c
and, especially, the dibenzofuran 27 were both potent
and moderately selective ECE inhibitors. The potency-
enhancing effect of a dibenzofuran at the P1′ position
was even more dramatically demonstrated in a series
of carboxylic acids. We have reported that the replace-
ment of the tetrazole heterocycle of CGS 26303 with a
carboxyl group resulted in a substantial loss of ECE
inhibition.30 By contrast, the dibenzofuran analogue 29
maintained potent ECE inhibitory activity while its
NEP activity was only in the micromolar range, leading
to an ECE selectivity of about 104, i.e. a 43000-fold
improvement over CGS 26303 (Table 2). Terminal
amides 30 and 31 were found to be weak ECE inhibi-
tors, a remarkable observation considering that similar
derivatives bearing a 2-fluorophenylacetylene P1′ sub-
stituent maintained significant ECE inhibitory activity.
Little emphasis was devoted to dipeptide analogues, but
among the few compounds prepared, such as 32-36,
those featuring large hydrophobic P2′ substituents,
including the 3-dibenzofuranyl group itself, produced
a
(a) 1-Naphthyl-CHO, NEt3, Na2SO4; (b) BF3‚OEt2, P(OMe)3;
(c) DBU then separate diastereomers on Lichroprep DIOL; (d)
HBr, AcOH; (e) NaOH then CHP-20.
known a priori. Unfortunately, in these diastereomers,
we were unable to unambiguously assign the configu-
ration of the R-aminophosphonate carbon. This R-ami-
nophosphonate synthesis could also be carried out with
the cyanoethyl-protected tetrazole analogue 24, provid-
ing an access to the two diastereomers 43a and 43b
(Scheme 7).
Resu lts a n d Discu ssion
In Vitr o Activity. The incorporation of a local
conformational constraint into a ligand can be a suc-
cessful strategy to increase its binding and selectivity
toward a particular molecular target. We have applied
this approach to the biphenyl group of CGS 26303,
aiming for a minimal yet significant alteration of the
original inhibitor core structure. The replacement of the
biphenyl group of CGS 26303 by a fluorene moiety
provided early support to this strategy (Table 1), as-
suming that the inhibitory activities (ECE and NEP)
of the phosphonic acids reside essentially in a single S
enantiomer, as suggested from previous SAR studies in
this series. Indeed, not only was 11a , as a racemate, a
slightly more potent ECE inhibitor than racemic CGS
26303, but its intrinsic NEP affinity was substantially

494 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
De Lombaert et al.
Ta ble 2. In Vitro ECE and NEP Inhibitory Potencies of
Phosphonomethyl 3-Dibenzofuranyl Alanine Derivatives
F igu r e 1. Inhibition of the increase in mean arterial pressure
elicited by big ET-1 in anesthetized, ganglion-blocked rats.
Rats were injected intravenously with the test compounds (10
mg/kg) or the vehicle (0.05 N NaOH, 1 mL/kg), then challenged
with big ET-1 (1 nmol/kg, iv) 15 and 90 min later. SB 209670,
phosphoramidon (PHOS), CGS 31447, CGS 34043 (27), and
CGS 35066 (29) attenuated the increase in mean arterial
pressure when compared to vehicle. Results are expressed as
percent inhibition of the big ET-1-induced pressor response
and listed on top of bars. Values are the mean ( SEM (n ) 4).
a
% Inhibition of ECE activity at a concentration of 1 µM of test
compound. Sodium salt. c ND, not determined. ECE selectivity
b
d
mals were challenged with big ET-1 (1 nmol/kg, iv) 15
and 90 min after intravenous administration of the
inhibitor (10 mg/kg). It had been demonstrated previ-
ously that, in the absence of ECE inhibitor, the pressor
response lasted for about 1 h and that the mean arterial
pressure had returned to control levels by 90 min. Many
peptidic ECE inhibitors, including the recently described
phenylacetylene di- and tripeptides 2a and 2b, while
functionally active at early time points (15 min), were
unable to block the pressor response substantially at
90 min (results not shown). Even the potent non-
peptidic inhibitor CGS 31447 was also plagued with a
short duration of action (Figure 1). Considering that,
under testing conditions, the rat plasma levels of CGS
31447 were extremely high (about 20 µg/mL),43 we
suspect an exceedingly high plasma protein binding at
the equilibrium state (> 99%)43 to be responsible for
limiting the access of the inhibitors to the target
enzyme. Similarly, among the new, potent ECE inhibi-
tors described in this work, those featuring a P1 lipo-
philic side chain also typically displayed weak or short-
lasting in vivo activity (Table 4).
However, compounds lacking these structural attri-
butes fared much better in this in vivo test. In particu-
lar, after a single intravenous administration of a 10
mg/kg dose to rats, 27 and 29 significantly prevented
the big ET-1-induced pressor response at both 15 and
90 min and compared favorably with the potent ETA/
ETB receptor antagonist SB 209670 (Figure 1). It is
worthwhile noting that, even at this dose, a complete
blockade of the pressor response was never achieved
(maximum 90%) with either the selective ECE inhibi-
tors, the ECE/NEP inhibitors, or the ET receptor
antagonist.
is defined as the ratio IC50 NEP/IC50 ECE.
potent ECE inhibition. Interestingly, the presence of
this second amino acid residue, as in 36, also restored
NEP inhibition, thereby imparting mediocre selectivity
to these compounds (Table 2). By contrast, it is worth
noting the weak NEP inhibitory activity, or even lack
thereof, of the dipeptides 34 and 35, considering that
structurally very close analogues, differing only by the
absence of a bridging oxygen in the biaryl P1′ group,
potently inhibited NEP at low nanomolar concentra-
tion.39
Since addition of a lipophilic P1 substituent had
resulted in an increase of ECE inhibitory activity in a
related biphenyl series (e.g. CGS 31447), substituted
derivatives of 27 were prepared (Table 3). A naphthyl-
ethyl substituent was originally selected for direct
comparison with CGS 31447. Although this modification
failed to provide a compound (40a ) with enhanced
potency, the selectivity toward ECE was increased.
Other substituents, potentially acting as naphthyl sur-
rogates but conferring lower lipophilicity and possibly
enhanced water solubility at physiological pH, were
selected from methoxy-substituted phenyl groups and
quinolyl moieties (40c-n ). In general, one of the dia-
stereomers produced was a potent (14-40 nM) and
highly selective ECE inhibitor. Methyl, isobutyl, ami-
nopropyl, and aminobutyl side chains, albeit tolerated,
were not optimal for ECE inhibition (40o-v). On the
basis of previous SAR considerations, the combination
of a 1-naphthylethyl P1 side chain with a 3-dibenzo-
furanyl substituent in P1′ and a terminal tetrazole group
was expected to be a very potent ECE inhibitor. Indeed,
the diastereomer 43a was the most potent inhibitor of
the recombinant human ECE-1 reported to date.
Con clu sion
In Vivo Activity. The functional activity of the
selective ECE inhibitors was evaluated according to
their ability to inhibit the big ET-1-induced pressor
response in anesthetized, ganglion-blocked rats. Ani-
In the present study, we have demonstrated that
replacement of the P1′ biphenyl substituent of CGS
26303 by a conformationally restricted 3-dibenzofuranyl

Non-Peptidic Inhibitors of ECE-1
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 495
Ta ble 3. In Vitro ECE and NEP Inhibitory Potencies of Phosphonoalkyl 3-Dibenzofuranyl Alanine Derivatives and Tetrazole
Analogues
a
b
Diastereomer A is obtained from the least polar aminophosphonate 37 (highest Rf by TLC) according to Scheme 6. Chemical yield,
after separation by flash chromatography, of aminophosphonate diastereomer 37 according to Scheme 6. c % Inhibition of ECE activity
at a concentration of 1 µM of test compound. Single determination. e Due to a low recovery yield, this aminophosphonate was not converted
d
to the final aminophosphonic acid. f NA, not available. ND, not determined. Yield of N-phthalimido-protected aminophosphonate
g
h
i
j
k
precursor. Contains 43% of diastereomer 37u . Contains 43% of diastereomer 40u . Chemical yield, after separation on Lichroprep
DIOL, of aminophosphonate diastereomers 42a and 42b according to Scheme 7. Sodium salt. ECE selectivity is defined as the ratio
l
m
IC50 NEP/IC50 ECE.
group leads to more potent and more selective ECE
inhibitors, such as 27. The remarkable effect of this P1′
modification allowed for the first time phosphonometh-
ylcarboxylic acids, such as 29, to display both potent
(IC50 ) 22 nM) and selective (104-fold vs NEP) ECE
inhibition. Although additional gains in intrinsic po-
tency could occasionally be achieved by addition of a P1
side chain, these analogues (e.g. 43a ) showed poor
functional activity in vivo in the big ET-1 pressor test.
Phosphonoalkyl dipeptides featuring 3-dibenzofuranyl
groups in both the P1′ and P2′ positions were also very
potent ECE-1 inhibitors, albeit lacking the desired
selectivity against NEP. On the basis of the unique
binding mode of CGS 26303 to ECE, relative to other
dipeptidic congeners, it remains possible that the non-
peptidic compounds presented in this study share a
common SAR which differs from that of the dipeptide
analogues.44 Functionally, 27 and 29 turned out to be
the two most efficacious compounds from this study,
producing sustained inhibition of ECE-1 activity in rats,
as measured by their ability to block the hypertensive
effects induced by big ET-1. This profile was similar to
that of a potent ETA/ETB dual receptor antagonist, SB
209670. At this time, due to their favorable in vitro and
in vivo profiles, 27 and 29 constitute new pharmacologi-
cal tools useful in assessing the role of ECE-1 in
pathological conditions. Additional pharmacological char-

496 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
De Lombaert et al.
Ta ble 4. Inhibition of the Big ET-1-Induced Pressor Response
by Phosphonoalkyl 3-Dibenzofuranyl Alanine Derivatives
min later. The maximum increase in MAP produced by big
ET-1 in rats treated with test compounds or vehicle was
determined at each time point. Results are expressed as
percent inhibition of the big ET-1-induced pressor response
in animals treated with ECE inhibitors as compared to vehicle.
N-(2-Cya n oet h yl)-2-(1,3-d ioxo-1,3-d ih yd r oisoin d ol-2-
yl)a ceta m id e (3). To a stirred solution of 3-aminopropionitrile
(0.89 mL, 12.1 mmol) in DMF (27 mL) was added N-phthal-
oylglycine (1.86 g, 9.06 mmol) followed by HOBt (1.26 g, 9.3
mmol). The reaction mixture was cooled to 0 °C with an ice
bath and EDC (2.1 g, 10.9 mmol) was added. After stirring
for 1 h at 0 °C then for 1.5 h at ambient temperature, the
reaction mixture was poured into ice-cold H2O and extracted
with EtOAc. The combined organic extracts were washed
successively with 1 N HCl, H2O, 8% NaHCO3 solution, H2O,
and brine. The organic layer was dried over Na2SO4 and
concentrated under reduced pressure to give 3 as a white solid
(62%): mp 187-189 °C; IR (Nujol) 3302, 2243, 1776, 1720,
1
1657, 1566, 956, 716 cm-1; H NMR (DMSO-d6, 300 MHz) δ
2.63 (2H, t, J ) 6.5 Hz), 3.30 (2H, q, J ) 6.3 Hz), 4.20 (2H, s),
7.85-7.93 (4H, m), 8.62 (1H, t, J ) 5.4 Hz); MS (ES1+) m/z
458 (M + 1). Anal. (C13H11N3O3) C, H, N.
a
Results are expressed as percent inhibition of the maximum
3-[5-(1,3-Dioxo-1,3-dih ydr oisoin dol-2-ylm eth yl)tetr azol-
1-yl]p r op ion itr ile (4). Triflic anhydride (31.7 mL, 188 mmol)
was added to a stirred slurry of 3 (44.0 g, 171 mmol) and
sodium azide (12.2 g, 188 mmol) in MeCN (800 mL) at 0 °C
under N2. The mixture rapidly dissolved resulting in a clear
solution. After 16 h, the reaction mixture was poured into 8%
NaHCO3 solution and extracted with CH2Cl2. The organic
extracts were washed with 8% NaHCO3 solution and H2O, then
dried over Na2SO4 and concentrated to give a solid. Recrys-
tallization from MeOH gave 4 as colorless crystals: mp 165-
increase in mean arterial pressure produced by intravenous
administration of big ET-1 at 1 nmol/kg iv, relative to vehicle
(NaOH). Big ET-1 was administered 15 and 90 min after iv bolus
injection of the inhibitor (10 mg/kg). Phosphoramidon tested at
30 mg/kg iv.
b
acterization of 27 (CGS 34043), 29 (CGS 35066), and
their diphenyl phosphonate prodrugs will be reported
elsewhere.
167 °C (67%); IR (Nujol) 2250, 1772, 1725, 1395, 937, 713 cm-1
;
1H NMR (DMSO-d6, 250 MHz) δ 3.27 (2H, t, J ) 6.6 Hz), 4.84
(2H, t, J ) 6.6 Hz), 5.23 (2H, s), 7.86-7.96 (4H, m); MS (DCI)
m/z 283 (M + 1). Anal. (C13H10N6O2) C, H, N.
Exp er im en ta l Section
Melting points were determined on either a Thomas-Hoover
or Mel-Temp II melting point apparatus and are uncorrected.
Infrared (IR) spectra were recorded on a Nicolet 5SXB FTIR
spectrometer. 1H NMR spectra were recorded on either a
Bruker AC-250 or Bruker AC-300 using CDCl3, D2O, or DMSO-
d6 as internal standard. When needed, a few drops of trifluo-
roacetic acid was used to solubilize the phosphonic acids in
DMSO-d6. 31P NMR spectra were recorded on a Bruker AC-
300 spectrometer. 31P chemical shifts are reported relative to
85% aqueous phosphoric acid as external standard. Optical
rotations were measured with a J asco DIP-370 instrument.
Mass spectra were obtained on a MicroMass Platform II
spectrometer. Microanalyses were performed at Robertson
Laboratory, Inc., Madison, NJ . All organic solvents were of
anhydrous grade. Chromatographic separations were per-
formed on either SiO2 60 (flash grade), LiChroprep Diol (E.
Merck), or copolymeric hydrophobic resin CHP-20P (Mitsubishi
Chemical Industries). Some chromatographic separations were
performed on a Biotage Flash40 apparatus with SiO2. Alcalase
(food grade) was acquired from Novo Nordisk (Franklinton,
NC).
3-(5-Am in om et h ylt et r a zol-1-yl)p r op ion it r ile H yd r o-
ch lor id e (5). A mixture of 4 (4.51 g, 16 mmol) and anhydrous
hydrazine (1.0 mL, 32 mmol) in MeOH (100 mL) was stirred
at reflux for 16 h. The reaction mixture was cooled to ambient
temperature and filtered, and the filtrate was concentrated
under reduced pressure. The residue was diluted with CH2-
Cl2 and filtered. The filtrate was concentrated under reduced
pressure to give a yellow oil. The oil was dissolved in MeCN
and 4.5 mL of 3.4 N HCl in Et2O were added. The white
precipitate was collected by filtration, washed with MeCN, and
dried in vacuo (99%): mp 157-159 °C; IR (Nujol) 2900 (vs),
2259, 1611 cm-1; 1H NMR (DMSO-d6, 250 MHz) δ 3.24 (2H, t,
J ) 6.7 Hz), 4.51 (2H, s), 4.81 (2H, t, J ) 6.6 Hz), 9.04 (3H, s).
Anal. (C5H8N6‚HCl) C, H, N.
3-{5-[Ben zh yd r ylid en ea m in om eth yl]tetr a zol-1-yl}p r o-
p ion itr ile (6). Benzophenone imine (2.51 mL, 15.0 mmol) was
added to a stirred slurry of 5 (1.75 g, 9.31 mmol) in CH2Cl2
under N2. After stirring for 16 h, the reaction mixture was
filtered and the filtrate concentrated under reduced pressure
to give an oil. The oil was triturated with hot hexanes (50 mL)
and the resulting residue suspended in hexanes:Et2O (1:1, 100
mL) until a solid formed. The solid was collected by filtration
and dried in vacuo (1.6 g, 55%): mp 108-110 °C; IR (Nujol)
2243, 1626, 699 cm-1; 1H NMR (CDCl3, 250 MHz) δ 3.16 (2H,
t, J ) 7.0 Hz), 4.93 (2H, s), 5.05 (2H, t, J ) 7.1 Hz), 7.20-7.60
(10 H, m); MS (DCI) m/z 317 (M + 1). Anal. (C18H16N6) C, H,
N.
3-{5-[1-(Ben zh yd r ylid en ea m in o)-2-(9H -flu or en -2-yl)-
eth yl]tetr a zol-1-yl}p r op ion itr ile (7a ). Sodium bis(trimeth-
ylsilyl)amide (1.0 M solution in THF) was added slowly over
5 min to a stirred solution of 6 (0.56 g, 1.77 mmol) and
2-(bromomethyl)-9H-fluorene (0.47 g, 1.77 mmol) in THF (10
mL) at -78 °C under an argon atmosphere. After 1 h of stirring
at -78 °C, the reaction mixture was quenched by the addition
of MeOH, then warmed to ambient temperature. The reaction
mixture was diluted with EtOAc and washed with saturated
NH4Cl and brine. The aqueous layer was extracted with
EtOAc. The combined organic extracts were dried over MgSO4
En zym e Assa ys. The ECE-1 (from membranes of CHO cells
expressing human recombinant ECE-1a) and NEP 24.11 (from
rat kidney cortex membrane homogenates) assays were per-
formed as described previously.31,40
Big ET-1-In d u ced P r essor Resp on ses in An esth etized
Ra ts. Male Sprague-Dawley rats (275-450 g) were anesthe-
tized with Inactin (100 mg/kg ip) and instrumented with
catheters in the femoral artery and vein to measure mean
arterial pressure (MAP) and administer compounds, respec-
tively. A tracheotomy was performed and a cannula inserted
into the trachea to ensure airway patency. The body temper-
ature of the animals was maintained at 37 °C by means of a
heating blanket. Following surgery, MAP was allowed to
stabilize for 15 min before interrupting autonomic neurotrans-
mission with chlorisondamine (3 mg/kg iv). The rats were given
an additional 30 min to stabilize under the conditions of
autonomic blockade. Animals were then treated with a test
compound at 10 mg/kg iv or vehicle (0.05-0.1 NaOH at 1 mL/
kg iv) and challenged with big ET-1 (1 nmol/kg iv) 15 and 90

Non-Peptidic Inhibitors of ECE-1
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 497
and concentrated under reduced pressure. Flash chromatog-
raphy of the residue on SiO2 (hexanes:EtOAc, 3:1) afforded
recovered starting materials 6 and 2-(bromomethyl)-9H-fluo-
rene in 29% and 31% yield, respectively, as well as the desired
product 7a as an oil (0.27 g, 30 %): 1H NMR (CDCl3, 300 MHz)
δ 2.73-2.89 (2H, m), 3.19-3.30 (2H, m), 3.78 (2H, s), 4.69 (1H,
dt, J ) 14.3, 7.5 Hz), 4.95 (1H, dt, J ) 14.3, 7.5 Hz), 5.58 (1H,
t, J ) 7.5 Hz), 6.68 (2H, d, J ) 7.1 Hz), 6.69 (1H, d, J ) 8.3
Hz), 7.08 (1H, s), 7.25-7.68 (11H, m), 7.73-7.81 (2H, m).
as a fluffy white solid (0.074 g, 57 %): mp 255 °C dec; IR (KBr)
3400 (s), 1625, 1129, 1089, 981, 769, 738 cm-1; 1H NMR (D2O,
300 MHz) δ 2.65 (1H, t, J ) 12.8 Hz), 2.85 (1H, t, J ) 12.9
Hz), 3.33-3.43 (1H, m), 3.54-3.58 (1H, m), 3.62 (2H, d, J )
3.8 Hz), 4.91 (1H, dd, J ) 10.9, 4.8 Hz), 6.97 (1H, d, J ) 7.8
Hz), 7.13 (1H, s), 7.24 (2H, dt, J ) 15.9, 7.1 Hz), 7.44 (1H, d,
J ) 7.2 Hz), 7.52 (1H, d, J ) 7.8 Hz), 7.62 (1H, d, J ) 7.3 Hz);
31P NMR (CDCl3, 121 MHz) δ 8.25. Anal. (C17H15N5Na3O3P‚
3/2H2O) C; H: calcd, 3.91; found, 4.49; N: calcd, 15.08; found,
14.45.
3-{5-[1-Am in o-2-(9H -flu or en -2-yl)et h yl]t et r a zol-1-yl}-
p r op ion itr ile (8a ). To a stirred slurry of 7a (0.42 g, 0.85
mmol) in Et2O:EtOAc (1:1, 10 mL) was added 1 N HCl (5 mL).
The reaction mixture was stirred 16 h then diluted with Et2O,
and the organic solvents were decanted. The remaining residue
was triturated with Et2O. The resulting white solid was dried
in vacuo. The hydrochloride salt was diluted with 8% NaHCO3
solution and extracted with CH2Cl2. The combined organic
extracts were washed with brine, dried over MgSO4, and then
concentrated under reduced pressure to give 8a as an oil (0.18
g, 80%): 1H NMR (DMSO-d6, 250 MHz) δ 2.52-2.61 (1H, m),
2.63-2.87 (1H, m), 3.24-3.28 (1H, m), 3.50 (1H, dd, J ) 13.3,
5.9 Hz), 3.84 (2H, s), 4.38-4.53 (2H, m), 5.27 (1H, t, J ) 7.7
Hz), 7.08 (1H, d, J ) 7.6 Hz), 7.27-7.39 (3H, m), 7.56 (1H, d,
J ) 6.7 Hz), 7.80 (1H, d, J ) 7.9 Hz), 7.85 (1H, d, J ) 7.1 Hz),
8.93 (2H, bs).
{[1-[1-(2-Cya n oet h yl)-1H-t et r a zol-5-yl]-2-(9H-flu or en -
2-yl)eth yla m in o]m eth yl}p h osp h on ic Acid Dim eth yl Es-
ter (9a ). A solution of dimethylphosphonomethyl triflate (0.18
g, 0.68 mmol) in CH2Cl2 (2 mL) was added to a stirred solution
of 8a (0.18 g, 0.54 mmol) and EtN(iPr)2 (0.12 mL, 0.68 mmol)
in CH2Cl2 at 0 °C under an atmosphere of N2. The reaction
mixture was slowly warmed to ambient temperature and
stirred for 64 h. The reaction mixture was then diluted with
EtOAc, washed with brine, dried over MgSO4, and then
concentrated under reduced pressure. Flash chromatography
of the residue on SiO2 (CH2Cl2:MeOH, 95:5) gave 9a as an oil
(0.2 g, 81%): IR (thin film) 3053 (b), 2986, 1422, 1264, 1054
cm-1; 1H NMR (CDCl3, 300 MHz) δ 2.62 (1H, dt, J ) 16.9, 6.8
Hz), 2.76-2.88 (2H, m), 3.00 (1H, t, J ) 14.7 Hz), 3.20 (1H,
dd, J ) 13.3, 8.7 Hz), 3.42 (1H, dd, J ) 13.3, 6.6 Hz), 3.68
(3H, d, J ) 10.7 Hz), 3.76 (3H, d, J ) 10.7 Hz), 3.85 (2H, s),
4.27 (2H, t, J ) 7.0 Hz), 4.68 (1H, t, J ) 7.4 Hz), 7.06 (1H, d,
J ) 7.7 Hz), 7.23 (1H, s), 7.35 (3H, dt, J ) 19.5, 7.2 Hz), 7.54
(1H, d, J ) 7.2 Hz), 7.69 (1H, d, J ) 7.7 Hz), 7.76 (1H, d, J )
7.4 Hz); 31P NMR (CDCl3, 121 MHz) δ 27.10; MS (DCI) m/z
453 (M + 1). Anal. (C22H25N6O3P) C, H, N.
{[2-(9H-F lu or en -2-yl)-1-(1H-tetr a zol-5-yl)eth yla m in o]-
m eth yl}p h osp h on ic Acid Dim eth yl Ester (10a ). To a
stirred solution of 9a (0.17 g, 0.39 mmol) in CH2Cl2 (2 mL)
was added DBU (0.14 mL, 0.97 mmol). After 2 h, the reaction
mixture was diluted with EtOAc and washed with pH 4 buffer
(0.5 M NaH2PO4‚H2O) and brine. The organic layer was dried
over MgSO4, then concentrated under reduced pressure to give
10a as a white solid (0.14 g, 92%): IR (thin film) 2900, 1608,
1260, 1040, 825, 738 cm-1; 1H NMR (CDCl3, 250 MHz) δ 2.92
(2H, t, J ) 11.6 Hz), 3.13 (1H, dd, J ) 13.7, 8.6 Hz), 3.40 (1H,
dd, J ) 14.0, 5.5 Hz), 3.64 (3H, d, J ) 11.0 Hz), 3.69 (3H, d, J
) 10.7 Hz), 3.81 (2H, s), 4.59 (1H, dd, J ) 8.6, 5.5 Hz), 7.10
(1H, d, J ) 7.9 Hz), 7.28-7.38 (3H, m), 7.51 (1H, d, J ) 6.4
Hz), 7.63 (1H, d, J ) 7.9 Hz), 7.71 (1H, d, J ) 7.0 Hz); 31P
NMR (CDCl3, 121 MHz) δ 28.43 (92%), 27.23 (8%); MS (DCI)
m/z 400 (M + 1).
{[2-(9H-F lu or en -2-yl)-1-(1H-tetr a zol-5-yl)eth yla m in o]-
m eth yl}p h osp h on ic Acid Tr isod iu m Sa lt (11a ). Trimeth-
ylsilyl bromide (0.28 mL, 2.10 mmol) was added to a suspen-
sion of 10a (0.140 g, 0.351 mmol) in CH2Cl2 (6 mL). After 1 h,
the reaction mixture was concentrated under reduced pressure
to give a tan solid. The solid was diluted with H2O (15 mL)
and stirred for 0.5 h. The resulting white solid was collected
by filtration, washed with Et2O and CH2Cl2, and dried in
vacuo. The phosphonic acid was then dissolved in a minimal
amount of 1 N NaOH and the sodium salt was purified by
chromatography on CHP-20P gel34 (gradient: 100% H2O to 1:1
H2O:MeOH in 5% intervals) to give, after lyophilization, 11a
{[2-(9-Meth yl-9H-ca r ba zol-2-yl)-1-(1H-tetr a zol-5-yl)eth -
yla m in o]m eth yl}p h osp h on ic a cid tr isod iu m sa lt (11b)
was prepared according to the synthesis of 11a , using 2-bro-
momethyl-9-methyl-9H-carbazole: mp > 280 °C; 1H NMR
(D2O, 300 MHz) δ 2.20 (1H, t, J ) 13.2 Hz), 2.45 (1H, t, J )
13.8 Hz), 3.17-3.24 (1H, m), 3.30 (3H, d, J ) 10.3 Hz), 3.56
(3H, s), 4.32 (1H, dd, J ) 9.6, 5.5 Hz), 6.86 (1H, d, J ) 8.1
Hz), 6.96 (1H, s), 7.09 (1H, t, J ) 7.0 Hz), 7.34 (2H, t, J ) 7.4
Hz), 7.82 (1H, d, J ) 7.7 Hz), 7.91 (1H, d, J ) 7.7 Hz); 31P
NMR (D2O, 121 MHz) δ 23.13; MS (ES1-) m/z 385 (M - 1).
Anal. (C17H16N6Na3O3P‚5H2O) C; H: calcd, 3.91; found, 4.49;
N: calcd, 15.08; found, 14.45.
{[2-(9-Oxo-9H -flu or en -2-yl)-1-(1H -t et r a zol-5-yl)et h yl-
a m in o]m eth yl}p h osp h on ic a cid (11c) was prepared ac-
cording to the synthesis of 11a , using 2-bromomethylfluoren-
9-one: mp 234-235 °C; IR (KBr) 3350, 2770 (vs), 1715, 1608,
1168, 1107, 1056, 945, 741 cm-1 1H NMR (DMSO-d6, TFA,
;
300 MHz) δ 3.21 (1H, t, J ) 14.3 Hz), 3.36 (2H, t, J ) 13.4
Hz), 3.68 (1H, dd, J ) 13.3, 4.1 Hz), 5.22 (1H, dd, J ) 11.4,
4.4 Hz), 7.16 (1H, d, J ) 7.7 Hz), 7.32 (1H, t, J ) 7.7 Hz), 7.37
(1H, s), 7.52-7.61 (3H, m), 7.69 (1H, d, J ) 7.7 Hz); 31P NMR
(DMSO-d6, TFA, 121 MHz) δ 12.39; MS (ES1-) m/z 384 (M -
1). Anal. (C17H16N5O4P‚5/4H2O) C, H, N.
2-Acetyla m in o-3-d iben zofu r a n -3-yl Acr ylic Acid Meth -
yl Ester (12). To a stirred solution of 3-iododibenzofuran (10.0
g, 34 mmol) in anhydrous DMF (260 mL) was added methyl
2-acetamidoacrylate (6.8 g, 47.26 mmol), followed by Pd(OAc)2
(0.30 g, 1.36 mmol), n-Bu4NCl (9.45 g, 34 mmol), and triethyl-
amine (4.7 mL, 34 mmol). The brown solution was placed in a
preheated oil bath (85 °C) and stirred for 15 h. The dark
reaction mixture was cooled to ambient temperature and
poured into ice-cold H2O (300 mL) and 1 N HCl (60 mL) and
stirred for 30 min. The precipitate was collected and rinsed
with H2O. The filtrate was diluted with Et2O, stirred, and
filtered. The tan solid was dried under high vacuum at 50 °C
for 1 h and at ambient temperature overnight (8.78 g, 84%):
1
mp 217-218 °C; H NMR (DMSO-d6, 300 MHz) δ 2.05 (3H,
s), 3.34 (1H, s), 3.73 (3H, s), 7.34 (1H, s), 7.42 (1H, t, J ) 7.5
Hz), 7.53-7.58 (1H, m), 7.66-7.73 (2H, m), 8.00 (1H, s), 8.17
(2H, d, J ) 8.1 Hz); IR (KBr) 1715, 1656, 1523, 1243, 1204,
755 cm-1. Anal. (C18H15NO4) C, H, N.
2-Acetylam in o-3-diben zofu r an -3-ylpr opion ic Acid Meth -
yl Ester (13). To a solution of 12 (8.72 g, 28.19 mmol) in
MeOH (350 mL) and toluene (310 mL) was added 10% Pd/C
(5.5 g) under N2 atmosphere. The reaction mixture was shaken
under 50 psi H2 for 18 h. The reaction mixture was filtered
through a bed of Celite and rinsed with 25% MeOH:CH2Cl2
and concentrated in vacuo. The residue was triturated with
Et2O hexanes (1:1). The light tan solid was filtered and dried
under high vacuum at 50 °C (7.12 g, 81%): 1H NMR (CDCl3,
250 MHz) δ 1.99 (3H, s), 3.2-3.35 (2H, m), 3.73 (3H, s), 4.90-
4.98 (1H, m), 5.98-6.02 (1H, m), 7.04-7.08 (1H, m), 7.28-
7.35 (2H, m), 7.40-7.46 (1H, m), 7.52-7.55 (1H, m), 7.83-
7.91 (2H, m).
(S)-2-Acetyla m in o-3-d iben zofu r a n -3-ylp r op ion ic Acid
(14). To a mechanically stirred solution of 13 (11.5 g, 30.50
mmol) in MeCN (385 mL) and 0.2 M NaHCO3 (510 mL) was
added alcalase (1.2 mL) and the reaction was monitored by
HPLC (column: YMC ODS-A C18; eluent: 70% MeCN:30% H3-
PO4; isocratic; flowrate ) 1.5 mL/min). After 2 h, the reaction
mixture was diluted with CH2Cl2 and stirred for 10 min; then
the layers were separated. The organic layer was washed once
with H2O. The combined aqueous layers were washed several
times with CH2Cl2. The combined organic layers were dried

498 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
De Lombaert et al.
over MgSO4, then concentrated to give an orange solid corre-
sponding to (R)-2-acetylamino-3-dibenzofuran-3-ylpropionic
acid methyl ester 13(R) (6.64 g). This compound could be
racemized with NaOMe in MeOH (16 h at ambient tempera-
ture) and resubmitted to the enzyme resolution (see below for
the recycling procedure of the corresponding ethyl ester). The
aqueous layer was acidified (pH ) 1) with 1 N HCl; the
precipitate was filtered and dried under high vacuum at 50
°C for 4 h to give a white solid (4.51 g, 99.5%): mp 247-249
The organic layer was dried over MgSO4 and concentrated
under reduced pressure. The residue was recrystallized from
Et2O:hexanes (1:2) and dried at 50 °C under high vacuum for
2 h then at ambient temperature for 12 h (8.8 g): mp 138-
140 °C; IR (CH2Cl2) 1726, 1652, 1233, 1165 cm-1 1H NMR
;
(CDCl3, 300 MHz) δ 1.25 (9H, s), 3.12 (2H, ABX), 4.41 (1H,
m), 5.14 (1H, m), 7.02 (1H, dd), 7.15 (1H, m), 7.21-7.31 (2H,
m), 7.38 (1H, m), 7.70 (1H, d), 7.75 (1H, d); 13C NMR (CDCl3,
75 MHz) δ 28.22, 38.17, 54.45, 79.40, 111.43, 112.44, 120.26,
120.42, 122.64, 122.71, 123.93, 124.30, 126.85, 136.30, 155.09,
1
°C; H NMR (DMSO-d6, 300 MHz) δ 1.77 (3H, s), 2.96-3.04
(1H, m), 3.22 (1H, dd, J ) 4.8, 13.7 Hz), 4.46-4.54 (1H, m),
7.27 (1H, d, J ) 8.0 Hz), 7.35-7.40 (1H, m), 7.46-7.52 (1H,
m), 7.56 (1H, s), 7.67 (1H, d, J ) 8.2 Hz), 8.03 (1H, d, J ) 7.91
Hz), 8.09 (1H, d, J ) 7.5 Hz), 8.26 (1H, d, J ) 8.1 Hz);
156.06, 156.14, 173.34; [R]25 ) +47.56 (c ) 1, CH2Cl2); MS
D
(ES1-) m/z 354 (M - 1). Anal. (C20H21NO5) C, H, N.
2-Acetylam in o-3-(3-ph en oxyben zyl)pr opion ic Acid Eth -
yl Ester (19). To a mechanically stirred solution of 2-acetyl-
amino-2-(3-phenoxybenzyl)malonic acid diethyl ester36 (363.86
g, 0.91 mol) in DMF (1.8 L) were added LiBr (79 g, 0.91 mol)
and H2O (33 mL, 1.82 mol). The solution was heated to 138
°C (internal temperature) and stirred for 16 h. The heteroge-
neous mixture was cooled to 60 °C and concentrated in vacuo
at 70 °C. The residue was partitioned between Et2O (1 L),
EtOAc (500 mL), and ice-cold H2O (500 mL). The insoluble
material was filtered. The organic layer was washed with H2O
(2 × 500 mL) and brine (300 mL) and dried over MgSO4. The
solvent was evaporated in vacuo and the residue was crystal-
lized from tert-butyl methyl ether:hexanes. The white crystal-
line solid was filtered and dried under high vacuum at 40 °C
(254 g, 85%): 1H NMR (CDCl3, 300 MHz) δ 1.22 (3H, t), 1.97
(3H, s), 3.01-3.18 (2H, m), 4.02-4.21 (2H, m), 4.84 (1H, m),
6.0 (1H, d), 6.71 (1H, m), 6.84 (1H, d), 6.92 (1H, dd), 6.94-
7.02 (2H, m), 7.11 (1H, m), 7.25 (1H, m), 7.30-7.39 (2H, m).
[R]25 ) +25.102 (c ) 0.8, DMSO).
D
The method of Shapiro et al.38 was used to estimate the
enantiomeric purity of 14: acid 14 (14.5 mg) in DMSO-d6 (0.5
mL) was mixed with (R)-(+)-1-(1-naphthyl)ethylamine (31 µL)
for 2 min. NMR spectra were recorded at 500 MHz. Upon
spiking the solution of 14 with the racemate 13 (0.6 mg), a
new distinct set of peaks (dd) centered at 7.227 ppm was
observed next to the initial set (dd) centered at 7.234 ppm. By
comparing the relative peak heights of these signals, an
enantiomeric purity of at least 97% was determined.
(S)-2-Am in o-3-d iben zofu r a n -3-ylp r op ion ic Acid Hy-
d r och lor id e (15). A mechanically stirred suspension of 14
(6.27 g, 21.09 mmol) in concentrated HCl (211 mL) and AcOH
(71 mL) was heated at 115 °C for 16 h. The white slurry was
cooled to ambient temperature, then cooled in an ice bath. The
white solid was filtered and dried under high vacuum at 55
1
°C for 4 h (5.2 g, 85%): mp 280-281 °C; H NMR (DMSO-d6,
2-Acetyla m in o-3-diben zofu r a n -3-ylpr op ion ic Acid Eth -
yl Ester (20). To a stirred solution of 19 (50 g, 153 mmol) in
AcOH (500 mL) was added Pd(OAc)2 (51.4 g, 230 mmol). The
reaction mixture was heated to reflux temperature (130 °C
internal) for 19 h. The black solution was cooled to ambient
temperature, then filtered through Celite. The filtrate was
concentrated under reduced pressure. The black residue was
dissolved in CH2Cl2 (50 mL) and diluted with Et2O (500 mL).
The insoluble material was removed by filtration and washed
again with Et2O. The filtrate was concentrated in vacuo. Acetic
acid was removed by coevaporation with toluene. The black
oily residue was purified by flash chromatography on SiO2 (900
g) with a gradient of EtOAc in hexanes (40% to 50%). The pure
fractions, according to HPLC analysis (column: Waters sym-
metry C8; eluent: 50% MeCN:50% pH 2.4 buffer; isocratic;
flowrate: 1.5 mL/min; tR ) 10 min) were combined and
concentrated under reduced pressure. The residue was recrys-
tallized from EtOAc:hexanes and dried at 40 °C under high
vacuum to give 20 (27 g) contaminated with a minor amount
of regioisomeric ester 21 (14%). 20: 1H NMR (CDCl3, 250 MHz)
δ 1.26 (3H, t, J ) 7.0 Hz), 2.00 (3H, s), 3.23-3.37 (2H, m), 4.2
(2H, q, J ) 7.0, 14.4 Hz), 4.90-4.97 (1H, m), 5.97 (1H, d, J )
7.63 Hz), 7.09 (1H, dd, J ) 1.22, 7.63 Hz), 7.31-7.36 (2H, m),
7.41-7.48 (1H, m), 7.55 (1H, d, J ) 8.24 Hz), 7.86 (1H, d, J )
7.94 Hz), 7.92 (1H, d, J ) 7.33 Hz).
(S)-2-Acetyla m in o-3-d iben zofu r a n -3-ylp r op ion ic Acid
(14). To a stirred solution of 20 (25 g, 77 mmol) containing
14% of 21 (this byproduct was not enzymatically hydrolyzed)
in MeCN (990 mL) was added 0.2 M NaHCO3 (1.3 L), followed
by alcalase (2.6 mL). After 3 h, EtOAc (1.75 L) and H2O (750
mL) were added. The organic layer was washed with H2O (500
mL) and set aside for subsequent optional epimerization (see
below). The aqueous layer was acidified with 2 N HCl (140
mL) under stirring. The white precipitate was filtered, washed
with H2O (2 × 500 mL), and dried under high vacuum at 60
°C for 3 h and at ambient temperature for 15 h (9.28 g). The
above organic layer was dried over Na2SO4 and concentrated
in vacuo to give mostly (R)-2-acetylamino-3-dibenzofuran-3-
ylpropionic acid ethyl ester (20(R)) as a white solid (13 g).
TFA, 300 MHz) δ 3.23-3.37 (2H, m), 4.3-4.31 (1H, m), 7.29-
7.32 (1H, m), 7.36-7.42 (1H, m), 7.48-7.53 (1H, m), 7.62 (1H,
s), 7.69 (1H, d, J ) 8.1 Hz), 8.1-8.13 (2H, m), 8.35-8.5 (1H,
m); 13C NMR (DMSO-d6, TFA, 75 MHz) δ 39.7, 62.5, 117.6,
117.8, 121.9, 122.1, 122.7, 125.4, 127.1, 127.2, 137.4, 155.0,
155.1, 177.0; [R]25D ) +15.09 (c ) 0.8, DMSO + 2% TFA). Anal.
(C15H13NO3) C, H, N.
(S)-2-Am in o-3-d iben zofu r a n -3-ylp r op ion ic Acid Meth -
yl Ester Hyd r och lor id e (16). Through a stirred solution of
15 (2.58 g, 8.84 mmol) in MeOH (90 mL) at 0 °C was bubbled
HCl(g) until saturation. The solution was warmed to ambient
temperature and then placed in a preheated oil bath (70 °C)
for 2 h, cooled to ambient temperature, and stirred overnight.
The mixture containing some precipitate was concentrated in
vacuo. The solid residue was triturated with Et2O, filtered,
and dried under high vacuum at 60 °C for 1 h (2.78 g, 100%):
[R]25D ) +13.982 (c ) 0.8, MeOH); 1H NMR (CD3OD, 300 MHz)
δ 3.3-3.37 (1H, m), 3.46 (1H, dd, J ) 6.0, 14.4 Hz), 3.83 (3H,
s), 4.43 (1H, dd, J ) 6.0, 7.5 Hz), 7.26-7.29 (1H, m), 7.34-
7.47 (1H, m), 7.49-7.6 (3H, m), 8.02-8.05 (2H, m).
(S)-2-ter t-Bu toxyca r bon yla m in o-3-d iben zofu r a n -3-yl-
p r op ion ic Acid Meth yl Ester (17). To a stirred solution of
16 (15.7 g, 51.4 mmol) in CH2Cl2 (300 mL) was added
triethylamine (9.7 mL, 69 mmol), followed by a solution of di-
tert-butyl dicarbonate (14 g, 64 mmol) in CH2Cl2 (40 mL). A
clear solution resulted after 20 min, and the reaction mixture
was stirred for 18 h at ambient temperature. The solution was
washed with ice-cold H2O (2 × 100 mL), pH 4 (0.5 M) NaH2-
PO4 buffer (100 mL), and H2O (100 mL), dried over Na2SO4,
filtered through a plug of SiO2, and rinsed with EtOAc:hexanes
(1:1, 500 mL). The filtrate was concentrated in vacuo, taken
up in CH2Cl2, and concentrated again. The oil slowly crystal-
lized (21 g, 100%): 1H NMR (CDCl3, 300 MHz) δ 1.53 (9H, s),
3.2-3.33 (2H, m), 3.74 (3H, s), 4.66 (1H, m), 5.05 (1H, m), 7.3-
7.38 (2H, m), 7.45 (1H, m), 7.57 (1H, m), 7.87 (1H, d), 7.92
(1H, d).
(S)-2-ter t-Bu toxyca r bon yla m in o-3-d iben zofu r a n -3-yl-
p r op ion ic Acid (18). To a stirred solution of 17 (8.3 g, 22
mmol) in MeOH (100 mL) was added a solution of lithium
hydroxide monohydrate (1.85 g, 44 mmol) in H2O (40 mL) with
adequate cooling to prevent the reaction temperature from
rising above 20 °C. After 75 min, ice-cold 1 N HCl (1 mL) was
added and the solution was extracted with CH2Cl2 (3 × 50 mL).
(R)-2-Acetylamino-3-dibenzofuran-3-ylpropionic acid ethyl
ester (20(R)) containing the racemic regioisomer 21 (53 g, 160
mmol) was dried by azeotropic removal of H2O with toluene-
ethanol then dissolved in dry ethanol (450 mL). A solution of
sodium ethoxide, freshly prepared from sodium (0.2 g, 8 mmol)

Non-Peptidic Inhibitors of ECE-1
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 499
and ethanol (50 mL), was added slowly at ambient tempera-
ture. The reaction mixture was stirred at 60 °C for 3 h, then
at ambient temperature for 12 h. The solution was saturated
with HCl(g) and stirred for 1.5 h at ambient temperature.
Ethanol was removed by evaporation under reduced pressure.
The residue was partitioned between EtOAc (500 mL) and H2O
(500 mL). The organic layer was separated, washed succes-
sively with saturated NaHCO3 (250 mL), H2O (250 mL), and
brine (250 mL), and then dried over Na2SO4. The solution was
concentrated in vacuo and the residue was recrystallized from
EtOAc:hexanes to give racemic 20, which could be resubmitted
to the above-mentioned enzymatic resolution.
50 min. The reaction mixture was concentrated in vacuo to
remove most of the formic acid. The oil crystallized upon
addition of Et2O. More Et2O was added and the mixture was
cooled in an ice bath. The solid was filtered and dried under
high vacuum at 50 °C to give 24 (13.46 g, 98%): mp 173-174
1
°C; IR (KBr) 2203, 1632, 1557, 1190 cm -1; H NMR (DMSO-
d6, 300 MHz) δ 3.0-3.21 (2H, m), 4.58-4.73 (3H, m), 7.24 (1H,
DD, J ) 0.99, 7.9 Hz), 7.37 (1H, m), 7.48 (1H, m), 7.58 (1H, s),
7.66 (1H, d, J ) 8.1 Hz), 8.02 (1H, d, J ) 7.9 Hz), 8.09 (1H, d,
J ) 7.0 Hz), 8.15 (1H, s); [R]25 ) +11.850 (c ) 1, DMSO).
D
Anal. (C19H18N6O3) H, N; C: calcd, 60.31; found, 59.55.
(S)-1-[2-Diben zofu r a n -3-yl-1-(1-(2-cya n oeth yl)-1H-tet-
r a zol-5-yl)eth yla m in o]m eth ylp h osp h on ic Acid Dip h en yl
Ester (25). To a mixture of 24 (11.7 g, 35 mmol) in EtOAc
(200 mL) and CH2Cl2 (100 mL) cooled in an ice bath was added
formaldehyde (37% aqueous; 5.2 mL, 68.5 mmol). The mixture
was warmed to ambient temperature and stirred for 18 h, to
give a homogeneous solution. EtOAc (200 mL) was added and
the mixture was washed with H2O (2 × 50 mL). The organic
layer was dried over MgSO4, concentrated, and dried under
high vacuum to give the intermediate hexahydrotriazine 3-{5-
[1-(3,5-bis{1-[1-(2-cyanoethyl)-1H-tetrazol-5-yl]-2-dibenzofuran-
3-ylethyl}-[1,3,5]triazinan-1-yl)-2-dibenzofuran-3-ylethyl]tetrazol-
1-yl}propionitrile as a glassy solid: 1H NMR (CDCl3, 300 MHz)
δ 2.64 (1H, m), 2.83 (1H, m), 3.18-3.5 (2H, m), 3.97-4.2 (2H,
m), 4.48 (1H, m), 4.93 (1H, m), 6.85 (1H, d), 7.12 (1H, s), 7.29
(1H, m), 7.36-7.5 (2H, m), 7.68 (1H, d), 7.82 (1H, d).
(S)-2-ter t-Bu t oxyca r b on yla m in o-N-(2-cya n oet h yl)-3-
d iben zofu r a n -3-ylp r op ion a m id e (22). To a stirred solution
of 18 (17.3 g, 48.7 mmol) in anhydrous DMF (300 mL) under
N2 atmosphere was added a solution of 3-aminopropionitrile
(3.93 g, 56 mmol) in anhydrous DMF (50 mL) followed by BOP
reagent (26.9 g, 60.9 mmol) and triethylamine (17 mL, 121.8
mmol). The reaction was monitored by HPLC (column: YMC
ODS; 70% MeCN:30% 0.1% H3PO4 in 0.1 M NaClO4; isocratic;
flowrate ) 1.5 mL/min). After 2 h, the reaction mixture was
diluted with EtOAc (1 L) and added to ice-H2O (500 mL). The
aqueous layer was extracted with EtOAc (2 × 400 mL); the
combined organic layers were washed successively with H2O
(250 mL), 0.5 M pH 4 buffer (300 mL), saturated NaCl (300
mL), ice-cold saturated NaHCO3 (300 mL), and saturated NaCl
(300 mL), then dried over Na2SO4, and filtered through a plug
of SiO2 (washed with EtOAc:hexanes, 3:1, 1.5 L). The filtrate
was concentrated to give a gelatinous solid, which was treated
with tert-butyl methyl ether (200 mL), then heated to reflux.
After slow addition of hexanes (75 mL), a white solid formed
upon cooling (18.26 g, 92%): mp 165-166 °C; IR (KBr) 2245,
To a stirred suspension of the above hexahydrotriazine (35
mmol) in a mixture of toluene (150 mL) and THF (75 mL)
under N2 atmosphere was added diphenyl phosphite (75%
pure, 12 g, 39 mmol). The mixture was heated in an oil bath
at 65 °C to dissolve most of the solids. The reaction was
monitored by HPLC (column: YMC ODS; eluent: 70% MeCN:
30% 0.1% H3PO4 in 0.1 M NaClO4; isocratic; flowrate ) 1.5
mL/min). Some solid precipitated out of the reaction solution
after 35 min of heating. After 70 min of stirring, the reaction
mixture was cooled to ambient temperature and diluted with
Et2O (100 mL) and hexanes (100 mL). The precipitate was
filtered and rinsed with Et2O:hexanes (1:1). The solid was
dried under high vacuum at 45 °C for 2 h to give 25 (13.07 g,
64%): mp 177-179 °C; IR (KBr) 2245, 1587, 1264, 1186, 947,
769 cm-1; 1H NMR (DMSO-d6, 300 MHz) δ 2.95-3.1 (1H, m),
3.12-3.52 (5H, m), 4.59-4.7 (2H, m), 4.76 (1H, m), 6.93-7.08
(4H, m), 7.09-7.2 (2H, m), 7.2-7.31 (6H, m), 7.37 (1H, m),
7.48 (1H, m), 7.6-7.7 (2H, m), 7.98 (1H, d), 8.08 (1H, d); 31P
1673, 1658, 1528, 1176 cm-1 1H NMR (CDCl3, 300 MHz) δ
;
1.38 (9H, s), 2.38-2.6 (2H, m), 3.17-3.27 (2H, m), 3.36-3.48
(2H, m), 4.43 (1H, m), 5.21 (1H, m), 6.74 (1H, m), 7.17 (1H,
dd), 7.32 (1H, m), 7.38-7.48 (2H, m), 7.54 (1H, m), 7.87 (1H,
d), 7.91 (1H, d); 13C NMR (CDCl3, 75 MHz) δ 18.18, 28.25,
35.58, 38.75, 55.99, 80.63, 111.68, 112.35, 117.82, 120.58,
120.78, 122.82, 123.25, 123.91, 123.97, 127.15, 135.97, 155.55,
156.33, 156.43, 172.06; [R]25 ) +6.2778 (c ) 1, CHCl3). Anal.
D
(C23H25N3O4) C, H, N.
(S)-3-[5-(1-ter t-Bu toxyca r bon yla m in o-2-d iben zofu r a n -
3-yleth yl)tetr a zol-1-yl]p r op ion itr ile (23). To a mechani-
cally stirred suspension of 22 (18.2 g, 44.7 mmol) and triphen-
ylphosphine (29.3 g, 112 mmol) in ice-cold anhydrous MeCN
(350 mL) were added from two separate addition funnels, first,
diisopropyl azodicarboxylate (22 mL, 11.2 mmol) and, 2 min
later, trimethylsilyl azide (16 mL, 11.8 mmol) over 20 min.
The heterogeneous reaction mixture was allowed to warm to
ambient temperature over 30 min then stirred for 14 h. To
the mixture cooled to 0 °C was added, under stirring, a solution
of NaNO2 (3.1 g, 45 mmol) in H2O (15 mL). After 30 min, a
solution of ceric ammonium nitrate (25 g, 45 mmol) in H2O
(70 mL) was added over 10 min and the mixture was stirred
for 20 min. The mixture was added to ice-cold H2O (300 mL)
and extracted with CH2Cl2 (1 L then 2 × 250 mL). The
combined organic layers were washed with H2O (2 × 250 mL),
dried over MgSO4, and concentrated in vacuo. The yellow solid
residue was recrystallized from 2-propanol (350 mL). The solid
was filtered, washed successively with cold 2-propanol (100
mL) and Et2O:hexanes (1:1, 200 mL), and dried under high
vacuum at 60 °C (15.67 g, 81%): mp 201-202 °C; IR (KBr)
2203, 1680, 1508, 1249, 1233, 1163 cm-1; 1H NMR (DMSO-d6,
300 MHz) δ 1.22 (9H, s), 2.97-3.18 (2H, m), 3.35-3.51 (2H,
m), 4.62-4.72 (2H, m), 5.30 (1H, m), 7.29-7.42 (2H, m), 7.50
(1H, m), 7.62-7.71 (2H, m), 7.92 (1H, d), 8.05 (1H, d), 8.11
(1H, d); 13C NMR (DMSO-d6, 75 MHz) δ 17.76, 27.91, 42.33,
45.81, 78.86, 111.6, 112.52, 117.63, 120.67, 120.98, 122.14,
123.03, 123.46, 124.62, 127.31, 136.97, 155.36, 155.51, 155.57,
NMR (DMSO-d6, 121 MHz) δ 20.75; [R]25 ) -3.8 (c ) 1,
D
DMSO). Anal. (C31H27N6O4P) C, H, N.
(S)-1-[2-Diben zofu r an -3-yl-1-(1H-tetr azol-5-yl)eth ylam i-
n o]m eth ylp h osp h on ic Acid Dip h en yl Ester (26). To a
slurry of 25 (13 g, 24.5 mmol) in CH2Cl2 (300 mL) was added
DBU (24.2 mL, 161 mmol). After 5.5 h, the clear solution was
poured into a mixture of ice (100 g) and 1 N HCl (125 mL);
then CH2Cl2 (200 mL) was added. The mixture was filtered
through Celite to remove solid impurities. The organic layer
was separated, dried over MgSO4, and concentrated under
reduced pressure. The solid residue was triturated with Et2O:
hexanes (2:1), filtered, and dried under high vacuum at 45 °C
for 2 h then at ambient temperature for 16 h to give 26 (8.5 g,
66%): mp 141-142 °C; IR (KBr) 1589, 1205, 1185, 1160, 947,
757 cm-1 1H NMR (CDCl3, 300 MHz) δ 3.02-3.23 (3H, m),
;
3.30 (1H, m), 4.62 (1H, dd, J ) 5.5, 8.7 Hz), 6.43 (2H, m), 6.98-
7.21 (7H, m), 7.22 (2H, m), 7.43 (1H, m), 7.51 (1H, m), 7.79
(1H, d, J ) 7.9 Hz), 7.88 (1H, d, J ) 7.3 Hz); 13C NMR (CDCl3,
75 MHz) δ 41.37, 42.68 (d), 56.34 (d), 111.68, 112.34, 120.33,
120.39, 120.59, 120.83, 122.88, 123.35, 123.84, 125.86, 125.95,
127.24, 129.99, 130.12, 135.74, 149.33, 149.46, 149.66, 149.79,
156.32, 156.36, 157.63; 31P NMR (CDCl3, 121 MHz) δ 19.43;
[R]25 ) -41.81 (c ) 1, CHCl3); MS (ES1+) m/z 526 (M + 1).
D
Anal. (C28H24N5O4P) C, H, N.
156.29; [R]25 ) -5.41 (c ) 1, DMSO). Anal. (C23H24N6O3) C,
D
(S)-1-[2-Diben zofu r an -3-yl-1-(1H-tetr azol-5-yl)eth ylam i-
n o]m eth ylp h osp h on ic Acid (27). To a slurry of 26 (6.4 g,
12.2 mmol) in MeCN (60 mL) was added 1 N NaHCO3 (115
mL). The solution was heated at 50 °C for 2 h while H2O was
added periodically to maintain homogeneity (up to 50 mL). The
H, N.
(S)-3-[5-(1-Am in o-2-d iben zofu r a n -3-yleth yl)tetr a zol-1-
yl]p r op ion itr ile F or m a te Sa lt (24). A solution of 23 (15.6
g, 36.1 mmol) in formic acid (200 mL) was stirred at 48 °C for

500 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
De Lombaert et al.
solution was cooled to ambient temperature and extracted with
EtOAc (3 × 75 mL). The aqueous layer was carefully acidified
with 2 N HCl. The precipitate was filtered, washed with H2O
(2 × 50 mL), and dried under high vacuum at 50 °C for 2 h to
give 27: mp 260-261 °C dec; IR (KBr) 1605, 1498, 1207, 1149,
1084, 1020, 942, 753 cm-1; 1H NMR (DMSO-d6, TFA, 300 MHz)
δ 3.11-3.53 (3H, m), 3.82 (1H, dd, J ) 4.0, 13.1 Hz), 5.26 (1H,
dd, J ) 5.5, 8.7 Hz), 7.00 (1H, d, J ) 8.04 Hz), 7.27-7.39 (2H,
m), 7.47 (1H, m), 7.64 (1H, d, J ) 8.2 Hz), 7.96 (1H, d, J ) 7.9
Hz), 8.05 (1H, d, J ) 7.4 Hz); 13C NMR (DMSO-d6, TFA, 75
MHz) δ 36.41, 41.27 (d), 54.25 (d), 111.58, 112.15, 121.01,
121.12, 122.65, 123.13, 123.17, 124.14, 127.56, 134.30, 154.45,
155.42, 155.55; 31P NMR (DMSO-d6, TFA, 300 MHz) δ 12.16;
124.50, 127.53, 134.81, 155.51, 155.6, 169.21; 31P NMR (DMSO-
d6, TFA, 121 MHz) δ 12.13. Anal. (C16H13NO6P‚NaOH) C, H,
N.
The enantiomeric purity of 29 was determined as follows:38
racemic 3-dibenzofuran-3-yl-2-[phosphonomethylamino]propi-
onic acid (prepared from 13) (7 mg) and (R)-(+)-1-(1-naphthyl)-
ethylamine (100 µL) were mixed for 2 min then diluted with
methanol-d4 (0.5 mL). NMR analysis showed a 43-Hz baseline
separation of the two equally intense peaks corresponding to
the enantiotopic methine protons appearing at 3.725 ppm (S
enantiomer) and 3.638 ppm (R enantiomer). When this meth-
odology was applied to the sample of 29 obtained by enzymatic
resolution, peak integration indicated an enantiomeric of 99%.
(S)-3-Dib en zofu r a n -3-yl-2-[p h osp h on om et h yla m in o]-
p r op ion ic a cid 2-(4-bip h en ylyl)eth yla m id e (30): 1H NMR
(DMSO-d6, TFA, 300 MHz) δ 2.50-2.62 (2H, m), 2.75-3.26
(5H, m), 3.36-3.42 (2H, m), 6.97 (2H, d), 7.18 (1H, d), 7.26-
7.53 (10H, m), 7.56 (1H, d), 8.07 (2H, dd); 13C NMR (DMSO-
d6, TFA, 75 MHz) δ 34.29, 35.84, 42.70, 62.00 (d), 111.73,
112.60, 121.14, 122.90, 123.28, 123.57, 124.73, 126.45, 126.52,
127.25, 127.64, 128.91, 129.13, 134.74, 138.18, 138.34, 140.00,
155.77, 166.14; 31P NMR (DMSO-d6, TFA, 121 MHz) δ 12.07;
MS (ES1-) m/z 527 (M - 1). Anal. (C30H27N2O5PNa2‚5/4H2O)
C, H, N.
[R]25 ) +55.31 (c ) 0.9, NaOH 0.1 N); MS (ES1-) m/z 372
D
(M - 1). Anal. (C16H16N5O4P‚3/4H2O) C, H, N.
(S)-3-Diben zofu r a n -3-yl-2-[p h osp h on om et h yla m in o]-
p r op ion ic Acid Meth yl Ester (28). To a stirred suspension
of 16 (57.5 g, 188 mmol) in EtOAc (300 mL) and H2O (300
mL) was added powdered NaHCO3 (19 g, 0.226 mmol). To this
mixture cooled to 12 °C and vigorously stirred was added an
aqueous solution of 37% aqueous formaldehyde (18.4 mL, 244
mmol). The slurry was stirred at ambient temperature for 15
h. A mixture of H2O (600 mL), EtOAc (400 mL), and CH2Cl2
(50 mL) was added. The solid was filtered and collected. The
remaining portion of product was obtained by drying the
organic layer of the filtrate over MgSO4, then concentrating
it under reduced pressure. The solid fractions were combined
to give the intermediate hexahydrotriazine 2-[3,5-bis(2-diben-
zofuran-3-yl-1-methoxycarbonylethyl)-[1,3,5]triazinan-1-yl]-3-
dibenzofuran-3-ylpropionic acid as a white solid: 1H NMR
(CDCl3, 300 MHz) δ 2.91-3.05 (2H, m), 3.58 (3H, s), 3.73 (1H,
m), 3.84 (2H, s), 6.92 (1H, d), 7.21-7.31 (2H, m), 7.40 (1H,
m), 7.50 (1H, d), 7.53 (1H, d), 7.76 (1H, d).
(S)-3-Dib en zofu r a n -3-yl-2-[p h osp h on om et h yla m in o]-
p r op ion ic a cid 2-p h en eth yla m id e (31): mp > 200 °C dec;
1H NMR (DMSO-d6, TFA, 500 MHz) δ 2.39-2.48 (1H, m),
2.50-2.57 (1H, m), 2.96-3.06 (2H, m), 3.15-3.22 (2H, m),
3.28-3.37 (2H, m), 4.17 (1H, dd, J ) 5.4 and 8.9 Hz), 6.93-
6.94 (2H, m), 7.02-7.09 (3H, m), 7.14-7.16 (1H, m), 7.32-
7.35 (1H, m), 7.41-7.47 (1H, m), 7.45 (1H,s), 7.61 (1H, d, J )
8.2 Hz), 7.99 (1H, d, J ) 7.9 Hz), 8.05 (1H, d, J ) 7.2 Hz); 13
C
NMR (DMSO-d6, TFA, 126 MHz) δ 35.22, 36.42, 40.57, 42.19
(d, J C-P ) 143.9 Hz), 62.59 (d, J C-P ) 7.9 Hz), 112.07, 113.11,
121.42, 123.38, 123.59, 124.04, 125.13, 126.60, 127.94, 128.69,
128.96, 135.12, 139.46, 156.27, 156.34, 166.57; 31P NMR
(DMSO-d6, TFA, 202 MHz) δ 11.81; MS (ES1-) m/z 451 (M -
1). Anal. (C24H23N2O5PNa2‚3/2NaOH) C, H, N.
To a stirred solution of the above hexahydrotriazine (52.8
g, 180 mmol) in toluene (650 mL) under nitrogen atmosphere
was added diphenyl phosphite (76% pure, 69.6 g, 220 mmol),
followed by toluene (100 mL). The solution was stirred at 70
°C (preheated bath) for 2 h, then cooled to ambient tempera-
ture, and concentrated under reduced pressure to a volume of
300 mL. Hexanes (150 mL) were added, and the precipitate
was filtered, then washed with Et2O:hexanes. The solid was
collected and dried under high vacuum at 40 °C to give 28
(78.93 g, 81%): mp 123-124 °C; 1H NMR (CDCl3, 300 MHz) δ
3.03-3.24 (3H, m), 3.43 (1H, m), 3.69 (3H, s), 3.80 (1H, m),
7.03-7.16 (7H, m), 7.16-7.30 (4H, m), 7.33 (1H, m), 7.45 (1H,
m), 7.57 (1H, d, J ) 8.1 Hz), 7.85 (1H, d, J ) 8.1 Hz), 7.92
(1H, dd, J ) 0.74 and 7.72 Hz); 13C NMR (CDCl3, 75 MHz) δ
39.82, 43.37 (d), 63.50 (d), 111.67, 112.36, 120.54, 120.60,
120.65, 122.76, 123.08, 124.06, 125.23, 127.04, 129.76, 136.50,
150.09, 150.22, 156.34, 156.42, 173.89; 31P NMR (CDCl3, 121
(S)-3-Diben zofu r a n -3-yl-2-[p h osp h on om et h yla m in o]-
p r op ion ic a cid (S)-p h en yla la n in a m id e (32): mp > 200 °C
dec; 1H NMR (DMSO-d6, TFA, 500 MHz) δ 2.64-2.81 (3H, m),
3.06-3.10 (1H, m), 3.22-3.26 (1H, m), 3.32-3.36 (1H, m),
4.20-4.23 (1H, m), 4.50-4.53 (1H, m), 7.08-7.20 (6H, m),
7.31-7.34 (1H, m), 7.42-7.46 (1H, m), 7.50 (1H, s), 7.59 (1H,
d, J ) 8.2 Hz), 7.95 (1H, d, J ) 7.9 Hz), 8.02-8.04 (1H, m);
13C NMR (DMSO-d6, TFA, 126 MHz) δ 36.61, 37.50, 42.42 (d,
J C-P ) 144 Hz), 54.08, 62.81, 113.25, 121.38, 121.46, 123.39,
123.54, 124.17, 125.23, 127.23, 127.88, 128.78, 129.71, 134.96,
137.66, 156.40, 166.54, 172.35; 31P NMR (DMSO-d6, TFA, 202
MHz) δ 11.77. Anal. (C25H22N2O7PNa3‚NaOH) C, H, N.
(S)-3-Diben zofu r a n -3-yl-2-[p h osp h on om et h yla m in o]-
p r op ion ic a cid (S)-leu cin a m id e (33): mp 233-235 °C dec;
1H NMR (DMSO-d6, TFA, 300 MHz) δ 0.77 (3H, d, J ) 5.5
Hz), 0.87 (3H, d, J ) 5.6 Hz), 1.45-1.57 (3H, m), 3.05 (2H, d,
J ) 13.6 Hz), 3.25-3.28 (2H, m), 4.18-4.21 (1H, m), 4.34-
4.38 (1H, m), 7.3 (1H, d, J ) 8.0 Hz), 7.34-7.39 (1H, m), 7.46-
7.51 (1H, m), 7.6 (1H, s), 7.66 (1H, d, J ) 8.2 Hz), 8.03 (1H, d,
J ) 7.9 Hz), 8.09 (1H, d, J ) 7.2 Hz); 13C NMR (DMSO-d6,
MHz) δ 18.60; [R]25 ) +10.17 (c ) 1, CHCl3); MS (ES1+) m/z
D
515 (M + 1). Anal. (C29H26NO6P) C, H, N.
(S)-3-Diben zofu r a n -3-yl-2-[p h osp h on om et h yla m in o]-
p r op ion ic Acid (29). A stirred suspension of 28 (78.8 g, 153
mmol) in AcOH (670 mL) and 9 N HCl (2.5 L) was heated to
100 °C (internal temperature) for 2 h. The reaction mixture
was cooled by addition of ice-cold H2O (2 L). The solid was
filtered, then washed successively with H2O (1L) and Et2O
(1L). The solid was slowly added to a solution of NaHCO3 (51.4
g, 612 mmol) in H2O (1.5 L). The turbid solution was extracted
with EtOAc (4 × 400 mL). The aqueous layer was clarified by
filtration through microfiber glass. To this stirred solution
were cautiously added 2 N HCl (400 mL) and Et2O to control
foaming. The gelatinous white precipitate was filtered, col-
lected, and then heated at 70 °C in H2O (1 L). The white
powder was filtered, washed with H2O (5 × 500 mL), and dried
at 50 °C under high vacuum for 18 h to yield 29 (50.2 g, 94%):
mp >250 °C; 1H NMR (DMSO-d6, TFA, 300 MHz) δ 3.19-3.34
(3H, m), 3.48-3.55 (1H, m), 4.39-4.44 (1H, m), 7.29 (1H, d,
J ) 8.0 Hz), 7.36-7.41 (1H, m), 7.50 (1H, t, J ) 7.2 Hz), 7.61
(1H, s), 7.68 (1H, d, J ) 8.2 Hz), 8.06-8.12 (2H, m); 13C NMR
(DMSO-d6, TFA, 75 MHz) δ 34.93, 41.72 (d, J ) 142 Hz), 60.88
(d, J ) 7 Hz), 111.62, 112.53, 121.04, 122.64, 123.16, 123.33,
TFA, 75 MHz) δ 20.83, 22.59, 23.98, 35.70, 41.57 (d, J C-P
)
144 Hz), 41.57, 50.40, 61.26 (d, J C-P ) 6.5 Hz), 111.49, 112.65,
120.81, 120.90, 122.58, 122.96, 123.36, 124.64, 127.32, 134.18,
155.56, 166.14, 172.86; 31P NMR (DMSO-d6, TFA, 121 MHz)
δ 12.23. Anal. (C21H27N2O7P‚1/4H2O) C, H; N: calcd, 6.16;
found, 5.59.
(S)-3-Dib en zofu r a n -3-yl-2-[p h osp h on om et h yla m in o]-
p r op ion ic a cid â-a la n in a m id e (34): mp >275 °C dec; 1H
NMR (DMSO-d6, TFA, 300 MHz) δ 2.16-2.19 (1H, m), 2.26-
2.30 (1H, m), 3.09 (2H, d, J ) 13.6 Hz), 3.20 (3H, s), 3.36-
3.37 (1H, m), 4.21 (1H, s), 7.19 (1H, d, J ) 6.7 Hz), 7.30-7.35
(1H, m), 7.40-7.50 (1H, m), 7.51 (1H, s), 7.61 (1H, d, J ) 7.9
Hz), 8.00 (1H, d, J ) 7.4 Hz), 8.04 (1H, d, J ) 6.6 Hz); 13C
NMR (DMSO-d6, TFA, 75 MHz) δ 33.64, 35.18, 36.35, 42.14
(d, J C-P ) 143.8 Hz), 62.33 (d, J C-P ) 7.2 Hz), 112.06, 113.08,
121.36, 121.43, 123.30, 123.52, 124.00, 125.06, 127.87, 135.04,

Non-Peptidic Inhibitors of ECE-1
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 501
156.21, 156.29, 166.75, 173.06; 31P NMR (DMSO-d6, TFA, 121
MHz) δ 11.85. Anal. (75% C19H18N2O7PNa3 + 25% C19H19N2O7-
PNa2) C, H, N.
mL) was added TMSBr (0.35 mL, 2.7 mmol). The solution was
stirred under nitrogen atmosphere for 5 h then concentrated
under reduced pressure at ambient temperature. Water (3 mL)
was added to the yellow foam and the solution was placed in
the refrigerator (5 °C) for 14 h. The white solid was filtered,
washed with water (3 mL), and dried under high vacuum at
65 °C for 1 h. The white solid (0.16 g) was dissolved in 1 N
NaOH (1 mL, 1 mmol) and placed on a column filled with MCI
CHP-20 gel (75-150 µL). The product was eluted with water
(20 mL), then with a gradient of methanol (5% to 40%) in
water. The pure fractions, according to HPLC analysis (col-
umn: YMC ODS; eluent: 65% MeCN:35% 0.1% H3PO4 in
NaClO4 0.1 M; isocratic; flowrate ) 1.5 mL/min), were com-
bined and lyophilized to provide a white solid (0.09 g, 47%):
mp 275-280 °C dec; IR (KBr) 1635, 1597, 1204, 1126, 1078
(S)-3-Diben zofu r a n -3-yl-2-[p h osp h on om et h yla m in o]-
1
p r op ion ic a cid glycin a m id e (35): mp 253-255 °C dec; H
NMR (DMSO-d6, TFA, 300 MHz) δ 3.02-3.20 (2H, m), 3.26-
3.42 (2H, m), 3.70-3.84 (2H, m), 4.31 (1H, dd, J ) 5.2 and 8.2
Hz), 7.26 (1H, d, J ) 8.0 Hz), 7.38 (1H, t, J ) 7.5 Hz), 7.47-
7.52 (1H, m), 7.57 (1H, s), 7.68 (1H, d, J ) 8.2 Hz), 8.05 (1H,
d, J ) 7.9 Hz), 8.11 (1H, d, J ) 7.6 Hz); 31P NMR (DMSO-d6,
TFA, 121 MHz) δ 12.04; MS (ES1-) m/z 405 (M - 1). Anal.
(C18H19N2O7P‚HCl) C, H, N.
(S)-3-Diben zofu r a n -3-yl-2-[p h osp h on om et h yla m in o]-
p r op ion ic a cid (S)-(3-d iben zofu r a n -3-yl)a la n in a m id e
(36): IR (KBr) 1604, 1205, 1128, 985 cm-1; 1H NMR (DMSO-
d6, TFA, 300 MHz) δ 2.92 (2H, dd), 3.06 (1H, dd), 3.24-3.38
(3H, m), 4.29 (1H, t), 4.57 (1H, dd), 7.14 (1H, d), 7.00 (1H, d),
7.31-7.43 (2H, m), 7.45-7.50 (4H, m), 7.60 (2H, dd), 7.87 (1H,
d), 7.95-7.97 (2H, m), 8.03 (1H, d); 13C NMR (DMSO-d6, TFA,
75 MHz) δ 36.09, 37.20, 41.46, 42.61, 54.08, 62.11 (d), 111.79,
112.33, 122.82, 114.08, 120.08, 121.12, 121.19, 122.44, 122.97,
123.17, 123.26, 123.72, 123.76, 124.55, 124.84, 127.43, 127.61,
134.61, 137.40, 155.89, 166.39, 171.86; 31P NMR (DMSO-d6,
TFA, 121 MHz) δ 11.81; MS (ES1-) m/z 585 (M - 1). Anal.
(C31H24N2O8PNa3 + 14% inorganic byproduct) C, H, N.
(S)-3-Diben zofu r an -3-yl-2-(3-n aph th alen -1-yl-1-dim eth -
ylp h osp h on op r op yla m in o)p r op ion ic Acid Meth yl Ester
(37a ,b). To a stirred suspension of 16 (0.3 g, 1.00 mmol) in
CH2Cl2 (3.5 mL) under nitrogen was added triethylamine (0.14
mL, 1.00 mmol), followed by 1-naphthyl-3-propionaldehyde (0.2
g, 1.1 mmol) and Na2SO4 (0.6 g, 4 mmol). The heterogeneous
mixture was stirred for 1 h and filtered directly into another
flask, while washing the cake with CH2Cl2 (2 mL). The filtrate
was maintained under nitrogen while being cooled with an ice
bath. To the cold solution was added trimethyl phosphite (0.19
mL, 1.6 mmol), followed by BF3‚Et2O (0.14 mL, 1.1 mmol). The
reaction mixture was stirred at ambient temperature for 14
h. The solution was diluted with CH2Cl2 (5 mL) and washed
with water (10 mL). The organic layer was dried over MgSO4
and concentrated. The diastereomers were separated by flash
chromatography on SiO2 (150 g), eluting with EtOAc:hexanes
(7:3). The less polar diastereomer (37a ) was obtained as a
white solid (0.225 g, 47%). The more polar diastereomer (37b)
was obtained as a pale yellow oil (0.169 g, 31%).
1
cm-1; H NMR (D2O, 300 MHz) δ 1.82 (1H, m), 2.17 (1H, m),
2.64-3.26 (5H, m), 3.9 (1H, s), 6.74 (1H, m), 6.88-7.72 (12H,
m), 7.8 (1H, m); 13C NMR (D2O: DMSO-d6, 75 MHz) δ 29.42,
29.95, 36.61, 57.08 (d, J ) 127.5 Hz), 62.88, 112.30, 112.63,
121.61, 121.79, 123.58,123.90, 124.23, 124.36, 124.88, 126.45,
126.61, 126.86, 127.06, 127.52, 128.21, 129.47, 131.91, 134.22,
135.96, 137.83, 156.46, 156.50, 164.84, 174.05; MS (ES1-) m/z
502 (free acid, M - 1); [R]25 ) +51.47 (c ) 0.76, H2O). Anal.
D
(C28H23NO6PNa3) C, H, N.
The other analogues 40b-v were prepared similarly.
40b: mp 270-273 °C dec; IR (KBr) 1635, 1597, 1204, 1077
1
cm-1; H NMR (D2O, 300 MHz) δ 1.32 (1H, m), 1.76 (1H, m),
2.62-2.96 (4H, m), 3.15 (1H, m), 3.67 (1H, m), 6.78 (1H, d),
6.9-7.35 (10H, m), 7.42-7.58 (2H, m), 7.66 (1H, m); 13C NMR
(D2O, 75 MHz) δ 29.33, 29.63, 38.06, 58.41 (d), 65.17, 112.08,
112.50, 121.53, 121.63, 123.44, 123.54, 123.88, 124.11, 124.56,
125.84, 126.16, 126.36, 126.78, 127.26, 127.89, 129.34, 131.67,
133.95, 136.08, 138.04, 156.23, 156.31, 175.14; 31P NMR (D2O,
121 MHz) δ 12.38; MS (ES1-) m/z 502 (free acid, M - 1). Anal.
(C28H23NO6PNa3) C, H, N.
(S)-3-Diben zofu r a n -3-yl-2-[3-(2,3-d im eth oxyp h en yl)-1-
p h osp h on op r op yla m in o]p r op ion ic a cid (40c): mp 211-
213 °C; 1H NMR (DMSO-d6, TFA, 300 MHz) δ 2.03-2.18 (2H,
m), 2.78-2.84 (2H, m), 3.25-3.36 (2H, m), 3.52-3.57 (1H, m),
3.70 (3H, s), 3.77 (3H, s), 4.76-4.8 (1H, m), 6.78 (1H, d, J )
7.2 Hz), 6.88 (1H, d, J ) 7.7 Hz), 6.95-7.00 (1H, m), 7.3 (1H,
d, J ) 7.9 Hz), 7.39 (1H, t, J ) 7.4 Hz), 7.48-7.53 (1H, m),
7.61 (1H, s), 7.68 (1H, d, J ) 8.2 Hz), 8.08-8.13 (2H, m); 13C
NMR (DMSO-d6, TFA, 75 MHz) δ 26.27, 29.6, 35.98, 54.3 (d,
37a : mp 112-113 °C; IR (CH2Cl2) 1734, 1239, 1032 cm-1
;
J
) 149 Hz), 55.49, 60.13, 60.22, 111.05, 111.64, 112.52,
1H NMR (CDCl3, 300 MHz) δ 1.97 (1H, m), 2.25 (1H, m), 3.00-
3.42 (5H, m), 3.61 (3H, d), 3.65 (3H, d), 3.66 (3H, s), 4.17 (1H,
m), 7.22 (1H, dd), 7.28-7.37 (3H, m), 7.39-7.58 (5H, m), 7.69
(1H, d), 7.80-7.94 (3H, m), 8.02 (1H, d); 13C NMR (CDCl3, 75
MHz) δ 29.31 (d), 31.52, 40.10, 51.93, 52.68, 52.74, 53.15, 53.26,
55.22, 61.53, 111.68, 112.43, 120.44, 120.54, 122.73, 122.96,
123.69, 124.03, 124.16, 125.50, 125.93, 125.98, 126.83, 127.00,
128.75, 131.73, 133.86, 136.50, 137.45, 156.33, 156.40, 174.38;
121.05, 121.28, 122.63, 123.17, 123.33, 123.86, 124.42, 127.54,
134.10, 135.01, 146.63, 152.44, 155.51, 155.61, 169.52; 31P
NMR (DMSO-d6, TFA, 121 MHz) δ 15.35; MS (ES1-) m/z 512
(M - 1). Anal. (C26H28NO8P) C, H, N.
40d (contains 20% of 40c): mp >250 °C; 1H NMR (DMSO-
d6, TFA, 300 MHz) δ 1.9-2.15 (2H, m), 2.55-2.7 (1H, m),
2.75-2.85 (1H, m), 3.23-3.41 (2H, m), 3.55-3.62 (1H, m), 3.67
(3H, s), 3.75 (3H, s), 4.44-4.48 (1H, m), 6.72-6.75 (1H, m),
6.85-6.89 (1H, m), 6.93-7.00 (1H, m), 7.29 (1H, d, J ) 7.9
Hz), 7.39 (1H, t, J ) 7.5 Hz), 7.48-7.53 (1H, m), 7.60 (1H, s),
7.68 (1H, d, J ) 8.2 Hz), 8.07-8.12 (2H, m); 13C NMR (DMSO-
d6, TFA, 75 MHz) δ 26.23, 28.32, 35.2, 53.54 (d, J ) 146 Hz),
55.39, 59.53, 60.08, 110.97, 111.57, 112.30, 120.99, 121.2,
122.61, 123.09, 123.26, 123.83, 124.33, 127.48, 134.00, 134.88,
146.50, 152.35, 155.47, 155.57, 169.36; 31P NMR (DMSO-d6,
31P NMR (CDCl3, 121 MHz) δ 28.9; [R]25 ) -3.00 (c ) 1,
D
CHCl3). Anal. (C31H32NO6P) C, H, N.
37b: 1H NMR (CDCl3 + 2 drops DMSO-d6, 300 MHz) δ 1.69
(1H, m), 1.92 (1H, m), 2.65-2.80 (2H, m), 2.86 (1H, m), 2.97-
3.11 (2H, m), 3.48-3.70 (10H, m), 6.91 (1H, d), 7.02-7.34 (8H,
m), 7.39 (1H, d), 7.48 (1H, d), 7.61-7.82 (4H, m).
(S)-3-Diben zofu r an -3-yl-2-(3-n aph th alen -1-yl-1-dim eth -
ylp h osp h on op r op yla m in o)p r op ion ic Acid (38a ). To a
stirred solution of 37a (0.24 g, 0.44 mmol) in a mixture of THF
(3 mL) and methanol (0.5 mL) was added 1 N NaOH (0.88
mL, 0.88 mmol). After 3 h, 1 N HCl (0.75 mL) was added. The
organic solvents were evaporated in vacuo and the residue was
diluted CH2Cl2 (10 mL) and water (10 mL). The aqueous layer
was extracted with CH2Cl2 (3 × 15 mL). The combined organic
phases were dried over MgSO4 and concentrated in vacuo to
give 38a as a glassy solid (0.20 g) which was used directly in
the next step. Partial hydrolysis of the dimethyl phosphonate
is sometimes observed as a side reaction.
TFA, 121 MHz) δ 15.24; MS (ES1-) 512 (M - 1). Anal. (C26H25
NO8PNa3) C, H, N.
-
3-Diben zofu r a n -3-yl-2-(S)-[3-(3,4-d im eth oxyp h en yl)-1-
p h osp h on op r op yla m in o]p r op ion ic a cid (40e): mp >250
°C; 1H NMR (DMSO-d6, TFA, 300 MHz) δ 1.99-2.19 (2H, m),
2.75 (1H, t, J ) 7.7 Hz), 3.25-3.32 (2H, m), 3.5-3.56 (1H, m),
3.7 (3H, s), 3.73 (3H, s), 4.78 (1H, dd, J ) 5.0, 8.0 Hz), 6.70-
6.72 (1H, m), 6.78-6.79 (1H, m), 6.84 (1H, d, J ) 8.2 Hz), 7.29
(1H, d, J ) 8.0 Hz), 7.39 (1H, t, J ) 7.4 Hz), 7.48-7.53 (1H,
m), 7.61 (1H, s), 7.68 (1H, d, J ) 8.2 Hz), 8.08-8.13 (2H, m);
13C NMR (DMSO-d6, TFA, 75 MHz) δ 30.67, 31.39, 36.06, 54.02
(d, J ) 145 Hz), 55.45, 55.55, 60.30, 111.73, 112.01, 112.32,
112.59, 120.27, 121.15, 122.75, 123.27, 123.43, 124.50, 127.64,
133.12, 135.08, 147.32, 148.77, 155.62, 155.72, 169.54; 31P
(S)-3-Diben zofu r an -3-yl-2-(3-n aph th alen -1-yl-1-ph osph o-
n op r op yla m in o)p r op ion ic Acid Tr isod iu m Sa lt (40a ). To
a stirred solution of 38a (0.18 g, 0.339 mmol) in CH2Cl2 (0.5

502 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
De Lombaert et al.
NMR (DMSO-d6, TFA, 121 MHz) δ 15.3. Anal. (75% C26H26
NO8PNa2 + 25% C26H25NO8PNa3) C, H, N.
-
8.5 Hz), 7.30 (1H, d, J ) 7.9 Hz), 7.39 (1H, t, J ) 7.5 Hz),
7.48-7.53 (1H, m), 7.61 (1H, s), 7.69 (1H, d, J ) 8.1 Hz), 8.07-
8.13 (2H, m); 31P NMR (DMSO-d6, TFA, 121 MHz) δ 15.41;
13C NMR (DMSO-d6, TFA, 75 MHz) δ 26.13, 28.54, 35.44, 53.66
(d, J ) 143 Hz), 55.68, 59.68, 60.12, 60.70, 107.71, 111.60,
112.30, 121.02, 122.54, 123.13, 123.28, 123.51, 124.38, 126.18,
127.50, 135.18, 141.74, 151.22, 151.93, 155.47, 155.56, 169.65.
Anal. (50% C27H27NO9PNa3 + 50% C27H28NO9PNa2) C, H, N.
40f: 1H NMR (DMSO-d6, TFA, 300 MHz) δ 2.05-2.15 (2H,
m), 2.57-2.75 (2H, m), 3.23-3.30 (2H, m), 3.54 (1H, dd, J )
4.8, 14.0 Hz), 3.67 (3H, s), 3.70 (3H, s), 4.48 (1H, dd, J ) 5.2,
9.2 Hz), 6.65-6.68 (1H, m), 6.76-6.81 (2H, m), 7.28 (1H, d,
J ) 8.83 Hz), 7.35-7.40 (1H, m), 7.47-7.52 (1H, m), 7.61 (1H,
s), 7.67 (1H, d, J ) 8.1 Hz), 8.06 (1H, d, J ) 8.1 Hz), 8.10 (1H,
d, J ) 7.0 Hz); 13C NMR (DMSO-d6, TFA, 75 MHz) δ 29.25,
31.41, 35.2, 53.34 (d, J ) 144 Hz), 55.2, 55.29, 59.55, 111.54,
111.74, 111.98, 112.32, 120.03, 120.97, 122.6, 123.08, 123.25,
124.36, 127.47, 132.94, 134.85, 147.14, 148.62, 155.47, 155.56,
169.35; 31P NMR (DMSO-d6, TFA, 121 MHz) δ 15.11. Anal.
(50% C26H25NO8PNa3 + 50% C26H26NO8PNa2) C, H, N.
(S)-3-Diben zofu r a n -3-yl-2-(3-qu in olin -8-yl-1-p h osp h o-
n op r op yla m in o)p r op ion ic a cid tr isod iu m sa lt (40m ): mp
286-287 °C; IR (KBr) 1600, 1204, 1128, 982, 796, 744 cm-1
;
1H NMR (DMSO-d6, TFA, 300 MHz) δ 2.20-2.28 (2H, m),
3.32-3.57 (5H, m), 4.80 (1H, t), 7.28-8.15 (11H, m), 9.01 (1H,
d), 9.09 (1H, d); 13C NMR (DMSO-d6, TFA, 75 MHz) δ 27.87,
29.64, 36.34, 54.02 (d), 60.53, 111.93, 113.06, 121.32, 122.17,
123.11, 123.46, 123.71, 124.90, 127.84, 128.13, 129.07, 129.50,
133.89, 134.37, 135.06, 138.87, 145.39, 146.84, 155.95, 156.03,
(S)-3-Diben zofu r a n -3-yl-2-[3-(3,4,5-tr im eth oxyp h en yl)-
1-p h osp h on op r op yla m in o]p r op ion ic a cid tr isod iu m sa lt
(40g): mp >250 °C; 1H NMR (DMSO-d6, TFA, 300 MHz) δ
2.02-2.56 (2H, m), 2.77 (2H, t, J ) 7.6 Hz), 3.25-3.34 (2H,
m), 3.53 (1H, dd, J ) 5.0, 14.5 Hz), 3.60 (3H, s), 3.74 (6H, s),
4.80 (1H, dd, J ) 5.1, 8.0 Hz), 6.49 (2H, s), 7.28-7.31 (1H, m),
7.34-7.39 (1H, m), 7.45-7.51 (1H, m), 7.61-7.66 (2H, m),
8.05-8.10 (2H, m); 13C NMR (DMSO-d6, TFA, 75 MHz) δ 30.21,
31.81, 35.86, 53.68 (d, J ) 145 Hz), 55.53, 59.70, 60.09, 105.46,
111.48, 112.43, 120.86, 122.61, 123.00, 123.27, 124.32, 127.38,
134.82, 135.77, 136.16, 152.78, 155.49, 155.58, 169.33; 31P
NMR (DMSO-d6, 121 MHz) δ 15.24. Anal. (50% C27H27NO9-
PNa3 + 50% C27H28NO9PNa2) C, H, N.
(S)-3-Diben zofu r a n -3-yl-2-[3-(3,4,6-tr im eth oxyp h en yl)-
1-p h osp h on op r op yla m in o]p r op ion ic a cid tr isod iu m sa lt
(40i): mp >250 °C; 1H NMR (DMSO-d6, TFA, 300 MHz) δ 1.8-
2.0 (1H, m), 2.05-2.20 (1H, m), 2.66-2.76 (2H, m), 3.01-3.07
(1H, m), 3.21-3.28 (1H, m), 3.54-3.73 (10H, m), 4.67-4.71
(1H, m), 6.61 (1H, s), 6.78 (1H, s), 7.29 (1H, d, J ) 8.0 Hz),
7.36 (1H, t, J ) 7.4 Hz), 7.45-7.50 (1H, m), 7.60-7.65 (2H,
m), 7.99 (1H, d, J ) 7.9 Hz), 8.06(1H, d, J ) 7.6 Hz); 31P NMR
(DMSO-d6, TFA, 121 MHz) δ 15.67; 13C NMR (DMSO-d6, TFA,
75 MHz) δ 26.04, 29.88, 36.44, 38.57, 55.17 (d, J ) 143.2 Hz),
55.78, 55.99, 56.26, 61.03, 98.35, 111.57, 112.46, 114.64,
120.30, 120.79, 120.94, 122.32, 123.07, 123.41, 124.50, 127.37,
135.81, 142.30, 147.76, 151.20, 155.50, 155.54, 157.82, 170.75;.
Anal. (90% C27H28NO9PNa2 + 10% C27H27NO9PNa3) C, H, N.
169.89; 31P NMR (DMSO-d6, TFA, 121 MHz) δ 14.02; [R]25
)
D
+28.60 (c ) 1.05, H2O); MS (ES1-) m/z 503 (M - 1). Anal. (70%
27H22N2O6PNa3 + 30% C27H23N2O6PNa2) C, H, N.
40n : mp >300 °C; IR (KBr) 1603, 1198, 775, 757 cm-1; H
C
1
NMR (DMSO-d6, TFA, 300 MHz) δ 2.17-2.25 (2H, m), 3.20-
3.59 (5H, m), 4.53 (1H, dd), 7.23-8.17 (11H, m), 9.04 (1H, d),
9.17 (1H, d); 13C NMR (DMSO-d6, TFA, 75 MHz) δ 27.78, 28.35,
35.41, 53.21 (d, J ) 141.9 Hz), 59.87, 111.86, 112.64, 121.23,
121.27, 122.13, 122.95, 123.38, 123.59, 124.68, 127.77, 128.10,
129.04, 129.40, 133.96, 134.12, 135.16, 136.78, 138.61, 145.50,
146.75, 155.81, 155.92, 169.77; 31P NMR (DMSO-d6, TFA, 121
MHz) δ 14.39; [R]25 ) -10.00 (c ) 0.95, H2O); MS (ES1-) m/z
D
503 (M - 1). Anal. (C27H22N2O6PNa3‚H2O) C, H, N.
(S)-3-Diben zofu r a n -3-yl-2-(4-a m in o-1-p h osp h on obu tyl-
a m in o)p r op ion ic a cid tr isod iu m sa lt (40o): mp 209-211
°C; 1H NMR (DMSO-d6, TFA, 300 MHz) δ 1.21 (4H, s), 1.76-
1.88 (2H, m), 2.75 (2H, s), 3.17-3.32 (2H, m), 3.5-3.58 (1H,
m), 4.76-4.8 (1H, m), 7.3 (1H, d, J ) 8.0 Hz), 7.36-7.41 (1H,
m), 7.48-7.53 (1H, m), 7.62 (1H, s), 7.68 (1H, d, J ) 8.1 Hz),
7.7-7.8 (1H, m), 8.07-8.12 (2H, m); 13C NMR (DMSO-d6, TFA,
75 MHz) δ 22.84, 26.42, 27.98, 35.85, 38.14, 54.22 (d, J ) 145
Hz), 60.17, 111.55, 112.50, 120.94, 122.59, 123.10, 123.28,
124.39, 127.47, 134.83, 155.46, 155.56, 169.38; 31P NMR
(DMSO-d6, TFA, 121 MHz) δ 15.07. MS (ES1-) m/z 419 (M -
1). Anal. (50% C20H24N2O6PNa + 50% C20H23N2O6PNa2) C, H,
N.
40j: mp >250 °C; 1H NMR (DMSO-d6, TFA, 500 MHz) δ
1.92-2.13 (2H, m), 2.56-2.62 (1H, m), 2.69-2.75 (1H, m), 3.2-
3.30 (2H, m), 3.55 (1H, dd, J ) 4.7, 13.8 Hz), 3.65 (3H, s), 3.68
(3H, s), 3.73 (3H, s), 4.38 (1H, dd, J ) 4.8, 9.3 Hz), 6.61 (1H,
s), 6.73 (1H, s), 7.29 (1H, dd, J ) 1.1, 8.0 Hz), 7.37-7.40 (1H,
m), 7.49-7.52 (1H, m), 7.59 (1H, s), 7.68 (1H, d, J ) 8.2 Hz),
8.07 (1H, d, J ) 7.9 Hz), 8.10-8.12 (1H, m); 31P NMR (DMSO-
d6, TFA, 202 MHz) δ 14.82; 13C NMR (DMSO-d6, TFA, 125
MHz) δ 26.20, 27.65, 35.56, 53.68 (d, J ) 143.7 Hz), 55.86,
56.05, 56.38, 59.60, 98.56, 111.68, 112.38, 114.79, 119.64,
121.11, 122.65, 123.21, 123.38, 124.44, 127.58, 135.28, 142.48,
148.11, 151.27, 155.51, 155.66, 169.58. Anal. (50% C27H27NO9-
PNa3 + 50% C27H28NO9PNa2) H, N; C: calcd, 54.20; found,
54.67.
40p : mp >250 °C; 1H NMR (DMSO-d6, TFA, 300 MHz) δ
1.35-1.6 (4H, m), 1.75-1.9 (2H, m), 2.15-2.8 (2H, m), 3.22-
3.3 (2H, m), 3.58 (1H, dd, J ) 4.8, 13.6 Hz), 4.47-4.52 (1H,
m), 7.3 (1H, d, J ) 8.0 Hz), 7.38 (1H, t, J ) 7.5 Hz), 7.47-7.53
(1H, m), 7.61 (1H, s), 7.67 (1H, d, J ) 8.2 Hz), 8.06-8.11
(2H,m); 13C NMR (DMSO-d6, TFA, 75 MHz) δ 23.15, 26.72,
26.78, 26.92, 35.40, 38.44, 38.53, 53.76 (d, J ) 144 Hz), 59.77,
111.81, 112.59, 121.23, 122.91, 123.35, 123.53, 124.64, 127.74,
135.08, 155.76, 155.86,169.64; 31P NMR (DMSO-d6, TFA, 121
MHz) δ 15.06; MS (ES1-) m/z 419 (M - 1). Anal. (25%
C
20H23N2O6PNa2 + 75% C20H24N2O6PNa) C, N; H: calcd, 5.34;
found, 4.74.
(S)-3-Diben zofu r a n -3-yl-2-[3-(2,3,4-tr im eth oxyp h en yl)-
1-p h osp h on op r op yla m in o]p r op ion ic a cid tr isod iu m sa lt
(40k ): mp >250 °C; 1H NMR (DMSO-d6, TFA, 300 MHz) δ
1.98-2.49 (2H, m), 2.74 (2H, t, J ) 7.8 Hz), 3.25-3.38 (2H,
m), 3.53 (1H, dd, J ) 4.9, 13.3 Hz), 3.71 (3H, s), 3.73 (3H, s),
3.76 (3H, s), 4.77 (1H, dd, J ) 5.0, 8.0 Hz), 6.68 (1H, d, J )
8.6 Hz), 6.85 (1H, d, J ) 8.5 Hz), 7.29 (1H, d, J ) 8.0 Hz),
7.35-7.40 (1H, m), 7.46-7.51 (1H, m), 7.61 (1H, s), 7.66 (1H,
d, J ) 8.2 Hz), 8.06-8.11 (2H, m); 31P NMR (DMSO-d6, TFA,
121 MHz) δ 15.48; 13C NMR (DMSO-d6, TFA, 75 MHz) δ 26.08,
29.56, 35.92, 54.10 (d, J ) 145 Hz), 55.57, 60.08, 60.15, 60.64,
107.59, 111.55, 112.49, 120.94, 122.65, 123.08, 123.32, 123.54,
124.38, 126.11, 127.44, 134.90, 141.82, 151.35, 152.03, 155.52,
155.62, 169.39;. Anal. (50% C27H28NO9PNa2 + 50% C27H27NO9-
PNa3) C, H, N.
(S)-3-Dib en zofu r a n -3-yl-2-(4-a m in o-1-p h osp h on op r o-
p yla m in o)p r op ion ic a cid tr isod iu m sa lt (40q): mp >300
°C; IR (KBr) 1633, 1603, 1206, 1127, 1080, 743 cm-1; 1H NMR
(DMSO-d6, TFA, 300 MHz) δ 1.72-2.06 (4H, m), 2.51-2.64
(2H, m), 3.25-3.33 (2H, m), 3.53 (1H, dd), 4.77 (1H, dd), 7.27-
7.37 (2H, m), 7.46 (1H, t), 7.61-7.63 (1H, m), 8.01-8.07 (2H,
m); 31P NMR (DMSO-d6, TFA, 121 MHz) δ 14.157; 13C NMR
(DMSO-d6, TFA, 75 MHz) δ 24.78, 26.28, 36.42, 54.59, 60.84,
112.07, 113.21, 121.43, 123.33, 123.60, 123.93, 125.03, 127.97,
135.22, 156.16, 156.26, 169.99; [R]25D ) +15.644 (c ) 1.1, H2O).
Anal. (C19H20N2O6PNa3) C, H, N.
40r : mp >300 °C; IR (KBr) 1602, 1583, 1457, 1205, 1129,
1
1104, 975 cm-1; H NMR (DMSO-d6, TFA, 300 MHz) δ 1.64-
2.0 (4H, m), 2.7-2.87 (2H, s), 3.17-3.42 (2H, m), 3.61 (1H,
dd), 4.46 (1H, dd), 7.28-7.48 (2H, m), 7.47 (1H, t), 7.50-7.65
(2H, m), 8.06 (2H, t); 31P NMR (DMSO-d6, TFA, 121 MHz) δ
14.41; 13C NMR (DMSO-d6, TFA, 75 MHz) δ 24.61, 24.79,
35.49, 53.46, 59.90. 59.95, 111.92, 112.71, 121.28, 123.04,
40l: mp >250 °C; 1H NMR (DMSO-d6, TFA, 300 MHz) δ
1.85-2.15 (2H, m), 2.5-2.62 (1H, m), 2.65-2.80 (1H, m), 3.23-
3.30 (2H, m), 3.52-3.58 (1H, m), 3.68 (3H, s), 3.72 (6H, s),
4.39-4.43 (1H, m), 6.66 (1H, d, J ) 8.6 Hz), 6.78 (1H, d, J )

Non-Peptidic Inhibitors of ECE-1
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 503
123.44, 123.71, 124.81, 127.81, 135.31, 155.94, 156.03, 169.83;
7.28-7.41 (2H, m), 7.42-7.58 (4H, m), 7.70-7.93 (5H, m); 31
NMR (CDCl3, 121 MHz) δ 20.51.
P
[R]25 ) +7.3939 (c ) 1, H2O). Anal. (C19H20N2O6PNa3) C, H,
D
N.
42b: 1H NMR (CDCl3, 300 MHz) δ 1.92 (1H, m), 2.17 (1H,
m), 2.76-2.97 (2H, m), 3.18 (1H, m), 3.50 (1H, m), 3.62-3.97
(1H,m), 3.72 (3H, d), 3.86 (3H, d), 5.02 (1H, m), 6.83 (1H, m),
7.08 (1H, m), 7.17-7.41 (5H, m), 7.40-7.53 (3H, m), 7.58 (1H,
d), 7.64 (1H, d), 7.74 (1H, d), 7.78-7.90 (2H, m); 31P NMR
(CDCl3, 121 MHz) δ 26.56.
(S)-3-Diben zofu r a n -3-yl-2-(4-m eth yl-1-p h osp h on op en -
tyla m in o)p r op ion ic a cid tr isod iu m sa lt (40s): mp >250
°C; 1H NMR (DMSO-d6, TFA, 300 MHz) δ 0.84 (3H, d, J ) 3.9
Hz), 0.86 (3H, d, J ) 3.9 Hz), 1.37-1.53 (3H, m), 1.72-1.9
(2H, m), 3.15-3.32 (2H, m), 3.51-3.57 (1H, m), 4.80 (1H, dd,
J ) 5.0, 8.0 Hz), 7.3 (1H, d, J ) 7.9 Hz), 7.36-7.41 (1H, m),
7.47-7.53 (1H, m), 7.62-7.69 (2H, m), 8.09 (2H, t, J ) 8.0
Hz); 13C NMR (DMSO-d6, TFA, 75 MHz) δ 22.09, 22.11, 26.62,
27.33, 34.65 (d, J ) 5.1 Hz), 35.93, 54.9 (d, J ) 144.1 Hz),
60.36, 111.60, 112.49, 120.98, 122.60, 123.13, 123.35, 124.40,
127.49, 135.10, 155.52, 155.61, 157.62, 158.11, 158.61, 159.11,
169.48; 31P NMR (DMSO-d6, TFA, 121 MHz) δ 15.3. Anal. (40%
(S)-[1-[2-Dib en zofu r a n -3-yl-1-(1H -t et r a zol-5-yl)et h yl-
a m in o]-3-n a p h th a len -1-ylp r op yl]p h osp h on ic Acid So-
d iu m Sa lt (43a ,b). Dimethyl phosphonate 42a (70 mg, 0.126
mmol) was dissolved in a 30% solution of HBr in AcOH (1.5
mL) for 3.5 h. Water (7 mL) was added causing the product to
precipitate. The mixture was stirred for 5 min then filtered.
The precipitate was washed with water (5 mL) then dried at
50 °C under high vacuum, affording the desired phosphonic
acid (61 mg). The solid was dissolved in 1 N NaOH (0.35 mL)
and the solution was placed on a column filled with MCI CHP-
20 gel (75-150 µm). The product was eluted with water (25
mL) then with a gradient of methanol (10% to 50%) in water.
The pure fractions, according to HPLC analysis (column: YMC
ODS; eluent: 65% MeCN:35% H3PO4 0.1% in NaClO4 0.1 M;
isocratic; flowrate ) 1.5 mL/min), were combined and lyoph-
ilized to provide a white solid (0.042 g, 51%): mp > 300 °C
dec; IR (KBr) 1635, 1204, 1075 cm-1; 1H NMR (D2O, 300 MHz)
δ 1.70-1.80 (1H, m), 2.00-2.20 (1H, m), 2.50-2.70 (1H, m),
2.78-3.05 (2H, m), 3.10-3.26 (2H, m), 4.50-4.60 (1H, m),
6.70-6.80 (1H, m), 6.92 (1H, s), 7.00-7.10 (1H, m), 7.12-7.42
(7H, m), 7.45-7.58 (2H, m), 7.65-7.70 (1H, m), 7.80-7.90 (1H,
m); 13C NMR (D2O, 75 MHz) δ 30.08, 30.18, 41.35, 55.77 (d,
J ) 137.3 Hz), 111.97, 112.42, 121.21, 122.93, 123.49, 124.06,
124.47, 124.66, 126.44, 126.50, 126.54, 126.92, 127.20, 127.74,
129.30, 131.90, 134.15, 137.31, 139.13, 156.28; 31P NMR (D2O,
121 MHz) δ 16.64; MS (ES1-) m/z 526 (free acid, M - 1). Anal.
(C28H23N5O4PNa3‚3/4H2O) C, N; H: calcd, 4.07; found, 4.51.
43b: starting from 42b, the above procedure afforded 43b
C
21H24NO6PNa2 + 60% C21H23NO6PNa3) C, H, N.
(S)-3-Dib en zofu r a n -3-yl-2-(1-p h osp h on oet h yla m in o)-
p r op ion ic a cid (40u ): mp 235-236 °C; IR (KBr) 1736, 1613,
1204, 1128, 942, 747, 721 cm-1; 1H NMR (DMSO-d6, TFA, 300
MHz) δ 1.42 (3H, dd), 3.26 (1H, dd), 3.40-3.54 (2H, m), 4.70
(1H, dd), 7.27 (1H, d), 7.37 (1H, t), 7.49 (1H, t), 7.59 (s, 1H),
7.66 (1H, s), 8.08 (2H, t); 13C NMR (DMSO-d6, TFA, 75 MHz)
δ 13.98, 36.01, 48.93, 50.87, 59.92, 112.80, 117.76, 121.15,
121.18, 122.83, 123.31, 124.65, 127.69, 134.78, 155.65, 155.75,
169.55; 31P NMR (DMSO-d6, TFA, 121 MHz) δ 15.49; [R]25
)
D
+43.538 (c ) 1.19, 1 N NaOH); MS (ES1-) m/z 362 (M - 1).
Anal. (C17H18NO6P) C, H, N.
40v (contains 43% of 40u ): mp 228-230 °C; IR (KBr) 1746,
1
1603, 1208, 1160, 1068, 753, 726 cm-1; H NMR (DMSO-d6,
TFA, 300 MHz) δ 1.43 (3H, dd), 3.22 (1H, dd), 3.42-3.60 (2H,
m), 4.63 (1H, dd), 7.27 (1H, d), 7.37(1H, t), 7.49 (1H, t), 7.59
(s, 1H), 7.66 (1H, s), 8.08 (2H, t); 13C NMR (DMSO-d6, TFA,
75 MHz) δ 12.79, 35.27, 48.75, 49.91, 59.56, 112.56, 117.76,
121.15, 121.23, 122.88, 123.39, 124.65, 127.75, 135.21, 155.65,
155.75, 169.55; 31P NMR (DMSO-d6, TFA, 121 MHz) δ 14.91;
MS (ES1-) m/z 362 (M - 1). Anal. (C17H18NO6P) C, H, N.
as a white solid; mp > 250 °C dec; IR (KBr) 1580, 1105 cm-1
;
(S)-[1-[2-Diben zofu r a n -3-yl-1-(1-(2-cya n oeth yl)-1H-tet-
r a zol-5-yl)et h yla m in o]-3-n a p h t h a len -1-ylp r op yl]p h os-
p h on ic Acid Dim eth yl Ester (41a ,b). To a stirred suspen-
sion of formate salt 24 (0.195 g, 0.51 mmol) in CH2Cl2 under
nitrogen atmosphere was added triethylamine (0.072 mL, 0.51
mmol). To this homogeneous solution was added 1-naphthyl-
3-propionaldehyde (0.11 g, 0.57 mmol) in CH2Cl2 (2 mL),
followed by Na2SO4 (0.3 g, 2.1 mmol). The reaction mixture
was stirred for 75 min, then transferred after filtration and
washing with CH2Cl2 (1 mL) directly into another flask. The
solution was maintained under nitrogen while being cooled
with an ice bath. To this cold solution was added, under
stirring, trimethyl phosphite (0.11 mL, 0.9 mmol), followed by
BF3‚Et2O (0.07 mL, 0.57 mmol). The solution was allowed to
warm to ambient temperature for 2 h then stirred for 18 h.
The reaction mixture was diluted with CH2Cl2 and washed
with ice-cold water. The organic extracts were dried over
MgSO4 then concentrated. The residue was purified by flash
chromatography using a gradient of EtOAc in hexanes (70%
to 90%) to provide a 1:1 mixture of diastereomers 41a and 41b
(0.21 g, 67%): 31P NMR (CDCl3, 121 MHz) δ 26.59 and 26.32.
This mixture was used directly in the next step.
(S)-[1-[2-Dib en zofu r a n -3-yl-1-(1H -t et r a zol-5-yl)et h yl-
a m in o]-3-n a p h t h a len -1-ylp r op yl]p h osp h on ic Acid Di-
m eth yl Ester (42a ,b). A 1:1 mixture of diastereomers 42a
and 42b (0.21 g, 0.345 mmol) in CH2Cl2 (3 mL) was treated
with DBU (0.21 mL, 1.38 mmol) and stirred for 2.5 h. The
mixture was diluted with EtOAc (20 mL) and washed with
pH 4 buffer (20 mL). The aqueous layer was extracted with
EtOAc (10 mL). The combined organic layers were dried over
MgSO4 and concentrated. The residue was chromatographed
on Lichroprep DIOL (E. Merck) eluting with a gradient of
EtOAc in hexanes (70% to 80%). The more polar diastereomer
(42a ) was obtained as a white powder (0.072 g, 38%). The less
polar diastereomer (42b) was also a white solid (0.077 g, 40%).
1H NMR (D2O, 300 MHz) δ 1.61 (1H, m), 2.10 (1H, m), 2.70-
2.90 (3H, m), 3.18-3.42 (2H, m), 4.65-4.80 (1H, m, masked),
6.7 (1H, d), 6.92 (1H, s), 7.00-7.17 (2H, m), 7.17-7.34 (3H,
m), 7.36-7.57 (3H, m), 7.56-7.68 (2H, m), 7.78 (1H, d), 7.84
(1H, d); 13C NMR (D2O, 75 MHz) δ 33.86, 33.95, 35.26, 43.76,
60.75 (d), 115.95, 116.42, 116.48, 125.12, 125.18, 126.82,
127.50, 128.00, 128.46, 128.56, 130.47, 130.54, 130.76, 130.98,
131.28, 131.76, 133.34, 135.80, 138.18, 140.59, 142.27, 160.22,
164.60; 31P NMR (D2O, 121 MHz) δ 15.94; MS (ES1-) m/z 526
(free acid, M - 1). Anal. (C28H23N5O4PNa3‚3/4H2O) C, H, N.
Ack n ow led gm en t . We thank Drs. Gary Ksander
and J ames Stanton for their support and encourage-
ment throughout the completion of this work, Dr.
Fariborz Firooznia for stimulating discussions, and Mr.
Mauricio Loo, Mr. Michael Moskal, and Mr. J oel Cohen
for their experimental assistance. We gratefully ac-
knowledge the members of the Novartis Analytical
Chemistry Staff (Summit) for their help in the physi-
cochemical characterization of some of the compounds
presented, in particular Mr. Karl Gunderson for the
determination of the enantiomeric purities.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and characterization of 2-bromomethyl-9-methyl-9H-
carbazole, 2-bromomethyl-fluoren-9-one, and 3-iododibenzo-
furan, the starting materials for the preparation of 7b, 7c, and
12, respectively. This material is available free of charge via
the Internet at http://pubs.acs.org.
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(1) Yanagisawa, M.; Kurihara, H.; Kimura, S.; Tomobe, Y.; Koba-
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potent vasoconstrictor peptide produced by vascular endothelial
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(2) Cheng, X.-M.; Nikam, S. S.; Doherty, A. M. Development of
agents to modulate the effects of endothelin. Curr. Med. Chem.
1994, 1, 271-312.
42a : 1H NMR (CDCl3, 300 MHz) δ 2.13-2.52 (2H, m), 3.08
(1H, m), 3.21 (1H, m), 3.34-3.52 (3H, m), 3.73 (3H, d), 3.84
(3H, d), 5.61 (1H, dd), 6.90 (1H, d), 7.07 (1H, d), 7.22 (1H, m),

504 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
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(3) Goto, K.; Hama, H.; Kasuya, Y. Molecular pharmacology and
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