Design, synthesis, and docking studies of novel benzimidazoles for the treatment of metabolic syndrome

Article abstract of DOI:10.1021/jm901272d

In addition to lowering blood pressure, telmisartan, an angiotensin (AT₁) receptor blocker, has recently been shown to exert pleiotropic effects as a partial agonist of nuclear peroxisome proliferator-activated receptor γ (PPARγ). On the basis of these findings and docking pose similarity between telmisartan and rosiglitazone in PPARγ active site, two classes of benzimidazole derivatives were designed and synthesized as dual PPARγ agonist/angiotensin II antagonists for the possible treatment of metabolic syndrome. Compound 4, a bisbenzimidazole derivative showed the best affinity for the AT₁ receptor with a K_i=13.4 nM, but it was devoid of PPARγ activity. On the other hand 9, a monobenzimidazole derivative, showed the highest activity in PPARγ transactivation assay (69% activation) with no affinity for the AT₁ receptor. Docking studies lead to the designing of a molecule with dual activity, 10, with moderate PPARγ activity (29%) and affinity for the AT₁ receptor (K_i=2.5 μM).

Full text of DOI:10.1021/jm901272d

1076 J. Med. Chem. 2010, 53, 1076–1085
DOI: 10.1021/jm901272d
Design, Synthesis, and Docking Studies of Novel Benzimidazoles for the Treatment
of Metabolic Syndrome
Cassia S. Mizuno,† Amar G. Chittiboyina,*,† Falgun H. Shah,† Akshay Patny,† Theodore W. Kurtz,
Harrihar A. Pershadsingh,^ Robert C. Speth,#,3 Vardan T. Karamyan,#,O Paulo B. Carvalho,† and Mitchell A. Avery*,†,‡,§,3
†Department of Medicinal Chemistry, School of Pharmacy, ‡National Center for Natural Products Research, §Department of Chemistry &
Biochemistry, The University of Mississippi, University, Mississippi 38677, Department of Laboratory Medicine, University of California,
San Francisco, California 94122, ^Department of Family Medicine, University of California, Irvine, California 92668, #Department of
3
Pharmacology, University of Mississippi, University, Mississippi 38677-1848, and Research Institute of Pharmaceutical Sciences, School of
Pharmacy, University of Mississippi, University, Mississippi 38677. OPresent address: Department of Pharmaceutical Sciences,
School of Pharmacy, Texas Tech University, Amarillo, Texas 79106.
Received August 25, 2009
In addition to lowering blood pressure, telmisartan, an angiotensin (AT1) receptor blocker, has recently
been shown to exert pleiotropic effects as a partial agonist of nuclear peroxisome proliferator-activated
receptor γ (PPARγ). On the basis of these findings and docking pose similarity between telmisartan and
rosiglitazone in PPARγ active site, two classes of benzimidazole derivatives were designed and
synthesized as dual PPARγ agonist/angiotensin II antagonists for the possible treatment of metabolic
syndrome. Compound 4, a bisbenzimidazole derivative showed the best affinity for the AT1 receptor
with a Ki =13.4 nM, but it was devoid of PPARγ activity. On the other hand 9, a monobenzimidazole
derivative, showed the highest activity in PPARγ transactivation assay (69% activation) with no affinity
for the AT1 receptor. Docking studies lead to the designing of a molecule with dual activity, 10, with
moderate PPARγ activity (29%) and affinity for the AT1 receptor (Ki = 2.5 μM).
Introduction
codynamic and pharmacokinetic profile as a consequence of
administering only one drug; better compliance to the ther-
apy5 and lower risk of drug-drug interactions.6 Because
DML are typically larger and more flexible, it suffers from a
poor pharmacokinetics profile which could compromise oral
bioavailability.7 The discovery of the DML occurred mostly
in the fields of angiotensin, thromboxane A2, cyclooxygenase,
histamine, PPARs, serotonin receptors, kinases, and nitric
oxide releasing conjugates.8 One of the first DML targeting
hypertension was Omapatrilat, a dual angiotensin converting
enzyme (ACE)/neutral endopeptidase (NEP) inhibitor.8 A
dual ACE/NEP inhibitor was expected to produce a synergis-
tic effect in the treatment of hypertension and heart failure.
However, the development of Omapatrilat was not further
pursued due to the occurrence of severe angioedema in treated
patients.
Dual PPARR/γ agonists have shown potential in the treat-
ment of metabolic syndrome. The increased insulin sensitivity
seen upon activation of PPARγ and the increased lipid
oxidation and anti-inflammatory activities shown by PPARR
agonists are a very attractive combination for the treatment of
metabolic syndrome. In addition, the combined activity of a
dual agonist is expected to reduce weight gain associated with
PPARγ activation through the simultaneous stimulation of
lipid oxidation and decreased adiposity seen when PPARR is
activated.
Metabolic syndrome (MetSa) is a multifactorial disease
that, in spite of the efforts of academia and pharma, remains
inadequately treated and debilitates millions of people each
year. It is characterized as a cluster of metabolic disorders that
includes elevated blood pressure, dyslipidemia, insulin resis-
tance, and central obesity.1 Thus, individuals with metabolic
syndrome are 2-3 times more likely to suffer a cardiovascular
event and 5-9 times more likely to have type 2 diabetes.1-3
Two decades ago, only individuals in industrialized countries
were affected, however, today, this syndrome affects people
worldwide and has become a major concern for healthcare
systems.4
The development of multifactorial diseases has challenged
medicinal chemists to adopt new strategies in drug discovery.
While very successful drugs were discovered through the “one
target, one disease” approach, multifactorial diseases such as
metabolic syndrome cannot be treated using the same method.
Recently there has been an increased interest in designing
drugs that modulate multiple targets simultaneously. The
advantages of designed multiple ligands (DML) compared
to combination therapy include a more predictable pharma-
*To whom correspondence should be addressed. For A.G.C.: 431
Faser Hall; phone, (662) 915-7555; fax, (662) 915-5638; E-mail, amar@
olemiss.edu. For M.A.A.: 417 Faser Hall; phone, (662) 915-5879; fax,
(662) 915-5638; E-mail, mavery@olemiss.edu.
aAbbreviations: AT1R, angiotensin-1 receptor, type I; PPAR, per-
oxisome proliferator-activated receptor; MetS, metabolic syndrome;
DML, designed multiple ligands; ACE, angiotensin converting enzyme;
NEP, neutral endopeptidase; MW, microwave; LBD, ligand binding
domain.
Metaglidasen (see Chart 1), a PPARγ partial agonist, is the
most advanced insulin sensitizer that is currently in phase III
clinical trials. The results of phase II clinical trials showed that
metaglidasen, a prodrug ester that is rapidly and completely
modified in vivo to its free acid form, significantly improved
r
pubs.acs.org/jmc
Published on Web 01/14/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1077
Figure 1. Proposed small molecules based on a benzimidazole scaffold.
Chart 1. Representative Examples of PPAR Agonists and an ACE Inhibitor
metabolic parameters without the side effects of fluid reten-
tion/edema or weight gain.9
agonists/angiotensin II antagonists (Figure 1). Along with
docking studies of the selected derivatives into both PPARγ
and AT1 receptor homology models, their biological activities
are also presented.
Rational design of multiple ligands targeting the angio-
tensin10-12 system to treat hypertension or PPARγ receptors
to treat metabolicdisease13,14 has beensuccessfully attempted;
however, these ligands were directed attargets within the same
superfamily (e.g., ACE/NEP in the case of hypertension or
PPARR, γ, and δ in the case of metabolic disorders). The
design of a multiple ligands targeting two different families of
receptors has proven more difficult to achieve.
Currently available antihypertensive drugs were designed
with the goal of reducing blood pressure and were not intended
to address the association between hypertension and insulin
resistance. Therefore, the ability of antihypertensive drugs to
ameliorate the metabolic disturbances known to be associated
with hypertension is limited.
As part of our ongoing effort to identify small molecule leads
for the treatment of metabolic syndrome, we observed that the
FDA approved angiotensin antagonist, telmisartan, functioned
as a partial PPARγ agonist15 in a human PPARγ-GAL-4 trans-
activation assay. These findings provided the framework for the
possibility of developing a dual PPARγ agonist/angiotensin II
antagonist. Identificationofsuchagentswillprovidea strategic
platform for designing prototypes of a new class of PPAR ligands
capable of antagonizing AT1R for broadly targeting cardio-
metabolic diseases for which therapy is presently insufficient
or nonexistent. Recently, we have made several unsuccessful
attempts16,17 to identify a molecule with such dual activity.
In this work, we have designed and synthesized two classes
of benzimidazole-based compounds as potential dual PPARγ
Chemistry
The synthesis of target compounds, 1-3 commenced with
the conversion of commercially available 3-methylbenzene-
1,2-diamine 13 to benzimidazole 14 using a modified method
of Chakravarty et al.18 where microwave (MW) irradiation
was used as an alternative heat source (Scheme 1). Alkylation
of benzimidazole 14 with 1-bromo-4-iodobenzene, followed
by Heck coupling19 with acrylonitrile or methylacrylate,
gave corresponding compounds 16 and target compound 1.
Hydrogenation of the double bond of 1 and intermediate 16
gave 2 and 17, respectively. Treatment of the resulting nitrile
17 with sodium azide20 furnished another target compound 3
in moderate yield.
For SAR studies around the benzimidazole ring, the bisben-
zimidazole derivative 4 was designed and synthesized. Com-
pound 19 was obtained via Heck coupling between 4-iodo-
1-methylbenzene 18 and acrylonitrile under microwave conditions
(Scheme 2). Selective benzylic bromination of 19 with NBS,21
followed by alkylation with bisbenzimidazole 21, afforded the
required intermediate 22 in good yield (two steps). Selective
double bond reduction followed by sodium azide treatment
furnished target derivative 4 in moderate yield.
To introduce fibrate-like pharmacophore in designed mo-
lecules, compounds 28 and 29 were prepared (Scheme 3).
Commercially available p-cresol 24 was O-alkylated in the

1078 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Mizuno et al.
Scheme 1. Synthesis of Monobenzimidazole-Based Compounds 1 and 2
Reagents and conditions: (a) butyric acid, polyphosphoric acid, MW irradiation, 150 ꢀC, 10 min, 57%; (b) 1-bromo-4-iodobenzene, Cs2CO3, DMF,
60 ꢀC, 3 h, 84%; (c) acrylonitrile, TEA, Pd(OAc)2, MW irradiation, 100 ꢀC, 5 min, DMF, 52%; (d) methylacrylate, Pd(OAc)2, MW irradiation, TEA,
DMF, 60%; (e) Pd/C, H2, MeOH, rt, 12 h, 70%; (f) NaN3, NH4Cl, DMF, 100 ꢀC, 40 h, 52%.
Scheme 2. Synthesis of Tetrazole-Based Benzimidazole 4
Reagents and conditions: (a) acrylonitrile, TEA, Pd(OAc)2, DMF, MW irradiation, 100 ꢀC, 5 min, 96%; (b) NBS, benzoyl peroxide, CCl4, reflux, 3 h,
65%; (c) Cs2CO3, DMF, 60 ꢀC, 3 h, 60%; (d) Pd/C, H2, MeOH, rt, 12 h, 71%; (e) NaN3, NH4Cl, DMF, reflux, 48 h, 50%.
Scheme 3. Synthesis of Fibrate-Based Benzimidazoles
presence of corresponding alkyl bromide and afforded com-
pounds 25-27, respectively. Selective benzylic bromination
with NBS followed by N-alkylation with benzimidazole 14
yielded corresponding target compounds 5, 6, and intermedi-
ate 31 (Scheme 4). The intermediate 31 was further subjected to
sodium azide conditions to yield required tetrazole derivative 7.
To study the effects of the spacer between the benzimida-
zole nitrogen and benzene ring fragments, 36 and 37 were
designed and synthesized (Scheme 5).
Alkylation of benzimidazole 14 and 21 with ethyl bromo-
acetate afforded compounds 32 and 33 in good yields. Reduc-
tion of esters with LiAlH4, followed by mesylation of the
corresponding alcohols, gave mesylates 36 and 37 in reason-
ably good yields.
The synthesis of the second fragment for 8 is depicted in
Scheme 6. Condensation of ethyl isobutyrate 38 and benzal-
dehyde 39 was carried out using LDA in THF.13 Treatment of
Reagents and conditions: (a) ethyl bromoacetate (for 25), ethyl-
2-bromoisobutyrate (for 26) NaH, THF, 0 ꢀC f rt, 12 h; 2-bromoaceto-
nitrile (for 27), Cs2CO3, DMF, 60 ꢀC, 3 h; (b) NBS, benzoyl peroxide,
CCl4, reflux, 3 h.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1079
Scheme 4. Benzimidazoles Coupling with Fibrate-like Moieties
Reagents and conditions: (a) Cs2CO3, DMF, 60 ꢀC, 3 h; (b) NaN3, NH4Cl, THF, reflux, 3 h, 90 ꢀC, 31%.
Scheme 5. N-Substituted Mono(bis)-benzimidazoles
Reagents and conditions: (a) ethyl bromoacetate, NaH, THF, 0 ꢀC, 12 h; (b) LiAlH4, THF, 0 ꢀC, 30 min; (c) MsCl, TEA, DCM, rt, 12 h.
Scheme 6. Synthesis of 4-Substituted Phenol Fragments
membranes. All samples were evaluated for the activation of
PPARγ using the human PPARγ-GAL-4 cell-based transac-
tivation assay. The results are summarized in Table 1.
Surprisingly, the compound that showed the best results in
an AT1R radioligand binding assay (4, Ki =13.4 nM) was
inactive in a PPARγ transactivation assay, whereas the
compound 9 was found to be superior in PPARγ activity
(69%), was inactive in an AT1R radioligand binding assay.
To explore the structure-activity relationship (SAR) of
this series, we synthesized several telmisartan-like analogues
with mono- and bis-benzimidazole moieties with different
acidic (tetrazole, carboxylate) substitutions. Several structural
features necessary for AT1R affinity as well as PPARγ agonist
activity were revealed in the present study. The tetrazole
containing molecules, 3, 4, 7, and 12 showed better activity
in an AT1R radioligand binding assay compared to corre-
sponding acid and ester analogues. This is further supported
by comparing the binding affinities of ester analogue, 2 (IC50>
10 μM), and corresponding tetrazole analogue, 3 (Ki=2.84 μM).
The same trend was observed when the activities of fibrate-like
derivatives [5 and 6 (IC50>10 μM)] were compared to the tetra-
zole analogue, 7 (Ki=5.06 μM). In contrast to their good affinity
toward AT1R, unfortunately, the compounds 3, 4, 7, and 12
found to be inactive in the PPARγ transactivation assay.
Reagents and conditions: (a) LDA, THF, -78 ꢀC f rt, 66%; (b)
3
BF3 Et2O, Et3SiH, DCM, rt, 2 h, 90%; (c) Pd/C, EtOAc, rt, 12 h, 93%.
40 with deoxygenation conditions,22 followed by hydrogeno-
lysis, furnished the required phenol derivative in 55% yield
(for three steps).
Coupling of benzimidazole 36 with fragment 42 and 43 under
basic conditions (Scheme 7) furnished 8 and 9, respectively. On
the other hand, coupling of bisbenzimidazole 37 and fragment
43 afforded another target compound 10 in moderate yield.
Hydrolysis of ester in 10 using alkaline methanol gave the
corresponding acid 11 as shown in Scheme 8.
For comparison purposes, the length of 4 was increased to
result bisbenzimidazole derivative 12 (Scheme 9). Heck reac-
tion of 4-iodophenol 44 with acrylonitrile, coupled with
bisbenzimidazole 37, afforded the required intermediate 46.
Reduction of the resulting double bond, followed by tetrazole
formation, afforded another target compound 12.
In the monobenzimidazole series, elongation of the spacer
between the nitrogen of the proximal benzimidazole moiety
and aryl ring by three atoms did not improve the affinity for
the AT1 receptor. For example, compounds 6 with one
methylene and 8 with 2 methylenes and one oxygen spacer
were inactive in AT1R assay, whereas the same compounds
showed moderate activity against PPARγ. Another interest-
ing key feature of the current SAR study is where derivatives
Results and Discussion
The binding affinity of the compounds for AT1 receptors
was measuredbytheirabilitytocompetewith125I-sarcsosine,1
isoleucine,8 angiotensin II (125I-SI Ang II) binding to rat liver

1080 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Mizuno et al.
Scheme 7. Synthesis of R-Aryloxy-carboxy Benzimidazoles 8-10
Reagents and conditions: Cs2CO3, DMF, 60 ꢀC, 3 h.
PPARγ ligand binding domain as shown in Figure 4. From
the docking pose, it is evident that the short length of the
phenethyl tetrazole arm, and as a result, lack of interactions
with key residues near AF-2helix, might be responsible for the
inactivity of 4 in the PPARγ transactivation assay.
Scheme 8. Synthesis of R-Aryloxy Carboxy Benzimidazoles 10
and 11
Elongation of compound 4 with one methylene and one
oxygen spacer between the nitrogen from the benzimidazole
and the aromatic ring yielded 12. The resultant compound
displayed moderate binding affinity for the AT1R(Ki=1.62μM).
The possible explanation for a lower affinity of 12 compared to
telmisartan (Ki = 0.23 nM) and 4 in AT1R might be a loss
of the key interaction of tetrazole with Lys199 as shown
in Figure 3. Other H-bonding interactions with Asn 195, Asn
111, and His 256 were retained. Docking studies of 12 in
PPARγ (shown in Figure 4) depicted H-bond interac-
tion with backbone NH of Ser 342 residue. Also, the center
phenyl ring of 12 exhibited Van der Walls interactions with Cys
285 and Leu 330. The loss of key interactions in AF-2 helix
might be one of the reasons for the loss of its PPARγ activity.
The compound 9 showed the best result inthe PPARγ assay
with an activation of 69% and an EC50 of 2.3 μM (pioglita-
zone EC50= 0.9 μM) as a partial agonist. When tested against
other isoforms of PPAR, 9 showed an EC50 of 0.3 μM (WY
14643 EC50= 0.7μM) with93%activation inPPARRasa full
agonist, whereas in PPARδ did not show any activity. Thus,
the phenoxyderivative 9 found tobea dual PPARR/γ agonist.
To study the effect of stereochemistry of compounds 9 and 10,
we have undertaken docking studies of both R and S isomers
in AT1 and PPARγ receptor. In the case of compound 9, the
R isomer showed reasonable interactions with residues near
AF2 helix. Figure 5 shows the proposed binding mode of
R-isomer of 9 in the PPARγ LBD. The benzimidazole accep-
tornitrogenatom forms aH-bond withthe Ser 342residueata
Reagents and conditions: (a) KOH, MeOH, rt, 24 h.
bearing bisbenzimidazole moiety showed better activity in
AT1R than the corresponding monobenzimidazoles. This
result is supported by the comparison of AT1R affinities of
monobenzimidazoles 3 (Ki =2.84 μM) and 9 (IC50 >10 μM)
with bisbenzimidazole derivatives 4 (Ki=13 nM) and 10 (Ki=
2.53 μM). These results further suggest that bisbenzimidazole
unit is crucial pharmacophore for maintaining AT1 affinity.
Docking studies were performed to evaluate the binding
mode of compounds in AT1R using a homology model23 and
rosiglitazone-bound crystal structure for PPARγ (PDB code:
2PRG) (Figure 2). Previous SAR and mutation studies re-
vealed His 256, Asn 111, Asn 295, and Lys 199 as key amino
acid residues for the binding of AT1R antagonists.24-26 For
PPARγ, interactions with key polar residues near AF-2 helix,
Tyr 473, His 449, and His 323 are crucial for full activation of
the receptor,27 whereas several hydrophobic interactions
mainly with residues of H3, H6, and H7 helices, accounts
for partial activation.28
The bisbenzimidazole analogue, 4, displayed potent bind-
ing affinity for the AT1R (Ki = 13.4 nM). The compound 4
also showed all key interactions important for AT1R affinity
as depicted in docking studies. As shown in Figure 3, the
nitrogen on the distal benzimidazole ring of 4 interacts with
N(δ) of His 256 through hydrogen bonding within a distance
˚
distance of 2 A. The phenoxy moiety of R isomer of 9 displa-
yed a H-bond with His 323, and furthermore it was stabilized
by hydrophobic interactions with Cys 285, Leu330, Phe 363,
and Tyr 473. These favorable interaction profiles of 9 in the
PPARγ LBD may explain its higher activity in PPARγ
transactivation assay.
In the AT1R model, docking of 9 (Figure 6) revealed the
absence of key interactions with His 256. The molecular
modeling studies suggested that the lack of affinity of 9 for
the AT1R might be due to the absence of a second (distal)
benzimidazole ring. Therefore, we designed a new compound
by appending a second benzimidazole ring to monobenzimi-
dazole 9. The resultant compound 10 exhibited moderate
affinity (2.53 μM) for the AT1R as anticipated. As shown in
Figure 7, the interaction of the distal benzimidazole moiety
with His 256 helped to regain affinity for the angiotensin
receptor. The proximal benzimidazole ring showed H-bond
interactions with Asn 295 and Asn 111. In addition, phenyl
˚
of 1.7 A. Additionally, the acceptor nitrogen atom of the
proximal heterocyclic benzimidazole ring of 4 formed a
hydrogen bond (H-bond) with Asn 111 and Asn 295 with a
˚
˚
distance of 2.5 A and 2.2 A, respectively. Also, free rotation of
the tetrazole moiety in the active site (linked by two methylene
units to a phenyl ring) helped to attain a favorable conforma-
tion to form an ionic bond with Lys 199 with a distance of
˚
2.3 A, an important interaction for AT1 affinity.
The presence of these interactions with key residues of
AT1R active site explains the potency of 4 in binding assay.
However, the compound 4 was inactive in the PPARγ trans-
activation assay. This result was in agreement with the recent
findings reported by Goebel et al.,29 where introduction of a
second benzimidazole group to monobenzimidazole com-
pound diminished the PPARγ activation. To explain the
inactivity of 4, the benzimidazole derivative was docked into

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1081
Scheme 9. Synthesis of Tetrazole-Based Bisbenzimidazole 12
Reagents and conditions: (a) acrylonitrile, TEA, Pd(OAc)2, MW irradiation, 100 ꢀC, 5 min, DMF, 96%; (b) Cs2CO3, DMF, 60 ꢀC, 3 h, 96%; (c) Pd/C,
EtOAc, rt, 12 h, 93%; (d) NaN3, NH4Cl, DMF, 100 ꢀC, 40 h, 22%.
Table 1. Biological Results of Novel Bisbenzimidazoles for PPARγ and
AT1R
was greater than thiazolidinedione-induced activation. On the
basis of this study, 10 may have the same effects on the
transcriptional control as telmisartan showing moderate acti-
vation of the receptor without directly interacting with
the residues near AF-2 helix, important for full activation of
the receptor. Inaddition, 10alsoshowed modestactivity in the
PPARR assay (15% activation).
Hydrolysis of the ester in 10 gave the corresponding acid
derivative 11, which did not have a major effect on binding
affinity toward the AT1R because the carboxylic acid ana-
logue, 11 (Ki=2.23 μM) exhibited affinity close to that of the
parent ester analogue 10 (Ki = 2.53 μM). However, this acid
analogue was completely devoid of PPARγ activity.
% of maximum
PPARγ activation
at 10 μMb
AT1R
Ki ( SEM (nM)a
compd
1
2
3
4
5
6
7
8
9
>10000
>10000
4
2
2840 ( 272
13.4 ( 4.6
>10000
>10000
21
5060 ( 1023
>10000
>10000
10
69 (93)c
29 (15)c
12
10
11
2534 ( 661
2226 ( 149
1618 ( 27
0.23 ( 0.09
Conclusion
12
telmisartan
In summary, we have discovered a dual angiotensin II
antagonist/PPARγ agonist molecule 10. In addition to the
dual activity, the benzimidazole derivative 10 also exhibited
moderate activity against PPARR. Despite the weaker bind-
ing affinity of 10 in AT1R, its additional PPARR activity
makes it useful starting point for design of multiple ligands.
During the course ofthiswork, significantly activenonpeptide
AT1 antagonist, 4, was synthesized together with a PPARR/γ
dual agonist 9. Efforts to increase PPARγ activity of AT1
antagonist 4 was found to be unsuccessful. However, mod-
ification of dual PPAR R/γ activator 9 generated another
interesting compound 10, with moderate activity against
PPARR, PPARγ, and AT1 receptor. Thus, the multiple
activity of 10 at AT1 receptor and PPARR/γ suggests that
phenoxy benzimidazole 10 can be used as a starting point for
the development of multiple AT1R antagonists and PPARR/γ
agonists for the treatment of multifactorial disorders such as
metabolic syndrome. To achieve dual activity against PPARγ
and AT1 R, further efforts for the structural modifications of
10 are currently underway according to insights gained from
the present study.
21.9
a Values >10000 are IC50 values. b % of maximum PPARγ activation
achieved by full agonist. c Number in parentheses represents for % of
maximum PPARR activation achieved with full agonist at 10 mM.
sidechainof10exhibited the additional π-π interactionswith
Phe 261 (not shown), which might account for its modest
affinity toward AT1R. However, both enantiomers of 10 did
not show any interaction with key Lys 199 residue in our
docking studies (Figure 8).
Compound 10 exhibited lower activity (29.6%) in the
PPARγ transactivation assay compared to the parent com-
pound 9 (69%). One possible explanation for the lower
activity of 10 might be improper placement of the distal
benzimidazole moiety in the downward arm of the distal
hydrophobicpocket whichmight interferewithproperfolding
of the PPARγ receptor necessary for activation. Both enan-
tiomers of 10 are mainly stabilized by hydrophobic interac-
tions with the residues (Cys 285, Leu 330, Phe 363, and Tyr
473) of PPARγ LBD with no polar interactions with residues
near AF-2 helix. Tagami et al.30 have studied the difference on
the transcriptional control between telmisartan and thiazoli-
dinediones using PPARγ mutants. Thus, it was observed that
the activation of the receptor by thiazolidinediones was im-
paired in the H323Y, S342A, H449A, and Y473A mutants.
However, activation stimulated by telmisartan was retained
in all four mutants, and in the Y473A mutant, the activation
Experimental Section
Materials and Methods. All solvents that could not be bought
anhydrous were distilled and/or stored with molecular sieves or
sodium prior to utilization. All reactions were run under an inert
atmosphere unless aqueous. All round-bottom flasks were dried

1082 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Mizuno et al.
Figure 2. (a) Telmisartan-bound AT1R homology model; (b) rosiglitazone-bound crystal structure of PPARγ (PDB code: 2PRG).
Figure 5. The proposed binding mode of the R isomer of 9 in the
PPARγ (PDB code: 2PRG). Only important residues of PPARγ
LBD is shown (colored by atom types). Hydrogen bonds are
Figure 3. The proposed binding mode of 4 and 12 in the AT1R
homology model. Only key residues of AT1R are shown. Hydrogen
bonds are denoted as yellow dotted lines. The benzimidazole 4 is
shown in orange and 12 is shown in pink (colored by atom types).
denoted as yellow dotted lines.
Figure 6. The proposed binding mode of R isomer of 9 in the AT1R
homology model with only important residues are shown (colored
by atom types). Hydrogen bonds are denoted as yellow dotted lines.
under vacuum and heat prior to use. Melting points were obta-
ined on an OptiMelt capillary melting apparatus. All NMR data
were obtained on a Bruker 500 MHz, 400 MHz, or Bruker 400 MHz
Ultra Shield and the analyses conducted in CDCl3, MeOD, or
DMSO-d6. Flash chromatography was done using silica gel
Sorbent Technologies (230 ꢀ 400 mesh). IR spectra were obta-
ined on a Bruker Tensor 27. HRMS were obtained on a Waters
Micromass Q-TOF micro mass spectrometer, and LCMS were
Figure 4. The proposed binding mode of 4 and 12 in the PPARγ
receptor (PDB code 2PRG). Only important residues of the protein
are shown. Hydrogen bonds are denoted as yellow dotted lines. The
compound 4 is shown in orange and 12 is shown in pink (colored by
atom types).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1083
51.6, 106.9, 118.2, 122.2, 122.6, 126.6 (2C), 128.5 (2C), 129.3,
134.0, 134.8, 138.4, 141.9, 143.8, 154.4, 167.1. HRMS: calcd for
C22H25N2O2 [M þ H] 349.1916, found 349.1910; mp 124.6 ꢀC.
IR: 3387, 2961, 1716, 1637, 1207 cm-1. UPLC (method 1): 98%
pure (1.02 min). UPLC (method 2): 97% pure (0.61 min).
Methyl 3-(4-((4-Methyl-2-propyl-1H-benzo[d]imidazol-1-yl)-
methyl)phenyl)-propanoate (2). To a solution of acrylate 1
(0.15 g, 0.47 mmol) in MeOH (10 mL) was added catalytic
amounts of 10% Pd/C and the reaction mixture was hydro-
genated with balloon pressure for overnight at room tempera-
ture. The reaction mixture was filtered, and the filtrate was
concentrated under vacuum. The residue was purified by flash
chromatography over silica gel eluting with CHCl3:MeOH (9:1)
1
yielded 0.11 g (70% yield) of 2. H NMR (CDCl3, 400 MHz):
δ 1.01 (t, 3H, J = 7.30 Hz), 1.80 (dd, 2H, J1,2 = 7.2 Hz, J1,3
=
15.2 Hz), 2.60 (t, 2H, J=7.8 Hz), 2.05 (s, 3H), 2.86 (t, 2H, J=
8.0 Hz), 2.92 (t, 2H, J=7.5 Hz). 3.66 (s, 3H), 5.29 (s, 2H), 6.97
(d, 2H, J=7.8 Hz), 7.01-7.07 (m, 3H), 7.13 (d, 2H, J=7.8 Hz).
13C NMR (CDCl3, 100 MHz): δ 14.0, 16.7, 21.8, 29.7, 30.4, 35.4,
46.8, 51.5, 107.0, 122.0, 122.4, 126.3 (2C), 128.8 (2C), 129.1,
134.2, 135.0, 140.1, 141.9, 154.5, 173.0. HRMS: calcd for
C22H26N2O2 351.2073 [M þ H], found 351.2058; mp 66.6 ꢀC.
IR: 3387, 2960, 1736, 1514, 1436 cm-1. UPLC (method 1): 97%
pure (1.05 min). UPLC (method 2): 97% pure (0.61 min).
General Procedure for Synthesis of 3, 4, 7, and 12. To a solution
of 17 (0.2 g, 0.62 mmol) in DMF (10 mL), NaN3 (0.16 g, 2.49 mmol)
and NH4Cl (66.8 mg, 1.24 mmol) were added and the reaction
mixture heated to 100 ꢀC. After stirring at 40 h, the suspension was
cooled, poured into ice-water, and extracted with EtOAc. The
combined organic phases were dried over MgSO4, evaporated, and
the crude was purified by silica gel column chromatography eluting
with chloroform/methanol (9:1) to give 3 in 52% yield. 1H NMR
Figure 7. The proposed binding mode of R and S isomers of 10 in
the AT1R homology model. Only important residues of the AT1R
are shown. Hydrogen bonds are denoted as yellow dotted lines.
R and S isomer of 10 is shown in pink and orange, respectively
(colored by atom types).
(CDCl3, 500 MHz): δ 0.93 (t, 3H, J=7.5 Hz), 1.72 (dd, 2H, J1,2
=
7.5 Hz, J1,3=15 Hz), 2.52 (s, 3H), 2.79 (t, 2H, J=8 Hz), 3.00 (t, 2H,
J=7.8 Hz), 3.15 (t, 2H, J=8 Hz), 5.40 (s, 2H), 6.95 (d, 1H, J=7
Hz), 6.99 (d, 2H, J= 8 Hz), 7.03 (t, 1H, J=7.5 Hz), 7.16 (d, 2H,
J=8 Hz), 7.21 (d, 1H, J=7.5Hz). 13CNMR(CDCl3, 125 MHz): δ
14.6, 17.2, 21.5, 25.2, 29.4, 33.0, 46.5, 108.1, 121.9, 122.1, 126.8 (2C),
128.2, 128.9 (2C), 135.1, 135.5, 139.4, 141.8, 154.4, 155.6. HRMS:
calcd for C21H25N6 361.2141 [M þ H], found 361.2132; mp 184.3
ꢀC. IR: 3404, 3204, 2927, 1656, 1606 cm-1. UPLC (method 1): 99%
pure (0.50 min). UPLC (method 2): 99.4% pure (0.46 min).
30-(4-(2-(1H-Tetrazol-5-yl)ethyl)benzyl)-1,70-dimethyl-20-pro-
pyl-1H,30H-2,50-bibenzo[d]-imidazole (4). Yield 50%. 1H NMR
(CDCl3, 400 MHz): δ 1.03 (t, 3H, J=6 Hz), 1.84 (dd, 2H, J1,2
=
5.6 Hz, J1,3 = 12 Hz), 2.71 (s, 3H), 2.88-2.91 (m, 2H), 2.98 (t,
2H, J = 5.6 Hz), 3.14 (t, 2H, J = 5.2 Hz), 3.81 (s, 3H), 5.26 (s,
2H), 6.83 (s, 4H), 7.23 (s, 1H), 7.26-7.33 (m, 4H), 7.40 (d, 1H,
J = 6 Hz), 7.65 (d, 1H, J = 6.4 Hz). 13C NMR (CDCl3, 100
MHz): δ 14.4, 17.3, 22.2, 26.0, 30.0, 32.1, 34.1, 47.6, 109.7, 110.0,
118.2, 122.3, 122.9, 123.1, 133.3, 126.4 (2C), 128.8 (2C), 129.4,
133.4, 134.8, 135.8, 139.5, 140.9, 143.21, 154.2, 156.0, 156.6.
HRMS: calcd for C29H31N8 491.2672 [M þ H], found 491.2687;
Figure 8. The proposed binding mode of the R and S isomers of 10
in the PPARγ (PDB code: 2PRG). Only important residues of
PPARγ LBD are shown. Hydrogen bonds are denoted as yellow
dotted lines. The R and S isomer of 10 is shown in pink and orange,
respectively (colored by atom types).
obtained on a Waters AcQuity Ultra Performance LC. Purity of
the final compounds (g95%) was established using a Waters
HPLC 2695.
mp 97 ꢀC. IR: 2962, 2931, 2872, 1653, 1597, 1454, 1417 cm-1
.
UPLC (method 1): 96.3% pure (0.50 min). UPLC (method 2):
97.0% pure (0.53 min).
Ethyl 3-(4-((4-Methyl-2-propyl-1H-benzo[d]imidazol-1-yl)methyl)-
phenyl)acrylate (1). In a 5 mL reaction vial, iodide 15 (0.21 g,
0.53 mmol), methyl acrylate (60 μL, 0.66 mmol), Pd(OAc)2
(catalytic), TEA (74 μL, 0.53 mmol), and DMF (500 μL) were
added. The contents of the flask were irradiated, at 100 ꢀC,
20 psi, and 100 W for 5 min. After cooling, the mixture was poured
into water and extracted with DCM. The organic phase was
concentrated under vacuum. The resulting crude was purified by
silica gel flash chromatography eluting with hexanes:EtOAc
(3:2) and provided the target compound 1 in 0.11 g (60% yield).
1H NMR (CDCl3, 400 MHz): δ 1.01 (t, 3H, J=7.2 Hz), 1.82 (dd,
2H, J1,2 =7.6 Hz, J1,3 =15.2 Hz), 2.71 (s, 3H), 2.85 (t, 2H, J=
8 Hz), 3.80 (s, 3H), 5.34 (s, 2H), 6.40 (d, 1H, J = 16 Hz),
6.99-7.11 (m, 5H), 7.45 (d, 2H, J = 8 Hz), 7.65 (d, 1H, J = 16
Hz). 13C NMR (CDCl3, 100 MHz): δ 14.0, 16.7, 21.8, 29.7, 46.8,
1-(4-((1H-Tetrazol-5-yl)methoxy)benzyl)-4-methyl-2-propyl-
1H-benzo[d]imidazole (7).Yield 31%. 1H NMR (DMSO, 500 MHz):
δ 0.94 (t, 3H, J=3.6 Hz), 1.72 (dd, 2H, J1,2 =6 Hz, J1,3 =12 Hz),
2.51 (s, 3H), 2.82 (t, 2H, J=6.4 Hz), 5.25 (s, 2H), 5.37 (s, 2H), 7.022
(m, 6H), 7.23 (d, 1H, J=6.4 Hz). 13C NMR (DMSO, 125 MHz): δ
14.6, 17.2, 21.5, 29.4, 46.3, 61.0, 108.1, 115.3 (2C), 122.0, 122.1, 128.1
(2C), 129.9 (2C), 135.0, 141.6, 154.4 (2C), 157.5. HRMS: calcd for
C20H23N6O 363.1933 [M þ H], found 363.1934; mp 104.7 ꢀC. IR:
3382, 2960, 2926, 1662, 1611, 1511 cm-1. UPLC (method 1): 99.3%
pure (0.51 min).; UPLC (method 2): 100% pure (0.47 min).
30-(2-(4-(2-(1H-Tetrazol-5-yl)ethyl)phenoxy)ethyl)-1,70-dimethyl-
1
20-propyl-1H,30H-2,50-bibenzo[d]imidazole (12). Yield 22%. H
NMR (CDCl3, 400 MHz): δ 1.07 (t, 3H, J = 7.5 Hz), 1.91 (dd,
2H, J1,2 =7.3 Hz, J1,3 =15.1 Hz), 2.67 (s, 3H), 2.75 (t, 2H, J=

1084 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Mizuno et al.
7.0 Hz), 2.93-3.01 (m, 4H), 3.90 (s, 3H), 4.00 (t, 2H, J=4.5 Hz),
4.49 (t, 2H, J=4.2 Hz), 6.28 (d, 2H, J=8.5 Hz), 6.57 (d, 2H, J=
8.3 Hz), 7.28 (s, 1H), 7.30-7.36 (m, 2H), 7.41-7.43 (m, 1H),
7.76-7.79 (m, 2H). 13C NMR (CDCl3, 100 MHz): δ 14.0, 16.9,
21.8, 25.6, 29.4, 31.9, 32.9, 43.6, 65.7, 109.1, 110, 114 (2C), 118.6,
122.6, 123, 123.2, 123.5, 129.1 (2C), 129.3, 132.2, 134.7, 136.1,
141.3, 143.1, 154.5, 156, 156.1, 157.5. HRMS: calcd for
C30H33N8O 521.2777 [M þ H], found 521.2787. IR: 3383,
2962, 2930, 1511, 1454, 1243 cm-1. UPLC (method 1): 96%
pure (0.50 min). UPLC (method 2): 95% pure (0.46 min).
General Procedure for the Synthesis of 5, 6, 8, 9, and 10. To a
solution of benzimidazole 14 (0.1 g, 0.57 mmol) in 10 mL of
DMF was added Cs2CO3 (0.28 g, 0.85 mmol), the mixture was
stirred for 30 min at 60 ꢀC, and ethyl 2-(4-(bromomethyl)phe-
noxy)acetate 28 (0.15 g, 0.69 mmol) was added. After stirring for
3 h, the mixture was cooled, poured into water, and extracted
with EtOAc. The organic phase was dried using MgSO4 and
evaporated. The residue was purified by silica gel column
chromatography eluting with ether/hexanes (7:3) to give 0.14 g
501.2729. IR: 2962, 2872, 1733, 1598, 1511, 1237 cm-1. UPLC
(method 2): 100% pure (0.55 min). HPLC (method 3): 96.9%
pure (1.42 min).
Ethyl 3-(4-(2-(1,70-Dimethyl-20-propyl-1H,30H-2,50-bibenzo-
[d]imidazol-30-yl)ethoxy)-phenyl)-2-methyl-2-phenoxypropanoate
(10). Yield 30%. 1H NMR (CDCl3, 400 MHz): δ 1.13 (t, 3H J=
7.3 Hz), 1.21 (t, 3H, J=7.0 Hz), 1.36 (s,3H), 1.99 (dd, 2H, J1,2
7.5 Hz, J1,3=15.3 Hz), 2.75 (s, 3H), 3.02-3.08 (m, 3H), 3.25 (d,
1H, J=13.8 Hz), 3.90 (s, 3H), 4.19 (dd, 2H, J1,2=7.3 Hz, J1,3
=
=
14.3 Hz), 4.29 (t, 2H, J = 5.2 Hz), 4.59 (t, 2H, J = 5.2 Hz),
6.73 (d, 3H, J=8.5 Hz), 6.81 (d, 1H, J=7.5 Hz), 6.97 (t, 1H, J=
7.3 Hz), 7.13 (d, 1H, J=8.8 Hz), 7.21 (t, 2H, J=7.5 Hz), 7.28 (s,
1H), 7.32-7.35 (m, 2H), 7.38-7.41 (m, 2H), 7.70 (s, 1H),
7.83-7.86 (m, 1H). 13C NMR (CDCl3, 100 MHz): δ 14.0,
14.1, 16.8, 20.6, 21.8, 29.6, 31.9, 43.4, 44.6, 61.3, 65.9, 81.8,
108.5, 109.5, 113.9 (2C), 114.2, 119.3 (2C), 119.6, 122.3, 133.5,
123.8, 128.5, 129.1 (2C), 129.3, 130.5, 131.7 (2C), 134.7, 136.7,
143.0, 143.2, 154.8, 155.3, 156.8, 157.0, 173.6. HRMS: calcd for
C39H43N4O4 631.3284 [M þ H], found 631.3275. IR: 3385, 2933,
1733, 1610, 1597, 1510, 1488 cm-1. UPLC (method 1): 96% pure
(1.97 min). UPLC (method 2): 94% pure (1.83 min).
1
(67% yield) of 5. H NMR (CDCl3, 400 MHz): δ 1.01 (t, 3H,
J=7.3 Hz), 1.29 (t, 3H, J=7.1 Hz), 1.8 (dd, 2H, J1,2=7.6Hz,J1,3=
15.6 Hz), 2.7 (s, 3H), 2.85 (t, 2H, J=7.9 Hz), 4.26 (dd, 2H, J1,2=
3-(4-(2-(1,70-Dimethyl-20-propyl-1H,30H-2,50-bibenzo[d]imidazol-
30-yl)ethoxy)-phenyl)-2-methyl-2-phenoxypropanoic acid (11).
Potassium hydroxide (50 mg) was added to a solution of 10
(15 mg, 0.016 mmol) in MeOH (5 mL). After stirring for 24 h at
room temperature, the organic phase was evaporated under
reduced pressure and pH was adjusted to 7. The aqueous layer
was extracted with EtOAc. The organic layer was dried using
MgSO4 and evaporated. The crude mixture was purified by
silica gel column chromatography eluting with chloroform/
methanol (9:1) and afforded 8 mg (88% yield) of 11. 1H NMR
(MeOD, 500 MHz): δ 1.09 (t, 3H, J=7.5 Hz), 1.25 (s, 3H), 1.94
(dd, 2H, J1,2 = 7.5 Hz, J1,3 =15.1 Hz), 2.68 (s, 3H), 2.99-3.08
(m, 3H), 3.21 (d, 1H, J=13.8 Hz), 3.85 (s, 3H), 4.67 (t, 2H, J=
4.2 Hz), 4.69 (t, 2H, J = 4.5 Hz), 6.72 (d, 2H, J = 8.3 Hz),
6.85-6.90 (m, 3H), 7.10-7.18 (m, 4H), 7.30-7.36 (m, 2H), 7.42
3.8 Hz, J1,3 = 14 Hz), 4.58 (s, 2H), 5.27 (s, 2H), 6.83 (d, 2H,
J=8.5 Hz), 6.98 (d, 2H, J=8.3 Hz), 7.02-7.11 (m, 3H). 13CNMR
(CDCl3, 100 MHz): δ 14.0, 14.1, 16.7, 21.8, 29.7, 46.5, 61.3, 65.4,
107.1, 115.1 (2C), 122.0, 122.45, 127.4 (2C), 129.1, 129.4, 134.9,
141.9, 154.5, 157.4, 168.6. HRMS: calcd for C22H27N2O3
367.2022 [M þ H], found 367.2018; mp 79.89 ꢀC. IR: 3384,
2963, 2931, 2872, 1757, 1512 cm-1. UPLC (method 1): 95% pure
(1.11 min). UPLC (method 2): 97% pure (0.61 min).
Ethyl 2-Methyl-2-(4-((4-methyl-2-propyl-1H-benzo[d]imidazol-
1-yl)methyl)-phenoxy)-propanoate (6). Yield 26%. 1H NMR
(CDCl3, 400 MHz): δ 1.00 (t, 3H, J = 7.5 Hz), 1.23 (t, 3H, J =
7.0 Hz), 1.57 (s, 6H), 1.77 (dd, 2H, J1,2=7.6 Hz, J1,3=15.6 Hz),
2.7 (s, 3H), 2.85 (t, 2H, J=7.8 Hz), 4.21 (dd, 2H, J1,2 =7.2 Hz,
J1,3=14 Hz), 5.26 (s, 2H), 6.77 (d, 2H, J=8.8 Hz), 6.94 (d, 2H,
J=8.8 Hz), 7.06 (m, 3H). 13C NMR (CDCl3, 100 MHz): δ 14.0
(2C), 16.7, 21.8, 25.3 (2C), 29.7, 46.5, 61.4, 79.2, 107.0, 119.4
(2C), 122.0, 122.3, 127.1 (2C), 129.1, 129.7, 135.0, 141.9, 154.5,
155.0, 173.9. HRMS: calcd for C24H31N2O3 395.2335 [M þ H],
(s, 1H), 7.50-7.52 (m, 1H), 7.68-7.70 (m, 1H), 7.77 (s, 1H). 13
C
NMR (MeOD, 125 MHz): 12.9, 15.4, 20.0, 21.3, 28.7, 30.8, 43.3,
43.8, 66.0, 82.2, 109.4, 110.0, 113.5 (2C), 117.8, 118.8 (2C),
121.0, 122.5, 122.7, 122.9, 123.7, 128.5 (2C), 128.6, 129.3, 131.5
(2C), 134.3, 136.1, 141.3, 142.1, 154.4, 155.9, 157.0, 157.6,
158.61, 178.6. HRMS: calcd for C37H39N4O4 603.2971 [M þ
found 395.2352. IR: 3384, 2959, 1724, 1609, 1509, 1142 cm-1
.
UPLC (method 1): 100% pure (1.85 min). UPLC (method 2):
100% pure (0.69 min).
H], found 603.2981. IR: 3385, 2925, 1577, 1458, 1225 cm-1
.
UPLC (method 1): 94% pure (1.72 min). UPLC (method 2):
93% pure (1.80 min).
Radioligand binding assays of AT1 receptors were carried out
as described previously31 using 125I-sar1ile8 angiotensin II pre-
pared as described previously.32
Ethyl 2,2-Dimethyl-3-(4-(2-(4-methyl-2-propyl-1H-benzo[d]-
imidazol-1-yl)ethoxy)-phenyl)pro-panoate (8). Yield 84%. 1H
NMR (CDCl3, 400 MHz): δ 1.10 (t, 3H, J = 7.3 Hz), 1.14 (s,
6H), 1.23 (t, 3H, J=7.3 Hz), 1.94 (dd, 2H, J1,2 =7.5 Hz, J1,3
=
15.3 Hz), 2.68 (s, 3H), 2.78 (s, 2H), 2.99 (t, 2H, J=8.0 Hz), 4.11
(dd, 2H, J1,2 =7.0 Hz, J1,3 =14.1 Hz), 4.24 (t, 2H, J=5.7 Hz),
4.51 (t, 2H, J=5.5 Hz), 6.71 (d, 2H, J=8.8 Hz), 7.00 (d, 2H, J=
8.5 Hz), 7.05 (d, 1H, J=7.0 Hz), 7.13-7.21 (m, 2H). 13C NMR
(CDCl3, 100 MHz): δ 14, 14.1, 16.7, 20.9, 24.8 (2C), 29.5, 43.2,
43.4, 45.3, 60.3, 65.8, 106.6, 113.8 (2C), 121.9, 122.4, 129.3,
130.9, 131.1 (2C), 134.5, 142.1, 154.8, 156.7, 177.3. HRMS:
calcd for C26H35N2O3 423.2648 [M þ H], found 423.2642. IR:
3386, 2967, 2932, 2872, 1724, 1610, 1511 cm-1. UPLC (method 1):
74% pure (0.57 min). UPLC (method 2): 93% pure (0.64 min).
Ethyl 2-Methyl-3-(4-(2-(4-methyl-2-propyl-1H-benzo[d]imidazol-
Acknowledgment. This investigation was conducted in a
facility constructed with support from research facilities im-
provement program grant number C06 Rr-14503-01 from the
National Center for Research Resources, National Institutes
of Health. NIH grant 2R42AR44767-02A2 for Bethesda
Pharmaceuticals is also acknowledged for the partial support.
Supporting Information Available: Procedures and character-
ization data for all the intermediate compounds; procedures for
biochemical assays and computational methods. This material
is available free of charge via the Internet at http://pubs.acs.org.
1
1-yl)ethoxy)-phenyl)-2-phe-noxypropanoate (9). Yield 49%. H
NMR (CDCl3, 400 MHz): δ 1.12 (t, 3H, J=7.3 Hz), 1.23 (t, 3H,
J=7.0 Hz), 1.40 (s, 3H), 1.95 (dd, 2H, J1,2=7.5 Hz, J1,3= 15.1
Hz), 2.70 (s, 3H), 2.98-3.03 (m, 2H), 3.11 (d,1H, J=13.8 Hz),
3.29 (d, 1H J=13.8 Hz), 4.19-4.28 (m, 4H), 4.50-4.53 (t, 2H,
J =5.2 Hz), 6.71 (d, 2H, J= 8.5 Hz), 6.76 (d, 1H, J =8.0 Hz),
6.98-7.01 (m, 2H), 7.06 (d, 1H, J=7.0 Hz), 7.15-7.18 (m, 3H),
7.21-7.26 (m, 2H), 7.28 (s, 1H). 13C NMR (CDCl3, 100 MHz): δ
14.0, 14.1, 16.7, 20.6, 22.0, 29.5, 43.2, 44.6, 53.4, 65.9, 81.9,
106.6, 113.9 (2C), 114.2, 119.3 (2C), 122.2, 128.4, 129.1 (2C),
129.3, 130.57, 131.8 (2C), 134.5, 142.1, 154.8, 155.4, 157.1,
173.6. HRMS: calcd for C31H37N2O4 501.2753 [M þ H], found
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