The Design, Synthesis, and Characterizations of Spore Germination Inhibitors Effective against an Epidemic Strain of Clostridium difficile

Article abstract of DOI:10.1021/acs.jmedchem.8b00632

Clostridium difficile infections (CDI), particularly those caused by the BI/NAP1/027 epidemic strains, are challenging to treat. One method to address this disease is to prevent the development of CDI by inhibiting the germination of C. difficile spores. Previous studies have identified cholic amide m-sulfonic acid, CamSA, as an inhibitor of spore germination. However, CamSA is inactive against the hypervirulent strain R20291. To circumvent this problem, a series of cholic acid amides were synthesized and tested against R20291. The best compound in the series was the simple phenyl amide analogue which possessed an IC₅₀ value of 1.8 μM, more than 225 times as potent as the natural germination inhibitor, chenodeoxycholate. This is the most potent inhibitor of C. difficile spore germination described to date. QSAR and molecular modeling analysis demonstrated that increases in hydrophobicity and decreases in partial charge or polar surface area were correlated with increases in potency.

Full text of DOI:10.1021/acs.jmedchem.8b00632

Subscriber access provided by Kaohsiung Medical University
Article
The Design, Synthesis and Characterizations of Spore Germination
Inhibitors Effective against an Epidemic Strain of Clostridium difficile
Shiv K. Sharma, Christopher Yip, Emilio Xavier Esposito, Prateek V.
Sharma, Matthew P. Simon, Ernesto Abel-Santos, and Steven M Firestine
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00632 • Publication Date (Web): 13 Jul 2018
Downloaded from http://pubs.acs.org on July 13, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The Design, Synthesis and Characterizations of Spore Germination Inhibitors Effective
against an Epidemic Strain of Clostridium difficile
1
2
3
1
Shiv K. Sharma , Christopher Yip , Emilio Xavier Esposito , Prateek V. Sharma , Matthew P.
1
2
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Simon , Ernesto AbelꢀSantos , and Steven M. Firestine *
1
Department of Pharmaceutical sciences, Eugene Applebaum College of Pharmacy and Health
Sciences, Wayne State University, 259 Mack Avenue, Detroit, Michigan, 48201
2
Department of Chemistry and Biochemistry, University of Nevada at Las Vegas, 4505 S.
Maryland Pkwy., Las Vegas, Nevada, 89154
3
exeResearch LLC, 32 University Drive, East Lansing, Michigan, 48823
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract
Clostridium difficile infections (CDI), particularly those caused by the BI/NAP1/027
epidemic strains, are challenging to treat. One method to address this disease is to prevent the
development of CDI by inhibiting the germination of C. difficile spores. Previous studies have
identified cholic amide mꢀsulfonic acid, CamSA, as an inhibitor of spore germination. However,
CamSA is inactive against the hypervirulent strain R20291. To circumvent this problem, a series
of cholic acid amides were synthesized and tested against R20291. The best compound in the
series was the simple phenyl amide analog which possessed an IC value of 1.8 ꢀM, more than
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
225ꢀtimes as potent as the natural germination inhibitor, chenodeoxycholate. This is the most
potent inhibitor of C. difficile spore germination described to date. QSAR and molecular
modeling analysis demonstrated that increases in hydrophobicity and decreases in partial charge
or polar surface area were correlated with increases in potency.
2
ACS Paragon Plus Environment

Page 3 of 75
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
8
9
0
1
2
3
Introduction
Clostridium difficile infection (CDI) is primarily a nosocomial disease correlated mainly
with antibioticꢀassociated diarrhea. These infections are caused by Clostridium difficile, an
obligate anaerobic, gramꢀpositive, spore forming bacterium that is found in the gastrointestinal
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
tract and can be transmitted between humans via the fecalꢀoral route. In the United States alone,
there are roughly 500,000 cases of CDI annually with associated costs estimated to be
2
ꢀ4
approximately $3 billion.
1
CDI begins with the ingestion of the C. difficile spores. These spores are highly resistant
to harsh environmental factors such as stomach acid, extreme temperatures, and
5
1
1
1
1
pharmaceutically relevant antibiotics. As the spores travel through the gastrointestinal tract,
various endogenous bile salts stimulate the spores to germinate into vegetative, toxin producing
1, 5ꢀ10
C. difficile cells.
In most cases, the vegetative C. difficile cells are able to produce two
exotoxins, TcdA and TcdB, that damage the intestinal walls leading to pseudomembraneous
1
1ꢀ13
14
colitis and, in severe cases, death.
1
1
5
6
CDIs are particularly challenging to treat. Approximately 25% of patients treated for CDI
will relapse within 30 days and the majority of these patients (45ꢀ65%) will have additional
1
4ꢀ15
17
recurrences.
Patients experiencing CDI relapse have a 30% higher mortality rate than those
1
6
1
1
2
2
2
2
8
9
0
1
2
3
successfully treated. In addition, there has been an increase in outbreaks of severe disease
which have been attributed to soꢀcalled hypervirulent strains, specifically those of the
1
7
BI/NAP1/027 designation. Epidemiological studies have shown that infections by these strains
1
7ꢀ18
are associated with higher mortality and now account for approximately 20ꢀ75% of all CDI.
While many theories have been postulated, the exact reason for the increased virulence in these
1
9
strains is still not fully understood.
24
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
8
9
0
1
2
3
Treatments for CDI usually require a rigorous regimen, which normally combines
4
decontamination of the local environment along with antibiotic therapy. However, due to C.
1, 5ꢀ10
difficile’s ability to form spores, complete decontamination is often difficult.
CDI is treated
predominately by three antimicrobial agents ꢀ metronidazole, oral vancomycin and fidaxomicin
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
(Figure 1). Metronidazole is considered the first line therapy for mild to moderate CDI;
however, several clinically relevant C. difficile strains have begun to show resistance to
2
0
metronidazole. Several studies have suggested that vancomycin may be superior to the azole
4
, 20
given its pharmacokinetic parameters and absence of any reported resistance.
Fidaxomicin
was approved in 2011 as a newer antiꢀCDI treatment but its usage has been limited by drug
2
1
1
1
1
1
costs. Finally, fecal transplantation for CDI relapse has been shown to be an effective, nonꢀ
2
2ꢀ23
antibiotic therapy, although its use is limited in the US.
While there are a number of risk factors associated with CDI, current or recent antibiotic
1
6
use is strongly correlated with the disease. Under normal circumstances, bacteria found
1
, 5ꢀ10
14
naturally in the gastrointestinal tract provide a barrier against C. difficile colonization.
Upon
1
1
5
6
exposure to antibiotics, the normal gut microbiome becomes disrupted. This perturbation leads to
6
ꢀ8, 24ꢀ25
elevated levels of taurocholate, a bile salt produced naturally in the gastrointestinal tract.
17
In conjunction with free amino acids, taurocholate triggers the germination of C. difficile spores
7, 25
1
1
2
2
2
8
9
0
1
2
and ultimately the colonization of the human gastrointestinal tract by C. difficile.
Chenodeoxycholate (CDCA), another bile salt found naturally in the gastrointestinal tract, has
7
ꢀ9
been shown to inhibit the germination of C. difficile spores. It is thought that the balance
between chenodeoxycholate and taurocholate is responsible for the control of spore germination.
1, 5ꢀ10, 24ꢀ25
4
ACS Paragon Plus Environment

Page 5 of 75
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
8
9
0
1
2
3
Current treatments for CDI, as well as those being clinically investigated, are antibiotics
that target the vegetative form of C. difficile. However, one potential approach to combat CDI
2
6
would be to prophylactically treat highꢀrisk patients to prevent the onset of disease.
CDI prophylaxis may be achieved by potentially inhibiting C. difficile spore germination.
We envisioned that inhibiting the binding of taurocholate with its putative receptor should
prevent the germination of C. difficile spores. In a previous study, one of our labs explored more
than a dozen bile salt analogs with modifications in the side chain and/or in the cholate
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
6
scaffold. From this, Cholic amide metaꢀSulfonic Acid (CamSA), a taurocholate analog (Figure
2), was identified which inhibited spore germination at concentrations almost four times lower
8
, 27
1
1
1
1
than the natural inhibitor, chenodeoxycholate in vitro.
prevent CDI in mice by inhibiting spore germination in vivo.
as inhibitors of spore germination has been utilized by others and in at least one case, it was
CamSA was subsequently shown to
2
8ꢀ29
The use of modified bile salts
2
8, 30
shown that a bile salt could also be used to treat C. difficile ileal pouchitis in a human.
14
Although CamSA has many attractive properties, it has an IC value in the micromolar
5
0
1
1
5
6
range against C. difficile type strain 630 spore germination. Interestingly, CamSA was unable to
inhibit spore germination of R20291, a BI/NAP1/027 epidemic strain of C. difficile (see Table
1). Thus, we wanted to explore new compounds with activity against the epidemic C. difficile
spores. Our approach examines modifications of the amide region of CamSA by exploring a
variety of aromatic and aliphatic replacements. Here we report the synthesis of CamSA analogs,
the analysis of these agents as inhibitors of C. difficile spore germination and QSAR studies on
these agents to identify important parameters needed for activity. This study has identified
analogs that are up to 225ꢀtimes more potent than chenodeoxycholate, the natural germination
inhibitor, against R20291. Several analogs reported in this study account for some of the most
potent antiꢀgerminants described to date so far against C. difficile.
17
1
1
2
2
2
2
8
9
0
1
2
3
24
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
ACS Paragon Plus Environment

Page 7 of 75
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
8
9
0
1
2
Results
Compound Design. Previous studies have postulated that CspC is the receptor responsible
for triggering spore germination and thus we hypothesized that CspC is the likely site of action
3
1ꢀ32
for CamSA.
Given this, we examined the sequences of CspC, from R20291, an epidemic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
strain that is not inhibited by CamSA and 630, a nonꢀepidemic strain that is inhibited by CamSA
with an IC of 58 µM. To our surprise, we found that the sequences were 99.3% conserved
5
0
between both strains. An examination of the sequence differences revealed that none occurred at
3
1
residues previously identified as being critical for germination activity. In addition, CspC has
not been crystallized and thus, structureꢀguided drug design is not currently possible. Given this,
we decided to embark on an empiric investigation of a series of cholic acid amides to find agents
with activity against R20291. We focused our study on amides comprised of aromatic, aliphatic
and benzylic groups.
1
1
1
1
3
Synthesis of the bile salt analogs. The synthesis is outlined in Scheme 1. Cholic acid (7)
was preꢀactivated with HBTU/NMM in DMF at room temperature and was subsequently treated
in situ with a variety of substituted or unsubstituted aniline analogs (8) that produced Nꢀ
14
1
1
5
6
3
3ꢀ35
arylcholanꢀ24ꢀamides (12aꢀn) in 74ꢀ94% yields.
The isolation and purification of the
17
products (12aꢀj) were carried out by stirring each concentrated postꢀreaction mixture with ice
cold 2ꢀ5% aqueous HCl. For compounds which contained acid labile functionalities, such as the
ester or Boc group (12kꢀn), crude mixtures were precipitated with cold water and the products
were further purified, if necessary, by column chromatography. The reaction between cholic acid
and 4ꢀaminophenol proceeded smoothly and produced Nꢀ(4ꢀhydroxylphenyl) cholanꢀ24ꢀamide
12f while the same compound was not obtained via demethylation of methoxy groups (12d)
using BBr /CH Cl . The generation of amine substituted products were done via deprotection of
1
1
2
2
2
2
8
9
0
1
2
3
3
2
2
24
tertꢀbutyloxycarbonyl (Boc) groups of 12m and 12n with 1M HCl in ethyl acetate at room
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
6ꢀ37
1
2
3
4
temperature to produce the corresponding chloride salts (12o and 12p).
The directly linked
heterocycles as pyridine and thiozole substituted cholanꢀ24ꢀamides (12q and 13) were prepared
from the activated ester of cholic acid 7 with 4ꢀaminopyridine 8 and 2ꢀaminoꢀ4ꢀmethylthiazole 9
in 70% and 76% yields respectively.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
6
7
8
9
0
1
2
3
The synthesis of benzyl and aliphatic analogs is also shown in Scheme 1. The Nꢀbenzyl
and Nꢀpyridinomethyl analogs of cholanꢀ24ꢀamides (14aꢀg and 14jꢀk) were synthesized in 64ꢀ
90% yields by treating the HBTU/NMM activated ester of cholic acid (7) with the corresponding
benzylamines or pyridinomethylamines (10). The nitro group of compounds 14f and 14g was
selectively reduced to give the desired amino functionality (14h and 14i) in 85% and 80%
3
8ꢀ39
1
1
1
1
respectively by high pressure hydrogenation in the presence of 10% Pd/C in MeOH.
Aliphatic substituted cholanꢀ24ꢀamides (15aꢀe) and mixed arylalkylꢀsubstituted cholanꢀ24ꢀ
amides (15fꢀh) were synthesized by the same coupling method and generated the desired
products in 61ꢀ92% yields. The terminal functional groups (alkyne, cyanoꢀ, hydroxylꢀ, esterꢀ,
and bocꢀaminoꢀ) containing alkyl substituted cholanꢀ24ꢀamides (15iꢀn, 15p and 15rꢀs) were
synthesized similarly in good yields. The methyl ester group in compound 15n was selectively
14
1
1
5
6
4
0ꢀ42
cleaved with an aqueous THF LiOH solution to give the free acid 15o in 76% yield.
The
17
benzyl groups of 15p were selectively removed by Pd/C catalyzed hydrogenation in MeOH to
4
3ꢀ44
1
1
2
2
2
2
8
9
0
1
2
3
generate the diacid 15q in 74% yield.
The Boc groups in compound 15r and 15s were
selectively cleaved by 1M HCl in ethyl acetate to yield the chloride salts of cholanꢀ24ꢀamides
3
6ꢀ37
15t and 15u in 86% and 89% yields respectively.
The terminally substituted hetrocyclic
(pyrrolidine, thiophene, dioxoisoindolin and dioxoindolin) alkyl cholanꢀ24ꢀamides (15vꢀ15z)
were prepared from the activated ester of cholic acid 7 with the appropriate amine in 65ꢀ84%
yields.
8
ACS Paragon Plus Environment

Page 9 of 75
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
8
9
0
1
2
3
Biological Activity. All compounds were analyzed as inhibitors of spore germination using
a standard optical density assay which measured germination as a decrease in absorbance at 580
2
6ꢀ29
nm.
A twoꢀstep process was taken for the analysis of the biological activity of the
compounds. Compounds were analyzed for their ability to inhibit spore germination of C.
difficile R20291 at a single concentration of 125 µM. Compounds were assessed for solubility at
this concentration by visual inspection and those which were not soluble were discarded.
Compounds that were able to slow spore germination >40% compared to untreated samples were
then reanalyzed at different concentrations to determine their IC values. CamSA was shown to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
be inactive against strain R20291 under these conditions (Table 1).
1
1
1
1
The biological activities of the compounds are shown in Tables 1ꢀ3. Not surprisingly
since CamSA is aromatic, the majority of active compounds were obtained with aromatic cholanꢀ
24ꢀamides. The most potent compound was 12a, which is simply the unsubstituted aniline
derivative (Table 1, Figure 3). Compound 12a possessed an IC of 1.8 µM in the spore
5
0
14
germination assay conducted in sodium phosphate media containing 6 mM taurocholate and 12
mM glycine (Figure 3A,B). Examination of the inhibition of spore germination by 12a in the
complex BHIS media containing 6 mM taurocholate gave an IC value (1.8 µM) identical to
1
1
5
6
5
0
17
that in sodium phosphate buffer (Figure 3C,D). Given this, all spore germination assays were
1
8
conducted in sodium phosphate buffer.
1
2
9
0
We examined the effects of electron donating or electron withdrawing substituents on the
aromatic ring on the inhibitory activity of C. difficile spore germination via compounds 12bꢀp
(Scheme 1). None of the substituents that we examined improved potency beyond 12a, although
electron donating substituents generally gave more potent compounds than electron withdrawing
substituents 12b (IC of 9.8 ꢀM) compared to 12g (IC of 16 ꢀM). Substitution at the ortho
21
2
2
2
3
5
0
50
24
position yielded the most potent substituted aromatic compound (12b) with substitution on the
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
para position being the weakest (12d). Addition of an electron donating group with Hꢀbonding
potential (eg. hydroxyl) on the para position regained some activity (12f, IC of 36 ꢀM),
5
0
although the presence of an amino group at this position was inactive (12p). The data suggests
that sterically larger groups on the metaꢀ and paraꢀ positions of the aromatic ring are not tolerated
for activity (12d vs 12f vs 12p).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
7
We examined a limited number of disubstituted derivatives (12e, 12h, and 12i). Unlike
12b, the dimethoxy compound 12e was inactive, possibly due to the large group on the paraꢀ
position. Compounds 12h and 12i have both electron donating and electron withdrawing groups.
These compounds are more potent (12h IC of 24 ꢀM, and 12i IC of 14 ꢀM) than 12e, but
8
9
5
0
50
10
essentially as potent at the metaꢀsubstituted fluorine compound 12g (Table 1). This suggests that
1
1
the fluorine group plays a dominate role in the activity of these agents.
1
1
2
3
We examined two aromatic heterocycles (12q, and 13) and found that only 12q, which
has a pyridine substituent with the aromatic nitrogen at positionꢀ4 in the aromatic ring was
active. This compound is able to inhibit C. difficile spore germination with IC at 30.1 ꢀM,
14
5
0
1
1
5
6
which is 14ꢀfold less active than 12a (table 1). The aromatic nitrogen in compound 12q causes
the aromatic group to be electron deficient and 12q displays an IC is a similar range as many of
50
17
the electron deficient aromatic substitutions (i.e. 12g, 12h, etc). Like 12f and 12p, the presence
1
8
of a hydrogen bond acceptor at this location does not enhance activity.
1
2
9
0
We also examined aliphatic and benzylic substitutions on the amide nitrogen (Table 2
and 3). All benzylic and pyridinomethyl analogs 14aꢀk were inactive, although we obtained an
IC value for 14f to be used in our QSAR studies discussed below. All cyclic aliphatic
21
5
0
2
2
2
3
compounds 15aꢀc displayed moderate activity though not as good as for 12a. The cyclohexyl
analog 15a inhibited spore germination with an IC of 95.4 ꢀM (fiftyꢀthree fold higher than
50
10
ACS Paragon Plus Environment

Page 11 of 75
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
8
9
12a). Decreasing the size of the ring increased activity as seen in compounds 15b (cyclopentyl),
and 15c (cyclopropyl)) which displayed IC values of 34.1 ꢀM and 25 ꢀM respectively.
5
0
Placement of a methylene group between the amide bond and cyclopropyl ring (15d) did not
alter the inhibition; however, branching the aliphatic group decreased potency (15e IC at 154
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀM). This suggested that there is a size constraint for the binding site of the amide nitrogen
substituent. An examination of straight or branched chain containing alkyl amides with a variety
of terminal functional groups such as arylꢀ, alkyneꢀ, cyanoꢀ, hydroxylꢀ, estersꢀ, acidsꢀ, aminoꢀ
and heterocyclic moieties (15fꢀz) generated inactive compounds with the exception of 15i (IC50
at 137 ꢀM) which was only modestly active.
10
QSAR and molecular modeling. The analysis of the activity of the compounds failed to
generate a clear SAR. There are a number of reasons for this including the fact that a number of
compounds were insoluble and the assay is a phenotypic assay that included many factors
besides strictly binding to the target receptor. Thus, we decided to examine the data by structural
alignment of active versus inactive compounds followed by quantitative structure activity
relationship (QSAR) studies to determine whether activity could be predicted based upon a set of
molecular descriptors.
1
1
1
2
13
1
1
4
5
16
17
Compounds with IC values 36 µM or below (12a, 12b, 12f, 12g, 12h, 12i, 15b, 15c,
5
0
1
1
8
9
15d) were aligned along with a selected number of inactive or weakly active compounds (12c,
12d, 12e, 12j, 12k, 12l, 14a, 14f, 15a, 15f) using the flexible alignment protocol (using the
45
20
MMF94x molecular force field and atomic partial charges with Born solvation ) executed in the
Molecular Operating Environment (MOE; version 2016.0802, Chemical Computing Group)
program. The molecular surface for each class was also calculated. The alignments are shown in
2
2
1
2
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
Figure 4. All compounds, both active and inactive, aligned completely with the cholic acid
scaffold. The only differences, as expected, were noted in the amide region of the molecule.
3
4
5
6
7
8
9
0
A comparison of the active versus inactive molecules reveals that the active agents
occupy a smaller volume than the inactive species. All active agents fit within the molecular
volume for the phenyl ring. Substituents on the orthoꢀposition are tolerated, but not in the metaꢀ
or paraꢀposition. The molecular surface of the inactive agents is larger suggesting that the
binding site for the agents is confined. An examination of the lipophilicity of the active versus
inactive agents reveals that the active agents are more lipophilic (darker green color) than the
inactive agents. Taken together, this analysis indicates that the size as well as lipophilicity of the
amide substituent plays a role in the activity of the agents as spore germination inhibitors.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
1
To quantitate the effect of properties on activity, we conducted a QSAR study on the 16
compounds with which we have IC values. Unfortunately, this low number of compounds
12
5
0
1
1
3
4
precluded the use of a training and test set of data and thus validation was done using a leaveꢀ
oneꢀout calculation. Three hundred and fortyꢀthree 2D and 3D molecular descriptors were
4
6
15
calculated for each compound using MOE. The IC values were converted to negativeꢀlog10
50
1
1
6
7
values and the molecular descriptor values were normalized (meanꢀcentered and divided by the
descriptor’s standard deviation). A set of models containing 2ꢀ7 descriptors were generated by
4
7
18
application of a genetic function algorithm via the Opdagelse Predictive Modeling Toolkit
4
8
49
2
1
2
2
9
0
1
(version 1.1.0) as executed in R . Models for each number of descriptors were ranked by Q
values and the top 20 models in each set were selected. Each model was analyzed by a series of
2
2
statistical methods to generate R , Q , Fꢀtest, mean absolute error (MAE), pꢀvalue as well as
adj
22
variance inflation factors (VIF) and pꢀvalues for each variable in the equations.
12
ACS Paragon Plus Environment

Page 13 of 75
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
We focused on models with 4 descriptors since these models are close to the 5ꢀ7
compounds per descriptor rule of thumb frequently cited for QSAR studies and have
2
2
substantially better R (Average 0.706 vs 0.865) and Q (Average 0.648 vs 0.824) values than
adj
5
0
the 3ꢀdescriptor models. Furthermore, there is no evidence of overfitting of these models based
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
on VIF and pꢀvalues of the terms.
6
7
8
9
0
Five distinct, 4 descriptor models along with their statistics are given in Table 4. These
models were used to generate a consensus model in which the predicted pIC values are the
50
2
2
mean predicted value of each QSAR model. The consensus model gave an R of 0.98, R of
adj
2
0.973 and Q of 0.927 (Figure 5). The MAE and RMSE of the consensus model was 0.081 and
1
0.1 respectively. Taken together, the consensus model accurately predicts the potency of the
molecules against the R20291 strain.
11
12
Analysis of the molecular descriptors in the models indicates, broadly, that activity is
dependent upon a mixture of charge (or partial charge) and hydrophobicity. Increases in
hydrophobicity (SlogP, GCUT_SLOP1_1) are positively correlated with increases in potency
and an analysis of the percentage of importance of these descriptors indicates that between 29ꢀ
38% of the variance captured by the model can be explained by these terms. ASA_H, which
measures the water assessable surface area of hydrophobic atoms, is an indirect measure of this
as well. The presence of Gasteiger partial atomic charge or polar van der Waals surface area
1
1
3
4
15
1
1
6
7
18
5
1
1
2
2
9
0
1
(PEOE_VSA_FPNEG, RPCneg, PEOE_VSA_FNEG, PEOE_VSA_FNEG) is negatively
correlated with potency and these terms generally account for the second largest source of
variance in the data. The presence of positive charge (PEOE_VSA+0, PEOE_VSA_POS) is also
negatively correlated with potency. The remaining terms in the models represent molecular
flexibility (KierFlex, bꢀsingle). Increasing the number of single bonds decreases activity as does
22
2
2
3
4
5
2ꢀ53
an increase in flexibility as measured by the method of Kier and Hall.
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
8
9
0
1
2
3
Discussion
Previously we disclosed a cholic acid analog, CamSA, which was a potent inhibitor of C.
difficile spore germination and also prevented the development of CDI in a murine model of the
2
6, 29
disease.
In these studies, CamSA was tested against C. difficile strains 630 and VPI 10463.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To our surprise, CamSA showed no inhibitory activity against an epidemic strain, R20291. The
inability of CamSA to work on this strain, coupled with an increased prevalence of these strains
in clinical settings, necessitated additional analogs of CamSA to be identified. Unfortunately, the
target of action for CamSA is unknown and there is little structural information on any proteins
believed to be involved in spore germination in C. difficile, thus, an empirical approach was
necessary for identifying new agents.
1
1
1
1
An analysis of a variety of cholanꢀ24ꢀamide analogs as spore germination inhibitors in
strain R20291 revealed that the best inhibitor in our series was 12a, which is the simple phenyl
5
4
derivative in which the sulfate group of CamSA was removed. In general, the loss of functional
groups with the potential for significant biomolecular interactions (i.e., the sulfate group) tends
to result in the loss of activity. An alignment of active versus inactive compounds indicates that
active agents are smaller and more hydrophobic than inactive agents. Our QSAR analyses also
indicate that increasing partial charge or polar van der Waals surface area decreases activity.
A key question is why does CamSA work against strain 630 and not R20291? One
possibility could be changes to the target of these agents. The CspA, CspB and CspC proteins
have been implicated in spore germination in C. difficile and mutational analyses have suggested
14
1
1
5
6
17
1
1
2
2
2
2
8
9
0
1
2
3
6
, 10, 31
CspC as the likely taurocholate germination receptor.
However, sequence analyses of
CspA, CspB and CspC reveal that these proteins greater than 98.7% identical between the 630
and R20291 strains. Thus, the inactivity of CamSA against the R20291 strain is likely not due to
alterations to a binding site in any of these proteins.
24
14
ACS Paragon Plus Environment

Page 15 of 75
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
8
9
0
1
2
3
The spore germination assays are spore (cell)ꢀbased assays. Thus, differences in activity
could be due to changes in transport of the molecule to the site of action. Problems with
transport, either due to the lack of active uptake, the presence of efflux pumps or poor
5
5ꢀ57
physicochemical properties necessary for passive transport, are common in cellꢀbased assays.
For example, vancomycin is inactive against gramꢀnegative bacteria due to its inability to cross
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
8
the outer membrane. Recent studies on drug transport in gramꢀnegative bacteria have shown
that changes in drug structure can convert an agent that is selective for gramꢀpositive into one
5
9
that works on gramꢀnegative agents by facilitating transport.
Unfortunately, there is little information on transport of molecules into spores and even less
1
1
1
1
for C. difficile spores. It has been postulated that the CspA, B, and C proteins are found in the
6
0
cortex layer of the bacterial spore. Thus, small molecules that bind to CspC must pass through
the exosporium, coat and outer membrane layer to reach the protein. One possible solution is that
there exist proteins that enable the transport of small molecules into the cortex to facilitate
germination. In B. subtilis, GerP has been shown to enhance permeability of nutrients into the
14
61
1
1
5
6
spore. No such protein has yet been identified in C. difficile; however, an AAA+ ATPase has
6
2ꢀ63
been recently suggested to facilitate nutrient transport.
However, it is possible that the
17
activity of the germination inhibitors could be the result of inhibiting such transporters.
Regardless of whether there is a change in passive or active transport in different strains
of C. difficile, it is clear that small molecule inhibitors must be tailored to initially reach the site
of action, and then to inhibit germination. Our data indicates that the overall size and shape of
the substitution on the amine side of the amide is fairly specific and our active compounds are
more hydrophobic than CamSA.
1
1
2
2
2
2
8
9
0
1
2
3
24
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
8
9
0
1
Conclusions
We have synthesized and examined a series of cholic acid amides as inhibitors of C.
difficile spore germination in the epidemic strain R20291. We have found that 12a is the most
potent known inhibitor of spore germination described to date with an IC of 1.8 µM. We have
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
conducted modeling and QSAR studies which indicate that an increase in hydrophobicity and a
decrease in size of the molecule enhances activity in this strain. This work also suggests that
there may be strain differences in response to spore germination inhibitors and that differences in
transport of molecules into the spore could be the reason for this difference. Future work will
examine the in vivo activity of 12a in animal models of spore germination as well as the use of
our QSAR model to identify potent analogs for future testing. Ultimately, we hope that these
agents will find utility in the prevention of C. difficile infections in patients.
1
1
1
2
Experimental Section
1
1
1
1
3
4
5
6
General Comments. Cholic acid (3α, 7α, 12αꢀTrihydroxyꢀ5βꢀcholanꢀ24ꢀoic acid) was
purchased from MP Biomedical, and silica gel for column chromatography from Sorbent
technologies, Inc. All reagents and dry solvents (DMF and EtOAc) were purchased from either
SigmaꢀAldrich, Acros Organics, TCI Chemicals or ChemꢀImpex International and were used
without further purification. Thin layer chromatography (TLC) were performed on preꢀcoated
(0.25 mm) silica gel plate (Sorbtech, 60 Fꢀ254), and visualization was done either by UV (254
nm), iodine staining or ninhydrin staining. Column chromatographic purifications of compounds
17
1
1
8
9
1
13
20
were performed on silicaꢀgel (Sorbtech, 60ꢀ230 mesh, 0.063ꢀ0.20mm). H and C NMR spectra
were recorded on a Varian VNMRS 600 MHz spectrometer by dissolving the compounds in
deuterated solvents as chloroformꢀd (CDCl ), methanolꢀd (CD OD) or dimethyl sulfoxideꢀd
2
2
1
2
3
4
3
6
23
(DMSOꢀd ) and all peaks were referenced with TMS as an internal standard. Some of the
6
16
ACS Paragon Plus Environment

Page 17 of 75
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
8
9
0
compound’s spectra were recorded in multiple solvents for clarity of the aliphatic region.
Chemical shifts are expressed in ppm (δ) whereas coupling constants (J) are listed in hertz (Hz)
and the multiplicities are recorded by following abbreviations: s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet), and br (broad signal). Highꢀresolution mass spectra (HRMS) were
recorded on a Kratos MS 80 RFA (ESI and CI) spectrometer. The purities of all testing
compounds were determined to be >95% by High Performance Liquid Chromatography (HPLC)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TM
on Beckman Coulter XꢀBridge REH130 C18 column (size 4.6 x 150 mm) with a flow rate of
0.80 mL/min using mobile phase consisted of methanol/acetonitrile and water, and monitoring
with UV lamp at wavelengths (223/234/254 nm). Melting points were determined using Melꢀ
temp II apparatus by Laboratory Device, in open capillaries and are uncorrected.
1
11
Coupling of Cholic acid with Aniline (MethodꢀA): Nꢀ(Phenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ
5βꢀcholanꢀ24ꢀamide (12a). To a 100 mL round bottom flask, cholic acid (7, 2.085 g, 5.10
mmol) and HBTU (1.987 g, 5.23 mmol) were dissolved in anhydrous DMF (12 mL) at room
temperature. NMM (0.60 mL, 5.45 mmol) was added to the solution and the reaction content was
allowed to stir for 30 min in order to generate an activated ester. During this time, the solution
became light yellow. Aniline (8, 612 mg, 6.57 mmol) and an additional aliquot of NMM (0.60
mL, 5.45 mmol) were added to the above reaction mixture, and the reaction was stirred for 48 h
at room temperature. The reaction mixture was concentrated using a high vacuum rotary
evaporator to produce a viscous lightꢀyellow material. Ice cold aqueous 5% HCl solution (200
mL) was added to the residue and the mixture was subjected to sonication for 5ꢀ15 minutes. A
resulting white precipitate was formed and the top aqueous layer was carefully decanted and
discarded. The treatment with 5% HCl and sonication was repeated 2ꢀ3 times. The white
1
1
1
1
1
2
3
4
5
6
17
1
1
8
9
20
2
2
2
2
1
2
3
4
precipitate was collected by filtration through a sintered glass funnel, washed with cold water
o
and dried under vacuum to produce 12a (2.241 g, 91 % yield). mp. 255ꢀ256 C; TLC R . 0.44
f
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
2
3
4
5
6
7
8
9
0
1
(AcOEt:CH OH, 95:5); H NMR (CD OD, 600 MHz): δ 7.51 (d, 2H, J = 7.8 Hz), 7.27 (t, 2H, J
3
3
= 7.8 Hz), 7.05 (t, 1H, J = 7.2 Hz), 3.94 (s, 1H), 3.78 (s, 1H), 3.36 (m, 1H), 2.44ꢀ2.40 (m, 1H),
2.30ꢀ2.22 (m, 3H), 2.01ꢀ1.85 (m, 5H), 1.80ꢀ1.72 (m, 2H), 1.63 (d, 1H, J = 13.2 Hz), 1.60ꢀ1.50
(m, 5H), 1.48ꢀ1.27 (m, 5H), 1.13ꢀ1.09 (m, 1H), 1.05 (d, 3H, J = 6.0 Hz), 0.97 (dt, 1H, J = 10.8
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Hz), 0.90 (s, 3H), 0.70 (s, 3H); H NMR (DMSOꢀd , 600 MHz): δ 9.82 (s, 1H), 7.55 (d, 2H, J =
6
7.8 Hz), 7.23 (t, 2H, J = 7.2 Hz), 6.96 (t, 1H, J = 7.2 Hz), 3.91 (br, 3H), 3.76 (s, 1H), 3.58 (s,
1H), 3.15 (s, 1H), 2.30 (m, 1H), 2.19ꢀ2.12 (m, 3H), 1.96 (q, 1H, J = 11.4 Hz), 1.82ꢀ1.72 (m, 4H),
1.62 (m, 2H), 1.42ꢀ1.38 (m, 3H), 1.32ꢀ1.14 (m, 8H), 0.94 (d, 4H, J = 6.0 Hz), 0.82ꢀ0.77 (m, 3H),
1
3
0.55 (s, 3H); C NMR (CD OD, 150 MHz): δ 173.8, 138.6, 128.3, 123.6, 119.8, 72.6, 71.4,
3
1
1
67.6, 46.2, 46.1, 41.8, 41.6, 39.6, 39.0, 35.6, 35.1, 34.5, 34.5, 33.6, 31.7, 29.8, 28.2, 27.3, 26.4,
1
3
22.8, 21.8, 16.4, 11.6; C NMR (DMSOꢀd , 150 MHz): δ 172.2, 139.9, 129.0, 123.3, 119.4,
6
12
71.4, 70.9, 66.7, 46.5, 46.2, 42.0, 41.8, 35.7, 35.6, 35.3, 34.8, 33.9, 31.9, 30.8, 29.0, 27.7, 26.6,
+
1
1
3
4
23.2, 23.1, 17.6, 12.8. HRMS (ESI, m/z): calcd for C H NO Na [M + Na ] 506.3246, found
3
0
45
4
506.3239.
15
Nꢀ(2’ꢀMethoxyphenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (12b). This
1
1
6
7
compound was prepared from cholic acid 7 and 2ꢀmethoxyaniline by following methodꢀA in
o
1
93% yield. mp. 207ꢀ209 C; TLC R . 0.61 (CH Cl :CH OH:NH OH, 89:10:1); H NMR (CD OD,
f
2
2
3
4
3
18
600 MHz): δ 7.86 (d, 1H, J = 7.2 Hz), 7.08 (t, 1H, J = 7.2 Hz), 6.97 (d, 1H, J = 7.8 Hz), 6.88 (t,
1H, J = 7.2 Hz), 3.95 (s, 1H), 3.85 (s, 3H), 3.78 (s, 1H), 3.35 (m, 1H), 2.52ꢀ2.44 (m, 1H), 2.37ꢀ
2.33 (m, 1H), 2.30ꢀ2.24 (m, 2H), 2.01ꢀ1.87 (m, 5H), 1.79 (d, 1H, J = 14.4 Hz), 1.76ꢀ1.72 (m,
1
2
9
0
21
1H), 1.63 (d, 1H, J = 13.2 Hz), 1.57ꢀ1.49 (m, 5H), 1.46ꢀ1.31 (m, 5H), 1.14ꢀ1.05 (m, 4H), 0.99 (t,
1
2
2
2
3
1H, J = 13.2 Hz), 0.90 (s, 3H), 0.70 (s, 3H); H NMR (DMSOꢀd , 600 MHz): δ 8.98 (s, 1H), 7.84
6
(d, 1H, J = 7.8 Hz), 7.01ꢀ6.95 (m, 2H), 6.83 (t, 1H, J = 7.8 Hz), 4.44 (d,1H, J = 4.2 Hz), 4.12 (d,
1H, J = 3.6 Hz), 4.02 (d, 1H, J = 3.0 Hz), 3.78 (s, 5H), 3.17ꢀ3.12 (m, 1H), 2.38ꢀ2.34 (m, 1H),
24
18
ACS Paragon Plus Environment

Page 19 of 75
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
2.26ꢀ2.07 (m, 3H), 1.92 (q, 1H, J = 12.0 Hz), 1.78ꢀ1.70 (m, 4H), 1.62ꢀ1.57 (m, 2H), 1.43ꢀ1.38
13
(m, 3H), 1.34ꢀ1.12 (m, 8H), 0.92 (d, 4H, J = 6.0 Hz), 0.85ꢀ0.75 (m, 4H), 0.54 (s, 3H); C NMR
(CD OD, 150 MHz): δ 173.8, 150.2, 126.8, 124.7, 122.2, 120.0, 110.4, 72.6, 71.4, 67.4, 54.8,
3
46.7, 46.1, 41.7, 41.6, 39.6, 39.0, 35.5, 35.1, 34.5, 33.5, 31.7, 29.7, 28.1, 27.3, 26.4, 22.8, 21.8,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
16.4, 11.6; C NMR (DMSOꢀd , 150 MHz): δ 172.5, 150.1, 127.7, 124.7, 122.5, 120.6, 111.4,
6
71.6, 70.9, 66.7, 56.0, 46.6, 46.2, 41.9, 41.8, 40.1, 35.6, 35.2, 34.8, 33.7, 32.0, 30.7, 28.9, 27.7,
26.6, 23.3, 23.0, 17.5, 12.8.
8
9
Nꢀ(3’ꢀMethoxyphenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (12c). This
compound was prepared from cholic acid 7 and 3ꢀmethoxyaniline by following methodꢀA in
o
1
10
87% yield. mp. 260ꢀ262 C; TLC R . 0.59 (CH Cl :CH OH:NH OH, 89:10:1); H NMR (CD OD,
f 2 2 3 4 3
1
1
1
1
1
1
1
1
1
2
3
4
5
6
7
8
600 MHz): δ 7.26 (t, 1H, J = 2.4 Hz), 7.16 (t, 1H, J = 7.8 Hz), 7.04 (d, 1H, J = 7.8 Hz), 6.62 (dd,
1H, J = 8.4 and 2.4 Hz), 3.94 (s, 1H), 3.77 (d, 1H, J = 2.4 Hz), 3.75 (s, 3H), 3.38ꢀ3.29 (m, 1H),
2.42ꢀ2.39 (m, 1H), 2.30ꢀ2.22 (m, 3H), 2.01ꢀ1.84 (m, 5H), 1.77ꢀ1.71 (m, 2H), 1.63 (d, 1H, J =
14.4 Hz), 1.58ꢀ1.49 (m, 5H), 1.45ꢀ1.28 (m, 5H), 1.13ꢀ1.07 (m, 1H), 1.05 (d, 3H, J = 6.0 Hz),
1
0.96 (dt, 1H, J = 14.4 Hz), 0.89 (s, 3H), 0.70 (s, 3H); H NMR (DMSOꢀd , 600 MHz): δ 9.81 (s,
6
1H), 7.26 (s, 1H), 7.13 (t, 1H, J = 8.4 Hz), 7.07 (d, 1H, J = 7.8 Hz), 6.56 (dd, 1H, J = 8.4 and 1.8
Hz), 4.29 (d, 1H, J = 4.2 Hz), 4.08 (d, 1H, J = 3.0 Hz), 3.98 (d, 1H, J = 3.0 Hz), 3.75 (s, 1H),
3.67 (s, 3H), 3.57 (s, 1H), 3.14 (m, 1H), 2.31ꢀ2.26 (m, 1H), 2.21ꢀ2.09 (m, 3H), 1.98ꢀ1.93 (m,
19
1H), 1.82ꢀ1.69 (m, 4H), 1.60 (d, 1H, J = 13.2 Hz), 1.43ꢀ1.38 (m, 3H), 1.34ꢀ1.12 (m, 8H), 0.96ꢀ
13
2
2
2
0
1
2
0.87 (m, 4H), 0.83ꢀ0.77 (m, 4H), 0.55 (s, 3H); C NMR (CD OD, 150 MHz): δ 173.8, 160.1,
3
139.7, 129.0, 111.9, 109.1, 105.5, 72.6, 71.4, 67.4, 54.2, 46.6, 46.1, 41.8, 41.6, 39.6, 39.0, 35.5,
35.1, 34.5, 34.4, 33.6, 31.7, 29.8, 28.2, 27.3, 26.4, 22.8, 21.7, 16.4, 11.6.
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
8
9
0
1
Nꢀ(4’ꢀMethoxyphenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (12d). This
compound was prepared from cholic acid 7 and 4ꢀmethoxyaniline by following methodꢀA in
o
1
94% yield. mp. 213ꢀ214 C; TLC R . 0.51 (CH Cl :CH OH:NH OH, 89:10:1); H NMR (CD OD,
f
2
2
3
4
3
600 MHz): δ 7.40 (d, 2H, J = 9.0 Hz), 6.84 (d, 2H, J = 9.0 Hz), 3.94 (s, 1H), 3.78 (m, 1H), 3.74
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(s, 3H), 3.34 (m, 1H), 2.40ꢀ2.37 (m, 1H), 2.30ꢀ2.21 (m, 3H), 2.01ꢀ1.83 (m, 5H), 1.80ꢀ1.71 (m,
2H), 1.64ꢀ1.49 (m, 6H), 1.45ꢀ1.29 (m, 5H), 1.12ꢀ1.07 (m, 1H), 1.05 (d, 3H, J = 6.6 Hz), 0.96 (dt,
1
1H, J = 13.8 and 3.6 Hz), 0.89 (s, 3H), 0.70 (s, 3H); H NMR (DMSOꢀd , 600 MHz): δ 9.67 (s,
6
1H), 7.45 (d, 2H, J = 8.4 Hz), 6.81 (d, 2H, J = 8.4 Hz), 4.09 (br s, 3H), 3.76 (s, 1H), 3.67 (s, 3H),
3.58 (s, 1H), 3.15 (s, 1H), 2.30ꢀ2.08 (m, 4H), 1.98ꢀ1.93 (m, 1H), 1.80ꢀ1.68 (m, 4H), 1.64ꢀ1.58
(m, 2H), 1.43ꢀ1.38 (m, 3H), 1.34ꢀ1.10 (m, 8H), 0.94 (d, 4H, J = 5.4 Hz), 0.82ꢀ0.77 (m, 4H), 0.55
1
1
1
3
(s, 3H); C NMR (CD OD, 150 MHz): δ 173.6, 156.4, 131.5, 121.7, 113.5, 72.6, 71.4, 67.6,
3
12
54.4, 46.6, 46.1, 41.8, 41.6, 39.6, 39.0, 35.6, 35.1, 34.5, 34.4, 33.4, 31.8, 29.8, 28.2, 27.3, 26.4,
22.8, 21.8, 16.4, 11.6.
1
1
3
4
Nꢀ(2’, 4’ꢀDimethoxyphenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (12e). This
15
compound was prepared from cholic acid 7 and 2,4ꢀdimethoxyaniline by following methodꢀA in
o
1
1
1
6
7
91% yield. mp. 203ꢀ204 C; TLC R . 0.69 (CH Cl :CH OH:NH OH, 89:10:1); H NMR (CD OD,
f 2 2 3 4 3
600 MHz): δ 7.67 (s, 1H), 6.87 (d, 1H, J = 9.0 Hz), 6.61 (d, 1H, J = 8.4 Hz), 3.94 (s, 1H), 3.80
(s, 3H), 3.77 (s, 1H), 3.71 (s, 3H), 3.67 (m, 1H), 2.52ꢀ2.44 (m, 1H), 2.36ꢀ2.31 (m, 1H), 2.29ꢀ2.20
(m, 2H), 2.01ꢀ1.84 (m, 5H), 1.79ꢀ1.72 (m, 2H), 1.63 (d, 1H, J = 12.6 Hz), 1.58ꢀ1.49 (m, 5H),
18
1
2
9
0
1.46ꢀ1.30 (m, 5H), 1.10 (m, 1H), 1.05 (d, 3H, J = 6.0 Hz), 0.97 (t, 1H, J = 13.8 Hz), 0.89 (s, 3H),
1
21
0.69 (s, 3H); H NMR (DMSOꢀd , 600 MHz): δ 8.96 (s, 1H), 7.66 (s, 1H), 6.88 (d, 1H, J = 8.4
6
2
2
2
3
Hz), 6.55 (d, 1H, J = 8.4 Hz), 4.08 (br s, 3H), 3.76 (s, 1H), 3.73 (s, 3H), 3.63 (s, 3H), 3.58 (s,
1H), 3.15 (s, 1H), 2.39 (s, 1H), 2.27ꢀ2.10 (m, 3H), 1.96 (q, 1H, J = 10.8 Hz), 1.77ꢀ1.60 (m, 6H),
1
3
24
1.41ꢀ1.15 (m, 11H), 0.92 (d, 4H, J = 5.4 Hz), 0.82ꢀ0.77 (m, 4H), 0.56 (s, 3H); C NMR
20
ACS Paragon Plus Environment

Page 21 of 75
1
Journal of Medicinal Chemistry
2
3
4
5
6
1
(CD OD, 150 MHz): δ 173.8, 153.5, 144.0, 127.6, 111.0, 108.5, 108.3, 72.6, 71.4, 67.6, 55.3,
3
2
54.6, 46.7, 46.1, 41.8, 41.6, 39.6, 39.0, 35.5, 35.1, 34.5, 34.4, 33.5, 31.7, 29.8, 28.2, 27.3, 26.4,
22.8, 21.8, 16.4, 11.6.
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5
6
7
8
9
0
1
Nꢀ(4’ꢀHydroxyphenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (12f). This
compound was prepared from cholic acid 7 and 4ꢀaminophenol by following methodꢀA in 80%
o
1
yield. mp. 140ꢀ142 C; TLC R . 0.59 (AcOEt:CH OH, 90:10); H NMR (CD OD, 600 MHz): δ
f
3
3
7.30 (d, 2H, J = 7.2 Hz), 6.71 (d, 2H, J = 7.2 Hz), 3.94 (s, 1H), 3.77 (s, 1H), 3.34 (s, 1H), 2.37
(s, 1H), 2.28ꢀ2.23 (m, 3H), 1.98ꢀ1.82 (m, 5H), 1.80ꢀ1.70 (m, 2H), 1.63 (d, 1H, J = 11.4 Hz),
1.56ꢀ1.49 (m, 5H), 1.42ꢀ1.29 (m, 5H), 1.16ꢀ1.11 (m, 4H), 0.95 (t, 1H, J = 13.8 Hz), 0.89 (s, 3H),
1
3
1
1
0.69 (s, 3H); C NMR (CD OD, 150 MHz): δ 173.5, 153.9, 130.4, 122.0, 114.8, 72.7, 71.5,
3
67.6, 46.6, 46.1, 41.7, 41.6, 39.6, 39.0, 35.6, 35.1, 34.5, 34.5, 33.4, 31.9, 29.8, 28.2, 27.3, 26.4,
22.8, 21.8, 16.4, 11.6.
12
13
Nꢀ(3’ꢀFluorophenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (12g). This compound
was prepared from cholic acid 7 and 3ꢀfluoroaniline by following methodꢀA, and after regular
work up procedure, the product was purified by column chromatography eluted from
1
1
4
5
16
AcOEt:CH OH (95:5, 90:10; 80:20 and 75:25) to afford white powder (83% yield). mp. 231ꢀ
3
o
1
1
1
7
8
233 C; TLC R . 0.39 (AcOEt:CH OH, 95:5); H NMR (CD OD, 600 MHz): δ 7.51 (d, 1H, J =
f 3 3
14.4 Hz), 7.27ꢀ7.21 (m, 2H), 6.77 (t, 1H, J = 8.4 Hz), 3.93 (s, 1H), 3.77 (s, 1H), 3.35 (m, 1H),
2.45ꢀ2.40 (m, 1H), 2.30ꢀ2.22 (m, 3H), 2.00ꢀ1.83 (m, 5H), 1.79ꢀ1.72 (m, 2H), 1.63 (d, 1H, J =
19
2
2
2
0
1
2
12.0 Hz), 1.56ꢀ1.27 (m, 10H), 1.11ꢀ1.08 (m, 1H), 1.04 (d, 3H, J = 5.4 Hz), 0.96 (t, 1H, J = 14.4
13
Hz), 0.89 (s, 3H), 0.69 (s, 3H); C NMR (CD OD, 150 MHz): δ 173.9, 140.5, 129.7, 129.7,
3
114.9, 109.9, 109.7, 106.7, 106.5, 72.6, 71.5, 67.6, 46.6, 46.1, 41.7, 41.6, 39.6, 39.0, 35.5, 35.1,
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
34.5, 33.6, 31.6, 29.8, 27.3, 26.4, 22.8, 21.8, 16.4, 11.6. HRMS (ESI, m/z): calcd for
+
C H FNO Na [M + Na ] 524.3152, found 524.3130.
30
44
4
3
4
Nꢀ(4’ꢀFluoroꢀ2’ꢀmethylphenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (12h). This
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compound was prepared from cholic acid 7 and 4ꢀfluoroꢀ2ꢀmethylaniline by following methodꢀA
o
5
to afford a light pink colored powder (91% yield). mp. 134ꢀ135 C; TLC R . 0.64
f
1
6
(CH Cl :CH OH:NH OH, 89:10:1); H NMR (DMSOꢀd , 600 MHz): δ 9.24 (s, 1H, 1H), 7.27 (t,
2
2
3
4
6
7
1H, J = 6.6 Hz), 7.01 (d, 1H, J = 9.6 Hz), 6.94 (t, 1H, J = 7.8 Hz), 4.32 (s, 1H), 4.10 (s, 1H),
3.99 (s, 1H), 3.77 (s, 1H), 3.58 (s, 1H), 3.13 (m, 1H), 2.34ꢀ2.28 (m, 1H), 2.22ꢀ2.10 (m, 6H), 1.97
(q, 1H, J = 11.4 Hz), 1.82ꢀ1.70 (m, 4H), 1.61 (d, 2H, J = 10.8 Hz), 1.44ꢀ1.36 (m, 3H), 1.35ꢀ1.13
8
9
1
3
10
(m, 8H), 0.96ꢀ0.92 (m, 4H), 0.83ꢀ0.77 (m, 4H), 0.56 (s, 3H); C NMR (DMSOꢀd , 150 MHz): δ
6
1
1
1
2
172.2, 160.5, 158.9, 135.3, 135.3, 133.2, 127.7, 127.6, 116.9, 116.8, 112.8, 112.7, 71.5, 70.1,
66.7, 46.6, 46.2, 41.9, 41.8, 35.7, 35.6, 35.3, 34.8, 33.2, 32.1, 30.8, 29.0, 27.8, 26.6, 23.2, 23.0,
1
3
13
18.3, 17.5, 12.8; C NMR (CD OD, 150 MHz): δ 174.5, 161.6, 160.0, 136.1, 136.1, 131.6,
3
1
1
4
5
127.8, 127.7, 116.4, 116.3, 112.4, 112.3, 71.6, 71.4, 67.6, 46.7, 46.1, 41.8, 41.6, 39.6, 39.0, 35.5,
35.1, 34.5, 34.5, 32.8, 31.9, 29.8, 28.2, 27.3, 26.5, 22.8, 21.8, 16.9, 16.4, 11.6.
16
Nꢀ(5’ꢀFluoroꢀ2’ꢀmethylphenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (12i). This
compound was prepared from cholic acid 7 and 5ꢀfluoroꢀ2ꢀmethylaniline by following methodꢀA
1
1
7
8
o
1
in 77% yield. mp. 206ꢀ207 C; TLC R . 0.65 (CH Cl :CH OH:NH OH, 89:10:1); H NMR
f
2
2
3
4
19
(CD OD, 600 MHz): δ 7.18 (m, 2H), 6.83 (t, 1H, J = 7.2 Hz), 3.95 (s, 1H), 3.78 (s, 1H), 3.60 (t,
3
2
2
0
1
1H, J = 10.8 Hz), 2.50ꢀ2.44 (m, 1H), 2.37ꢀ2.32 (m, 1H), 2.30ꢀ2.23 (m, 3H), 2.19 (s, 3H), 2.02ꢀ
1.87 (m, 5H), 1.80ꢀ1.73 (m, 3H), 1.64 (d, 1H, J = 12.6 Hz), 1.57ꢀ1.30 (m, 9H), 1.11ꢀ1.06 (m,
1
22
4H), 0.98ꢀ0.90 (m, 4H), 0.71 (s, 3H); H NMR (DMSOꢀd , 600 MHz): δ 9.22 (s, 1H, 1H), 7.34
6
2
3
(d, 1H, J = 7.8 Hz), 7.15 (s, 1H), 6.82 (s, 1H), 4.11 (br s, 3H), 3.76 (s, 1H), 3.57 (s, 1H), 3.15 (s,
22
ACS Paragon Plus Environment

Page 23 of 75
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
1H), 2.35 (s, 1H), 2.28ꢀ2.13 (s, 6H), 1.96 (s, 1H), 1.82ꢀ1.68 (m, 4H), 1.61 (s, 2H), 1.52ꢀ1.10 (m,
13
11H), 1.02ꢀ0.77 (m, 8H), 0.56 (s, 3H); C NMR (DMSOꢀd , 150 MHz): δ 172.4, 161.3, 159.7,
6
138.3, 138.2, 131.7, 131.6, 126.7, 111.3, 111.1, 71.5, 70.9, 66.7, 46.6, 46.2, 42.0, 41.8, 35.7,
35.6, 35.3, 34.8, 33.3, 32.0, 30.8, 29.0, 27.8, 26.6, 23.2, 23.0, 17.6, 17.6, 12.8.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
6
Nꢀ(3’ꢀAminocarbonylphenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (12j ). This
compound was prepared from cholic acid 7 and 3ꢀaminobenzamide by following methodꢀA in
o
1
7
74% yield. mp. 130ꢀ132 C; TLC R . 0.55 (AcOEt:CH OH, 85:15); H NMR (CD OD, 600
f 3 3
8
MHz): δ 8.05 (s, 1H), 7.72 (d, 1H, J = 7.8 Hz), 7.55 (d, 1H, J = 7.8 Hz), 7.39 (t, 1H, J = 7.8 Hz),
3.94 (s, 1H), 3.77 (d, 1H, J = 1.8 Hz), 3.36 (m, 1H), 2.46ꢀ2.42 (m, 1H), 2.33ꢀ2.21 (m, 3H), 2.00ꢀ
1.85 (m, 5H), 1.79ꢀ1.72 (m, 2H), 1.63 (d, 1H, J = 12.6 Hz), 1.61ꢀ1.49 (m, 5H), 1.44ꢀ1.29 (m,
9
10
1
1
1
1
1
1
1
2
3
4
5
6
5H), 1.10ꢀ1.07 (m, 1H), 1.04 (d, 3H, J = 6.0 Hz), 0.96 (t, 1H, J = 13.8 Hz), 0.88 (s, 3H), 0.69 (s,
1
3H); H NMR (DMSOꢀd , 600 MHz): δ 10.05 (s, 1H), 8.02 (s, 1H), 7.73 (d, 1H, J = 5.4 Hz),
6
7.47 (d, 1H, J = 5.4 Hz), 7.29 (s, 1H), 3.75 (s, 1H), 3.56 (s, 1H), 3.14 (s, 1H), 2.31 (s, 1H), 2.24ꢀ
2.06 (m, 3H), 1.94 (m, 1H), 1.80ꢀ1.66 (m, 4H), 1.60 (s, 2H), 1.44ꢀ1.10 (m, 11H), 0.92 (s, 4H),
13
0.82ꢀ0.74 (m, 4H), 0.53 (s, 3H); C NMR (DMSOꢀd , 150 MHz): δ 172.4, 168.5, 139.8, 135.3,
6
128.8, 122.2, 122.1, 119.1, 72.5, 70.9, 66.7, 46.6, 46.2, 41.9, 41.8, 35.7, 35.0, 35.3, 34.8, 33.9,
31.9, 30.8, 28.1, 27.7, 26.6, 23.2, 23.0, 17.6, 12.8. HRMS (ESI, m/z): calcd for C H N O Na
17
3
1
46
2
5
+
1
1
8
9
[M + Na ] 549.3304, found 549.3270.
Coupling of Cholic Acid with Ethyl 3ꢀaminobenzoate (MethodꢀB): Nꢀ(3’ꢀ
20
Ethoxycarbonylphenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (12k). A mixture of
Cholic acid (7, 826 mg, 2.02 mmol) and HBTU (957 mg, 2.53 mmol) was dissolved in
anhydrous DMF (6 mL) followed by the addition of NMM (0.25 mL, 2.27 mmol). The reaction
was stirred at room temperature for 50 min to generate the activated ester. Ethyl 3ꢀ
2
2
2
1
2
3
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
8
9
0
1
2
3
aminobenzoate (418 mg, 2.53 mmol) and NMM (0.20 mL) were added to the reaction and the
reaction was stirred for 72 h at room temperature. After completion, the reaction was
concentrated using a high vacuum rotary evaporator to give a viscous material residue. Iceꢀcold
water (120 mL) was added to the residue and the mixture was subjected to sonication for 5ꢀ10
minutes to produce a white precipitate. The aqueous layer was carefully decanted and the water
wash and sonication was repeated. The resulting white precipitate was collected by filtration,
washed with cold water and dried under vacuum to produce 12k (976 mg, 87% yield). mp. 106ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
1
108 C; TLC R . 0.60 (AcOEt:CH OH, 90:10); H NMR (CD OD, 600 MHz): δ 8.22 (s, 1H), 7.80
f
3
3
(d, 1H, J = 7.8 Hz), 7.72 (d, 1H, J = 7.8 Hz), 7.39 (t, 1H, J = 7.8 Hz), 4.34 (q, 2H, J = 7.2 Hz),
3.94 (s, 1H), 3.77 (d, 1H, J = 2.4 Hz), 3.34 (m, 1H), 2.45ꢀ2.42 (m, 1H), 2.32ꢀ2.23 (m, 3H), 2.01ꢀ
1
1
1
1
1.85 (m, 5H), 1.80ꢀ1.72 (m, 2H), 1.63 (d, 1H, J = 13.2 Hz), 1.58ꢀ1.49 (m, 6H), 1.48ꢀ1ꢀ1.27 (m,
13
7H), 1.13ꢀ1.04 (m, 4H), 0.96 (t, 1H, J = 14.4 Hz), 0.89 (s, 3H), 0.70 (s, 3H); C NMR (CD OD,
3
150 MHz): δ 174.0, 166.3, 139.0, 130.8, 128.5, 124.4, 124.0, 120.5, 72.6, 71.4, 67.6, 60.8, 46.6,
46.0, 41.8, 41.6, 39.6, 39.0, 35.5, 35.1, 34.5, 34.4, 33.5, 31.6, 29.8, 28.2, 27.3, 26.5, 22.8, 21.7,
14
+
1
1
5
6
16.4, 13.2, 11.6. HRMS (ESI, m/z): calcd for C H NO Na [M + Na ] 578.3458, found
3
3
49
6
578.3458.
1
1
1
7
8
9
Nꢀ(4’ꢀEthoxycarbonylphenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (12l). This
compound was prepared from cholic acid 7 and ethyl 4ꢀaminobenzoate by following methodꢀB
o
1
in 83% yield, mp. 106ꢀ108 C; TLC R . 0.50 (AcOEt:CH OH, 95:5); H NMR (CD OD, 600
f
3
3
20
MHz): δ 7.93 (d, 2H, J = 7.8 Hz), 7.67 (d, 2H, J = 7.8 Hz), 4.31 (b, 2H), 3.93 (s, 1H), 3.77 (s,
2
2
1
2
1H), 3.35 (s, 1H), 2.48ꢀ2.42 (m, 1H), 2.33ꢀ2.23 (m, 3H), 1.99ꢀ1.87 (m, 5H), 1.79ꢀ1.72 (m, 2H),
1.63 (d, 1H, J = 12.6 Hz), 1.60ꢀ1.49 (m, 5H), 1.44ꢀ1.26 (m, 8H), 1.10 (m, 1H), 1.05 (d, 3H, J =
1
23
4.8 Hz), 0.98ꢀ89 (s, 4H), 0.69 (s, 3H);); H NMR (DMSOꢀd , 600 MHz): δ 10.19 (s, 1H), 7.86
6
2
4
(d, 2H, J = 9.0 Hz), 7.69 (d, 2H, J = 8.4 Hz), 4.30 (s, 1H), 4.25 (q, 2H, J = 6.6 Hz), 4.10 (d, 1H, J
24
ACS Paragon Plus Environment

Page 25 of 75
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
= 3.0 Hz), 4.00 (d, 1H, J = 2.4 Hz), 3.76 (s, 1H), 3.58 (s, 1H), 3.15 (s, 1H), 2.37ꢀ2.30 (m, 1H),
2.25ꢀ2.09 (m, 3H), 1.97 (m, 1H), 1.82ꢀ1.72 (m, 3H), 1.62 (m, 2H), 1.43ꢀ1.39 (m, 3H), 1.35ꢀ1.12
1
3
(m, 12H), 0.95ꢀ88 (s, 4H), 0.82ꢀ0.78 (m, 4H), 0.56 (s, 3H); C NMR (DMSOꢀd , 150 MHz): δ
6
174.0, 166.3, 143.2, 131.0, 130.1, 125.0, 118.7, 112.9, 72.6, 71.4, 66.6, 60.6,46.6, 46.1, 41.7,
41.6, 39.6, 39.0, 35.5, 35.1, 34.5, 33.2, 31.5, 29.8, 28.2, 27.3, 26.4, 22.8, 21.8, 16.4, 13.3, 11.7.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
7
Nꢀ(3’ꢀtertꢀButyloxycarbonylaminophenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide
(12m). This compound was prepared from cholic acid 7 and 3ꢀ(tertꢀ
o
8
butyloxycarbonylamino)aniline by following methodꢀB in 83% yield. mp. 134ꢀ136 C; TLC R .
f
1
9
0.37 (AcOEt:CH OH, 95:5); H NMR (CD OD, 600 MHz): δ 7.67 (s, 1H), 7.18ꢀ7.14 (m, 2H),
3
3
10
7.08 (d, 1H, J = 7.2 Hz), 3.94 (s, 1H), 3.77 (s, 1H), 3.35 (t, 1H, J = 10.8 Hz), 2.42ꢀ2.38 (m, 1H),
2.29ꢀ2.21 (m, 3H), 2.00ꢀ1.85 (m, 5H), 1.79ꢀ1.72 (m, 2H), 1.63ꢀ1.51 (m, 6H), 1.49 (s, 9H), 1.44ꢀ
1.29 (m, 5H), 1.11ꢀ1.08 (m, 1H), 1.04 (d, 3H, J = 6.0 Hz), 0.96 (t, 1H, J = 12.6 Hz), 0.89 (s, 3H),
1
1
1
1
1
1
2
3
4
5
1
3
0.69 (s, 3H); C NMR (CD OD, 150 MHz): δ 173.8, 153.7, 139.6, 139.0, 128.5, 114.3, 114.2,
3
110.5, 79.4, 72.7, 71.5, 67.7, 46.6, 46.1, 41.7, 41.6, 39.6, 39.0, 35.5, 35.1, 34.5, 33.6, 31.8, 29.8,
28.2, 27.4, 27.3, 26.4, 22.9, 21.9, 16.8, 11.7.
1
6
Nꢀ(4’ꢀtertꢀButyloxycarbonylaminophenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide
17
(12n). This compound was prepared from cholic acid 7 and 4ꢀ(tertꢀ
o
1
1
8
9
butyloxycarbonylamino)aniline by following methodꢀB in 84% yield. mp. 152ꢀ153 C; TLC R .
f
1
0.38 (AcOEt:CH OH, 95:5); H NMR (CD OD, 600 MHz): δ 7.41 (d, 2H, J = 9.0 Hz), 7.30 (d,
3
3
20
2H, J = 8.4 Hz), 4.61 (s, 1H), 3.94 (s, 1H), 3.78 (s, 1H), 3.35 (m, 1H), 2.44ꢀ2.38 (m, 1H), 2.29ꢀ
2.23 (m, 3H), 2.00ꢀ1.85 (m, 5H), 1.80ꢀ1.72 (m, 2H), 1.64 (d, 1H, J = 12.0 Hz), 1.56ꢀ1.52 (m,
3H), 1.49 (s, 9H), 1.45ꢀ1.28 (m, 6H), 1.11ꢀ1.08 (m, 1H), 1.05 (d, 3H, J = 6.6 Hz), 0.96 (t, 1H, J =
2
2
2
1
2
3
1
3
14.4 and 2.4 Hz), 0.89 (s, 3H), 0.70 (s, 3H); C NMR (CD OD, 150 MHz): δ 173.6, 154.0,
3
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
135.3, 133.5, 120.5, 118.7, 79.4, 72.7, 71.5, 67.7, 46.6, 46.1, 41.7, 41.6, 39.6, 39.0, 35.5, 35.1,
34.5, 34.4, 33.5, 31.8, 29.7, 28.1, 27.3, 27.3, 26.4, 22.8, 21.8, 15.4, 11.6.
3
4
5
6
7
8
9
0
Cleavage of Boc group (MethodꢀC): Nꢀ(3’ꢀAminophenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀ
cholanꢀ24ꢀamide Hydrochloride (12o). Into a reaction mixture of 12m (230 mg, 0.386 mmol)
in anhydrous AcOEt (20 mL) under argon, 1M HCl in AcOEt (0.5 mL) was added dropwise. The
reaction mixture became clear after a few minutes followed by the formation of a white solid.
The reaction was stirred at room temperature and upon completion (as determined by TLC), the
solid was collected by filtration. The solid was washed with AcOEt and dried under high vacuum
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
to give 12o (163 mg, 80% yield); TLC R . 0.56 (CH Cl :CH OH:NH OH, 85:14:1); H NMR
f
2
2
3
4
1
(CD OD, 600 MHz): δ 8.00 (s, 1H), 7.48 (d, 1H, J = 7.8 Hz), 7.44 (t, 1H, J = 7.8 Hz), 7.10 (d,
3
11
1H, J = 7.2 Hz), 3.94 (s, 1H), 3.78 (s, 1H), 3.36 (m, 1H), 2.48ꢀ2.44 (m, 1H), 2.34ꢀ2.23 (m, 3H),
1.97ꢀ1.70 (m, 8H), 1.62ꢀ1.28 (m, 10H), 1.11ꢀ1.04 (m, 4H), 0.96 (t, 1H, J = 12.6 Hz), 0.89 (s,
1
1
2
3
1
3
3H), 0.69 (s, 3H); C NMR (CD OD, 150 MHz): δ 174.2, 140.5, 130.8, 130.1, 119.5, 117.6,
3
14
114.0, 72.6, 71.5, 67.7, 46.6, 46.1, 41.7, 41.6, 39.6, 39.0, 35.6, 35.1, 34.5, 33.6, 31.6, 29.7, 28.2,
1
1
1
5
6
27.3, 26.5, 22.8, 21.8, 16.4, 11.62. NMR data for free base H NMR (CD OD, 600 MHz): δ 7.04
3
(s, 1H), 7.00 (t, 1H, J = 7.8 Hz), 6.80 (d, 1H, J = 7.8 Hz), 6.45 (d, 1H, J = 7.8 Hz), 3.94 (s, 1H),
3.77 (s, 1H), 3.35 (m, 1H), 2.38 (m,1H), 2.29ꢀ2.23 (m, 2H), 2.00ꢀ1.84 (m, 5H), 1.79ꢀ1.73 (m,
2H), 1.63 (d, 1H, J = 12.6 Hz), 1.58ꢀ1.49 (m, 5H), 1.46ꢀ1.27 (m, 6H), 1.09 (m, 1H), 1.04 (d, 3H,
17
1
1
8
9
1
3
J = 5.4 Hz), 0.95(t, 1H, J = 13.2 Hz), 0.89 (s, 3H), 0.69 (s, 3H); C NMR (CD OD, 150 MHz): δ
3
20
173.7, 147.5, 139.2, 128.9, 111.3, 110.1, 107.3, 72.7, 71.5, 67.6, 48.5, 46.6, 46.1, 41.7, 41.6,
39.6, 39.0, 35.5, 35.1, 34.5, 34.5, 33.6, 31.8, 29.8, 28.2, 27.3, 26.4, 22.8, 21.8, 16.4, 11.6.
2
2
1
2
Nꢀ(4’ꢀAminophenyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide Hydrochloride (12p).
It was prepared from 12n in a manner similar to that of the compound 12o by following methodꢀ
23
26
ACS Paragon Plus Environment

Page 27 of 75
1
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
C in 84% yield. TLC R . 0.51 (CH Cl :CH OH:NH OH, 85:14:1); H NMR (CD OD, 600 MHz):
f 2 2 3 4 3
δ 7.73 (d, 2H, J = 9.0 Hz), 7.32 (d, 2H, J = 8.4 Hz), 3.94 (s, 1H), 3.78 (s, 1H), 3.36 (m, 1H),
2.48ꢀ2.43 (m, 1H), 2.33ꢀ2.23 (m, 3H), 1.96ꢀ1.84 (m, 5H), 1.80ꢀ1.74 (m, 2H), 1.63 (d, 1H, J =
13.2 Hz), 1.57ꢀ1.28 (m, 10H), 1.11ꢀ1.09 (m, 1H), 1.05 (d, 3H, J = 6.0 Hz), 0.97 (t, 1H, J = 15.0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
Hz), 0.90 (s, 3H), 0.70 (s, 3H); C NMR (DMSOꢀd , 150 MHz): δ 172.5, 139.7, 126.3, 124.1,
6
120.2, 71.4, 70.9, 66.7, 46.5, 46.2, 41.9, 41.8, 40.5, 35.7, 35.6, 35.3, 34.8, 33.9, 31.8, 30.8, 29.0,
27.7, 26.6, 23.2, 23.0, 17.6, 12.8.
8
9
Nꢀ(Pyridineꢀ4’ꢀyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (12q). This compound
was prepared from cholic acid 7 and 4ꢀaminopyridine by following methodꢀB in 70% yield. mp.
o
1
10
230ꢀ232 C; TLC R . 0.28 (CH Cl :CH OH:NH OH, 89:10:1); H NMR (CD OD, 600 MHz): δ
f
2
2
3
4
3
1
1
1
2
8.37 (s, 2H), 7.68 (s, 2H), 3.92 (s, 1H), 3.76 (s, 1H), 3.34 (s, 1H), 2.46ꢀ2.25 (m, 4H), 2.02ꢀ1.22
1
3
(m, 18H), 1.03ꢀ0.88 (m, 8H), 0.68 (s, 3H); C NMR (CD OD, 150 MHz): δ 174.6, 148.5, 147.5,
3
13
113.7, 72.6, 71.4, 67.6, 46.6, 46.1, 41.8, 41.6, 39.6, 38.0, 35.5, 35.1, 34.5, 33.7, 31.2, 29.8, 28.2,
27.3, 26.5, 22.8, 21.8, 16.4, 11.6.
1
1
4
5
Nꢀ(4’ꢀMethylthiozolꢀ2’ꢀyl) ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (13). This
compound was prepared from cholic acid 7 and 2ꢀaminoꢀ4ꢀmethylthiazole 9 by following
16
o
1
1
1
7
8
methodꢀB in 76% yield. mp. 237ꢀ238 C; TLC R . 0.39 (CH Cl :CH OH:NH OH, 89:10:1); H
f 2 2 3 4
NMR (CD OD, 600 MHz): δ 6.60 (s, 1H), 3.93 (s, 1H), 3.77 (s, 1H), 3.34 (m, 1H), 2.52ꢀ2.49 (m,
3
19
1H), 2.40ꢀ2.36 (m, 1H), 2.27ꢀ2.21 (m, 5H), 2.00ꢀ1.86 (m, 5H), 1.79ꢀ1.72 (m, 2H), 1.63 (d, 1H, J
2
2
0
1
= 12.6 Hz), 1.58ꢀ1.49 (m, 5H), 1.46ꢀ1.38 (m, 3H), 1.35 (m, 1H), 1.28 (m, 1H), 1.10ꢀ1.07 (m,
1
1H), 1.04 (d, 3H, J = 5.4 Hz), 0.95 (t, 1H, J = 14.4 Hz), 0.88 (s, 3H), 0.68 (s, 3H); H NMR
22
(DMSOꢀd , 600 MHz): δ 11.90 (s, 1H), 6.63 (s, 1H), 4.40 (s, 1H), 4.11 (s, 1H), 4.00 (s, 1H), 3.73
6
2
3
(s, 1H), 3.14 (s, 1H), 2.36 (m, 1H), 2.25ꢀ2.04 (m, 6H), 1.92 (m, 1H), 1.73ꢀ1.52 (m, 5H), 1.45ꢀ
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
1
2
3
4
5
6
7
8
9
0
1
1.04 (m, 12H), 0.89 (s, 4H), 0.82ꢀ0.74 (m, 4H), 0.52 (s, 3H); C NMR (CD OD, 150 MHz): δ
3
172.8, 158.1, 146.8, 107.5, 72.6, 71.4, 67.6, 46.6, 46.1, 41.8, 41.6, 39.6, 39.0, 35.4, 35.1, 34.5,
1
3
32.2, 31.2, 29.8, 28.2, 27.3, 26.4, 22.8, 21.8, 16.3, 16.6, 11.6; C NMR (DMSOꢀd , 150 MHz): δ
6
172.2, 157.8, 146.9, 107.8, 71.5, 70.9, 66.7, 46.5, 46.2, 41.9, 41.8, 35.7, 35.6, 35.2, 34.8, 32.5,
31.6, 30.7, 28.9, 27.7, 26.6, 23.2, 23.0, 17.4, 17.3, 12.7.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Nꢀ(Benzyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (14a). This compound was
prepared from cholic acid 7 and benzylamine 10 by following methodꢀA in 90% yield. mp. 236ꢀ
o
1
238 C; TLC R . 0.58 (AcOEt:CH OH, 90:10); H NMR (DMSOꢀd , 600 MHz): δ 8.27 (t, 1H, J
f
3
6
= 6.0 Hz), 7.27 (t, 2H, J = 7.8 Hz), 7.20 (t, 3H, J = 7.2 Hz), 4.30 (s, 1H), 4.24ꢀ4.20 (m, 2H),
1
1
4.08 (s, 1H), 3.99 (s, 1H), 3.75 (s, 1H), 3.58 (s, 1H), 3.15 (br, 1H), 2.21ꢀ2.09 (m, 3H), 2.04ꢀ1.99
(m, 1H), 1.97ꢀ1.92 (m, 1H), 1.77ꢀ1.60 (m, 6H), 1.43ꢀ1.38 (m, 3H), 1.35ꢀ1.30 (m, 3H), 1.28ꢀ1.05
1
12
(m, 5H), 0.95ꢀ0.91 (m, 4H), 0.83ꢀ0.73 (m, 4H), 0.55 (s, 3H); H NMR (CD OD, 600 MHz): δ
3
1
1
3
4
7.30ꢀ7.25 (m, 4H), 7.21 (t, 1H, J = 6.6 Hz), 4.33 (dd, 2H, J = 15.0 Hz), 3.92 (s, 1H), 3.78 (s, 1H),
3.35 (m, 1H), 2.30ꢀ2.22 (m, 3H), 2.17ꢀ2.13 (m, 1H), 2.00ꢀ1.91 (m, 2H), 1.88ꢀ1.77 (m, 4H), 1.74ꢀ
1.71 (m, 1H), 1.64 (d, 1H, J = 13.2 Hz)), 1.59ꢀ1.50 (m, 5H), 1.44ꢀ1.29 (m, 4H), 1.27ꢀ1.22 (m,
1H), 1.10ꢀ1.04 (m, 1H), 1.01 (d, 3H, J = 6.6 Hz), 0.96 (t, 1H, J = 14.4 Hz), 0.90 (s, 3H), 0.67 (s,
15
1
1
1
1
6
7
8
9
1
3
3H); C NMR (CD OD, 150 MHz): δ 175.2, 138.8, 128.1, 127.2, 126.7, 72.6, 71.4, 67.6, 46.7,
3
46.1, 42.6, 41.8, 41.6, 39.6, 39.0, 35.4, 35.1, 34.5, 32.7, 32.0, 29.8, 28.2, 27.3, 26.4, 22.8, 21.8,
+
16.3, 11.6. HRMS (ESI, m/z): calcd for C H NO Na [M + Na ] 520.3403, found 520.3392.
3
1
47
4
2
0
Nꢀ(2’,4’ꢀDimethoxybenzyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (14b). This
compound was prepared from cholic acid 7 and 2,4ꢀdimethoxybenzylamine by following
methodꢀA. The product was further purified by stirring with anhydrous diethyl ether, and
insoluble material was collected and vacuum dried to furnish a white powder in 85% yield. mp.
21
2
2
2
3
28
ACS Paragon Plus Environment

Page 29 of 75
1
Journal of Medicinal Chemistry
o
1
2
3
4
1
222ꢀ223 C; TLC R . 0.54 (CH Cl :CH OH:NH OH, 89:10:1); H NMR (CD OD, 600 MHz): δ
f
2
2
3
4
3
2
7.08 (s, 1H), 6.48 (s, 1H), 6.42 (s, 1H), 4.23 (s, 2H), 3.90 (s, 1H), 3.79 (s, 3H), 3.75 (s, 4H), 3.34
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
4
5
6
7
8
9
(s, 1H), 2.23 (br s, 3H), 2.11 (s, 1H), 1.94ꢀ1.66 (m, 7H), 1.64ꢀ1.50 (m, 6H), 1.44ꢀ1.14 (m, 6H),
1
1.10ꢀ0.82 (m, 8H), 0.61 (s, 3H); H NMR (DMSOꢀd , 600 MHz): δ 7.97 (s, 1H), 6.99 (d, 1H, J =
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8.4 Hz), 6.48 (s, 1H), 6.41 (d, 1H, J = 8.4 Hz), 4.08 (br s, 2H), 3.73 (s, 4H), 3.69 (s, 3H), 3.57 (s,
1H), 3.14 (br m, 1H), 2.10ꢀ2.05 (m, 2H), 2.02ꢀ1.95 (m, 2H), 1.90ꢀ1.56 (m, 6H), 1.43ꢀ1.02 (m,
1
3
12H), 0.90 (d, 4H, J = 3.6 Hz), 0.82ꢀ0.77 (m, 4H), 0.53 (s, 3H); C NMR (DMSOꢀd , 150
6
MHz): δ 173.0, 160.0, 158.0, 128.9, 119.7, 104.6, 98.5, 71.5, 70.9, 66.7, 55.8, 55.6, 46.6, 46.2,
42.0, 41.8, 37.1, 35.7, 35.6, 35.3, 34.8, 32.9, 32.3, 30.8, 29.0, 27.7, 26.6, 23.3, 23.1, 17.5, 12.8.
1
1
1
1
1
0
1
2
3
4
Nꢀ(3’,5’ꢀDimethoxybenzyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (14c). This
compound was prepared from cholic acid 7 and 3,5ꢀdimethoxybenzylamine by following
o
methodꢀA in 79% yield. mp. 88ꢀ91 (softening) and 110ꢀ112 C; TLC R . 0.57
f
1
(CH Cl :CH OH:NH OH, 89:10:1); H NMR (CD OD, 600 MHz): δ 6.41 (s, 2H), 6.32 (s, 1H),
2
2
3
4
3
4.25 (s, 2H), 3.90 (s, 1H), 3.71 (s, 6H), 3.34 (s, 1H), 2.93 (s, 1H), 2.40ꢀ2.20 (br, 4H), 1.92ꢀ1.23
1
15
(m, 18H), 1.00ꢀ0.88 (2 peaks, 8H), 0.64 (s, 3H); H NMR (DMSOꢀd , 600 MHz): δ 8.25 (s, 1H),
6
1
1
6
7
6.35 (s, 2H), 6.31 (s, 2H), 4.14 (s, 2H), 3.76 (s, 3H), 3.70 (s, 1H), 3.67 (s, 6H), 3.57 (s, 1H), 3.15
(s, 1H), 2.22ꢀ2.08 (m, 3H), 2.04ꢀ1.90 (m, 2H), 1.76ꢀ1.59 (m, 6H), 1.44ꢀ1.18 (m, 10H), 1.13ꢀ1.06
1
3
18
(m, 1H), 0.90 (d, 4H, J = 4.8 Hz), 0.84ꢀ0.77 (m, 4H), 0.53 (s, 3H); C NMR (CD OD, 150
3
1
2
9
0
MHz): δ 175.4, 161.0, 140.9, 105.1, 98.6, 72.6, 71.5, 67.7, 54.4, 46.7, 46.1, 42.8, 41.7, 41.5,
39.6, 39.0, 35.4, 35.1, 34.5, 32.7, 32.1, 29.8, 28.2, 27.3, 26.4, 22.9, 21.9, 16.4, 11.7.
21
Nꢀ(3’,4’ꢀDichlorobenzyl)ꢀ3α,7α,12αꢀtrihydroxyꢀ5βꢀcholanꢀ24ꢀamide (14d). This
compound was prepared from cholic acid 7 and 3,4ꢀdichlorobenzylamine by following methodꢀ
A. The product was further purified by stirring with anhydrous diethyl ether, and insoluble
2
2
2
3
29
ACS Paragon Plus Environment