Structure-Activity Relationship of Antischistosomal Ozonide Carboxylic Acids

Article abstract of DOI:10.1021/acs.jmedchem.0c00069

Semisynthetic artemisinins and other bioactive peroxides are best known for their powerful antimalarial activities, and they also show substantial activity against schistosomes - another hemoglobin-degrading pathogen. Building on this discovery, we now de

Full text of DOI:10.1021/acs.jmedchem.0c00069

Subscriber access provided by UNIV OF YORK
Article
Structure-Activity Relationship of Antischistosomal Ozonide Carboxylic Acids
Jianbo Wu, Xiaofang Wang, Francis C. K. Chiu, Cecile Haeberli, David M. Shackleford, Eileen
Ryan, Sriraghavan Kamaraj, Vivek J. Bulbule, Alexander Wallick, Yuxiang Dong, Karen
White, Paul H Davis, Susan A. Charman, Jennifer Keiser, and Jonathan L. Vennerstrom
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00069 • Publication Date (Web): 05 Mar 2020
Downloaded from pubs.acs.org on March 6, 2020
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 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Structure-Activity Relationship of Antischistosomal Ozonide Carboxylic Acids
†
†
§
‡,£
§
Jianbo Wu , Xiaofang Wang , Francis C. K. Chiu , Cécile Häberli , David M. Shackleford ,
§
†
†
∞
†
Eileen Ryan , Sriraghavan Kamaraj , Vivek J. Bulbule , Alexander I. Wallick , Yuxiang Dong ,
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
§
∞
§
‡,£
Karen L. White , Paul H. Davis , Susan A. Charman , Jennifer Keiser , and Jonathan L.
Vennerstrom†*
†College of Pharmacy, University of Nebraska Medical Center, 986125 Nebraska Medical
Center, Omaha, NE, United States
§Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash
University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia
‡Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
£University of Basel, CH-4003 Basel, Switzerland
∞Department of Biology, University of Nebraska at Omaha, Omaha, NE, United States
*J.L.V.: Phone: 402 559 5362. E-mail: jvenners@unmc.edu
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 56
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
The semisynthetic artemisinins and other bioactive peroxides are best known for their powerful
antimalarial activities, but also have substantial activity against schistosomes - another
hemoglobin-degrading pathogen. Building on this discovery, we now describe the initial
structure-activity relationship (SAR) of antischistosomal ozonide carboxylic acids OZ418 (2)
and OZ165 (3). Irrespective of lipophilicity, these ozonide weak acids have relatively low
aqueous solubilities and high protein binding values. Ozonides with para-substituted
carboxymethoxy and N-benzylglycine substituents had high antischistosomal efficacies. It was
possible to increase solubility, decrease protein binding and maintain high antischistosomal
activity in mice infected with juvenile and adult Schistosoma mansoni by incorporating a weak
base functional group in these compounds. In some cases, adding polar functional groups and
heteroatoms to the spiroadamantane substructure increased solubility and metabolic stability, but
in all cases, decreased antischistosomal activity.
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
Keywords: 1,2,4-trioxolane, ozonide, antischistosomal, artemisinin, OZ418
2
ACS Paragon Plus Environment

Page 3 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Introduction
Artemisinin and its semisynthetic derivatives dihydroartemisin, artemether and artesunate
1
,2
(Figure 1) are best known for their powerful antimalarial activities, but also have substantial
activity against schistosomes - another hemoglobin-degrading pathogen.3-7 With their high
activity against juvenile stage schistosomes, the semisynthetic artemisinins have promise for the
chemoprophylaxis and prevention of schistosomiasis.4,6,8 In contrast, praziquantel, the only drug
available for treatment of this disease, has little activity against the young developmental stages
of the parasite and is rarely curative.4,7,8 Abundant data indicates that the pharmacophoric
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
9
peroxide bond of semisynthetic artemisinins and other bioactive peroxides undergoes one-
electron reduction by heme released during parasite hemoglobin digestion10,11 to produce carbon-
centered radicals that alkylate heme and parasite proteins.10,12-21
Figure 1. Artemisinin (ART) and its semisynthetic derivatives dihydroartemisinin (DHA),
artemether (AM), and artesunate (AS).
We have shown that one class of synthetic peroxides - ozonides (1,2,4-trioxolanes) - have
5
promising antischistosomal activity. The most active of these were OZ418 (2) and OZ165
(
3),22-24 carboxylic acid analogs of next-generation antimalarial ozonide OZ439 (1)
(artefenomel).25 For example, single 400 mg/kg oral doses of 2 administered to Schistosoma
mansoni-, S. japonicum- or S. haematobium-infected mice reduced adult worm burden by 80, 69
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 56
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
and 86%, respectively.23,24 Like the semisynthetic artemisinins, ozonides 2 and 3 are even more
effective against the juvenile form of the parasite; single 200 mg/kg oral doses of 2 and 3
administered to S. mansoni-infected mice reduced juvenile worm burden by 84 and 100%,
respectively.24 However, with IC values of 44 and 39 μM against ex vivo S. mansoni and S.
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
japonicum, 2 has very weak in vitro activity.23,26 The structure-activity relationship (SAR) of
antischistosomal ozonides has demonstrated that: 1) the spiroadamantane ring system and
peroxide bond are essential for activity; 2) the core 1,2,4-trioxolane is superior to the
corresponding 1,2,4-trioxane or 1,2,4,5-tetraoxane isosteres; and 3) ozonides with carboxylic
acid, but not neutral or basic, functional groups have the highest antischistosomal activity. For
example, 1 has no antischistosomal activity.24 In this paper, we profile ozonides 4-25 to expand
the structure-activity relationship (SAR) of 2 and 3.
Figure 2. Ozonides OZ439 (1), OZ418 (2), and OZ165 (3).
Chemistry
Ozonides 4-25 were prepared as described in Schemes 1-12. Ozonide carboxylic acids 9 and 10
2
7
were prepared in 77 and 72% yield by a one-pot alkylation of ozonide phenol 26 with the
corresponding bromoalkyl esters followed by hydrolysis (Scheme 1). Ozonide acylsulfonamide
1
3 was prepared in 62% yield by a DCC-mediated coupling of 2 with methanesulfonamide
(Scheme 2). HOBt/EDC-mediated condensation of 2 with the ethyl ester of glycine followed by
ester hydrolysis afforded ozonide carboxyamide 14 in 91% yield (Scheme 2).
4
ACS Paragon Plus Environment

Page 5 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Scheme 1a
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
aReagents and conditions: 4-bromobutanoate (a) or 5-bromopentanoate (b), powdered NaOH,
tetrabutylammonium hydrogensulfate, dimethoxyethane, rt to 60 °C, 12 h, then aq. AcOH.
Scheme 2a
a
Reagents and conditions: (a) methanesulfonamide, DCC, DMAP, CH Cl , rt, 24 h; (b) glycine
2
2
ethyl ester hydrochloride, HOBt, EDC, DIPEA, DMF, rt, 12 h; (c) 2% aq. NaOH, EtOH, rt, 12 h,
then aq. AcOH.
27
Reaction of ozonide acetate 27 with N-chloroethylpiperidine 28 according to the method of
2
5
Charman et al. furnished ozonide ester 29 in 45% yield; hydrolysis of 29 gave ozonide
piperidine carboxylic acid 15 as its sodium salt in 86% yield (Scheme 3). Alkylation of ozonide
25
piperidine 30 with ethyl bromoacetate produced ozonide ester 31 in 69% yield (Scheme 4).
Hydrolysis of 31 afforded ozonide piperidine carboxylic acid 16 in 84% yield.
Scheme 3a
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 56
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
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
a
Reagents and conditions: (a) powdered NaOH, tetrabutylammonium hydrogensulfate, CH CN,
3
rt to 60 °C, 12 h; (b) powdered NaOH, EtOH, rt, 24 h.
Scheme 4a
a
Reagents and conditions: (a) ethyl bromoacetate, K CO , 12:1 THF:H O, rt, 12 h; (b) 1 M aq.
2
3
2
NaOH, EtOH, 50 °C, 20 h, then aq. AcOH.
Ozonide esters 33 and 34 were formed in 99-100% yield by alkylation of ozonide benzyl
2
8
chloride 32 with the ethyl esters of glycine and azetidine-3-carboxylic acid, respectively
Scheme 5). Ester hydrolysis of 33 and 34 followed by treatment with methanesulfonic acid
(
afforded the aza ozonide carboxylic acids 17 and 18 as their mesylate salts in 77 and 84% yield,
respectively.
Scheme 5a
6
ACS Paragon Plus Environment

Page 7 of 56
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
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
aReagents and conditions: glycine ethyl ester hydrochloride (a) or ethyl azetidine-3-carboxylate
hydrochloride (b), DIPEA, DMA, 50 °C, 5 h; (c) 1 M NaOH, aq. THF, rt, 12 h, then aq. AcOH;
(
d) methanesulfonic acid, Et O, rt, 1 h; (e) methanesulfonic acid, EA, rt, 1 h.
2
2
7
Chlorination of ozonide phenol 26 with one and nine molar equivalents of N-chlorosuccinimide
(
NCS) furnished the chlorinated phenol intermediates 35 (77%) and 36 (63%), respectively
Scheme 6). Alkylation of 35 and 36 with ethyl bromoacetate afforded a quantitative yield of
(
ozonide esters 37 and 38; ester hydrolysis of the latter produced the target ozonide carboxylic
acids 7 and 8 in 97 and 89% yield, respectively.
Scheme 6a
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 56
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
aReagents and conditions: (a) 1 eq. NCS, DMF rt, 24 h; (b) 9 eq. NCS, DMF rt, 24 h; (c) ethyl
bromoacetate, K CO , acetone, 60 °C, 12 h; (d) 1 M aq. NaOH, THF, rt, 12 h, then aq. AcOH.
2
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
Ozonide salicylic acid 5 was synthesized in a multistep sequence starting from phenol ketal 3929
(Scheme 7). Formylation of 39 led to a quantitative yield of salicylaldehyde 40 which then
underwent successive silver oxide oxidation and ketal deprotection to afford keto salicylate 41 in
9
9
7
6% yield. Successive Fisher esterification and acetylation of 41 furnished the keto diester 42 in
3
0
31
9% yield. Griesbaum coozonolysis of 42 and oxime ether 43 formed ozonide diester 44 in
0% yield. Ester hydrolysis of 44 afforded the desired ozonide salicylate 5 in quantitative yield.
Ozonide dicarboxylic acid 6 was obtained in 84% yield by alkylation of 5 with ethyl
bromoacetate followed by hydrolysis.
Scheme 7a
8
ACS Paragon Plus Environment

Page 9 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
Reagents and conditions: (a) paraformaldehyde, MgCl , Et N, THF, rt to reflux, 5 h; (b) Ag O,
2
3
2
H O, 100 °C, 12 h; (c) 6 N HCl, THF, rt, 4 h; (d) acetyl chloride, MeOH, 0 °C to reflux, 4 h; (e)
2
Ac O, pyridine, DCM, 0 °C to rt, 5 h; (f) O , 4:1 cyclohexane:DCM, 0 °C, 0.25 h; (g) KOH,
2
3
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
4
:4:1 H O:MeOH:THF, rt to 50 °C, 4 h; (h) ethyl bromoacetate, K CO , acetone, 60 °C, 12 h; (i)
2
2
3
1
M aq. NaOH, 5:1 THF:H O, rt, 12 h, then aq. AcOH.
2
3
0
28
Coozonolysis of oxime ether 47 and keto ester 46, formed by alkylation of keto phenol 45
with ethyl bromoacetate, furnished ozonide ester 48 in 15% yield (Scheme 8). Ester hydrolysis
of 48 afforded the desired ozonide carboxylic acid 19 in 93% yield.
Scheme 8a
a
Reagents and conditions: (a) ethyl bromoacetate, K CO , acetone, 60 °C, 12 h; (b) O , 5:2
2
3
3
cyclohexane:DCM, 0 °C, 0.25 h; (c) 15% aq. KOH, THF, rt, 12 h, then aq. AcOH.
As shown in Scheme 9, ozonide carboxylic acids 20 and 21 were synthesized from 53, a
common ozonide keto phenol precursor. The synthesis of 53 began with conversion of the
3
2
mono-ketal of adamantane-2,6-dione (49) to its oxime ether 50 in high yield. This was
30
27
followed by coozonolysis of 50 and keto ester 51 to form ozonide ester ketal 52 in 43% yield.
Successive ester and ketal deprotection of 52 furnished 53 in an overall yield of 89%. Alkylation
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 56
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
of 53 with ethyl bromoacetate to form ozonide ester 54 followed by hydrolysis afforded ozonide
carboxylic acid 20 in 95% overall yield. Reduction of 53 with sodium borohydride afforded the
corresponding secondary alcohol 55 in 99% yield. Alkylation of 55 with ethyl bromoacetate to
form ozonide ester 56 followed by hydrolysis afforded carboxylic acid 21 in 95% overall yield.
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
Scheme 9a
a
Reagents and conditions: (a) methoxyamine HCl, pyridine, EtOH, rt, 12 h; (b) O , 3:1
3
cyclohexane:DCM, 0 °C, 0.25 h; (c) 1 M aq. NaOH, THF, rt, 12 h; (d) 1 M MsOH, 5:1
acetone:H O, rt, 12 h; (e) ethyl bromoacetate, K CO , acetone, 55 °C, 12 h; (f) 5:1 1 M aq.
2
2
3
NaOH:THF, rt, 12 h, then aq. AcOH; (g) NaBH , MeOH, 0 °C, 2 h.
4
Reductive amination of ozonide ester 54 followed by acetylation afforded ozonide ester 57 in
8
0% yield (Scheme 10). Ester hydrolysis of 57 furnished ozonide acetamide carboxylic acid 22
in 94% yield. Similarly, Boc protection of the crude amino ester ozonide formed by reductive
amination of 54 afforded 58 in 69% yield. Ester hydrolysis of 58 followed by treatment with
methanesulfonic acid afforded ozonide amino acid 23 as its mesylate salt in 98% yield.
10
ACS Paragon Plus Environment

Page 11 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Scheme 10a
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
a
Reagents and conditions: (a) NaBH CN, NH OAc, AcOH, MeOH, rt, 12 h, then 1 M aq. NaOH;
3
4
(b) acetyl chloride, pyridine, DCM, rt, 6 h; (c) 1 M aq. KOH, THF, rt, 12 h, then aq. AcOH; (d)
Boc) O, DIPEA, DCM, 0 °C to rt, 12 h; (e) 1 M methanesulfonic acid in EtOAc, rt, 12 h.
(
2
The synthesis of ozonide 24 began with formation of oxime ether 60 from the Boc-protected
33
30
azaadamantanone 59 which then underwent coozonolysis with keto ester 51 to form ozonide
ester 61 in 36% yield (Scheme 11). Ester hydrolysis of 61 furnished ozonide phenol 62 in 98%
yield which was then alkylated with ethyl bromoacetate to form ozonide ester 63 in quantitative
yield. Ester hydrolysis of 63 produced ozonide carboxylic acid 64 in 90% yield. Boc
deprotection of 64 with methanesulfonic acid afforded the mesylate salt of ozonide carboxylic
acid 24 in quantitative yield.
Scheme 11a
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 56
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
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
aReagents and conditions: (a) methoxyamine hydrochloride, pyridine, EtOH, rt, 12 h; (b) O3,
1
0:1 cyclohexane:DCM, 0 °C, 0.25 h; (c) 15 % aq. KOH, THF, rt, 12 h, then aq. AcOH; (d) ethyl
bromoacetate, K CO , acetone, 60 °C, 12 h; (e) methanesulfonic acid, THF, rt, 12 h.
2
3
3
0
The synthesis of target ozonide 25 began with coozonolysis of oxime ether 60 and ketone 65 to
form ozonide 66 in 26% yield (Scheme 12). Alkylation of glycine ethyl ester with 66 afforded
ozonide ester 67 in 97% yield. Intermediate 67 then underwent successive ester hydrolysis and
Boc deprotection with methanesulfonic acid to afford the dimesylate salt of ozonide carboxylic
acid 25 in 82% yield. Ozonides 2-4, 11, and 12 were obtained as previously described. 34-36
Scheme 12a
12
ACS Paragon Plus Environment

Page 13 of 56
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
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
a
Reagents and conditions: (a) O , 10:1 cyclohexane:DCM, 0 °C, 0.25 h; (b) glycine ethyl ester
3
hydrochloride, DIPEA, DMA, 50 °C, 5 h; (c) 1 M aq. NaOH, THF, rt, 12 h, then aq. AcOH; (d)
methanesulfonic acid, THF, rt, 12 h.
Physicochemical, in vitro ADME, and Antischistosomal Properties
When considering the physicochemical and in vitro ADME properties of these ozonides (Tables
1-3), we note several overarching trends. First, the calculated polar surface area (PSA) values of
2
between 68 and 117 Å indicate that the polarity of these compounds is unlikely to be a rate-
limiting factor for membrane permeability and oral bioavailability.37 Second, most of the
compounds have low aqueous solubilities even those with LogD values ≤ 3. Third, these
7.4
compounds had high estimated protein binding values ranging from 94.2 to >99.5%, an
unsurprising result for these ozonide weak acids.
The data in Table 1 show the SAR of the phenyl substructure of 2 and 3. The only compounds
with high aq. solubilities at pH 6.5 were salicylate 5 and dicarboxylic acid 6; the latter was also
metabolically stable. Ozonide 7, the more lipophilic chloro analog of 2, was also metabolically
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 56
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
stable. Ozonide 4, the meta isomer of 3, was somewhat less stable to metabolism; interestingly,
addition of a phenol functional group in 5 improved metabolic stability in mouse microsomes,
but substantially decreased metabolic stability in human microsomes. Of these analogs (Table
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
), only 7 had in vivo activity against chronic S. mansoni infections equal or superior to that of
the two prototype ozonides 2 and 3. For comparison, at this same 400 mg/kg oral dose,
5
41
dihydroartemisinin and praziquantel reduce worm burden by 66 and 96%, respectively.
Table 1. Physicochemical, in vitro ADME, and antischistosomal data for ozonides 2-8.
Compd gLogD7.4a PSA Sol /Sol
h/m CLint
cPPB
S. mansoni
WBR (%)
1 x 400
2.0
6.5
2
b
c
e
(Å ) (µg/mL)
(µL/min/mg (%)
d
protein)
f
mg/kg po
i
2
3
4
5
6
7
8
2.9
3.0
3.0
3.0
1.9
3.2
3.3
77.1 <1.6/6.3-12.5
67.8 <3.1/<1.6
67.8 <1.6/<1.6
88.1 <1.6/>100
117.2 <3.1/>100
77.1 <3.1/3.1-6.3
77.1 <3.1/3.1-6.3
NDg
NDh
98.8
98.9
99.0
99.1
80 *
NDg
74i*
63
36
0
12/30
108/20
<7/8
<7/8
>99.5 84
ND 32
NDg
a
38
Log D values were estimated by chromatography
7
.4
bCalculated PSA values were generated using JChem for Excel.
cKinetic solubilities at pH 6.5 phosphate buffer and 0.01 M HCl (approx. pH 2.0)39
14
ACS Paragon Plus Environment

Page 15 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
d
In vitro intrinsic clearance (CL ) values in human and mouse liver microsomes.
int
eProtein binding values were estimated by chromatography.40
fGroups of five S. mansoni-infected NMRI mice were treated on day 49 post-infection with
ozonides dissolved or suspended in 7% v/v Tween 80, 3% v/v ethanol. At 28 d post-treatment,
animals were sacrificed and dissected to assess total worm burden reduction (WBR).
gAnalytical difficulties precluded measurement.
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
hPoor chromatography precluded determination.
idata from Keiser et al.24
*
p <0.02 from the Kruskal–Wallis test comparing the medians of the responses between the
treatment and control groups.
ND = not determined
The data in Table 2 show the SAR of the carboxymethoxy substructure of 2. Extending the
length of the side chain to carboxypropoxy (9) or carboxybutoxy (10) decreased aq. solubility,
increased protein binding, and decreased antischistosomal activity compared to 2. Compound
1
1, the gem-dimethyl analog of 2, also had very high protein binding and was less active than 2.
With the exception of ethyl ester 12 and glycine conjugate 14, the other compounds were
metabolically stable; 12 was rapidly converted to 2 in the both human and mouse liver
microsomes, whereas 14 was converted to 2 in human but not mouse liver microsomes. Of these
two compounds, only 14 had significant antischistosomal activity. Interestingly, 14 was more
and less soluble than 2 at pH 2.0 and 6.5, respectively. Acyl sulfonamide 13 had the same
solubility profile as 2, but had only modest antischistosomal activity. The amphoteric analogs
15-18 show the effects of adding a weak base functional group to the overall profiles of these
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 56
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
ozonides. Compound 17, an aza isostere of 9 had a much superior profile than the latter. For
example, 17 was less lipophilic, had lower protein binding and was much more active than 9
against S. mansoni in vivo. However, for the two piperidine carboxylic acids 15 and 16, only the
former had significant antischistosomal activity. Like 15, azetidine carboxylic acid 18 was less
lipophilic than 2, but had only weak antischistosomal activity.
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
Table 2. Physicochemical, in vitro ADME, and antischistosomal data for ozonides 9-18.
Compd gLogD7.4 PSA Sol /Sol
h/m CLint
(µL/min/mg (%)
protein)
cPPB S. mansoni
2.0
6.5
2
(Å ) (µg/mL)
WBR (%)
1 x 400
mg/kg po
9
1
1
1
1
1
1
1
1
3.6
4.0
3.1
>5.3
3.0
3.0
3.8
NDc
3.3
77.1 <3.1/<1.6
77.1 <1.6/<1.6
77.1 <1.6/3.1-6.3
63.2 <1.6/<0.78
97.4 <1.6/6.3-12.5
106.2 12.5-25/<3.1
81.5 3.1-6.3/<1.6
81.5 NDd
<7/8
>99.5 55
>99.5 51
>99.5 48
0
1
2
3
4
5
6
7
<7/13
<7/7
NDa
98.9
99.0
98.5
98.0
ND
60
<7/12
<7/CNDa,b
<7/9
53
76*
0
<7/<7
<7/10
75*
90*
84.4 <12.5/<6.3
97.6
16
ACS Paragon Plus Environment

Page 17 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
8
NDc
72.3 6.3-12.5/<1.6
7/10
ND
43
aapparent non-NADPH-mediated degradation evident
bPutative amide hydrolysis product 2 was detected in controls and NADPH samples.
cPoor chromatography precluded determination
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
dPoor solubility in DMSO (<1 mg/mL) precluded determination of solubility
*
p <0.02 from the Kruskal–Wallis test comparing the medians of the responses between the
treatment and control groups.
CND = could not determine
ND = not determined
The data in Table 3 summarize the SAR of the spiroadamantane substructure of 2. Replacing the
distal methylene carbon of 2 with a difluoromethylene (19) or a ketone (20) increased aq.
solubility, decreased protein binding, and decreased both metabolic stability and
antischistosomal activity. Decreasing lipophilicity by adding a secondary alcohol (21),
acetamide (22), or primary amine (23) to the distal methylene carbon of 2 increased aq. solubility
at pH 6.5 for 21 and 22, but abolished antischistosomal activity for all three ozonides.
Interestingly, the amphoteric 23 had the lowest protein binding (94.2%) for this series of
ozonides. Compounds 24 and 25, azaadamantane analogs of 2 and 17, show the consequences of
replacing the methylene carbon of 2 with a nitrogen atom. Unfortunately, 24 had low aq.
solubility, intermediate metabolic stability, and low antischistosomal activity. Even though 25
was metabolically stable and was the sole ozonide in this series with high aq. solubility at both
pH 2.0 and 6.5, it also had weak antischistosomal activity.
Table 3. Physicochemical, in vitro ADME, and antischistosomal data for ozonides 19-25.
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 56
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
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
Compd gLogD7.4 PSA Sol /Sol
h/m CLint
(µL/min/mg (%)
protein)
cPPB S. mansoni
2.0
6.5
2
(Å ) (µg/mL)
WBR (%)
1 x 400 mg/kg
po
57
1
2
9
0
2.9
1.9
1.7
1.8
1.4
1.3
1.6
77.1 <1.6/12.5-25
23/130
NDa
99.4
97.8
98.5
98.2
94.2
94.1 <1.6/50-100
97.3 <1.6/25-50
24
14
0
21
<7/<7
2
2
2
3
106.2 3.1-6.3/12.5-25 10/<7
104.7 6.3-12.5/<1.6
93.7 3.1-6.3/<1.6
101.0 >100/50-100
13/8
30
24
35/27
<7/<7
NDb 29
NDb
2
5
41
aapparent non-NADPH-mediated degradation
bpoor chromatography precluded determination
As the data in Tables 1-3 reveal, we identified 7, 14, 16, and 17 – four new ozonides with high
activity against adult S. mansoni in a mouse model. As a measure of their antischistosomal
selectivity, these ozonides were tested for cytotoxicity against four human cell lines: foreskin
fibroblast (HFF), osteosarcoma (U-2 OS), kidney (HEK 293T), and hepatocyte (HC-04).
None of the compounds inhibited these cell lines at concentrations up to 50 µM (data
not shown). Although our primary objective was to identify ozonides with high activity against
the adult stage of S. mansoni, we wanted to find compounds with high activities against both
adult and juvenile stages. Therefore, the most active ozonides were also tested for activity
18
ACS Paragon Plus Environment

Page 19 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
against juvenile S. mansoni in infected mice (Table 4). At a single 200 mg/kg dose, all of the
compounds reduced worm burden by more than 80% in mice with juvenile stage S. mansoni
infections (Table 4); of these, 2, 14, and 17 were the most effective with WBR values of 97-
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
1
00%.
Table 4. In vivo activity of selected ozonides against the juvenile stage of S. mansoni.
Compd
S. mansoni WBR (%)
x 200 mg/kg po
1
a
2
3
100
a
84
7
14
1
1
92*
97*
74*
97*
6
7
a
24
data from Keiser et al.
*
p <0.05 from the Kruskal–Wallis test comparing the medians of the responses between the
treatment and control groups.
Summary
In summary, these ozonide weak acids had relatively low aqueous solubilities and high protein
binding values that were independent of lipophilicity. It was possible to increase solubility,
decrease protein binding and maintain high antischistosomal activity in mice infected with
juvenile and adult Schistosoma mansoni by incorporating a weak base functional group in these
compounds. Ozonides with para-substituted carboxymethoxy and N-benzylglycine substituents
had high antischistosomal activities. These are exemplified by 7, 14, 16, and 17 – four new
ozonides with LogD values ranging from 3.0 to 3.3, similar to that of prototype ozonide 2
7
.4
(
LogD of 2.9). Adding polar functional groups and heteroatoms to the spiroadamantane
7.4
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 56
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
substructure could increase solubility and metabolic stability, but in all cases, decreased
antischistosomal activity.
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
Experimental Section
1
13
General. Melting points are uncorrected. Unless otherwise noted, 1D H and C NMR spectra
were recorded on a 500 MHz spectrometer using CDCl or DMSO-d as solvents. All chemical
3
6
1
shifts are reported in parts per million (ppm) and are relative to internal (CH ) Si (0 ppm) for H
3
4
1
3
and CDCl (77.2 ppm) or DMSO-d (39.5 ppm) for C NMR. Silica gel (sg) particle size 32−63
3
6
μm was used for all flash column chromatography. Reported reaction temperatures are those of
the oil bath. GCMS data was generated with an Agilent 6890-5973N GC-MS system using a
DB-5 column and helium flow rate of 1 mL/min. Combustion analysis confirmed that all target
compounds have a purity of at least 95%. X-ray crystallographic data for all ozonides with 8’-
aryl substituents (like 2-25) demonstrate that they have cis configurations.27,42
5
-(Dispiro[adamantane-2,3'-[1,2,4]trioxolane-5',1''-cyclohexan]-4''-yl)-2-hydroxybenzoic
acid (5). Step 1. To a mixture of anhydrous magnesium chloride (12.21 g, 128.2 mmol),
paraformaldehyde (5.78 g, 192.3 mmol) and triethylamine (17.9 ml, 128.2 mmol) in anhydrous
THF (150 mL) at rt was added dropwise a solution of 4-(1,4-dioxaspiro[4.5]decan-8-yl)phenol
2
9
(
39) (15.0 g, 64.1 mmol) in THF (100 mL) under Ar. The reaction mixture was then refluxed
for 5 h. After completion of the reaction, an insoluble solid was filtered and washed with THF (2
x 25 mL). After solvent removal in vacuo, the residue was dissolved in DCM (150 mL), washed
with 10% HCl (3 x 25 mL), water (3 x 25 mL) and dried over MgSO . After filtration, the
4
solvent was removed in vacuo to afford 2-hydroxy-5-(1,4-dioxaspiro[4.5]decan-8-
+
1
yl)benzaldehyde (40) as viscous oil (16.8 g, 100%). GCMS: m/z: 262 [M ], H NMR (CDCl ) δ
3
20
ACS Paragon Plus Environment

Page 21 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
.66–1.90 (m, 8H), 2.50–2.60 (m, 1H), 3.98 (s, 4H), 6.90–6.92 (m, 1H), 7.40–7.42 (m, 2H), 9.85
1
3
(s, 1H), 10.86 (s, 1H). C NMR (CDCl ) δ 31.4, 34.8, 41.8, 64.1, 108.1, 117.3, 120.2, 131.1,
3
135.9, 137.99, 159.79, 196.5. Step 2. To a stirred solution of silver nitrate (7.13 g, 41.98 mmol)
in water (25 mL) was added dropwise a solution of NaOH (3.35 g, 83.96 mmol) in water (25
mL) at 0 ºC. After stirring for 5 min, silver oxide was obtained as a brown semisolid. To this
mixture, a solution of 40 (5.5 g, 20.99 mmol) in water (25 mL) was added and the reaction
mixture was stirred at 100 ºC for 12 h. After filtration of the black silver suspension, the aq.
solution was neutralized (pH = ~7) with 10% HCl at 0 ºC to obtain a solid that was filtered,
washed with water and dried to afford 2-hydroxy-5-(1,4-dioxaspiro[4.5]decan-8-yl)benzoic acid
as a colorless solid (5.6 g, 96%). To a stirred solution of hydroxy-5-(1,4-dioxaspiro[4.5]decan-8-
yl)benzoic acid (5.6 g, 20.1 mmol) in THF (75 mL) was added 6 N HCl (20 mL) dropwise at rt.
After completion of the reaction, the solvent was removed in vacuo and the residue was
dissolved in DCM (100 mL), washed with water (3 x 25 mL) and dried over anhydrous MgSO4.
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
After filtration, the solvent was removed in vacuo to afford 2-hydroxy-5-(4-
1
oxocyclohexyl)benzoic acid (41) as a colorless solid (4.69 g, 100%). H NMR (DMSO-d ) δ
6
1
1
.78–1.90 (m, 2H), 2.02–2.08 (m, 2H), 2.22–2.30 (m, 2H), 2.52–2.62 (m, 2H), 3.01–3.08 (m,
H), 6.92 (d, J = 8.42 Hz, 1H), 7.47 (dd, J = 1.47, 8.42 Hz, 1H), 7.71 (d, J = 1.47 Hz, 1H), 11.02
1
3
(bs, 1H), 14.0 (bs, 1H). C NMR (DMSO-d ) δ 33.6, 40.8, 40.9, 112.8, 117.4, 128.1, 134.5,
6
1
36.2, 159.9, 172.2, 210.4. Step 3. To a solution of 41 (6.54 g, 27.9 mmol) in MeOH (200 mL)
was added acetyl chloride (2 mL) dropwise at 0 ºC. Then the reaction mixture was heated to
reflux for 4 h. Solvents were then removed in vacuo and the residue was dissolved in DCM (100
mL), washed with water (3 x 25 mL) and dried over anhydrous MgSO . After filtration, the
4
solvent was removed in vacuo to afford methyl 2-hydroxy-5-(4-oxocyclohexyl)benzoate as a
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 56
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
+
viscous oil (6.98 g, 100%). GCMS: m/z: 248 [M ]. Successive dropwise additions of pyridine
(
11.28 mL, 139.6 mmol) and acetic anhydride (13.2 ml, 139.6 mmol) were made to a solution of
methyl 2-hydroxy-5-(4-oxocyclohexyl)benzoate (6.98 g, 28.2 mmol) in DCM (50 mL) at 0 ºC.
The reaction mixture was stirred at rt for 5 h before solvent removal in vacuo. The residue was
cooled to 0 °C to obtain a solid that was filtered, washed with water (3 x 25 mL) and dried to
afford methyl 2-acetoxy-5-(4-oxocyclohexyl)benzoate (42) as a colorless solid (8.0 g, 99%).
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
GCMS: m/z: 290 [M ], H NMR (CDCl ) δ 1.80–1.90 (m, 2H), 2.02–2.08 (m, 2H), 2.26 (s, 3H),
3
2
8
.38–2.46 (m, 4H), 2.96–3.04 (m, 1H), 3.79 (s, 3H), 6.98 (d, J = 8.30 Hz, 1H), 7.35 (dd, J = 1.95,
.30 Hz, 1H), 7.81 (d, J = 1.95 Hz, 1H). Step 4. A solution of 42 (5.89 g, 20.29 mmol) and O-
3
1
methyl-2-adamantanone oxime (43) (5.45 g, 30.44 mmol) in cyclohexane (320 mL) and DCM
(
80 mL) at 0 °C was treated with ozone according to the method of Dong et al.42 Solvents were
removed in vacuo and the residue was triturated with 95% aq. EtOH to obtain a solid that was
filtered, washed with 95% aq. EtOH and dried to afford methyl 2-acetoxy-5-
(
dispiro[adamantane-2,3'-[1,2,4]trioxolane-5',1''-cyclohexan]-4''-yl)benzoate (44) as a colorless
1
solid (6.5 g, 70%). mp 132–134 °C. H NMR (CDCl ) δ 1.68–2.08 (m, 22H), 2.34 (s, 3H), 2.56–
3
2
.62 (m, 1H), 3.86 (s, 3H), 7.01 (d, J = 8.30 Hz, 1H), 7.38 (dd, J = 2.44 & 8.30 Hz, 1H), 7.85 (d,
J = 1.95 Hz, 1H). Step 5. To a solution of 44 (2.0 g, 4.39 mmol) in 4:4:1 H O:MeOH:THF (45
2
mL) was added solid KOH (2.46 g, 43.9 mmol) at rt. After the addition, the reaction mixture was
stirred at 50 ºC for 4 h. After cooling to rt, the reaction mixture was concentrated to 10 mL in
vacuo, diluted with water (50 mL) and neutralized (pH = ~ 7) with 10% aq. HCl at 0 °C to obtain
a solid that was filtered, washed with water and dried to afford 5 (1.8 g, 100%) as a colorless
1
solid. mp 159–161 ºC. H NMR (DMSO-d ) δ 1.46–1.56 (m, 2H), 1.64–1.96 (m, 20H), 2.43–
6
2.52 (m, 1H), 6.55 (d, J = 8.30 Hz, 1H), 6.99 (dd, J = 2.44, 8.30 Hz, 1H), 7.52 (d, J = 1.95 Hz,
22
ACS Paragon Plus Environment

Page 23 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
13
1
H). C NMR (DMSO-d ) δ 26.0, 26.5, 31.7, 34.4, 34.5, 36.0, 36.3, 41.1, 108.5, 110.7, 115.8,
6
1
20.1, 127.8, 129.2, 133.4, 161.2, 171.8. Anal. calcd for C H NaO H CO : C, 59.50; H, 6.03.
2
3
27
6
2
3
Found: C, 60.09; H, 5.77.
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
cis-Adamantane-2-spiro-3'-8'-[3-carboxy-4-(carboxymethoxy)phenyl]-1',2',4'-
trioxaspiro[4.5]decane (6). Step 1. A mixture of 5 (200 mg, 0.50 mmol), ethyl bromoacetate
(
167 mg, 1.00 mmol) and potassium carbonate (208 mg, 1.50 mmol) in acetone (20 mL) was
stirred at 60 °C for 12 h. A solid was filtered and washed with acetone, and the filtrate was
concentrated in vacuo to afford cis-adamantane-2-spiro-3'-8'-[3-chloro-4-(2-ethoxy-2-
1
oxoethoxy)phenyl]-1',2',4'-trioxaspiro[4.5]decane as colorless oil (250 mg, 87%). H NMR
(CDCl ) δ 1.28 (m, 6H), 1.77–1.65 (m, 8H), 1.87–1.77 (m, 6H), 1.93 (d, J = 12.9 Hz, 2H), 2.07–
3
1
1
.97 (m, 6H), 2.54 (t, J = 12.3, 1H), 4.25 (m, 4H), 4.69 (s, 2H), 4.81 (s, 2H), 6.85 (d, J = 8.6 Hz,
1
3
H), 7.30 (d, J = 8.4, 1H), 7.78 (s, 1H). C NMR (CDCl ) δ 14.1, 26.5, 26.9, 31.4, 34.6, 34.8,
3
36.4, 36.8, 41.88, 61.0, 61.2, 61.3, 67.0, 108.2, 111.4, 114.9, 119.9, 130.6, 131.9, 139.5, 156.4,
64.8, 167.8, 168.6. Step 2. A mixture of cis-adamantane-2-spiro-3'-8'-[3-chloro-4-(2-ethoxy-2-
1
oxoethoxy)phenyl]-1',2',4'-trioxaspiro[4.5]decane (250 mg, 0.44 mmol) and 1 M aq. NaOH (2
mL) in THF (10 mL) was stirred at rt for 12 h. The solvent was removed in vacuo and the
residue was diluted with H O (10 mL) and acidified with AcOH (0.2 mL). The resultant
2
precipitate was collected by filtration and washed with cold water, dried in vacuo at 50 °C to
1
afford 6 as a white solid (190 mg, 95%). mp 164–165 °C. H NMR (DMSO-d ) δ 1.51 (q, J =
6
13.0, 2H), 1.98–1.62 (m, 20H), 2.59 (t, J = 12.3, 1H), 4.61 (s, 2H), 7.04 (d, J = 8.6 Hz, 1H), 7.28
13
(
d, J = 8.2 Hz, 1H), 7.39 (s, 1H). C NMR (DMSO-d ) δ 26.3, 26.7, 31.6, 34.5, 34.7, 36.2, 36.6,
6
4
0.9, 68.4, 108.5, 111.1, 116.2, 124.2, 128.9, 131.0, 139.4, 155.5, 168.3, 171.7. Anal. calcd for
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 56
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
+
C H O H O: C, 63.01; H, 6.77. Found: C, 63.38; H, 6.58. HRMS (ESI-TOF) m/z: [M + Na]
2
5
30
8
2
Calcd for C H O Na 481.1838; Found 481.1838.
25
30
8
cis-Adamantane-2-spiro-3'-8'-[3-chloro-4-(carboxymethoxy)phenyl]-1',2',4'-
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
trioxaspiro[4.5]decan (7). Step 1. A solution of N-chlorosuccinimide (1.25 g, 9.36 mmol) in
2
7
dry DMF (10 mL) was added to a solution of 26 (3.32 g, 9.31 mmol) in dry DMF (30 mL) and
stirred under nitrogen at rt for 24 h protected from light. The mixture was poured on ice water
and then extracted with EtOAc (2 x 50 mL). The combined organic layers were washed with
water (100 mL) and brine (25 mL), dried with MgSO and concentrated in vacuo to give a
4
semisolid that was purified by chromatography (sg, 9:1 hexane:ethyl acetate) to afford 2-chloro-
4-dispiro[adamantane-2,3'-[1,2,4]trioxolane-5',1''-cyclohexan]-4''-yl)phenol (35) (2.80 g, 77%) as
o
1
a white solid. mp 119–121 C. H NMR (CDCl ) δ 1.62–2.01 (m, 22H), 2.44–2.50 (m, 1H), 5.41
3
1
3
(
s, 1H), 6.92 (d, J = 8.0 Hz, 1H), 7.01 (dd, J = 8.0, 2.0 Hz, 1H), 7.15 (d, J = 2.0 Hz, 1H). C
NMR (CDCl ) δ 26.7, 27.1, 31.5, 31.7, 34.7, 34.8, 34.99, 35.00, 36.6, 37.0, 42.1, 108.4, 111.7,
3
1
16.2, 119.8, 126.8, 126.9, 127.3, 139.8, 149.7. Step 2. A mixture of 35 (210 mg, 0.54 mmol),
ethyl bromoacetate (180 mg, 1.08 mmol) and potassium carbonate (224 mg, 1.62 mmol) in
acetone (20 mL) was stirred at 60 °C for 12 h. The solid was filtered and washed with acetone
and the filtrate was concentrated in vacuo to afford cis-adamantane-2-spiro-3'-8'-[3-chloro-4-(2-
ethoxy-2-oxoethoxy)phenyl]-1',2',4'-trioxaspiro[4.5]decane (37) as a colorless oil (0.257 g,
1
1
00%). H NMR (CDCl ) δ 7.22 (d, J = 2.1 Hz, 1H), 7.01 (dd, J = 8.5, 2.1 Hz, 1H), 6.77 (d, J =
3
8.5 Hz, 1H), 4.66 (s, 2H), 4.25 (q, J = 7.1 Hz, 2H), 2.48 (t, J = 12.1 Hz, 2H), 2.07–1.97 (m, 6H),
13
1
.93 (d, J = 12.8 Hz, 2H), 1.87–1.59 (m, 15H), 1.28 (t, J = 7.2 Hz, 3H). C NMR (CDCl ) δ
3
1
4.1, 26.5, 26.9, 31.4, 34.6, 34.8, 36.4, 36.8, 41.8, 61.4, 66.5, 108.2, 111.4, 114.2, 123.1, 125.5,
129.0, 140.8, 151.9, 168.5. Step 3. A mixture of 37 (110 mg, 0.23 mmol) and 1 M aq. NaOH (1
24
ACS Paragon Plus Environment

Page 25 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mL) in THF (10 mL) was stirred at rt for 12 h. The solvent was removed in vacuo and the
residue was diluted with H O (10 mL) and acidified with AcOH (0.2 mL). The precipitate was
2
filtered, washed with water and dried in vacuo at 50 °C to afford 7 as a white solid (100 mg,
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
1
9
7%). mp 145–146 °C. H NMR (CDCl ) δ 2.12–1.54 (m, 22H), 2.48 (t, J = 12.4 Hz, 1H), 4.67
3
1
3
(
s, 2H), 6.79 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 7.23 (s, 1H), 8.07 (brs, 1H). C NMR
(
CDCl ) δ 26.5, 26.9, 31.4, 34.6, 34.8, 36.4, 36.8, 41.9, 66.2, 108.2, 111.5, 114.4, 123.2, 125.9,
3
1
6
29.1, 141.3, 151.4, 173.2. Anal. calcd for C H ClO H O: C, 61.73; H, 6.69. Found: C,
24 29 6 2
2.00; H, 6.40.
cis-Adamantane-2-spiro-3'-8'-[3,5-dichloro-4-(carboxymethoxy)phenyl]-1',2',4'-
2
7
trioxaspiro[4.5]decan (8). Step 1. To a solution of 26 (410 mg, 1.0 mmol) in dry DMF (5 mL)
was added dropwise a solution of N-chlorosuccinimide (1.25 g, 9.36 mmol) in dry DMF (3 mL).
The reaction mixture was stirred under nitrogen at rt for 24 h. The mixture was poured on ice and
then extracted with EtOAc (2 x 20 mL). The combined organic extracts were washed with water
(
30 mL) and brine (20 mL), dried over anhydrous MgSO and concentrated in vacuo. The crude
4
product was purified by chromatography (sg, hexane:EA, 25:1) to afford cis-adamantane-2-
spiro-3'-8'-(3,5-dichloro-4-hydroxyphenyl)-1',2',4'-trioxaspiro[4.5]decane (36) (270 mg, 63%) as
1
a white solid. mp 143–145 °C. H NMR (400 MHz, CDCl ) δ 1.61–1.87 (m, 14H), 1.90–2.08 (m,
3
1
3
8
2
H), 2.45 (tt, J = 12.5, 3.2 Hz, 1H), 5.70 (s, 1H), 7.10 (s, 2H); C NMR (100 MHz, CDCl ) δ
3
6.5, 26.9, 31.4, 34.5, 34.8, 36.4, 36.8, 41.8, 108.0, 111.6, 120.8, 126.6, 139.6, 146.0. Step 2. A
mixture of 36 (270 mg, 0.63 mmol), ethyl bromoacetate (159 mg, 0.95 mmol) and potassium
carbonate (175 mg, 1.26 mmol) in acetone (20 mL) was stirred at 60 °C for 12 h. The solid was
removed by filtration and washed with acetone and the filtrate was concentrated in vacuo to
afford cis-adamantane-2-spiro-3'-8'-[3,5-dichloro-4-(2-ethoxy-2-oxoethoxy)phenyl]-1',2',4'-
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 56
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
trioxaspiro[4.5]decane (38) (322 mg, 100%) as a white solid. mp 99–100 °C. H NMR (CDCl ) δ
3
1
.30 (t, J = 7.1 Hz, 3H), 1.58–1.69 (m, 6H), 1.69–1.84 (m, 8H), 1.87–1.93 (m, 2H), 1.94–2.05
1
3
(m, 6H), 2.46 (tt, J = 12.3, 3.3 Hz, 1H), 4.28 (q, J = 7.1 Hz, 2H), 4.56 (s, 2H), 7.11 (s, 2H); C
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
NMR (CDCl ) δ 14.1, 26.4, 26.8, 31.1, 34.4, 34.8, 36.4, 36.7, 41.9, 61.3, 69.3, 107.8, 111.5,
3
1
27.3, 128.8, 144.3, 148.4, 167.9. Step 3. A mixture of 38 (322 mg, 0.63 mmol) and 1 N aq.
NaOH (2 mL) in THF (20 mL) was stirred at rt for 12 h. Removal of the solvents in vacuo gave a
white residue which was suspended in H O (10 mL) and acidified with AcOH to pH 3. The
2
precipitate was filtered, washed with water and dried in vacuo at 50 °C to afford 8 (270 mg,
1
8
9%) as a white solid. mp 142–143 °C. H NMR (CDCl ) δ 1.61–1.87 (m, 14H), 1.90–2.09 (m,
3
1
3
8H), 2.49 (tt, J = 12.3, 3.4 Hz, 1H), 4.64 (s, 2H), 7.15 (s, 2H); C NMR (CDCl ) δ 26.5, 26.9,
3
3
1.2, 34.4, 34.8, 36.4, 36.8, 42.0, 68.9, 107.9, 111.6, 127.4, 128.7, 144.8, 148.0, 171.2. Anal.
calcd for C H Cl O6: C, 59.63; H, 5.84. Found: C, 59.89; H, 6.00.
2
4
28
2
cis-Adamantane-2-spiro-3'-8'-[4-(carboxypropoxy)phenyl]-1',2',4'-
27
trioxaspiro[4.5]decane (9). To a solution of 26 (250 mg, 0.70 mmol) in dry
dimethoxyethane (25 mL) were added powdered NaOH (168 mg, 4.20 mmol) and
Bu NHSO (48 mg, 0.15 mmol) and the resulting mixture was stirred at rt for 30 min
4
4
before addition of ethyl 4-bromobutanoate (205 mg, 1.05 mmol). The reaction mixture
was stirred at 60 °C for 12 h, then concentrated in vacuo. The residue was suspended in
H O (20 mL) and acidified with AcOH to pH 3. The precipitate was filtered, washed with
2
H O and dried in vacuo at 50 °C to afford 9 (240 mg, 77%) as a white solid. mp 148–149
2
1
°
C. H NMR (CDCl ) δ 1.87–1.57 (m, 14H), 1.94 (d, J = 12.8 Hz, 2H), 2.06 – 1.97 (m, 6H),
3
26
ACS Paragon Plus Environment

Page 27 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
6
2
1
.09 (p, J = 6.7 Hz, 2H), 2.49 (tt, J = 12.1, 3.3 Hz, 1H), 2.57 (t, J = 7.3 Hz, 2H), 3.98 (t, J =
13
.1 Hz, 2H), 6.81 (d, J = 8.3 Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H); C NMR (CDCl ) δ 24.5, 26.5,
3
6.9, 30.6, 31.7, 34.77, 34.83, 36.4, 36.8, 42.1, 66.5, 108.5, 111.4, 114.4, 127.6, 138.5, 157.1,
79.2. Anal. calcd for C H O : C, 70.56; H, 7.74. Found: C, 70.70; H, 7.75.
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
26
34
6
cis-Adamantane-2-spiro-3'-8'-[4-(carboxybutoxy)phenyl]-1',2',4'-
27
trioxaspiro[4.5]decan (10). To a solution of 26 (250 mg, 0.70 mmol) in dry
dimethyoxyethane (25 mL) were added powdered NaOH (168 mg, 4.20 mmol) and
Bu NHSO (48 mg, 0.15 mmol) and the resulting mixture was stirred at rt for 30 min
4
4
before addition of methyl 5-bromopentanoate (220 mg, 1.05 mmol). The reaction
mixture was stirred at 60 °C for 12 h, then concentrated in vacuo. The residue was
suspended in H O (20 mL) and acidified with AcOH to pH 3. The precipitate was filtered,
2
washed with H O and dried in vacuo at 50 °C to afford 10 (230 mg, 72%) as a white
2
1
solid. mp 144–145 °C. H NMR (CDCl ) δ 1.87–1.61 (m, 18H), 1.94 (d, J = 12.8 Hz, 2H),
3
2.06–1.97 (m, 6H), 2.43 (d, J = 6.9 Hz, 2H), 2.48 (tt, J = 15.7, 5.0 Hz, 1H), 3.93 (t, J = 5.3 Hz,
13
2H), 6.80 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H); C NMR (CDCl ) δ 21.5, 26.5, 26.9,
3
28.7, 31.7, 33.8, 34.77, 34.83, 36.4, 36.8, 42.1, 67.3, 108.5, 111.4, 114.4, 127.6, 138.3, 157.3,
179.3. Anal. calcd for C H O : C, 71.03; H, 7.95. Found: C, 70.84; H, 7.78.
27
36
6
cis-Adamantane-2-spiro-3'-8'-[4-[2-[methylsulfonamino]-2-oxoethoxy]phenyl]-1',2',4'-
trioxaspiro[4.5]decane (13). To a mixture of 2 (150 mg, 0.36 mmol), methanesulfonamide (47
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 56
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
mg, 0.40 mmol) and DMAP (10 mg, 0.07 mmol) in DCM (20 mL) was added DCC (82 mg, 0.40
mmol). The resulting mixture was stirred at rt for 24 h. The solid was filtered and the filtrate was
concentrated in vacuo to produce a residue that was purified by chromatography (sg, DCM) 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
1
afford 13 (110 mg, 62%) as a white solid. mp 148–149 °C; H NMR (CDCl ) δ 2.08–1.68 (m,
3
2
2H), 2.55 (td, J = 12 Hz, J = 3 Hz), 3.38 (s, 3H), 4.85 (s, 2H), 6.88 (d, J = 8.5 Hz, 2H), 7.21
1 2
1
3
(
d, J = 8.5 Hz, 2H), 8.84 (br, 1H); C NMR (CDCl ) δ 26.5, 26.9, 31.6, 34.7, 41.7, 42.1, 67.4,
3
1
08.3, 111.5, 114.7, 128.2, 140.9, 154.8, 167.7. Anal. calcd for C H NO S 0.25 H O: C,
25 33 7 2
6
0.53; H, 6.81; N, 2.82. Found: C, 60.87; H, 6.86; N, 2.84.
cis-Adamantane-2-spiro-3'-8'-[4-[2-[(carboxymethyl)amino]-2-
oxoethoxy]phenyl]-1',2',4'-trioxaspiro[4.5]decane (14). Step 1. To a mixture of 2
415 mg, 1.0 mmol) and HOBt (162 mg, 1.2 mmol) in DMF (10 mL) was added EDC (230
(
mg, 1.2 mmol) followed by addition of glycine ethyl ester hydrochloride (154 mg, 1.1
mmol) and DIPEA (155 mg, 1.2 mmol). The resulting mixture was stirred at rt for 12 h
and then diluted with EA (50 mL), washed with brine (2 x 50 mL), 1 N HCl (20 mL), 1 N
NaOH (20 mL) and brine (20 mL), dried over anhydrous MgSO , filtered and
4
concentrated in vacuo to give a colorless residue that was dissolved in a mixture of
ethanol (40 mL) and 2% aq. NaOH (4 mL). The resulting mixture was stirred at rt for 12 h
and then concentrated in vacuo to afford a white residue that was suspended in H O (50
2
mL), acidified with AcOH to pH 3, filtered, washed with H O, and dried in vacuo to afford
2
1
1
4 (430 mg, 91%) as a white solid. mp 147–148 °C; H NMR (DMSO-d ) δ 1.50–1.58 (m,
6
2H), 1.66–1.93 (m, 19H), 2.59 (t, J = 15 Hz, 1H), 3.81 (d, J = 7.2 Hz, 2H), 4.49 (s, 2H), 6.91
28
ACS Paragon Plus Environment

Page 29 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
3
(
d, J = 11.2 Hz, 2H), 7.15 (d, J = 11.2 Hz, 2H), 8.35 (t, J = 7.2 Hz, 1H), 12.63 (bs, 1H); C
NMR (DMSO-d ) δ 26.3, 26.7, 31.7, 34.6, 34.7, 36.3, 36.6, 40.9, 41.2, 67.4, 108.6, 111.0,
6
1
15.2, 127.9, 139.2, 156.5, 168.7, 171.4. Anal. calcd for C H NO : C, 66.23; H, 7.05; N,
26 33
7
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
2
.97. Found: C, 66.40; H, 6.96; N, 2.60.
Sodium 1-(2-(4-dispiro[adamantane-2,3'-[1,2,4]trioxolane-5',1''-cyclohexan]-4''-
yl)phenoxy)ethyl)piperidine-4-carboxylate (15). Step 1. To a solution of cis-adamantane-2-
2
7
spiro-3'-8'-(4'-acetoxyphenyl)-1',2',4'-trioxaspiro[4.5]decane (27) (430 mg, 1.1 mmol) in dry
acetonitrile (20 mL) were added powdered NaOH (259 mg, 6.5 mmol) and tetrabutylammonium
hydrogensulfate (110 mg, 0.3 mmol) according to the method of Charman et al.25 The mixture
4
3
was stirred at rt for 0.5 h. Then, ethyl 1-(2-chloroethyl)piperidine-4-carboxylate (28) (475 mg,
.2 mmol) was added and the reaction stirred at 60 ºC for 12 h. The inorganic solid was filtered
off and washed with CH Cl . After removal of the solvents in vacuo, the residue was dissolved in
2
2
2
EtOAc (50 mL). The organic layer was washed with water, brine, and dried over MgSO4.
Removal of the solvent in vacuo afforded ethyl 1-(2-(4-dispiro[adamantane-2,3'-
[1,2,4]trioxolane-5',1''-cyclohexan]-4''-yl)phenoxy)ethyl)piperidine-4-carboxylate (29) (260 mg,
1
4
2
5%) as a white solid. mp 70–72 °C; H NMR (CDCl ) δ 1.25 (t, J = 7.3 Hz, 3H), 1.69–2.04 (m,
3
6H), 2.15–2.19 (m, 2H), 2.26–2.30 (m, 1H), 2.46–2.51 (m, 1H), 2.77 (t, J = 5.9 Hz, 3H), 2.95
(brs, 2H), 4.07 (t, J = 5.9 Hz, 2H), 4.13 (q, J = 7.3 Hz, J = 14.2 Hz, 2H), 6.83 (d, J = 8.3 Hz, 2H),
13
7
4
.10 (d, J = 8.3 Hz, 2H); C NMR (CDCl ) δ 14.2, 26.5, 26.9, 28.2, 31.6, 34.7, 34.8, 36.4, 36.8,
3
1.0, 42.0, 53.4, 57.4, 60.3, 66.0. 108.4, 111.3, 114.4, 127.6, 138.5, 157.1, 175.0. Step 2. To a
solution of 29 (223 mg, 0.41 mmol) in EtOH (5 mL) was added powdered NaOH (50 mg, 1.2
mmol) and the reaction mixture was stirred for 24 h at rt. Filtration afforded 15 (190 mg, 86%)
29
ACS Paragon Plus Environment