Hit-to-Lead Studies for the Antimalarial Tetrahydroisoquinolone Carboxanilides

Article abstract of DOI:10.1021/acs.jmedchem.6b00752

Phenotypic whole-cell screening in erythrocytic cocultures of Plasmodium falciparum identified a series of dihydroisoquinolones that possessed potent antimalarial activity against multiple resistant strains of P. falciparum in vitro and show no cytotoxicity to mammalian cells. Systematic structure-activity studies revealed relationships between potency and modifications at N-2, C-3, and C-4. Careful structure-property relationship studies, coupled with studies of metabolism, addressed the poor aqueous solubility and metabolic vulnerability, as well as potential toxicological effects, inherent in the more potent primary screening hits such as 10b. Analogues 13h and 13i, with structural modifications at each site, were shown to possess excellent antimalarial activity in vivo. The (+)-(3S,4S) enantiomer of 13i and similar analogues were identified as the more potent. On the basis of these studies, we have selected (+)-13i for further study as a preclinical candidate.

Full text of DOI:10.1021/acs.jmedchem.6b00752

Subscriber access provided by Northern Illinois University
Article
Hit-to-Lead Studies for the Antimalarial
Tetrahydroisoquinolone Carboxanilides
David M. Floyd, Philip Stein, Zheng Wang, Jian Liu, Steve Castro, Julie A Clark, Michele
C. Connelly, Fangyi Zhu, Gloria Holbrook, Amy Matheny, Martina S. Sigal, Jaeki Min,
Rajkumar dhinakara, Senthil Krishnan, Sridevi Bashyam, Spencer Knapp, and R. Kiplin Guy
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00752 • Publication Date (Web): 09 Aug 2016
Downloaded from http://pubs.acs.org on August 11, 2016
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 free 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 accessible to all
readers and 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.
Journal of Medicinal Chemistry 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 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Hit-to-Lead Studies for the Antimalarial Tetrahydroisoquinolone
i
Carboxanilides
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
a
a
a
a
b
David M. Floyd, Philip Stein, Zheng Wang, Jian Liu, Steve Castro, Julie A. Clark,
b
b
b
b
b
Michele Connelly, Fangyi Zhu, Gloria Holbrook, Amy Matheny, Martina S. Sigal,
b
c
c
c
a
Jaeki Min, Rajkumar Dhinakaran, Senthil Krishnan, Sridevi Bashyum, Spencer Knapp,
b
and R. Kiplin Guy *
a
Department of Chemistry & Chemical Biology, Rutgers The State University of New Jersey, 610 Taylor Rd.,
Piscataway, NJ 08854 USA
b
Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny
Thomas Place, Memphis, TN 38105 US
c
Syngene International Ltd., Plot No. 2 & 3, Bommasandra IV Phase, Jigani Link Rd., Bangalore, India
560 099
i
This paper is respectfully dedicated to the memory of our friend and colleague Phil Stein.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 53
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
Phenotypic wholeꢀcell screening in erythrocytic coꢀcultures of Plasmodium falciparum identified a
series of dihydroisoquinolones that possessed potent antiꢀmalarial activity against multiple resistant
strains of P. falciparum in vitro and show no cytotoxicity to mammalian cells. Systematic structureꢀ
activity studies revealed relationships between potency and modifications at Nꢀ2, Cꢀ3 and Cꢀ4.
Careful structureꢀproperty relationship studies, coupled with studies of metabolism, addressed the
poor aqueous solubility and metabolic vulnerability, as well as potential toxicological effects,
inherent in the more potent primary screening hits such as 10b. Analogues 13h and 13i, with
structural modifications at each site, were shown to possess excellent antiꢀmalarial activity in vivo.
The (+)ꢀ(3S,4S) enantiomer of 13i and similar analogues were identified as the more potent. Based
on these studies, we have selected (+)ꢀ13i for further study as a preclinical candidate.
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
ACS Paragon Plus Environment

Page 3 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
Despite significant efforts over the last decade to identify and bring to market new
agents, malaria remains a significant health issue, with close to 50% of the world’s
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
population currently at risk of infection. Of the five species of plasmodium that cause
malaria in man, Plasmodium falciparum accounts for the majority of deaths associated
with the disease. In most cases, treatment of uncomplicated P. falciparum malaria results
in low mortality (ca 0.1%). However, in the absence of effective treatment the disease
rapidly progresses and, especially in children, mortality rises to approximately 20%. Drug
resistant strains of P. falciparum have emerged for every currently utilized antiꢀmalarial
2
agent. Therefore, there is a need for a sustained effort to identify new antimalarial agents
that are efficacious against existing resistant strains. Although multiple avenues for
addressing these issues are being followed, the use of highꢀthroughput phenotypic
screening to identify compounds that kill P. falciparum in human red blood cells has
3
, 4
proven to be a particularly effective approach. Phenotypic screens offer the potential to
identify novel antimalarial chemotypes that fall outside of the known antiꢀmalarial agents
and, as a consequence, increase the potential to identify compounds with previously
5, 6
unrecognized or unexploited molecular targets.
The highꢀthroughput phenotypic screening of over 300,000 compounds against
cultures of the 3D7 strain of P. falciparum in human red blood cells resulted in the
identification of three series of validated hits with chemical structures that are distinct
7
from previously reported agents possessing antiꢀmalarial activity. Subsequent reꢀsynthesis
and testing of selected examples from each hit series focused our attention on a series of
1,2,3,4ꢀtetrahydroꢀ1ꢀisoquinoloneꢀ4ꢀcarboxanilides of general structure 3 (Scheme 1) as
our highest priority for hitꢀtoꢀlead and lead optimization studies. The data available at the
initiation of hitꢀtoꢀlead indicated the compounds were generally potent against all tested
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 53
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
resistant strains of malaria and had significant effects on gametocyte development but not
8
on liver stages. We have previously described the clinical candidate (+)ꢀSJ733 (13i),
8
which arose from this series. In this report, we describe the primary structure activity
relationship (SAR) within the series of analogues 3 that resulted in the identification of
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
lead compounds and selection of a preclinical candidate.
RESULTS AND DISCUSSION
Various 1,2,3,4ꢀtetrahydroꢀ1ꢀisoquinoloneꢀ4ꢀcarboxylic acid analogues 6 were
prepared by the wellꢀdocumented condensation of an aldimine (3) with homophthalic
9
ꢀ11
anhydride (HPA, 4), which, in a single step, provides 5/6 (Scheme 1).
Depending on
the reaction conditions and the nature of the aldimine, the reaction affords predominantly
the 3,4ꢀcis product, predominantly the 3,4ꢀtrans product, or a mixture of both. However,
despite the potential for both possible stereoisomers, the cis product (5) can be efficiently
converted to the thermodynamically favored trans isomer (6) by heating at reflux in acetic
1
2
acid solution. Thus, the reaction sequence shown in Scheme 1 affords racemic 3,4ꢀtrans
carboxylic acids 6 with differing substituents at Nꢀ2 and Cꢀ3, from which various series of
carboxamide analogues 8 can be prepared by amide coupling reactions.
The general methods in Scheme 1 served to prepare the compounds discussed below.
Formation of the requisite aldimine (1) was generally accomplished by mixing amine 2
and aldehyde 1 in methanol solution with a catalytic amount of acetic acid, followed by
stirring at ambient temperature for 4–8 hours and then concentration (Method A).
Although this method did allow for the preparation of aldimines derived from many
amines, an alternate method was developed for 2,2,2ꢀtrifluoroethylamine, given the
eventual focus on this Nꢀ2 substituent (see below). In this case, a mixture of the amine
hydrochloride salt and the requisite aldehyde in toluene was treated with one equivalent of
50% aqueous sodium hydroxide (Method B). The mixture was then allowed to stir for
4
ACS Paragon Plus Environment

Page 5 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
several hours to give, after simple workup, the desired aldimine in high yield. As prepared
by either of these conditions, the aldimines are of sufficient purity to be used directly. The
preparation of carboxylic acid 6 was accomplished by the addition of HPA to a solution of
aldimine 3 in acetic acid solution at room temperature followed by heating at reflux for 16
hours. Concentration of the reaction mixture and either (1) trituration of the resulting
residue with a mixture of ethyl acetate / water followed by collection of the crude product
by filtration, or (2) extractive work up, generally gave the desired 3,4ꢀtransꢀ1,2,3,4ꢀ
tetrahydroꢀ1ꢀisoquinoloneꢀ4ꢀcarboxylates (6) in moderate but reproducible yields (30ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
0%). Crude trans acids 6 were normally used without further purification, although
purification by crystallization from isopropyl alcohol or ethyl acetate proved useful in
some cases. This procedure was used for preparation of all trans-acids used for
subsequent synthesis. Confirmation of the 3,4ꢀtrans configuration relied on analysis of
proton NMR spectra, wherein the J3ꢀ4 coupling constants are on the order of 1ꢀ2 Hz,
1
1, 13, 14
consistent with literature values.
For example, carboxylic acid 6 (X = i-Bu and Y =
thienꢀ2ꢀyl), exhibits J3ꢀ4 = 1.5 Hz, consistent with the assignment as the trans
diastereomer. In order to confirm the stereochemistry was as expected, alternate
conditions were used to produce the corresponding cis carboxylic acid (5, X = i-Bu and Y
=
thiophenꢀ2ꢀyl) by conducting the reaction between the aldimine and HPA in
o
dichloromethane solution at –30 C for several hours followed by additional stirring at
ambient temperature. Under these conditions, the cis acid precipitated and was obtained in
high purity and acceptable yield by simple filtration of the reaction mixture. The observed
J
3ꢀ4 (5.4 Hz) indicates the cis stereochemistry. These observations support the assumption
that, unless otherwise indicated, the compounds evaluated in this series are the 3,4ꢀtrans
carboxamides (8), consistent with the known thermodynamic preference and the general
reaction conditions employed to prepare analogues.
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 53
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
Treatment of a solution of a carboxylic acid (6, Scheme 1), 1.2 equivalents of the
2
requisite amine (7, ZꢀNH , generally an aniline), and 2 equivalents of triethylamine with
2
.5 equivalents of propanephosphonic acid anhydride (T3P) afforded, after
chromatographic purification, 20–70% yields of the desired corresponding carboxamide
(8, Method A). Under these conditions, small amounts of cisꢀamide (cisꢀ8) are sometimes
observed, possibly as the result of competitive ketene formation: addition of the amine to
an intermediate Cꢀ4 ketene can give after protonation some portion of cisꢀamide.
Additionally,
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
small amounts
of a product
arising
from
acylation of the
product
carboxamide
were observed
in some cases.
However,
Scheme 1. Synthesis of 1,2,3,4ꢀtetrahydroꢀ1ꢀisoquinoloneꢀ4ꢀ
carboxamides 8
treatment of the
crude reaction
mixture with methanolic potassium or sodium carbonate equilibrated the mixture to the
thermodynamically more stable 3,4ꢀtrans carboxamide 8, and also cleaved any bisꢀ
acylated adduct, and afforded pure 8 after purification by column chromatography.
Unless otherwise noted, Method A served to prepare all of the compounds described
below. However, in certain cases superior results were obtained by treating a mixture of
carboxylic acid 6 and 1.5–2.0 equiv. of the desired amine (7) with 1.1 equiv. of
6
ACS Paragon Plus Environment

Page 7 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
5
phosphorous oxychloride in acetonitrile solution at reflux (Method B). After extractive
workꢀup, the desired amides (8) were obtained in 60–95% yield. The use of the higher
temperature assured that any 3,4ꢀcis diastereomer was converted to the thermodynamically
favored 3,4ꢀtrans carboxamide, 8.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The initial high throughput screening campaign identified over 40 analogues of 8
covering more than a 100ꢀfold range in potency. Early hit validation studies confirmed the
antiꢀmalarial activity, lack of significant mammalian cytotoxicity, acceptable
7
pharmacokinetic properties, and preliminary indication of antiꢀmalarial activity in vivo. A
major driver for lead optimization of this series was the broad spectrum antiꢀmalarial
activity against drug resistant strains of malaria representing a range of resistance
mechanisms that was exhibited by the initial hits (< 5ꢀfold range across all genotypes) and
generally demonstrated by analogues prepared in this study (generally less than 3ꢀfold; for
occasional analogs larger, see Table SꢀI in Supporting Information).
Preliminary SAR trends were apparent from analysis of the reꢀsynthesized analogues
from the screening set. First, from the compounds in the screening set, those bearing a
paraꢀmethoxy substituent on the aromatic ring of the Cꢀ4 carboxamide substituent
(Scheme 1, 8, Z = m-MeOPh) were generally the most potent, although there were highly
potent compounds possessing alternate substitution patterns. Therefore, careful
exploration of the substitution of this ring was included in early plans (see Tables 1 and 2
below). Second, of the compounds represented in the initial screening set, virtually all
compounds with EC50 < 500 nM possessed a 2ꢀthienyl substituent at Cꢀ3 of the 1,2,3,4ꢀ
tetrahydroꢀ1ꢀisoquinolone ring (Scheme 1, 8, Y = thiophene). Given the physiochemical
and metabolic liabilities potentially embedded in the thiophene, replacing this ring became
an initial focus of work (see Table 3 below). Finally, all of the screening hits with subꢀ
micromolar potency possessed an isobutyl group at Nꢀ2 (Scheme 1, 8, X =
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 53
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
CH(CH
3
)CH
2
CH
3
), with analogues having smaller or larger substituents showing reduced
potency. Thus, based on the confirmation of structure and potency after the reꢀsynthesis of
multiple analogues, extensive structureꢀactivity relationship (SAR) studies were
undertaken with the series of 3,4ꢀtransꢀ1,2,3,4ꢀtetrahydroꢀ1ꢀisoquinoloneꢀ4ꢀcarboxamides,
8 to further explore these trends.
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
As noted above, compounds with a 4ꢀmethoxy substituent on the carboxanilide ring
were among the most potent screening hits. At the outset, this functionality presented a
clear metabolic liability, namely, the potential to generate toxic metabolites resulting from
Oꢀdemethylation followed by oxidation to an Nꢀacylꢀiminoquinone, a behavior
16
precedented by the metabolism of acetaminophen. Furthermore, all of the most potent
hits feature a carboxanilide at Cꢀ4, which presents the potential for metabolic amide
hydrolysis and the release of the aniline. Based on these considerations, a thorough
investigation of the Cꢀ4 carboxamide functionality became an early focus of the program,
with the objective of either completely replacing the aniline, or identifying anilines lacking
metabolic
liabilities and
mutagenic
properties.
We
carried
out
metabolism
Figure 1. Metabolism studies of transꢀ1,2,3,4ꢀtetrahydroꢀ1ꢀ
isoquinoloneꢀ4ꢀcarboxanilides in mouse microsomes.
and oxidative
stability studies
using microsomes from rat, dog, and human, as well as hepatocytes from these species
(data not shown). The metabolism patterns were consistent across all species, with mouse
8
ACS Paragon Plus Environment

Page 9 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
having the fastest rate. For this reason, and because the mouse is the species used for
primary efficacy testing in vivo, we report only the mouse microsomal metabolism data.
These studies confirmed that the methoxy substituent on the Cꢀ4 carboxanilide aromatic
ring of 10b was readily demethylated. Likewise, the 3ꢀcyano carboxanilide ring of 10d
was hydroxylated, although the specific site of oxidation was not determined. In both
compounds, significant oxidative metabolism of the Nꢀ2 isobutyl group was observed.
Thus, with respect to maximizing the potential for compounds in this series to express
efficacy in vivo, modifications of both the Cꢀ4 carboxamide and Nꢀ2 substituent would
likely be required.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We initially investigated changes to the carboxamide at Cꢀ4 (Tables 1 and 2).
Substitution of the aniline portion for a benzyl amine, as with Nꢀ(4ꢀmethoxybenzyl)
carboxamide (9a), resulted in more than a 100ꢀfold drop in potency compared to the 4ꢀ
methoxy carboxanilide (10i). Likewise, both simple alkyl (9b, 9c) or heteroalkyl (9d, 9e)
analogues were weakly potent (Table 1). Heteroaromatic surrogates for the Cꢀ4
carboxanilide (9f – 9i) were explored with a view toward improving the poor aqueous
solubility of the initial screening hits (ca 1–2 ꢀM at pH 7.4). Relative to the parent,
unsubstituted Cꢀ4 anilide 10a, the pyridinꢀ2ꢀyl amide (9f) was less potent, the pyridinꢀ3ꢀyl
amide 9g was equipotent, and the pyridinꢀ4ꢀyl carboxamide 9h was significantly less
potent. Incorporation of a cyano group meta to the amide on the pyridyl ring was also
explored. Compounds 9i and 9j nitrogen were modestly more potent than the respective
unsubstituted pyridyl analogues, 9f and 9g. On the other hand, the Nꢀ(4ꢀcyanoꢀ2ꢀpyridyl)ꢀ
carboxamide (9l) was significantly less potent than either 9i or 9f suggesting possibly
opposing drivers for binding to the area of the target accommodating this ring.
Surprisingly, the 6ꢀcyanopyridꢀ4ꢀyl derivative (9k) was substantially (73ꢀfold) more potent
than the corresponding unsubstituted pyridinꢀ4ꢀyl analogue (9h), and equipotent with the
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 53
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
3ꢀcyano carboxanilide (10d). Thus, with the exception of the Nꢀ(2ꢀcyanopyridꢀ4ꢀyl)
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 1. C-4 Carboxamide modifications.
a
Concentration causing 50% reduction in growth of P falciparum 3D7 coꢀcultured with human
erythrocytes; values shown are from two independent experiments done in triplicate.
analogue (9k), none of the pyridyl analogues were deemed sufficiently potent to merit
1
0
ACS Paragon Plus Environment

Page 11 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
further investigation. Although 9k possessed potency sufficient to make it attractive for
further development, the absence of clear lack of genotoxicity data in the literature for the
parent aniline and the tightness of the SAR kept it from frontrunner status. Generally,
these analogs maintained broad spectrum activity, although occasionally (10d, 10i) had
activity ranges as high as 15ꢀfold, with the V1/S strain consistently being the outlier.
Furthermore, the impact on solubility with this series was modest giving less than 10ꢀfold
improvements. Overall, these observations, coupled with the data from additional Cꢀ4
amides not described here, focused efforts on exploring aromatic substituent effects in a
series of carboxanilides. In other words, despite the examination of many possible
surrogates, few if any analogues lacking the Cꢀ4 carboxanilide functionality maintain
acceptable antiꢀmalarial potency in vitro.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Studies with a series of monoꢀsubstituted carboxanilides indicated that electron
donating substituents para (4' in Table 2) to the amide nitrogen were favored, whereas
Table 2. Carboxanilide substitution effects at Cꢀ4.
Me
Me
N
O
4
S
H (Me)
O
N
2
'
substituted phenyl
X
10
a
a
Cmpd.
X
H
ED50 (nM)
Cmpd.
X
EC50 (nM)
1300
10a
97
53
10o
10p
2'Cl
3'ꢀCl
10b
3'ꢀCl, 4'ꢀOMe
162
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 53
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
0c (N-Me)
3'ꢀCl, 4'ꢀOMe
>5000
10q
4'ꢀC
497
10d
3'ꢀCN
3'ꢀCN
3'ꢀCN
50
10r
10s
10t
3'ꢀOCF
3
3
57
10e (cis)
207
4'ꢀOCF
1022
136
1
0f (N-Me)
4115
3'ꢀCF
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
10g
2'ꢀCN
4'ꢀCN
4'ꢀOMe
3'ꢀOMe
2'ꢀOMe
2'ꢀF
1426
1134
65
10u
10v
3'ꢀCN, 4'ꢀOMe
3'ꢀCN, 4'ꢀF
3'ꢀCN, 4'ꢀF
3'ꢀCN, 4'ꢀCl
3'ꢀCN, 5'ꢀF
3'ꢀCN, 5'ꢀCl
3'ꢀCN, 5'ꢀCN
29
19
1
0h
0i
10j
0k
0l
10m
0n
1
10w (cis)
10x
146
157
58
190
943
475
89
1
10y
1
10z
25
3'ꢀF
10aa
1104
1
4'ꢀF
56
a
Concentration causing 50% reduction in growth of P falciparum 3D7 coꢀcultured with human
erythrocytes; values shown are from two independent experiments done in triplicate.
electronꢀwithdrawing substituents appeared to be preferred in the meta (3' in Table 2)
position relative to the carboxanilide nitrogen. In general, an ortho (2' in Table 2)
substituent caused diminished potency relative to the unsubstituted carboxanilide (10a).
Among monoꢀmethoxylated analogues, para-substituted analogue 10i was somewhat more
potent than its metaꢀsubstituted isomer 10j, and both were significantly more potent than
10k, which features an orthoꢀmethoxy carboxanilide. With an electron withdrawing cyano
substituent, metaꢀsubstituted analogue 10d was 20ꢀfold more potent than either the orthoꢀ
(
10g) or paraꢀsubstituted (10h) compounds. Similar effects were seen among a series of
ortho-, meta-, and paraꢀsubstituted fluoro (10l, 10m) and chloro (10o, 10p, and 10q)
analogues. Of note was 10n, a clear outlier in this trend, in which the fluoro substituent
appears to mimic the effects of the paraꢀmethoxy group of 10i. Similarly, comparing the
1
2
ACS Paragon Plus Environment

Page 13 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
potency of metaꢀtrifluoromethoxy derivative 10r with that of its paraꢀsubstituted analogue
(10s) revealed a 20ꢀfold difference in potency. The metaꢀtrifluoromethyl analogue, 10t,
was equipotent with 10p (mꢀCl), and slightly less potent than metaꢀcyano analogue 10d.
Thus, within this limited set of monoꢀsubstituted Cꢀ4 carboxanilides, the most potent
compounds possessed either an electronꢀdonating group such as methoxy para to the
anilide nitrogen (10i), or an electronꢀwithdrawing group such as cyano (see 10d) meta to
the carboxanilide nitrogen.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
While this data set did not completely define the nature of the observed substituent
effects on compound potency, the potencyꢀenhancing effects of certain para and metaꢀ
substituents were maintained in disubstituted analogues. Thus, 10u (EC = 29 nM), with
5
0
metaꢀcyano and paraꢀmethoxy carboxanilide substituents, was of similar potency to monoꢀ
substituted analogues 10i (EC50 = 65 nM) and 10d (EC50 = 50 nM). More encouraging,
10v (EC50 = 19 nM), with metaꢀcyano and paraꢀfluoro aromatic substituents, was
somewhat more potent than 10b (EC = 53 nM) or 10n (EC = 56 nM), and represented a
5
0
50
carboxanilide analogue with good potency without the requirement for the paraꢀmethoxy
substituent. Exchanging the paraꢀfluoro (10v) for the chloro substituent (10x) resulted in a
10ꢀfold reduction in potency, consistent with the trend seen with monoꢀsubstituted
analogues. Moving the fluoro substituent to a metaꢀposition (10y) led to a modest
reduction in potency compared to 10v (EC50 = 58 nM vs. EC50 = 19 nM respectively),
although exchanging the metaꢀfluoro of 10y for the chloro of 10z gave an analogue with
similar potency (EC50 = 25 nM). The bis-metaꢀcyano substitution in 10aa caused a 100ꢀ
fold drop in potency relative to 10v. Many additional disubstituted carboxanilides were
explored, although none proved to be as potent as either 10v or 10z. In this series of
compounds, the 3,4ꢀtransꢀconfiguration provides the highest potency with the cis isomers
10e and 10w being approximately 10ꢀfold less potent than their respective trans isomers
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 53
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
10d and 10v (see Table 2). Of these two substitution patterns, the metaꢀcyano-para-fluoro
carboxanilide was chosen for subsequent investigations of the effects of modification at Nꢀ
2
and Cꢀ3 of the 1,2,3,4ꢀtetrahydroꢀ1ꢀisoquinolone system. Important to this decision was
the observation that the parent 3ꢀcyanoꢀ4ꢀfluoroaniline was negative in the Ames' assay.
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
The importance of the Cꢀ4 carboxanilide NꢀH was probed by preparing two Nꢀmethyl
analogues. Based on the significant drop in respective potency of 10f and 10c relative to
10d and 10b, the amide NꢀH appears to be important to potency in this series.
Furthermore, the ester analogue of 10d (i. e., “O” replaces “NH”) was also prepared from
the corresponding carboxylic acid (see Supporting Information), and was devoid of
measurable activity in vitro (EC50 > 11 ꢀM).
Overall, the systematic study of the C4 carboxamide functionality supported a
preference for the carboxanilide as indicated by the significant loss of potency observed
for the various alkyl, heteroalkyl or heteroaryl amide alternatives. Substituent effects on
the carboxanilide appeared to correlate with the inductive effects of the substituent such
that electronꢀdonating paraꢀsubstituents and electronꢀwithdrawing metaꢀsubstituents are
preferred. These substituent effects could be moderately additive, as evidenced by the
potency of 10v compared with that of 10n and 10d; or 10u with 10i and 10d. The loss of
potency with the ester analogue of 10d as well as the reduction in potency associated with
Nꢀmethyl analogues and the reduction in relative potency observed with orthoꢀsubstituted
carboxanilides indicate the important role of the amide functionality in activity As noted
above, the majority of these analogs maintained broad spectrum activity, although
occasionally (10d, 10i) had moderately broader activity ranges. A more complete
understanding of the substituent effects and the role of the amide group in receptor binding
must await structural studies with the target protein.
1
4
ACS Paragon Plus Environment

Page 15 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
While exploration of the carboxanilide proved successful with respect to identifying
analogues possessing surrogates for the methoxy substituent on carboxanilide aromatic
ring and provided analogues with moderately improved potency, no analogues with
improved aqueous solubility were found. The majority of these compounds possessed
modest solubility (< 5 µM) and some were considerably worse. Consequently, we next
focused on modifications at Cꢀ3 in order to identify structural modifications improving
aqueous solubility (Table 3).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Small heteroaromatic rings appear to be preferred at Cꢀ3. Several alkyl substituents
were examined, which uniformly caused a large drop in potency as exemplified by 11a
and 11b and the piperidinꢀ3ꢀyl analogues (11c and 11d). Replacing the C3 thienꢀ2ꢀyl
substituent with a phenyl in 11e caused a 7ꢀfold drop in potency. While the thienꢀ3ꢀyl
compound 11f was similar in potency to the thienꢀ2ꢀyl analogue 10v (EC50 = 40 nM and
EC50 = 19 nM respectively), there was a small reduction in potency for furꢀ3ꢀyl analogue
11g and a much more pronounced loss of relative potency with the furꢀ2ꢀyl analogues
(11h). For a series of 5ꢀsubstituted thienꢀ2ꢀyl analogues, there is either no, or a small,
reduction in potency (5ꢀMeꢀ, 11i; 5ꢀOMeꢀ, 11j; 5ꢀCl – 11k), with the exception of the 5ꢀ
cyano analogue (11l), where an apparent increase in potency was observed.
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 53
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
Table 3. Modifications at C-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
1
6
ACS Paragon Plus Environment

Page 17 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
An attempt to mimic this effect by incorporating the 5ꢀcarboxamido functionality (11m)
resulted in a marked reduction in potency. The increase in potency was also lost with the
4ꢀcyanoꢀthienꢀ2ꢀyl Cꢀ3 substituent in 11n. Overall, this subseries of analogs maintained
broad spectrum activity with ranges of potency less than 3ꢀfold.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Despite the apparent small improvement in potency associated with analogues such as
1
1l, solubility in the series remained an issue. In this regard, the pyridinꢀ3ꢀyl analogue
(
11o) both retained good potency and demonstrated a marked improvement in aqueous
solubility (Table 3). For example, 10v, with a Cꢀ3 thienꢀ2ꢀyl substituent, was poorly
soluble in water (2 ꢀM at pH 7.4), whereas 11o exhibits an aqueous solubility of 59 uM at
pH 7.4. While maintaining improved aqueous solubility, the pyridinꢀ4ꢀyl analogue 11p
was somewhat less potent, indicating fairly strict structural requirements in this region of
the molecule. The addition of a cyano group to the 5ꢀposition of the pyridine ring (11q),
which is the same relative positioning in the 5ꢀcyanoꢀthienꢀ2ꢀyl analogue (11l), reduced
potency relative to 11o. Exchanging the Cꢀ3 pyridinyl group for a pyridazinꢀ4ꢀyl group
(
11r) resulted in a 6ꢀfold reduction in potency compared to 11o, a change that is perhaps
related to the difference in potency between the 3ꢀyl and 4ꢀyl Cꢀ3 pyridine analogues. The
pyrimidinꢀ4ꢀyl Cꢀ3 substituent (11s) reduced potency by more than 10ꢀfold relative to 11o
(
EC50 = 486 nM vs EC50 = 25 nM, respectively) but maintained enhanced aqueous
solubility, reflecting the sensitivity to structural changes in this region of the molecule.
There was also an improvement in solubility with a series of Cꢀ3 pyrazolyl analogues
(
Table 3). Given that pyrazole ring nitrogen (pK
a
of conjugate acid ~ 2.5) is not
~ 5.2), the
significantly protonated at physiological pH (compare the pyridine ring, pK
a
effects on aqueous solubility appear to be related to factors other than simple protonation,
such as hydration in this region of the molecule. Pyrazolꢀ4ꢀyl analogue 11t also
maintained excellent potency, and was equipotent with the 5ꢀcyanoꢀthienꢀ2ꢀyl analogue
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 53
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
(11l), yet much more soluble at pH
.4 (3 ꢀM vs. 57 ꢀM respectively).
NꢀMethylation of the pyrazole group
11u) had no impact on potency or
Table 4. Polar alkyl substituents at N-2.
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
aqueous solubility.
Additional
5ꢀmembered
heterocycles
nitrogenꢀcontaining
surveyed at Cꢀ3 included thiazoꢀ5ꢀyl
11v), isothiazoꢀ4ꢀyl (11w), and
(
isoxazoꢀ5ꢀyl (11x), all of which
displayed potency similar to that of
pyridinꢀ3ꢀyl analogue 11o and 5ꢀ8
fold less than C3 pyrazoꢀ4ꢀyl
analogue 11t. With the exception of
the thiadiazolꢀ4ꢀyl analogue 11y,
each of these analogues maintained
enhanced aqueous solubility.
In summary, modification of the
Cꢀ3 substituent identified analogues
such as 11o and 11t, with improved
aqueous solubility and good or even
slightly improved potency in vitro.
a
Concentration causing 50% reduction in growth of P
falciparum 3D7 coꢀcultured with human erythrocytes.
Values are from two independent experiments done in
The data also indicate a strict structural requirement for the Cꢀ3 substituent, with 5ꢀ and 6ꢀ
membered heterocyclic ring systems demonstrating moderate to good potency in vitro.
1
8
ACS Paragon Plus Environment

Page 19 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Within this set of analogues, modifications in the position of heteroatoms may have an
impact on potency.
The objectives for investigating modifications of the Nꢀ2 substituent were twoꢀfold:
improving aqueous solubility of the series, and reducing the metabolic liability associated
with oxidation on the Nꢀ2 alkyl functionality. Examples of early attempts to address the
solubility issue, shown in Table 4, demonstrated that polar functionality in the Nꢀ2 alkyl
group leads to a marked reduction in potency. Thus, compared to Nꢀ2 isobutyl analogue
10d (EC50 = 50 nM), hydroxyalkyl analogues 12a and 12b were significantly less potent,
as was 12c, the methyl ether of 12b. Consistent with these data, Nꢀ2 substituents such as
dimethylaminoethyl (12d), carboxymethyl (12e), and the methyl ester (12f), resulted in
compounds devoid of measurable activity. The loss of potency with the Nꢀ2 methyl
analogue 12g relative to 10d (Nꢀisobutyl) indicates an apparent requirement for a larger,
more lipophilic Nꢀ2 substituent. In fact, there are multiple examples of Nꢀ2 methyl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
compounds in the original hit set, all of which show very low potency relative to their Nꢀ2
isobutyl analogues.
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 53
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
Table 5. Modified alkyl substituents at N-2.
Further studies demonstrated
fairly strict structural requirements
for the lipophilic N2 substituent. As
shown in Table 5, the Nꢀ2 nꢀbutyl
analogue 13a was more than 20ꢀfold
less potent than the corresponding
isobutyl derivative, 11n. Similarly,
cyclopropylmethyl derivatives 13b
and 13c were significantly less potent
compared to the corresponding
compounds with isobutyl Nꢀ2
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
substituents,
11t
and
11n,
respectively. Nevertheless, 3ꢀpentyl
analogues 13d (EC50 = 22 nM) and
1
3e (EC = 11 nM) were equipotent
50
with their respective isobutyl
analogues. There was a slight drop in
potency with the cyclopentyl
modification (a “cyclized 3ꢀpentyl”)
in 13f (EC50 = 51 nM). Finally,
replacement of the isobutyl with
phenyl, as in 13g, resulted in a
significant drop in potency. These
data indicate that both the relative
lipophilicity and the shape of the Nꢀ2
a
Concentration causing 50% reduction in growth of P
falciparum 3D7 coꢀcultured with human erythrocytes;
values shown are from two independent experiments done
in triplicate.
2
0
ACS Paragon Plus Environment

Page 21 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
substituent are important to potency in this tetrahydroꢀ1ꢀisoquinolone series.
Early studies had shown that the metabolic oxidation of the Nꢀ2 isoꢀbutyl group
occurred predominately adjacent to nitrogen, which is consistent with the wellꢀ
1
7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
documented Nꢀdealkylation of amides. Incorporation of an electronꢀpoor Nꢀ2 substituent
could suppress the observed oxidative process; consequently, the effects of a 2,2,2ꢀ
trifluoroethyl substituent at Nꢀ2 were examined. In general, only a small reduction in
potency was observed relative to the corresponding isobutyl. Thus, among the Cꢀ3
pyrazolꢀ4ꢀyl compounds, 13h, which possesses a trifluoroethyl substituent, was only 4ꢀ
fold less potent than isoꢀbutyl derivative 11t (EC50 = 45 nM vs EC50 = 10 nM
respectively). For the corresponding Cꢀ3 pyridinꢀ3ꢀyl compounds, the trifluoroethyl
substituent had little impact on potency (EC50 = 25 nM for 11o vs. EC50 = 58 nM for 13i).
Again, relatively minor structural modifications appeared to have a significant impact on
potency. For example, extending the length of the alkyl chain in the Nꢀ2 substituent, as in
13j, resulted in a 20ꢀfold drop in potency in vitro; an even more pronounced effect on
potency (>50ꢀfold) was observed for the difluorocyclobutylmethyl group of 13k.
Subsequent metabolism studies in the mouse demonstrated the anticipated metabolic
stability of the Nꢀ2,2,2ꢀtrifluoroethyl substituent was realized, which is clearly reflected in
the relative antiꢀmalarial potency in vivo for these analogues, as described below.
Overall, this subseries of analogs maintained broad spectrum activity, with potency range
less than 4ꢀfold across all genotypes.
The data in Table 6 demonstrate that modification of the C4 carboxanilide aromatic
ring substituents and incorporation of the 2,2,2ꢀtrifluoroethyl Nꢀ2 substituent results in
improved metabolic stability in mouse liver microsomes. The T1/2 for 10b, which bears the
4'ꢀmethoxy C4 carboxanilide substituent, is lower than the initial 30ꢀminute time point in
the microsomal assay. Comparison of the kinetic parameters of 10d and 11t implicates
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 53
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
metabolism of the Nꢀ2 isoꢀbutyl group as a significant process, given that 11t possesses
the 3ꢀcyanoꢀ4ꢀfluoroꢀsubstituted carboxanilide aromatic ring and the pyrazolꢀ4ꢀyl
substituent at Cꢀ3 that are stable under these conditions as evidenced by the data with 13h.
Nevertheless, the Cꢀ3 5ꢀcyanoꢀthiophenꢀ2ꢀyl substituent of 11l does appear to result in
improved metabolic stability. Further improvement in stability is realized with analogue
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
13i, with the Nꢀ(2,2,2ꢀtrifluoroethyl) substituent, and 13h, which appears to be stable to
metabolism in vitro. As noted above, comparing the T1/2 of 11t to that of 13h, which is
completely stable in this assay, clearly indicates the improved metabolic stability afforded
by the Nꢀ2 trifluoroethyl substituent. The difference in metabolic stability between 13i and
13h is associated with oxidative metabolism of the Cꢀ3 pyridine ring of 13i. Overall,
modification of both the Nꢀ2 substituent and the Cꢀ4 carboxamide aromatic ring
substituents results in improved metabolic stability in the mouse.
In summary, the systematic modification of substituents at Nꢀ2, Cꢀ3, and Cꢀ4 in the
1
,2,3,4ꢀtetrahydroꢀ1ꢀ
isoquinoloneꢀ4ꢀ
Table 6. Metabolic Stability in Mouse Liver Microsomes
Cmpd.
T
1/2 (hr)
CLint'
carboxanilide
afforded analogues with
improved aqueous
series
(
ml/min/kg)
1
0d
1t
11l
3i
3h
0.28 +/ꢀ 0.01
0.33 +/ꢀ 0.02
1.21 +/ꢀ 0.11
1.63 +/ꢀ 0.10
>4
195.32 +/ꢀ 8.91
162 +/ꢀ 10.83
44.73 +/ꢀ 4.25
33.09 +/ꢀ 2.06
<13.5
1
solubility and addressed the
anticipated metabolic and
1
toxicological
while
liabilities,
1
maintaining
acceptable potency in vitro. Although highly polar and/or solubilizing functionality in the
substituents at Cꢀ4 or Nꢀ2 attenuates potency, incorporation of certain small nitrogenꢀ
containing heterocycles at Cꢀ3 provides analogues with improved aqueous solubility. The
2
2
ACS Paragon Plus Environment

Page 23 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
data reveal an apparent requirement for a liphophilic substituent at Nꢀ2, but seemingly
minor changes in structure result in a marked impact on analogue potency. Incorporation
of the 2,2,2ꢀtrifluoroethyl group at Nꢀ2, in place of the metabolically labile isoꢀbutyl
group, resulted in significantly increased microsomal stability while causing only a small
drop in potency and no decrease in compound solubility. Examination of the antiꢀmalarial
activity in vivo of several analogues in this series confirmed that the modifications noted
above afford compounds with improved physiochemical and pharmacological properties.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
18
Efficacy testing was performed in vivo using the standard 4ꢀday Peter's test in Swiss
Webster mice infected with Plasmodium berghei NK65 ANKA strain (Figure 2). The
studies were carried out in 2 phases: 1) a screening assay at a fixed dose of 50 mg/kg/d for
all compounds tested, and 2) a dose response up to 200 mg/kg/d as well as a single fixed
dose of 200
mg/kg for
select
compounds
of interest.
These
studies
demonstrate
d that the
Figure 2. Antiꢀmalarial activity of transꢀ1,2,3,4ꢀtetrahydroꢀ1ꢀ
combinatio
isoquinoloneꢀ4ꢀcarboxanilides in the mouse. q.d. X 4 days, S.D. =
n
of
single dose
improved
solubility and metabolic stability resulted in an increase in efficacy at the doses tested. In
this assay, 10d failed to demonstrate significant activity at either a 100 or 300 mg/kg oral
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 53
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
dose. Comparing doseꢀresponse data for 11l (EC50 = 8 nM) at 50–200 mg/kg oral doses
with that of 13i (EC50 = 58 nM) indicated that either the improved solubility of 13i or the
increased metabolic stability of the Nꢀ2 trifluoroethyl group (compared with the isoꢀbutyl
group of 11l) caused in improved potency. In contrast, 11l demonstrated only a small
increase in potency above the 100 mg/kg oral dose, with an oral dose of 200 mg/kg being
essentially equivalent to 13i at 50 mg/kg. For either compound, a single 200ꢀmg/kg dose
showed reductions in parasitemia similar to that observed with 50 mg/kg x 4ꢀday dosing
schedule. The effects of limiting metabolism at Nꢀ2 on efficacy in vivo, as shown in 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
6
, were clearly demonstrated by comparing 11t (EC = 10 nM) with 13h (EC = 45 nM).
50 50
At 50 mg/kg oral doses, 13h demonstrated a 99% reduction in parasitemia, whereas 11t
caused only a 50% reduction despite being 4ꢀfold more potent in vitro. For comparison,
amodiaquine caused a 99% reduction in parasitemia at a 5 mg/kg oral dose in this assay.
These data indicate that the improvement in analogue solubility and/or modifications that
mitigate metabolism of the Nꢀ2 substituent result in improved antimalarial activity in vivo
upon oral dose. It is important to note that no overt sign of toxicity was observed in any of
the efficacy studies that have been performed with this series of compounds.
The compounds described above were prepared and tested as racemates. In order to
define enantiomeric preference in the series, selected carboxamides, or the esters of acids
6, were separated by chiral SFC or HPLC to obtain individual enantiomers (See
Supporting Information). Evaluation of the resolved enantiomers shows the (+)ꢀisomers to
be the more potent for the compounds shown in Table 7, although the corresponding (ꢀ
)ꢀisomers retain measurable potency. Assignment of absolute configuration at Cꢀ3 and Cꢀ4
was accomplished through resolution of racemic 1,2,3,4ꢀtetrahydroꢀ1ꢀisoquinoloneꢀ4ꢀ
carboxylate 6 (X = 2,2,2ꢀtrifluoroethyl, Y = pyridinꢀ3ꢀyl) by chiral SFC to afford the
individual enantiomers. Subsequent crystallographic analysis of the crystalline salt formed
2
4
ACS Paragon Plus Environment

Page 25 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
between the (–)ꢀenantiomer ([α]
D
= –58; 98% ee by chiral HPLC analysis) and Rꢀ(–)ꢀ2ꢀ
aminoꢀ1ꢀphenylethanol demonstrates the (3R,4R) absolute configuration for this
19
carboxylic acid (Figure 3). Liberation of (–)ꢀ6 (X = 2,2,2ꢀtrifluoroethyl, Y = pyridinꢀ3ꢀ
yl) from this salt and subsequent amide formation gave (–)ꢀ13i with antiꢀmalarial potency
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 7. Comparative potencies of
characteristic of the less potent
enantiomer (EC50 = 587 nM). The more
potent (+)ꢀenantiomers in this series must
therefore possess the (3S,4S) configuration.
From among the compounds described
above, 13i and 13h were selected for
consideration as candidates for preclinical
development based on their physical
properties and potency in vivo. Although
the two compounds compare favorably in
most respects, especially with respect to
1,2,3,4ꢀtetrahydroꢀ1ꢀisoquinoloneꢀ4ꢀ
carboxanilide enantiomers.
a
2
5
Cmpd.
3i
13i
EC50 (nM)
[α]
D
c = 1
1
ꢀ131
+147
ꢀ154
+152
ꢀ175
+159
587
36
13h
189
21
13h
1
1o
1o
477
15
1
a
Concentration causing 50% reduction in growth
of P falciparum 3D7 coꢀcultured with human
erythrocytes; values shown are from two
independent experiments done in triplicate.
potency against P. falciparum in
a
20
humanized mouse model, the decision
8
was made to pursue 13i for preclinical development. This choice is based primarily on the
observation
that,
while
13i
demonstrated excellent dose related
exposure in the rat, 13h did not (data
not shown).
CONCLUSION
In the continuing effort to identify
new antiꢀmalarial agents, highꢀ
Figure 3. ORTEP representation of (–)ꢀ6 (X =
2,2,2ꢀtrifluoroethyl, Y = pyridinꢀ3ꢀyl) as the
2
5
ACS Paragon Plus Environment
Rꢀ(–)ꢀ2ꢀaminoꢀ1ꢀphenylethanol salt.

Journal of Medicinal Chemistry
Page 26 of 53
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
throughput phenotypic screening has proven useful in identifying novel chemotypes with
the added potential for identifying agents with novel mechanisms of action. This
undertaking resulted in the identification of a series of 1,2,3,4ꢀtetrahydroꢀ1ꢀisoquinoloneꢀ
4ꢀcarboxanilide hits from a highꢀthroughput phenotypic screen against P. falciparum in
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
human erythrocytes. Hit validation studies confirm that the series possesses potent antiꢀ
malarial activity without measurable cytotoxic activity, and also exhibits potency against a
broad range of resistant parasite strains. In preliminary investigations, the compounds were
shown to possess very low aqueous solubility, and to be extensively metabolized with the
possibility of generating potentially toxic metabolites. SAR studies to address these
liabilities focused on the systematic modification of functionality appended to Nꢀ2 and Cꢀ3
of the ring system, and addressed the nature of the Cꢀ4 carboxamide. Studies indicated that
a Cꢀ4 carboxanilide in which electronꢀdonating groups are para to the amide nitrogen and
electronꢀwithdrawing groups are meta to the amide nitrogen appears to provide the more
potent analogues. Attempts to improve aqueous solubility by modification in this region of
the molecule failed. It was found, however, that incorporation of certain nitrogenꢀ
containing rings at Cꢀ3, such as pyridinꢀ3ꢀyl or pyrazolꢀ4ꢀyl, improved solubility while
maintaining good potency in vitro. Attempts to incorporate polar functionality into the Nꢀ2
substituent to improve aqueous solubility resulted in a significant loss of potency.
Additionally, the data indicated a rather strict structural requirement for the lipophilic Nꢀ2
substituent. It was also observed that alkyl groups at Nꢀ2 were subject to significant
metabolic oxidation. Ultimately, a compromise was reached with the incorporation of an
Nꢀ(2,2,2ꢀtrifluoroethyl) substituent, which mitigated metabolism without affecting aqueous
solubility and has only a minor impact on potency in vitro. The data show that both
enantiomers in this series retain measurable activity, with the (+)ꢀenantiomers being
significantly more potent than the (ꢀ)ꢀisomers of several analogues. Associated
2
6
ACS Paragon Plus Environment

Page 27 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
crystallographic studies demonstrate that the more potent (+)ꢀenantiomers possess the
(3S,4S) absolute configuration. Finally, 13i and 13h demonstrate improved antiꢀmalarial
potency in vivo compared to analogues without either the Nꢀ(2,2,2ꢀtrifluoroethyl) group or
enhanced solubility characteristics. Taken together, these preliminary studies identified
13h and 13i as potential lead compounds, with (+)ꢀ13i ultimately being selected for
preclinical development.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 53
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
EXPERIMENTAL SECTION
Animal Studies Statement. All animal studies carried out to support this work were
executed under approved protocols coverned by the respective Institutional Animal Care
and Use Committees (IACUC).
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
General Procedure for the Preparation of Aldimines (3). A solution of the 1 equiv
of the aldehyde (1) and 1 equiv of the amine (2) in methanol (5 mL per mmol of aldehyde)
was treated with a few drops of glacial acid acid, and the resulting mixture was stirred at
25 °C for 4 h. The solution was concentrated, and the crude aldimine (3), containing no
1
more than a trace of aldehyde according to H NMR analysis, was used for the next step
without further purification. An alternate method was employed to prepare aldimine 3
from 2,2,2ꢀtrifluoroethyl amine. Generally, a slurry of aldehyde 1 and 2,2,2ꢀtrifluoroethyl
amine hydrochloride (1.5 equiv) in toluene (2 mmole aldehyde per mL toluene) was
cooled in an iceꢀbath and treated with 1.5 equiv of 50% aqueous sodium hydroxide. The
mixture was then allowed to warm to ambiant temperature and stirred for 12ꢀ16 hours.
After separation of the phases, the aqueous phase was extracted several time with small
amounts of toluene and the combined organic phases were dried over sodium sulfate and
concentrated to yield the desired aldimine 3 (X = 2,2,2ꢀtrifluoroethyl) in near quantative
yield of sufficient purity to be used without further purification.
General Procedure for the Reaction of Homophthalic Anhydride with Imines. A
solution of 1 equiv of aldimine 3 and 1 equiv of homophthalic anhydride (4) in glacial
acetic acid (2 mL per mmol of aldimine) was heated at reflux for 16 h. The solution was
concentrated, and the residue was (usually) crystallized from ethyl acetate to provide the
transꢀ1,2,3,4ꢀtetrahydroꢀ1ꢀisoquinoloneꢀ4ꢀcarboxylic acid product, 6. In some cases, the
product required chromatographic purification on silica with ethyl acetate / methanol as
2
8
ACS Paragon Plus Environment

Page 29 of 53
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the eluant.
General Procedures for Amide Coupling. Method A: A solution of 1 equiv of the
1,2,3,4ꢀtetrahydroꢀ1ꢀisoquinoloneꢀ4ꢀcarboxylic acid (6), 1.2 equiv of the amine (7), and 2
equiv of triethylamine in dichloromethane (5 mL per mmol of carboxylic acid) was stirred
at 0 °C. Propaneꢀ1ꢀphosphonic acid anhydride (T3P, 2.5 equiv) was added, and the
reaction mixture was allowed to warm to room temperature and stir for 16 h. The reaction
mixture was partitioned between dichloromethane and saturated aqueous sodium
bicarbonate, and the organic layer was washed sequentially with water and brine, and then
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
was dried over anhydrous sodium sulfate. In cases where H NMR analysis indicated the
presence of some cis amide formed during the reaction, the crude product mixture was
treated for 15 min with 0.5 mL of 1 N potassium carbonate and 2 mL of methanol per
mmol of starting carboxylic acid. This mixture was concentrated, diluted with water, and
then extracted with dichloromethane. Concentration and then chromatography on silica
with ethyl acetate/petroleum ether as the eluant afforded the trans amide, 8. LC analysis of
the amide products indicated >95% purity in each case.
Method B: Phosphoryl chloride (1.1 equiv) was added to a solution of carboxylic acid
6
and 1.5 equiv of amine 7. The resulting mixture was heated to reflux for 3 hr, cooled to
room temperature and quenched with iceꢀwater. Standard extractive workꢀup followed by
chromatographic purification as described in Method A afforded 60ꢀ95% yields of the
desired transꢀamides 8.
(3
S*,4
S
*)-2-iso-butyl-1-oxo-3-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic
21
acid (6, X = iꢀBu, Y = phenyl). The known aldimine,
(E)ꢀNꢀisoꢀbutylꢀ1ꢀ
phenylmethanimine, was prepared by the general procedure. A solution of the aldimine
1.3 g, 8.0 mmol) and homophthalic anhydride (1.3 g, 8.0 mmol, 1 equiv) in acetic acid
(19 mL) was heated at reflux for 16 h, and worked up according to the general procedure.
(
2
9
ACS Paragon Plus Environment