A Cinchona Alkaloid Antibiotic That Appears to Target ATP Synthase in Streptococcus pneumoniae

Article abstract of DOI:10.1021/acs.jmedchem.8b01353

Optochin, a cinchona alkaloid derivative discovered over 100 years ago, possesses highly selective antibacterial activity toward Streptococcus pneumoniae. Pneumococcal disease remains the leading source of bacterial pneumonia and meningitis worldwide. The structure-activity relationships of optochin were examined through modification to both the quinoline and quinuclidine subunits, which led to the identification of analogue 48 with substantially improved activity. Resistance and molecular modeling studies indicate that 48 likely binds to the c-ring of ATP synthase near the conserved glutamate 52 ion-binding site, while mechanistic studies demonstrated that 48 causes cytoplasmic acidification. Initial pharmacokinetic and drug metabolism analyses of optochin and 48 revealed limitations of these quinine analogues, which were rapidly cleared, resulting in poor in vivo exposure through hydroxylation pendants to the quinuclidine and O-dealkylation of the quinoline. Collectively, the results provide a foundation to advance 48 and highlight ATP synthase as a promising target for antibiotic development.

Full text of DOI:10.1021/acs.jmedchem.8b01353

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Article
A Cinchona Alkaloid Antibiotic that Appears to
Target ATP Synthase in Streptococcus pneumoniae
Xu Wang, Yuna Zeng, Li Sheng, Peter Larson, Xue Liu, Xiaowen Zou, Shufang Wang, Kaijing Guo, Chen
Ma, Gang Zhang, Huaqing Cui, David M. Ferguson, Yan Li, Jing-Ren Zhang, and Courtney C. Aldrich
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01353 • Publication Date (Web): 19 Feb 2019
Downloaded from http://pubs.acs.org on February 19, 2019
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A Cinchona Alkaloid Antibiotic that Appears to Target ATP Synthase in
Streptococcus pneumoniae
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a†
b†
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d
b
c
a
e
Xu Wang, Yuna Zeng, Li Sheng, Peter Larson, Xue Liu, Xiaowen Zou, Shufang Wang, Kaijing Guo,
e
a
a
d
c
b
Chen Ma, Gang Zhang, Huaqing Cui, David M. Ferguson, Yan Li, Jingren Zhang, * Courtney C.
Aldricha,d*
aDepartment of Synthetic Medicinal Chemistry, Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
bCenter for Infectious Disease Research, School of Medicine, Tsinghua University, Beijing 100084, People’s
Republic of China
cDepartment of Drug Metabolism, Institute of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100050, People’s Republic of China
dDepartment of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
eDepartment of Pharmaceutical Analysis, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of
China
†These authors contributed equally
*
Corresponding author’s email address:
*(
C.C.A.) E-mail: ccaldrich@imm.ac.cn and aldri015@umn.edu Phone: +86 17319037727.
*(J.Z.) E-mail: zhanglab@tsinghua.edu.cn Phone: +86 10 62795892.
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Abstract
Optochin, a cinchona alkaloid derivative discovered over 100 years ago, possesses highly selective
antibacterial activity towards Streptococcus pneumoniae. Pneumococcal disease remains the leading
source of bacterial pneumonia and meningitis worldwide. The structure activity relationships of
optochin were examined through modification to both the quinoline and quinuclidine subunits, which
led to the identification of analogue 48 with substantially improved activity. Resistance and molecular
modeling studies indicate 48 likely binds to the c-ring of ATP synthase near the conserved glutamate 52
ion binding site while mechanistic studies demonstrated 48 causes cytoplasmic acidification. Initial
pharmacokinetic and drug metabolism analysis of optochin and 48 revealed limitations of these quinine
analogues, which were rapidly cleared resulting in poor in vivo exposure through hydroxylation pendant
to the quinuclidine and O-dealklyation of the quinoline. Collectively, the results provide a foundation to
advance 48 and highlight ATP synthase as a promising target for antibiotic development.
Keywords: Streptococcus pneumoniae, pneumococcal disease, antibiotic, cinchona alkaloid, quinine,
optochin, ATP synthase.
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Introduction
Pneumococcal disease caused by the gram-positive bacterium Streptococcus pneumoniae remains
the leading source of bacterial pneumonia and meningitis worldwide. Additionally, S. pneumoniae is
responsible for bacteremia and many common upper respiratory tract infections including sinusitis and
otitis media.1,2 The pneumococcal conjugate vaccine PCV13 provides immunity to thirteen of the most
prevalent S. pneumoniae serotypes and has significantly reduced the global burden of pneumococcal
disease. The wide deployment of PCV13 and the heptavalent predecessor PCV7 has led to a
corresponding increase in drug-resistant S. pneumoniae (DRSP), thus complicating therapy for
individuals that are either unvaccinated or infected with any of the more than eighty serotypes not
covered by these vaccines.3-5 Narrow spectrum agents effective against DRSP are therefore desirable
and could complement existing vaccination programs to decolonize asymptomatic carriers. The concept
of narrow spectrum antibacterial agents, defined as active against a single or related species, is gaining
traction due to advances in rapid diagnostics in clinical microbiology labs coupled with the importance
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of the human microbiome in maintaining health and preventing disease. Moreover, narrow spectrum
agents are expected to have a smaller impact on human and environmental resistome, which serve as a
reservoir for transmissible resistance elements.7
8
The antibiotic optochin 1 (Figure 1A), a cinchona alkaloid derivative, possesses highly selective
pneumococcal activity and was also shown to effectively treat septicemia in animals and humans.9-11
However, development of optochin was discontinued because of toxicity observed in humans with
incidence of optical disturbances in up to 5% of patients given ~25 mg/kg/day and moderate activity in
treating lobar pneumonia, the most prevalent infection caused by S. pneumoniae.10 Clinical experience
indicated optochin appeared to be effective in the early course of pneumonia infections, but that S.
pneumoniae became “optochin-fast” or resistant during treatment, likely owing to sub-optimal exposure
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due to lack of pharmacokinetic (PK) and pharmacodynamic (PD) information and inabilty to administer
higher doses to compensate for the presumptive low lung exposure.10 It should be highlighted that all of
this aforementioned work was done over a century ago or more than three decades before penicillin
revolutionized the treatment of bacterial infections. The analytical tools available to perform proper
PK/PD studies were simply not available, the word “antibiotic” did not yet exist in the scientific lexicon,
and the concept of drug resistance was inchoate. Optochin, thus remains largely forgotten in the history
of antibiotics, but could be considered the very first antibiotic discovered if one defines an antibiotic as a
natural product or derivative thereof with antibacterial activity that is not a general cytotoxin.12,13
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A
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B
O
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6'
H
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OH
1, Optochin
N
OH
Br
N
O
2, Bedaquiline
Figure 1. A) Structures of optochin (1) and bedaquiline (2). The numbering of optochin is based on the
convention used for quinine. B) ATP synthase is a nanoscale rotary motor composed of two multimeric
subunits (the membrane bound F subunit and F subunit), which transforms proton motive force into
o
1
rotational energy that is used to phosphorylate ADP. The F subunit is a cylinder with a central pore
o
(the c-ring) comprised of multiple c-subunits (10-14 subunits in S. pneumoniae).
Given optochins remarkable selective antibacterial activity, it has been used in clinical microbiology
laboratories as an important diagnostic reagent for distinguishing pneumococcal strains from other
alpha-hemolytic streptococci for more than seventy years.14 The mechanism of action (MOA) of
optochin has been inferred through genetic studies that show optochin-resistance maps to atpE, which
encodes for the c-ring of ATP synthase (Figure 1B). ATP synthase is a multimeric complex and plays a
central role in energy metabolism by synthesizing a majority of the ATP in most bacteria.15 ATP
1
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synthase is essential in S. pneumoniae and has been chemically validated as a new antibacterial target
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with the recent FDA approval of bedaquiline 2, a diarylquinoline drug that selectively blocks ATP
production in Mycobacterium tuberculosis (Mtb) by coincidentally also binding to the c-ring of ATP
synthase.17 Bedaquiline achieves an extraordinary 10,000-fold selectivity for the mycobacterial ATP
synthase over human mitochondrial ATP synthase and other bacterial homologues demonstrating it is
possible to achieve high species selectivity with this new molecular target. Interestingly, ATP synthase
was recognized as the likely target of optochin nearly 10 years before the first report of bedaquiline, yet
none of the published work on bedaquiline or any derivative has mentioned this prior precedent.18
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Herein we have examined the structure–activity relationships (SAR) of optochin that govern anti-
pneumococcal activity and toxicity through the synthesis of a systematic series of analogues. Based on
the recent revelations into optochin’s MOA, we hypothesized the observed adverse effects in animals
and humans may have been caused by mechanism-based toxicity due to inhibition of human
mitochondrial ATP synthase. The high biochemical selectivity attained with bedaquiline suggests the
feasibility of separating potency and toxicity of ATP synthase inhibitors. Moreover, the 4-[(quinuclidin-
2
-yl)hydroxymethyl]quinoline scaffold of optochin has superior physicochemical properties (clogP 3.1,
MW 340) compared to bedquiline (clogP 7.3, MW 556) and thus represents a more attractive molecular
scaffold for development of new ATP synthase inhibitors.17
RESULTS
Chemistry. We began by exploring the SAR at C-6 of optochin (see Figure 1 for numbering of
optochin). Morgenroth and Levy reported in 1911 that an ethyl substituent was optimal and indicated
many homologues were less active; although information on many of the actual analogues prepared as
well as chemical and microbiological characterization by contemporary standards were not
provided.8,19,20 We therefore synthesized a systematic series of analogues at the C-6 position as shown
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in Scheme 1. Catalytic hydrogenation of quinine 3 afforded dihydroquinine 4 in 97% yield.21
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Demethylation was accomplished employing sodium thioethoxide in DMF at 100 °C to furnish phenol
5.22, 23 This nucleophilic cleavage condition was preferred over more traditional methods using Lewis
acids or potent electrophiles because of the nucleophilicity of the quinuclidine nitrogen atom. The
desired compounds were prepared by phenol alkylation of 5 with a variety of primary and secondary
alkyl bromides and iodides using cesium carbonate in DMF to afford optochin 1 and 6–14 in yields
ranging from 9–87%.24
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Scheme 1. Synthesis of C-6 analogues.
OMe
OMe
H
N
H
N
H2, Pd/C
EtOAc
EtSH, NaH
OH
OH
DMF
100 ºC, 13 h
79%
23 ºC, 3d
N
N
97%
3, Quinine
4
1
6
7
8
, 63%, R = Et
, 9%, R = CH2CF3
, 83%, R = n-Pr
, 81%, R = i-Pr
OH
H
OR
N
R-X, DMF
Cs2CO3
H
N
9, 68%, R = allyl
10, 87%, R = n-Bu
11, 63%, R = CH2c-Pr
R =
OH
OH
23 ºC, 3 h
1
1
2, 68%, R = c-Bu
3, 84%, R = c-Pen
N
N
5
1, 6–14
14, 83%, R = Bn
The vinyl group at C-3 of quinine provides another convenient synthetic handle to introduce
diversity, thus we next explored modification at this position. Ruthenium-catalyzed isomerization of the
vinyl moiety of quinine 3 provided exocyclic olefin 15 as an inseparable 3:1 mixture of geometric
isomers (Scheme 2A).25 Oxidation of the exocyclic olefin in 15 was accomplished by a two-step
procedure employing osmium tetroxide catalyzed dihydroxylation followed by oxidative cleavage of the
resultant diol to furnish ketone 16 in 56% overall yield.26, 27 Condensation of 16 with hydroxylamine in
refluxing methanol provided oxime 17.28 A wide variety of other oxime derivatives, 18–30 were
prepared analogously by condensation with the appropriate alkoxyamines. Interestingly, n-propyl 21, i-
propyl 22, cyclopropylmethyl 27, and cyclopentyl 28 were isolated as single undefined geometric
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isomers, whereas all of the other analogues were obtained as approximately 2:1 mixtures of geometric
isomers. The synthesis of the corresponding alkoxyamines was conveniently performed by alkylation of
N-hydroxyphthalimide 31 with various alkylbromides using DBU in DMF to afford 32a–l (Scheme
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B).29 Hydrazinolysis of 32a–l liberated the alkoxyamines, which were precipitated to provide the
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desired alkyoxyamine building blocks 33a–l as the HCl salts.30
Scheme 2. Synthesis of C-3 oxime analogues.
R1
R2
A
1
) OsO4, K2CO3
K3[Fe(CN)6]
t-BuOH–H2O
OMe
N
OMe
N
H
N
H
N
23 ºC, 16 h
RuCl3• 3H2O, H2SO4
OH
OH
2
) NaIO4
EtOH, Reflux, 4h
83% (3:1 Z:E)
t-BuOH–H O
2
23 ºC, 16 h
(E)-15, R1 = Me, R2 = H
Z)-15, R1 = H, R2 = Me
3
, Quinine
55% (2 steps)
(
17, 81%, R = H
1
8, 85%, R = Me
O
N
OR
19, 84%, R = Et
0, 74%, R = CH2CF3
2
OMe
N
OMe
21, 84%, R = n-Pr
22, 65%, R = i-Pr
23, 95%, R = allyl
24, 84%, R = propargyl
H
N
H
N
RO-NH2• HCl
R =
OH
MeOH, reflux, 1h
OH
2
2
5, 88%, R = n-Bu
6, 76%, R = c-Bu
N
16
17–30
2
27, 92%, R = CH c-Pr
28, 93%, R = c-Pen
29, 81%, R = -(CH2)3E
30, 81%, R = Bn
where, E = CO2Me
B
O
O
a) NH2NH2• HCl
EtOH, reflux
R-Br, DBU
N
OH
N
OR
RO-NH • HCl
2
DMF
23 ºC, 5h
b) conc HCl
EtOH, 23 ºC
30 min
O
33a–l
O
32a-l
31
For the next set of analogues the secondary alcohol of quinine 3 was TBS protected using standard
3
1
conditions to afford 34 (Scheme 3). Lemieux-Johnson oxidation by sequential dihydroxylation to 35
and sodium periodate oxidation furnished an intermediate aldehyde.27 Interestingly, competitive
oxidation of the quinuclidine nitrogen was observed when 35 was not purified. Wittig olefination
proceeded to afford 36 smoothly at 0 °C without significant epimerization of the aldehyde at C-3 and
32
subsequent TBAF deprotection yielded 37. Subjection of the intermediate diol 35 to TBAF similarly
provided 38.2
7,31
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Scheme 3. Modification of the vinyl moiety at C-3.
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OH
HO
OMe
1
) NaIO4
OMe
N
OMe
OsO4, K2CO3,
K3[Fe(CN)6]
t-BuOH–H O
23 ºC, 3 h
2
H
N
H
N
H
N
t-BuOH–H2O
2) CH2=PPh3,
THF, 0 ºC, 12 h
53% (2 steps)
OR
OTBS
OTBS
23
ºC, 16 h
8%
N
N
8
35
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TBSCl
imid., DMF
00 ºC, 5 h
3
, R = H
TBAF, THF
r.t., 5 h
68%
TBAF, THF
r.t., 5 h
91%
1
34, R = TBS
99%
OH
HO
OMe
OMe
N
H
N
H
N
OH
OH
N
3
8
37
As a result of the enhanced activity and reduced toxicity observed with compound 15 containing an
exocyclic olefin at C-3 (vide infra) this scaffold was prioritized for further SAR studies. Compound 15
contains a methyl ether at C-6 of the quinoline and our SAR studies with the optochin series of
compounds indicates this position can further modulate potency, thus a systematic series of analogues
was synthesized as shown in Scheme 4. Demethylation of 15 employing sodium thioethoxide smoothly
23
afforded apo-cupreine 39, which was reacted with a series of alkyl bromides and alkyl iodides to
2
4
furnish 40–50 (Scheme 4A). To investigatie the importance of the stereochemistry at C-8 and C-9 of
1
5, quinidine 51 was converted to the diastereomeric analogue 52 (Scheme 4B).25
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2
3
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6
7
8
9
1
1
1
1
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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
Scheme 4. apo-Cupreine analogues.
A
4
4
0, 93%, R = Et
1, 7%, R = CH2CF3
Me
Me
42, 81%, R = n-Pr
3, 73%, R = i-Pr
OMe
OR
4
H
N
H
N
44, 87%, R = allyl
EtSH, NaH
DMF
45, 62%, R =
OH
OH
OH
100 ºC,13 h
N
N
87%
46
,
87%, R = n-Bu
47, 78%, R = CH2c-Pr
8, 79%, R = c-Bu
49, 89%, R = c-Pen
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
15
39, R = H
R-X, Cs2CO3
DMF, 23 ºC, 3 h
4
4
0-50, R =
5
0, 81%, R = Bn
B
Me
OMe
OMe
N
OH
RuCl3• 3H2O, H2SO4
OH
N
EtOH, Reflux, 4h
88% (d.r. 4:1)
N
N
H
H
51, Quinidine
52
As a last series of analogues, fluorination of the C-9 alcohol of quinine 3 was explored as a model
substrate. In the absence of base, quinine 3 reacted with diethylaminosulfur trifluoride (DAST) to give a
mixture of C-9 fluorinated diastereomers 53 and the rearranged fluorinated product 54 (Scheme 5A).
3
3
The identity of the rearranged product 54 was previously established by X-ray crystallography. In
contrast, in the presence of pyridine, a single C-9 fluorinated diastereomer 53 and eliminated product 55
were obtained.34 Using this method, we then synthesized the fluorinated products 56, 57, and 58
respectively from 5, 15 and quinidine derivative 52 with yield ranging from 36% to 50% (Scheme 5B).
Scheme 5. Fluorinated derivatives.
A
N
OMe
OMe
OMe
N
H
N
DAST, THF
-20 ºC, 14 h
+
OH
F
F
N
N
N
H
3
, Quinine
9R/9S-53, 51%
54, 11%
B
OMe
OMe
OMe
DAST, Py,
CH2Cl2
H
N
N
N
+
OH
0 ºC, 2 h
F
N
N
N
3
, Quinine
9S-53, 33%
55, 49%
R
R
56, 50%, R = Et, 8R, 9R
OMe
OMe
N
DAST, Py,
CH2Cl2
N
8
9
N
F
8
9


57, 38%, R = ethylidene, 8R, 9R


OH
0 ºC, 2 h
58, 36%, R = ethylidene, 8S, 9S
N
5, 15, 52
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Micobiology. All optochin analogues were initially evaluated against S. pneumoniae strain D39
according to CLSI guidelines as described in the Experimental Section to determine the minimum
inhibitory concentrations (MIC) that resulted in complete inhibition of observable growth and for
mammalian toxicity against the HepG2 cell line to determine the concentration which resulted in 50%
decrease of cell viability (CC ). Optochin and quinine were included as controls and the relative
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
5
0
potencies of the compounds discussed below are with respect to optochin. The SAR at C-6 shows an
ethyl substituent is preferred as the MIC is 2–4 µg/mL for optochin 1 whereas the methyl analogue 4
loses 16-fold in potency and the unsubstituted phenol is 32-fold less active. The 2,2,2-trifluoroethyl
analogue 6, n-propyl 7, iso-propyl 8 and allyl 9 are equipotent with MICs of 4 µg/mL demonstrating
slightly larger groups are also tolerated. However, substituents beyond three carbons begin to show loses
in potency with n-butyl 10 and cyclopropylmethyl 11 displaying 2-fold loses in potency and benzyl 14 a
more pronounced 8–16 decrease in potency. We observed that more compact cycloalkyl substituents
including cyclobutyl 12 and cyclopentyl 13 were equipotent to optochin. The cytotoxicity loosely
tracked with antibacterial activity wherein weakly active compounds possessing MICs greater than 32
µg/mL exhibited no apparent toxicity at 100 µM, the highest concentration evaluated. On the other hand
compounds, whose MICs were between 2–16 µg/mL displayed cytotoxicities (CC ) from 41 to 86 µM.
50
Cyclopentyl 13 and optochin 1 deviated from this trend and lacked observable cytotoxicity despite their
potent antibacterial activities.
1
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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 1. SAR at C-6.
Et
R
O
N
OH
N
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
MIC
μg/mL
CC50
μM
Compound
R
b
(µM)
Optochin (1)
Et
n.a.a
Me
H
2–4 (6–12)
128 (395)
32 (98)
64 (204)
4 (10)
>100
>100
>100
>100
72
Quinine (3)
4
5
6
7
8
9
n-Pr
i-Pr
4 (11)
57
4 (11)
86
allyl
n-Bu
4–8 (11–22)
8 (22)
78
1
0
41
11
8 (22)
87
1
2
2 (5)
51
13
2 (5)
>100
1
4
benzyl
16–32
(40~80)
76
a
b
not applicable, see structure in Scheme 1. Standard deviation was less than 10% of the mean value for all compounds.
The SAR of the quinuclidine moiety at C-3 was performed with the C-6 position fixed as a methyl
ether (Table 2). The MIC of quinine 3 containing a vinyl moiety at C-3 was 128 µg/mL. Saturation of
the vinyl moiety in analogue 4 provided a 4-fold increase in potency while isomerization of the double
bond to exocylic olefin 15 or addition of a methyl in propenyl 37 resulted in a striking 32-fold increase
in activity relative to quinine. The ketone 16 arising from oxidative cleavage of 15 and diol 38 derived
from dihydroxylation of vinyl group of 3 were both devoid of activity suggesting polar functional
groups at C-3 are poorly tolerated. The exocyclic olefin in 15 orients the side chain at a completely
different vector, which may explain its enhanced activity. Unfortunately, access to diverse alkylidene
analogues at this position were synthetically challenging, thus isosteric oxime derivatives that are also
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sp hybridized at C-3 were explored. The unsubstitued oxime 17 was inactive; however, introduction of
O-alkyl groups on the oxime led to enhancements in potency providing MICs of 16–32 µg/mL with
methyl 18, n-propyl 21, iso-propyl 22, cyclopropylmethyl 27, cyclobutyl 26, cyclopentyl 28, and benzyl
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
3
0 oxime analogues. The SAR is relatively flat in this series of compounds, but some outliers were
observed including ethyl 19 and 2,2,2-trifluoroethyl 20 oximes, whose MICs were ≥ 128 µg/mL while
the MICs of allyl 23 and propargyl 24 oximes were 64 µg/mL. The n-butyl oxime derivative 25
exhibited the most potent activity of all oximes with an MIC of 8–16 µg/mL or 8-16-fold greater than
the parent compound quinine 3. The butyryl oxime 29 containing a terminal methyl ester in the carbon
chain abrogated the benefits found oxime butyl ether 25 shifting the MIC to 128 µg/mL. Overall,
compounds 15 containing an exocyclic olefin and 37 emerged as the most potent analouge from this
series with an MIC of 4 µg/mL followed by oxime butyl ether 25 with an MIC of 8–16 µg/mL. None of
the compounds except allyl 23 displayed any cytotoxicity at 100 µM, the highest concentration
evaluated.
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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 2. SAR at C-3.
R
OH
MeO
N
MIC
μg/mL
MIC
μg/mL
(µM)
CC50
CC50
(μM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound
R
Compound
R
(μM)
(µM)
128
32
(86)
Quinine (3)
>100
>100
>100
>100
>100
>100
>100
21
>100
(395)
3
2
16–32
4
22
23
24
25
26
27
>100
(98)
(43–86)
4
64
(174)
51a
1
5
(12)
4
6
4
3
1
3
7
6
8
>100
>100
>100
>100
(175)
(12)
>128
>410)
8–16
(
(
(
(21~42)
>
128
32
(84)
>357)
>
128
16–32
17
>391)
(42–84)
16–32
375–750)
16–32
(40–81)
1
8
>100
28
>100
(
128
128
(299)
1
2
9
0
>100
>100
29
30
>100
>100
(360)
>
128
32
(77)
(
>313)
aStandard deviation was less than 10% of the mean value.
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The promising activity of 15 combined with the ease of synthesis prompted optimization of the
substituent at C-6 because our SAR studies on the optochin scaffold indicated this position allows
further tuning of potency. The simple unsubstituted compound 39 containing a free phenol lost 4-fold in
potency relative to 15 while the ethyl analogue 40 realized a substantial 8-fold increase in activity
providing an MIC of 0.5 µM (Table 3). The iso-propyl analogue 43 was equipotent to 40, but most other
small substituents led to a slight loss of activity with MICs ranging from 2–4 µg/mL as observed with
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
2
,2,2-trifluoroethyl 41, n-propyl 42, allyl 44, 1-hydroxypropan-2-yl 45, n-butyl 46, cyclopropylmethyl
7, and cyclopentyl 49. However, cyclobutyl 48 broke this trend and yielded the most potent optochin
4
analogue yet described with an MIC of 0.25 µg/mL. Given the potency of 48, we sought to determine if
activity resided in one of the geometric isomers and thus separated the mixture by preparative reverse-
phase HPLC and assigned each isomer by NOE studies. Thus, irradiation of the C-2 methylene
of the major isomer led to an enhancement of the C-11 methyl group while irradiation
of the C-10 vinyl proton led to enhancement of the C-4 methine group, securing the
structural assignment as Z (note: the methyl group is in the plane with N-1, C-2, C-4
and directed towards the C-2 methylene) The minor isomer was assigned as E in a
similar manner. The E-isomer conferred optimal activity and was 2-fold more potent than (Z)-48.
Analogues with larger substituents progressively lost activity as illustrated by benzyl 50, whose MIC
increased to 16 µg/mL. The overall trends for this compounds series paralleled the SAR of our initial
optochin analogues (Table 1) with small alkyl substitutents between 2–5 carbons being preferred with
significant reductions in potency as the chain length decreased or increased beyond this narrow range.
Interestingly, the cytotoxicity did not track with antibacterial activity and several compounds including
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
9–41, 45, and 47 displayed no cytotoxicity while the remaining analogues exhibited modest
cytotoxicity with CC values ranging from 27 to 65 µM.
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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Table 3. SAR at C-6 of apo-cupreine analogues.
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
Me
R
O
N
OH
N
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
MIC
μg/mL
CC50a
Compound
39
R
(μM)
(µM)
H
Me
Et
16 (52)
4 (12)
>100
>100
>100
>100
51
1
5
40
0.5 (1)
2 (5)
4
1
42
n-Pr
i-Pr
1–2 (3–6)
0.5 (1)
2 (4)
4
3
54
44
allyl
54
4
5
2 (5)
>100
27
46
n-Bu
4 (11)
4
7
2 (5)
>100
56
48
0.25 (0.7)
2 (5)
4
9
48
50
benzyl
16 (40)
0.25 (0.7)
0.5 (1.4)
47
(E)-48
65
(Z)-48
59
bStandard deviation was less than 10% of the mean value for all compounds.
Our last series of analogues explored the importance of the stereochemical requirements of C-8 and
C-9 along with the role of the alcohol at C-9 (Table 4). Epimerization of the stereocenters at C-8 and C-
9
in 52 obliterated activity demonstratng a strict requirement for the native 8S, 9R-stereochemistry.
Bioisosteric replacement of the 9-hydroxy with a fluorine atom in the 9S-configuration with
dihydroquinine or apo-cupreine derivatives 57 and 58 ablated activity illustrating the importance of the
alcohol at this position. Finally, we obtained a small amount of a rearranged scaffold
azabicyclo[3.2.2]nonane 54 during our attempts to fluorinate the C-9 position of the model substrate
quinine. Intiguingly, this compound was equipotent to quinine suggesting 54 could be a viable scaffold
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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
for future medicinal chemistry efforts through appropriate modification at C-6 and reduction of the
olefin. We also tested 56 obtained during our studies, but this was inactive consistent with the SAR
trends observed in the previously described series of compounds. Taken together, our results indicate the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
S, 9R-stereochemistry of 48 is required for activity, the C-9 alcohol is also essential, and fluorination at
C-9 is poorly tolerated. None of these derivatives displayed any cytotoxicity at the highest concentration
evaluated (CC > 100 µM).
50
Table 4. SAR at C-8 and C-9 positions.
R'
O
R
N
MIC
CC50
Compound
52
R
R'
μg/mL
(μM)
(µM)
Me
>
128
Me
>100
N
(>395)
HO
H
H
N
1
28
5
4
Me
Me
>100
>100
F
(392)
N
>
128
56
F
(
>390)
H
Me
1
28
5
5
7
8
Me
Me
N
>100
>100
(392)
F
H
Me
>
128
N
(
>392)
F
H
The minimum bactericidal concentration (MBC) of selected compounds was evaluated against S.
pneumoniae D37 is shown in Table 5. Compound 48 demonstrated potent bactericidal activity with an
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MBC of 1-2 µg/mL followed closely by optochin 1, whose MBC was 4 µg/mL. The kinetics of bacterial
killing were assessed in vitro using time-kill assays by incubating 48 with S. pneumoniae at 1, 5 and
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
5
0 the MIC. However, a significant decrease in the number of bacteria as measured by OD620 or plating
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to determine the residual colony forming units (CFU) These experiments revealed 48 was bacteriostatic
during the first 18 hours (Figure 2). Experiments with higher concentrations of antibiotic (up to 1000
7
the MIC) as well as with a lower initial inoculum (10 CFU) yielded the same outcome. Vancomycin
was used as a positive control and displayed rapid bactericidal activity reducing the initial inoculum by
about 1,000-fold units within 3 hours at 5 the MIC. Attempts to extend the duration of the experiments
were confounded by well known autolytic behavior of S. pneumoniae that occurs in vitro as observed by
the decreased CFU of the untreated control after 5 hours (data not shown).
Figure 2. In vitro time-kill analysis of S. pneumoniae. Bacterial killing was monitored by measuring the
optical density at 620 nm (OD ) up to 18 hours. Four independent time-kill experiments were
6
20
performed, and one representative experiment is shown. The CFU at each time point was independently
determined by plating and the results paralleled the OD620 measurements. Compound 48 was evaluated
as a E/Z mixture.
Table 5. MBC of 1, 39, and 48 (E/Z mixture).
MIC
μg/mL)
MBC
(μg/mL)
Compound
(
Optochin (1)
2–4
16
4
apo-Cupreine (39)
16
4
8
0.25–0.5
1–2
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S. pnuemoniae mutants that were resistant to 48 were isolated with a frequency of ~1 in 10 CFU
when 48 was present at a concentration of 4 MIC (Figure 3). This frequency decreased to ~2 in 107
8
CFU at 8 MIC and to ~1 in 10 CFU at 16 MIC. Mutants resistant to optochin (1) were recovered at a
0
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0
similar frequency. For rifampicin used as positive control, we observed a frequency of resistance of
7
8
about 2 in 10 at 4 MIC, which decreased to 4 in 10 at 8 and 16 MIC.
Figure 3. Frequency of the emergence of spontaneous resistance in S. pneumoniae D39 on standard
solid medium in the presence of 48 (E:Z mixture) at concentrations 4, 8, and 16 the MIC (1, 2, and 4
µg/mL). Optochin 1 (8, 16, and 32 µg/mL) and rifampicin (0.125, 0.25, and 0.5 µg/mL) were used as
controls. Data were obtained twice independently with two to three replicates (except for the data for
optochin at 16 MIC, which was a single experiment with no recoverable colonies). The mean value is
8
given for platting 10 CFU and the error bars represent the standard error of the mean (SEM). Error bars
for compound 48 at 4 the MIC are not shown because only two data points were obtained (120 and 138
colonies were recovered). The individual data points are provided in Table S1 in the Supporting
Information.
We next evaluated 48 along with apo-cupreine 39 and optochin 1 against a panel of other S.
pneumoniae strains including drug-sensitive, multidrug resistant, and optochin-resistant strains (Table 6).
Compound 48 maintained fairly uniform activity against four S. pneumoniae multidrug resistant strains
including serotypes 6B, 19A, and 19F (Table 6, entries 3–6) with MICs ranging from 0.25–1 µg/mL.
Interestinly, apo-cupreine 39 that also contains the C-3 exocyclic olefin found in 48 lost considerable
activity against several of drug-resistant strains (Table 6, entries 4–5). We also generated two optochin-
resistant strains through point mutations (G142C and G145A) of atpE in S. pneumoniae strain R6 using
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an efficient PCR-based approach based on previously described resistant studies (Table 6, entries 7–
).18,35 The MIC values of optochin 1 and 48 were each shifted 16–32-fold confirming AtpE as a likely
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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2
2
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3
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3
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4
4
4
4
4
4
4
4
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5
6
8
molecular target of these compounds.
0
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2
3
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5
6
7
8
9
0
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2
3
4
5
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7
8
9
0
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8
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0
1
2
3
4
5
6
7
8
9
0
Table 6. Inhibitory activity against resistant S. pneumoniae.
MIC, μg/mL
Entry
Strain
Resistancea
Optochin (1)
apo-Cupreine (39)
48 (E:Z mixture)
1
2
3
4
5
6
7
8
D39
DS
2–4
2
8–16
n.d.
4
0.25–0.5
0.5
R6
DS
TH2784 (19F*)
TH2582 (19A)
TH2889 (6B)
APCET
APChEMC
APEErMoC
PEMT
2
0.5
2
64
0.25–0.5
0.5
2
128
n.d.
n.d.
n.d.
TH2863 (19F)
4–8
64
16
0.5–1
8–16
4
R6-atpEG142C
Optochin
R6-atpEG145A
Optochin
aAbbreviation: n.d.: not determined, DS: Drug Sensitive, A: Amoxicillin, P: Penicillin, C: Cefotaxime, E: Erythromycin, T: Tetracycline,
Ch: Chloramphenicol, M: Meropenem, Er: Ertapenem, Mo: Moxifloxacin.
To further validate atpE as the target, we sequenced the atpE gene of 29 mutants from the resistance
tests discussed in Figure 2 isolated at 4, 8, and 16 MIC of 48. Indeed all of the mutants contained
missense mutations in atpE as shown in Table 7. Interestingly, at 4 the MIC, all mutations (10/10)
mapped to G18S within the N-terminal helix of AtpE while at 8 the MIC, the mutations were found at
different positions (A31V and A49S). We also sequenced five mutants from multiple experiments
recovered at 16 the MIC of 48 and mutations mapped to F50L and F45C within the C-terminal helix of
AtpE. For comparison, 15 mutants resistant to optochin (1) were sequenced and mutations mapped to
similar positions (Table 7).
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6
Table 7. Mutational substitutions in S. pneumoniae atpE.
Amino
acid
sequence
DNA
sequence
# of
clones
Compounda
4
4
8 (4 MIC)
8 (8 MIC)
G52A
C92T
G18S
A31V
A49S
F50L
F45C
G18S
A49S
G14S
N24K
10
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
2
3
4
5
6
7
8
9
0
C145T
T148C
T134G
G52A
G145T
G49A
T72A
6
4
4
8 (16 MIC)
1
7
Optochin (1),
4 MIC)
(
1
5
Optochin (1),
8 MIC)
(
2
aThe value in parentheses, i.e. (4 MIC), refers to the concentration of compound used to select the isolated mutant.
Compound 48 was used as an E:Z mixture.
We hypothesized ATP synthase in S. pneumoniae is involved in pH homeostatsis as observed in other
fermentive microorganisms by operating in the reverse direction to regulate the intracellular pH (pHIB)
by consuming ATP.36-38 To explore the physiological consequences of inhibition of S. pneumoniae ATP
synthase, we determined the pH of strain D39 and two isogenic optochin-resistant mutants (D39-
IB
atpEG142C and D39-atpEG145A) using a pH-sensitive ratiometric GFP biosensor on a plasmid that allowed
non-invasive measurements of pHIB.3
9,40
The bacterial cells in medium at pH 7.2 were treated with 48,
DMSO only (negative control), or the proton uncoupler carbonyl cyanide-m-chlorophenylhydrazone
(CCCP) (positive control) and the pH was measured at 5 minute intervals (Figure 4).
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9
0
Figure 4. The impact of compound 48 treatment on intracellular pH of S. pneumoniae. D39 WT,
D39 atpEG142C, D39 atpEG145A were treated with vehicle control DMSO (0.5% v/v), compound 48, or
CCCP (10 µM) in PBS pH7.2 buffer supplemented with 10 mM glucose. pH measurements immediately
and at 5-min intervals after treatment were shown in A, B, C and D, respectively. Experiments were
performed two times independently in triplicate. Representative data from one independent experiment
is shown with mean ± standard deviation.
The basal pH of wild-type S. pneumoniae treated with DMSO was 7.34 ± 0.04. Treatment with 48
IB
resulted in a time and concentration-dependent decrease in pH (Figure 4). For example, at 5 minutes,
IB
the pH decreased to 7.02 ± 0.03 with 0.0625 µg/mL 48 (0.25×MIC) and to 6.82 ± 0.003 with 0.25
IB
µg/mL 48 (1×MIC), but then quickly began to level off reaching 6.66 ± 0.01 at 8 µg/mL 48 (32×MIC)
(Figure 4B). The impact of 48 on pH was rapid, thus addition of 48 to wild-type S. pneumoniae at 0.25
IB
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µg/mL (1×MIC) immediately lowered the pH to 7.11 ± 0.003 (pH = -0.23 compared to the DMSO
only control at the same time point), then to 6.82 ± 0.003 (pH = -0.47) at 5 minutes, followed by a
more gradual decline to 6.68 ± 0.01 (pH = -0.54) at 10 minutes that remained essentially unchanged at
0
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8
9
0
1
5 minutes (pH = -0.52) (Figures 4A-D). Addition of DMSO only (negative control) did not impact
pH whereas addition of 10 µM of the proton uncoupler CCCP (positive control) caused an immediate
IB
reduction of the intracellular pH to 5.43 ± 0.07 (pH = -1.9) that persisted for the duration of the
experiment. To provide support for the selectivity of this process, we also evaluated two isogenic S.
pneumoniae mutant strains D39-atpEG145A and D39-atpEG142C whose MICs of 2 and 8 µg/mL are shifted
8
and 32-fold, respectively. Treatment of the more resistant strain D39-atpEG142C with 0.25 µg/mL 48,
which is the MIC of the wild-type strain, had essentially no impact on pH at any time point (pH ≤
IB
0
.03). The mutant strain D39-atpEG142C did undergo cytoplasmic acidification with 48, but only at
substantially higher concentrations than required for the wild-type S. pneumoniae D39. The isogenic S.
pneumoniae mutant D39-atpEG145A that exhibits low-level resistance (MIC = 2 µg/ml) showed a
comparable time-dependent decrease in intracellular pH as the wild-type; but only at concentration of 48
equal to or greater than 2 µg/mL. Taken together these data show 48 results in mild cytoplasmic
acidification that is dependent on AtpE.
To confirm the microbiological selectivity, we evaluated 48 against a panel of gram positive and
negative organisms as well as a couple of other streptococcal species. As shown in Table 8, compound
4
8 was weakly active towards Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Enterobacter clocae
displaying MICs of ≥128 µg/mL. Compound 48 retained moderate activity against other pathogenic
streptococci with MICs of 32 µg/mL and 16 µg/mL against Streptococcus pyogenes and Streptococcus
mitis, respectively.
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Table 8. Susceptibility of gram positive and negative bacteria to 48 (E:Z mixture).
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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
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5
5
5
5
5
5
5
6
MIC
µg/mL)
Species
Strain
(
Staphylococcus aureus
Streptococcus pyogenes
Streptococcus mitis
TH4114
ST157
128
32
0
1
2
3
4
5
6
7
8
9
0
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2
3
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0
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2
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0
1
2
3
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8
9
0
ST138
16
Enterococcus faecium
Enterobacter clocaae
Escherichia coli
TH6064
TH11715
TH4746
ATCC13883
TH9781
TH4090
H37Rv
64–128
>128
>128
128
Klebsiella pneumoniae
Acinetobacter baumannii
Pseudomonas aeruginosa
Mycobacterium tuberculosis
128
>128
>256
The effect of four antibiotics (vancomycin, penicillin, levofloxacin and rifampicin) used to clinically
treat pneumococci was next examined in combination with 48 by checkboard assay with S. pneumoniae
D39. The fractional inhibition concentration indices (FICI) were calculated to assess drug interaction
between these compounds. The minimum FICI values for each combination ranged from 0.63 to 1.
m
indicating these antibiotics exhibit no interaction (FICI >1 to ≤ 4). Representative data from a single
experiment are shown in Figure 5.
A
1
.8
.6
B
1
0.8
0.6
0.4
0.2
0
C
1
0.8
0.6
0.4
0.2
0
0
0
0.4
0.2
0
0
0.2 0.4 0.6 0.8
8 (FIC)
1
0
0.2 0.4 0.6 0.8
1
0
0.2 0.4 0.6 0.8
48 (FIC)
1
4
48 (FIC)
Figure 5. Graphical representation of checkerboard assays used to determine drug interactions with 48
(E/Z mixture). Representative data is presented for 48 in combination with rifampicin (panel A),
penicillin (panel B), and levofloxacin (panel C). FICI , minimum value of FICI in the tested
m
combinations is 1.0 (panel A), 0.63 (panel B), and 1.0 (panel C). Representative data from at least
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two independent experiments are shown. FICI
m
, minimum value of FICI in the tested
combinations is shown. Synergy (FICI
m
ꢀ≤ꢀ0.5). No interaction (FICI ꢀ>ꢀ0.5–4). Three to four
m
independent checkerboard assays were performed, and one representative experiment is shown.
To assess the potential for mitochondrial toxicity of 48 caused by inhibition of ATP synthase, we
0
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performed cell viability assays employing glucose or galactose as the primary carbon sources following
4
1
the protocol described by Will and co-workers. Highly proliferative HepG2 cells have adapted their
metabolism under standard cell culture conditions to obtain ATP primarily from glycolysis and thus are
insensitive to inhibitors of ATP synthase. By contrast, metabolism of galactose does not generate ATP
by glycolysis; consequently, cells grown on galactose supplemented medium are highly sensitive to
inhibition of mitochondrial function because they are forced to rely on the mitochondrial oxidative
phosphorylation pathway to generate a proton motive force that in turn drives ATP synthase in the
mitochondrial membrane. The impact of optochin 1, apo-cupreine 39, and 48 on cell viability of HepG2
cells did not depend on carbon source (glucose or galactose) confirming these quinine derivatives do not
disrupt mitochondrial function (see Table 9 and a representative dose-response curve for 48 in Figure
6
A). By contrast, the positive control, oligomycin, a potent inhibitor of mammalian ATP synthase
inhibited cell viability more than 90% (CC ) at  0.3 µM in galactose medium. The CC value was
9
0
90
shifted by four orders of magnitude to 300 µM when grown in glucose-supplemented medium (Figure
B). The negative control tamoxifen, which does act on ATP synthase, displayed equipotent cytotoxicity
6
in both media (Figure 6C). Taken together, our results demonstrate that optochin 1 and derivatives 39
and 48 do not pertub mitochondrial function indicating these molecules likely do not inhibit mammalian
ATP synthase due to their intrinsic biochemical selectivity for the S. pneumoniae homologue.
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A
B
C
1
1
1
1
1
1
1
1
1
1
2
2
2
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2
2
2
2
3
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4
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0
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8
9
0
Figure 6. Dose-response curves for cell viabililty of HepG2 cells treated with compound 48 as an E:Z
mixture (panel A), oligomycin (panel B) and tamoxifen (panel C) for 24 hours using the CellTiter-
®
Glo luminescent cell viability assay. Cells were grown in DMEM medium either supplemented with 10
mM glucose (solid circles) or 10 mM galactose (solid squares).
Table 9. Mitochondrial Toxicity of HepG2 cells.a
CC90, +Glu
CC90, +Gal
Compound
(µM)
(µM)
Tamoxifen
Oligomycin
75
75
<0.3
300
300
150
300
300
Optochin (1)
Apo-Cupreine (39)
>300
4
8 (E/Z mixture)
150
aInhibition of 90% cell viability (IC90) measured against HepG2 cells grown in DMEM medium supplemented either with 10 mM glucose
+Glu) or 10 mM galactaose (+Gal)
(
Pharmacokinetics and Metabolism Studies. Each of the pure diasteromers Z-48 and E-48 along
with optochin 1 and apo-cupreine 39 were evaluated in single dose pharmacokinetic experiments using
male ICR mice administered as a bolus orally (p.o.) at 25 mg/kg and intravenously (i.v.) at 2.5 mg/kg.
The serum concentration-versus-time curves shown in Figure 7 were used to determine the
pharmacokinetic parameters (Table 10) by noncompartmental analysis. After intravenous (i.v.)
administration (2.5 mg/kg), all compounds exhibited biexponential kinetics with a rapid distribution
phase followed by a slow terminal elimination phase resulting in an extremely large steady state
volumes of distribution (VD ) ranging from 8.8 L/kg for E-48 to 20 L/kg for apo-cupreine 39 with
ss
optochin possessing an intermediate value of 12.7 L/kg. Each of the compounds also exhibited
–
1
–1
extremely high clearances (Cl) ranging from 172–274 mL·min ·kg , which indicates extra-hepatic
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6
–1
–1
metabolism since the clearance vastly exceeds mouse hepatic blood flow (90 mL·min ·kg ) suggesting
tissue oxidases may play a significant role in metabolism of these quinine derivatives. The high

clearance overrides the extensive tissue distribution resulting in short half-lives (t1/2 ) from 0.42 to 0.53
0
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hours for Z-48, E-48, and optochin 1. After oral administration of optochin 1, apo-cupreine 39, Z-48 and
E-48, the compounds reached peak plasma concentrations (Cmax) of 639, 619, 207 and 331 ng/mL. The
oral bioavailabilities (F) of optochin 1, apo-cupreine 39, Z-48 and E-48 calculated from the area-under-
the-curve (AUC) of the concentration-time plots shown in Figure 7 were 109%, 23%, 14% and 21%,
respectively. Based on the Cmax values, only E-48 exceeded its MIC when dosed orally. Even with its
impressive bioavailability optochin 1 was unable to approach its MIC. These preliminary PK studies
suggest the poor in vivo performance of optochin was likely caused by poor oral exposure as a result of
rapid clearance. While E-48 provides improved oral exposure as measured by the MIC/AUC ratio, it
also suffers from rapid clearance and low bioavailability.
Table 10. Pharmacokinetic parameters of 1, 39, Z-48, and E-48 after a single oral or i.v. administration
to male ICR mice.
Optochin (1)
apo-Cupreine (39)
Z-48
E-48
Parameters
Units
p.o.25
i.v.2.5
p.o.25
i.v.2.5
p.o.25
i.v.2.5
p.o.25
mg/kg
i.v.2.5
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
mg/kg
t
1/2β
h
h
1.00
0.5
639
1569
-
0.53
0.033
375
144
12.7
274
-
0.91
0.083
619
442
-
1.12
0.033
511
190
20.0
207
-
0.69
0.25
207
319
-
0.42
0.033
521
235
6.30
172
-
0.70
0.083
331
368
-
0.44
0.033
399
175
8.8
T
max
max
C
ng/mL
h×ng/mL
L/kg
AUC(0-t)
VDss
Cl
mL/min/kg
-
-
-
-
230
-
F%
109
23.3
13.6
21.0
2
7
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Page 29 of 93
Journal of Medicinal Chemistry
1
2
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9
p.o.
i.v.
9
7
6
00
50
00
700
Optochin
Apo-Cuperine
Z-48
6
5
4
3
00
00
00
00
1
1
1
1
1
1
1
1
1
1
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0
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0
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0
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0
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0
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0
E-48
450
3
1
00
50
0
200
1
00
0
0
2
4
6
8
0
1
2
3
4
Time (h)
Time (h)
Figure 7. Mean plasma concentration versus time profile after single p.o. (25 mg/kg) and i.v. (2.5 mg/kg)
bolus administration of 1, 39, Z-48, and E-48 to male ICR mice. Error bars represent standard deviation
of the mean (n = 3).
4
2
Based on the excellent pharmacokinetic behavior of quinine, we were surprised that the related
derivatives optochin (1), apo-cupreine 39, and 48, were so rapidly cleared. Given quinine primarily
undergoes hepatic metabolism, we incubated 1, 39, and Z-48 with ICR mouse liver microsomes (MLM)
and observed 87%, 89%, and 99.8% metabolism, respectively after 60 minutes. Further characterization
of the metabolites of Z-48 generated by MLMs using LC-MS/MS revealed the time-dependent
formation of seven metabolites (M1-M7) ranked in order of their relative abundance with M1-M3
accounting for 80% of the total abundance based on ion counts. The primary metabolite M1 was formed
by hydroxylation (+16 Da) pendant to the quinuclidine ring while M2 was formed by O-dealkylation of
the cyclobutyl group (-54 Da) and M3 resulted from hydroxylation of M2 on or pendant to the
quinuclidine ring (-54 Da + 16 Da). Further hydroxylation of M1 on the quinoline (+32 Da) provided
M4 and oxidation of M1 to a putative keto derivative (+16 Da – 2Da) yielded M7. Metabolism of M2
via glucuronidation afforded M5 (-54 Da + 176 Da) while oxidation of M2 furnished ketone M6 (-54 Da
–
2 Da). Putative structural assignments of M1-M7 are shown in Figure 8 based on accurate mass
2
8
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