Derivatives of Natural Product Agrimophol as Disruptors of Intrabacterial pH Homeostasis in Mycobacterium tuberculosis

Article abstract of DOI:10.1021/acsinfecdis.8b00325

This article reports the rational medicinal chemistry of a natural product, agrimophol (1), as a new disruptor of intrabacterial pH (pH_IB) homeostasis in Mycobacterium tuberculosis (Mtb). Through the systematic investigation of the structure-activity relationship of 1, scaffold-hopping of the diphenylmethane scaffold, pharmacophore displacement strategies, and studies of the structure-metabolism relationship, a new derivative 5a was achieved. Compound 5a showed 100-fold increased potency in the ability to reduce pH_IB to pH 6.0 and similarly improved mycobactericidal activity compared with 1 against both Mycobacterium bovis-BCG and Mtb. Compound 5a possessed improved metabolic stability in human liver microsomes and hepatocytes, lower cytotoxicity, higher selectivity index, and similar pK_a value to natural 1. This study introduces a novel scaffold to an old drug, resulting in improved mycobactericidal activity through decreasing pH_IB, and may contribute to the critical search for new agents to overcome drug resistance and persistence in the treatment of tuberculosis.

Full text of DOI:10.1021/acsinfecdis.8b00325

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Article
Derivatives of Natural Product Agrimophol as Disruptors of
Intrabacterial pH Homeostasis in Mycobacterium tuberculosis
Jie Wu, Ran Mu, Mingna Sun, NAN ZHAO, Miaomiao Pan, Hongshuang Li, Yi Dong,
Zhaogang Sun, Jie Bai, Minwan Hu, Carl F. Nathan, Babak Javid, and Gang Liu
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00325 • Publication Date (Web): 24 Apr 2019
Downloaded from http://pubs.acs.org on April 25, 2019
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Derivatives of Natural Product Agrimophol as Disruptors of
Intrabacterial pH Homeostasis in Mycobacterium tuberculosis
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Jie Wu a, #, Ran Mu b, #, Mingna Sun b, #, Nan Zhao , Miaomiao Pan , Hongshuang Li , Yi Dong
c
d
b
b, Zhaogang Sun , Jie Bai , Minwan Hu , Carl F. Nathan , Babak Javid , Gang Liu
e
f
f
c
d,*
a, b,*
a
b
c
d
Tsinghua-Peking Center for Life Sciences, Tsinghua University, Renhuan Building, Rm 311,
Haidian Dist., Beijing 100084, PR China
School of Pharmaceutical Sciences, Tsinghua University, Renhuan Building, Rm 311, Haidian
Dist., Beijing 100084, PR China
Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY
10065
Centre for Global Health and Infectious Diseases, Collaborative Innovation Centre for the
Diagnosis and Treatment of Infectious Diseases, Tsinghua University School of Medicine,
Beijing 100084, China
e
National Tuberculosis Clinical Laboratory, Beijing Chest Hospital, Capital Medical University,
and Beijing Key Laboratory in Drug Resistant Tuberculosis Research, Beijing Tuberculosis &
Thoracic Tumor Research Institute, 9 Beiguan Street, Tongzhou District, Beijing 101149,
China
f
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
College, 1 Xian Nong Tan Street, Beijing 100050, China
Corresponding Author: * G. L. E-mail: gangliu27@tsinghua.edu.cn and * B. J. E-mail:
bjavid@gmail.com
KEYWORDS: Agrimophol, Diphenylmethane scaffold, Mycobacterium tuberculosis,
Intrabacterial pH homeostasis, Coumarin, pharmacophores
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This article reports the rational medicinal chemistry of a natural product, agrimophol (1), as a
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new disruptor of intrabacterial pH (pH ) homeostasis in Mycobacterium tuberculosis (Mtb).
IB
Through the systematic investigation of the structure-activity relationship of 1, scaffold-hopping
of the diphenylmethane scaffold, pharmacophore displacement strategies, and studies of the
structure-metabolism relationship, a new derivative 5a was achieved. Compound 5a showed
1
00-fold increased potency in the ability to reduce pHIB to pH 4.5 and similarly improved
mycobactericidal activity compared with 1 against both Mycobacterium bovis-BCG and Mtb.
Compound 5a possessed improved metabolic stability in human liver microsomes and
hepatocytes, lower cytotoxicity, a higher selectivity index and a similar pKa value to natural 1.
This study introduces a novel scaffold to an old drug, resulting in improved mycobactericidal
activity through decreasing pH , and may contribute to the critical search for new agents to
IB
overcome drug resistance and persistence in the treatment of tuberculosis.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is an airborne infectious
disease that continues to represent the leading cause of death from a single pathogen, with 10.4
1
million new cases and 1.7 million deaths in 2016. An estimated one-quarter of the global
population is latently infected with Mtb. Meanwhile, increased incidence of drug-resistance to
first-line and second-line antitubercular antibiotics has necessitated the identification of new
targets and agents against Mtb.2,3
Upon aerosol infection, Mtb is phagocytosed by alveolar macrophages, the first line of lung
defense. One of the mechanisms that macrophages employ to destroy invading pathogens occurs
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via phagosomal maturation and fusion to lysosomes. Mtb is able to partially arrest these
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processes, although the phagosomal pH in interferon-γ-stimulated macrophages still falls to 4.5,
which is sufficient to cause growth arrest and multi-drug tolerance in Mtb.5,6 Although Mtb is
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able to resist acid stress and maintain its intrabacterial pH near neutrality via a transmembrane
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protease (i.e., Rv3671c) and potentially other mechanisms, these are still much less understood
than the acid-resistance mechanisms of enterobacteria.
To investigate the mechanisms involved in acid resistance in Mtb and to identify novel
compounds with activity against non-replicating Mtb, a whole-cell high throughput screening
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(
HTS) method was developed to identify compounds that interfere with Mtb pH homeostasis.
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Application of the HTS to a natural product library identified natural agrimophol (1) as a
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promising candidate (Figure 1A). Compound 1, bound to Rv3852, a membrane-anchored, DNA-
binding histone-like protein, was able to disrupt Mtb’s intracellular pH homeostasis and
1
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effectively kill the bacterium when it was exposed to acidic environments. Compound 1 has
been used in traditional Chinese medicine to treat helminthic and protozoal infections, with the
1
1
drawbacks of slow gastrointestinal absorption and low deworming rates. Here, we describe our
efforts to explore novel natural-like synthetic agrimophol analogs through scaffold-hopping,
hydrogen bond mimicking strategies and the integration of coumarin and phloroglucin
pharmacophores that can effectively target Mtb’s pH homeostasis and kill Mtb with superior
potency.
■
RESULTS AND DISCUSSION
Structure Activity Relationships (SARs) of Natural 1 and its Analogs. Natural products are
the main source of most antibiotics. The structural complexity of natural products imposes
challenges for chemical synthesis and derivatization during lead optimization, which leads to a
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high attrition rate during drug development. Natural agrimophol (1, Figure 1A) has a unique 6-
benzyl-6-methylcyclohexa-2,4-dienone scaffold and consists of two fully substituted cyclohexa-
2,4-dienone (A) and polyphenolic (B) rings, thus giving rise to four enantiomers with two chiral
carbon atoms. Due to stereoselective hindrance and lower electron density, the 6-methyl group of
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is extremely difficult to synthesize and the yield of synthetic agrimophol is very low. Studies
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of the physical properties of agrimophol using H nuclear magnetic resonance (NMR) and C
NMR revealed that natural 1 consists of an enantiomeric pair (6R, 2’S/6S, 2’R), whereas the
synthetic product 1 generated all four enantiomers: 6R, 2’S/6S, 2’R and 6R, 2’R/6S, 2’S (~2:2:1:1,
molar ratio). In addition, the results of single-crystal X-ray diffraction revealed that natural 1
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consists of four intramolecular hydrogen bonds (I, II, III and IV), which may account for its
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poor solubility and low intestinal absorption (Figure 1A). Comparing the activity of natural and
synthetic 1 in disrupting the pH of Mtb in vitro, natural 1 was approximately two-fold more
IB
potent than synthetic 1 (Figure 1B). These data suggested that the 6R, 2’R/6S, 2’S enantiomers of
synthetic 1, which are not present in natural 1, do not contribute to its activity in disrupting pHIB.
Natural polyphenols are generally considered to have a short half-life, low oral bioavailability
and metabolism-related toxic effects. However, they are still attractive for drug development
because their natural chemical structures are more bioactive than their metabolites, and
improvement of pharmaceutical formulations and some delivery systems can increase their
1
4
bioavailability. Through a scaffold-hopping principle, a diphenylmethane scaffold compound 2
was first synthesized. Surprisingly, the ability to disrupt the pH and the bactericidal activity
IB
against Mtb of compound 2 were similar to those of natural 1 (Figure 1C). The new analog with
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diphenylmethane scaffold, a privileged structure, was superior to agrimophol in potentially
druggable properties, relative ease of large-scale synthesis and relative metabolic stability of the
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fully substituted polyphenolic template, suggesting that a medicinal chemistry approach to
generate better agrimophol analogs was feasible.
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Figure 1. The structures of 1 and 2 and their impacts on pH and survival of Mtb. A) Scaffold-
IB
hopping from 1 containing four intramolecular hydrogen bonds to a fully substituted
diphenylmethane 2; B) Both natural and synthetic 1 reduced the pH of Mtb H37Rv-pHGFP.
IB
Bacteria were incubated with the compounds at the indicated concentrations in pH 4.5 buffer for
o
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days at 37 C. C) Viability by CFU (colony-forming units; left panel) and pH (right panel) of
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Mtb H37Rv-pHGFP following exposure to the indicated compounds and concentrations for 6
days at pH 4.5. The mean ± SD of biological triplicates is shown.
The structure activity relationships (SARs) of 2 were then fully investigated. During the first
round of optimization, forty analogs of 2 were designed (Figure 2, Tables S1 and S2).
Compounds 3a-3c and S1a-S1b (2-acyl side chain of ring C), 3d and S2a-S2e (4-position of ring
C), and 3e and S3a-S3c (2-nonacyl side chain of ring C) (Figure 2 and Table S1) were designed
to investigate the contributions of the length and branching of aliphatic substituents, cyclic
substituents, heteroatoms and isosteres. Halogenation has become increasingly important in
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medicinal chemistry and has attracted a great deal of attention in recent years for hit-to-lead-to-
candidate optimization, with the advantages of changing the electronic effects, hydrophobic
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effects and three-dimensional conformations of molecules. Replacement of the 4- and/or 10-
methyl group(s), which possessed lipophilic properties and could represent potential
metabolizable group(s) by liver microsomes, with a halogen atom (3e, S2b-S2e, Table S1) might
improve the physicochemical properties compared with the nonsubstituted compound 3d.
Modifications of all hydroxyl groups (Figure 2 and Table S2), including 1- and 7-hydroxyl
groups, which formed four intramolecular hydrogen bonds and conferred poor solubility and
weak absorption of 1, through functional alkylation (S4a-S4d), selective methylation (3f-3i, S4e-
S4f and S5a-S5d), selective acylation (S4g and S5e-S5g), deletion (3j), isosteric replacement
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(S6a) and alkylation (S5h) were made to understand the medicinal role of each group in the
molecule. Introduction of a methyl or phenyl group (S6b and S6c) into the 13-methylene linker
of the scaffold could block the potential metabolic site of the molecule and break the hydrogen
bond formation between the two polyphenolic fragments of 2.
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Figure 2. The structures of the selected diphenylmethane scaffold analogs (the concentration
indicates the minimum concentration able to decrease the pH ≤ 6.0; inactive compounds were
IB
tested at a maximum concentration of 12.5 µM).
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Testing the activity of the compounds in Figure 2 and Tables S1-S2 revealed key insights at this
primary SAR stage (Figure 3). For instance, it was observed that the 2-carbonyl aliphatic linear
chain (C ring, 3a-3c and S1a-S1c, Figure 2 and Table S1) of 2 affected the biological activity in
decreasing the pH value below 6.0 of both BCG and Mtb, and the molecule with the shortest
IB
chain 3a was relatively the most potent: with activity at a minimum concentration of 1.56 µM in
both BCG and Mtb (Figure 2) whereas 1 could only achieve similar activity at a concentration of
12.5 µM. The modification of any hydroxyl group resulted in loss of activity, with the exception
of 3h. Compound 3h still showed activity at 1.56 µM (Figure 2). Carefully surveying the
structural information in Figure 2 and Table S2, we concluded that either the 3-position of the C
ring or 9-position of the B ring must be methylated, e.g., with 3g and 3j; however, they should
not be simultaneously methylated (i.e., 3f).
Chiral center and the unsaturated
ketone ring are not essential(2).
O
O
OH
B
O
A
O
OH
OH OH
Agrimophol (1)
1
-Hydroxyl and 7-hydroxyl groups are
Introduction of substituents results
in loss of activity (S6b and S6c).
important. Replacement with hydrogen (3j)
or methyl (S6a) results in reduced activity.
Other isosteric replacements (S3a-S3c and
S4a-S4d) cause total loss of the ability
O
OH
B
OH O
1
3
1
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''
7
2'
Replacement of methoxy group with other
linear carbon chains (S4a) and introduction
of heteroatoms (S4b) or (3i) lead to lost
activity.
4''
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''
1''
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5
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3'
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4'
The chiral center in the side chain is not
essential(3c). Short linear chains greatly
increase activity (3a).
A
O 910
4
3 OH 5'
OH OH
2
Replacement of methyl groups with hydrogens
3d) and halogen (S2b and S2c) enables the
ability to decrease the pHIB.
3-Hydroxyl group (3h) is critical to activity.
Alkylation (3f) and acetylation (S4g) cause
loss of activity.
(
Figure 3. Structure activity relationships of 1 analogs in the first round optimization step.
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Scheme 1. Synthetic route of 1 and its diphenylmethane scaffold analoguesa.
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Reagents and conditions: (a) i) R12 = H, R CHO, CH COOH/H SO , rt-120 C; ii) R CHO, TsCl, DCE,
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reflux; iii) R = CH N(CH ) , ZnCl , dioxane, reflux.
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The general process of diphenylmethane scaffold compound synthesis involved a convergent
approach in which the two corresponding B and C ring fragments were synthesized individually
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and subsequently condensed (Scheme 1). When R was H, the compounds were prepared from
the corresponding B ring, C ring and corresponding formyl group in acetic acid16 or
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corresponding aldehyde with tosyl chloride in dichloroethane. To prevent dimerization, R of
the B ring, which includes a relatively highly reactive segment, was previously activated by
preparing N,N,N,N-tetramethylmethanediamine and then reacted with another C segment
through ZnCl catalysis in appropriate yields.
2
Scheme 2. Synthetic route of the corresponding fragments of diphenylmethane scaffold
analoguesa.
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OH O
MOMO
O
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OH O
O
OH
OH
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R1
R1
R
R1
R
CHO
OH
e
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c
d
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HO
OH
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MOMO
OH
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MOMO
HO
HO
OH
HO
HO
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i
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OH
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phloroglucinol
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OMOM
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d
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OMOM
OH
O
O
OH
OH
3
f
41
R5
43
44
42
O
O
R5
O
O
O
R1
R6
c
d
e
f
HO
O
HO
OH
HO
OH
5-47
HO
OMOM
OMOM
m
O
32,33
4
48
49
OH
50
OH
OH
OH
O
OH
OH
OH
OH O
R7
or
OH HO
k
R9
L
R8
n
L
R10
HO
OH
HO
OH HO
HO
OH
OH
HO
OH
COOCH3
HO
OH
HO
OH
Cl
62-67
7
7
R
R
Br
COOCH3
Cl
61
phloroglucinol
5
2
53-55
56-58
R = CH
17: R1 = CH(CH )CH CH , R = CH
59,60
R4 =CH3
R1 = H
R1 = CH3
R1 =CH(CH3)
16: R1 = CH CH CH ,
2
3
6
7
8
:
:
:
:
22: R1 = CH CH CH ,
2
56: R8 = CH3
2
2
3
3
3
R = CH
2
2
3
3
2
23: R1 = CH(CH )CH CH , R = CH
2
8
3
2
3
57: R = CH CH
2 3
3
2
3
3
18: R1 = CH CH CH ,
2
R = CH(CH )
24: R1 = CH CH CH ,
2
8
2
2
3
3 2
R = CH(CH )
58: R = CH(CH )CH CH
3 2 3
2
2
3
3 2
19: R1 = CH CH CH ,
2
2 2
25: R1 = CH CH CH ,
R = (CH ) CH Cl 59 R10 =CH(CH )CH CH
2
2
2
2
3
R = (CH ) CH Cl
2
2
2
3
2 2 2
3 2 3
9: R1 = CH CH(CH )
20: R1 = CH CH CH ,
2
R = (CH ) NHBoc
R2 = CH CCH
26: R1 = CH CH CH ,
2
10
2
3 2
2
2
3
2 3
R = (CH ) NHBoc 60: R =Cl
2
2
3
2 3
21: R1 = CH CH CH ,
62: R10 = H
63: R10 = CH3
1
1
1
1
0: R1 = CH(CH2)2
27: R1 = CH CH CH ,
R2 = CH CCH
2
2
3
2
2 2 3
2
1: R1 = CH CH CH
28,30,32: R1 = CH CH CH
45: R5 = H,
46: R5 = H,
6
2
2
3
2
2
3
R
= CH CH CH
2
2
3
2: R1 = (CH ) CH
29,31,33: R1 = CH(CH )CH CH
R6 = CH(CH )CH CH
64: R10 =CH CH CH
2
5
3
3
2
3
3
2
3
2 2 3
3: R1 = CH(CH )CH CH
37: R3 = CH CH CH
47: R5 = CH3, R6 = CH(CH )CH CH
52,53: R7 = Br
65: R10 = CH(CH )CH CH
3 2 3
2
2
3
3
2
3
3
2
3
38: R3
39: R4
14: R1 = CH CH CH
= CH(CH )CH CH
66: R10 = CH (CH CH )
3
2
3
2
2
3
2 2 3 2
15: R1 = CH(CH )CH CH
= CH CH CH
0: R4 = CH(CH )CH CH
54: R7 =CN
55: R7 =F
67: R10 =CH CH3
2
2
2
3
3
3
2
3
4
2
3
a
Reagents and conditions: (a) POCl , DMF, EA, rt; (b) NaBH CN, 2N HCl, THF, rt; (c) Acyl chlorides, 1,2-
3
3
dichloroethane, AlCl , 50 C; (d) MOMCl, K CO , acetone, rt; (e) i. Me SO , K CO , acetone, reflux; ii.
3
2
3
2
4
2
3
Alcohols, PPh , DEAD, THF, rt; (f) 2N HCl, MeOH, reflux; (g) POCl , DMF, dioxane, rt; (h) Zn/HgCl , conc.
3
3
2
HCl, dioxane, rt; (i) N,N,N',N'-tetramethylmethanediamine, DCM, rt; (j) Zn powder, conc. HCl, CH OH, rt; (k)
3
o
o
i. F-TEDA-BF , CH CN, -45 C-rt; ii. NH Br, oxone, rt; iii. NH Br, oxone, rt and CuCN, DMF, 130 C; (l) Acyl
4
3
4
4
o
chlorides, BF .Et O, 70 C; (m) SO Cl , ether; (n) N,N-dimethylaniline, 150°C.
3
2
2
2
To synthesize the designed diphenylmethane scaffold compounds, all other required
corresponding fragments (B and C segments) were initially prepared as shown in Scheme 2.
Various acyl group substituted intermediates 6-13 were prepared from phloroglucinol via a
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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
+
Vilsmeier–Haack formylated product (4), which gave intermediate (5) by NaBH CN/H
3
reduction, followed by a Friedel-Crafts (F-C) acylation catalyzed by aluminum chloride.18, 19
Through optimization of protective groups, the methoxymethyl (MOM) was finally employed.
The MOM reaction (14, 15) and the following methylation (16, 17) and deprotection steps were
carried out in sequence to successfully provide intermediates 22-27. Similarly, intermediates 32,
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
2
3, 43, and 50 were synthesized via alternative starting materials. Collectively, C ring segments
4-27 were synthesized from 16 via the Mitsunobu reaction with appropriate alcohols in the
presence of diethyl azodicarboxylate (DEAD) and triphenylphosphine (PPh ) after removal of
3
the protecting MOM group. Intermediates 37 and 38 were finally synthesized from
phloroglucinol under the reaction conditions of g, h (Clemmensen reduction is a key step in this
route) and c. Intermediates 56-58 were also attained from phloroglucinol via an oxidative
bromination (52, 53) or a nucleophilic substitution (54, 55) and then F-C acylation catalyzed by
BF ·Et O. Using 2,4,6-trihydroxybenzoic acid methyl ester as the starting material, intermediates
3
2
6
2-67 were gained through chloro substituted compounds (59, 60), decarboxylation reaction (61)
and the final F-C acylation.
Cyclization Strategy to Mimic Intramolecular Hydrogen Bonds of Natural 1. The poor
solubility and low absorption of natural 1 have been thought to be due to its intramolecular
hydrogen bonds. A cyclization strategy to mimic intramolecular hydrogen bonds was further
explored to overcome such non-druggable properties (Figure 4A). Meanwhile, computational
studies (Figure S1) indicated that some cyclic compounds (i.e., 4b, 4f, 4j, and 4k) had potentially
lower pKa values than the corresponding linear compounds (i.e., 3a, 3b) (Figure 4B).
A
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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
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
B
Compound
1
pKa
Compound
4j
pKa
4.5±1.0
5.9±0.4
3a
7.4±0.5
7.4±0.5
4k
4b
6.0±0.5
5.9±0.4
3
b
5
.7±0.4 (Acid)
4
f
4i
8.0±0.4
1.2±0.3 (Base)
Figure 4. The designed and calculated physicochemical properties of 1 and its analogs. A)
Cyclization to mimic intramolecular hydrogen bonds (I, II, III and IV); B) The pKa values were
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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
calculated by ACD/Percepta Platform software.20
Chroman-4-one is a well-known pharmacophore displaying multiple bioactivities, e.g.,
antibacterial and antiviral activities.21-23 To mimic the hydrogen bond I of 1, a chroman-4-one
via the 1,2-position of the C ring was constructed on the basic diphenylmethane scaffold (Table
S3). Via the 2,3-position of the C ring, a chroman-4-one F ring was built to mimic hydrogen
bond II (S9a-S9f, S10a-S10e and 4k, Table S4 and Figure 4A). Additional rings were also
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
4
designed, such as the E, G, I, J and K rings, which cooperated onto the basic diphenylmethane
scaffold B or C ring (Table S5). Meanwhile, several well-known medicinal chemistry concepts,
including but not limited to the following, were practiced: the introduction of a fluorine atom
(S11b, Table S5), which is known to increase some biological functions and enhance metabolic
stability,25 a cyano group (S11c, Table S5) with its electron withdrawing group and better
2
6
solubility, a nitrogen atom (4c, Figure 4A) to replace the oxygen atom with the aim of adjusting
the pKa and avoiding oxidative metabolism, a benzisoxazole pharmacophore (4f, Figure 4A)
because its nitrogen atom could be a hydrogen bond donor and a 2',3'-unsaturated ketone (4h,
Figure 4A) that might help the molecule to bind its target protein through a Michael addition
reaction due to rich electron density.24,27 Collectively, at this second optimization step, fifty-one
compounds were designed (Tables S3-S5). Screening the compounds for activity disclosed that
compounds 4f-4h showed improved activity in decreasing the pHIB of both BCG and Mtb,
demonstrating that the chroman-4-one (D ring) was useful via 1,2-position cooperation of the C
ring. Halogenation of the C ring (4b, 4c, S11a-S11b and 4h, Figure 4A and Tables S5) clearly
contributed to activity. The electron withdrawing cyano group contributed to biological function,
since S11c (3.13 μM, pH = 5.82) and S11g (0.78 μM, pH = 5.90) both showed the ability to
IB
IB
decrease the activity against BCG (Tables S5). Further cyclic H ring compounds were made that
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could break the hydrogen bonds III or IV (Figure 4A). A chroman-4-one C ring was also
cooperated onto the new scaffold (S12a-S12e, Table S6). All compounds in Table S6 were
inactive, which strongly implied that hydrogen bonds III and/or IV were essential for activity.
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
Cyclization to form benzisoxazole
may increase bioactivity (4f)
O 1'
O
OH
O2'
5' 6'
4'
1
3
J
1
''
7
1
2''
8
3'
3
'
2' 4'' 3''
6
5
2
N
O
O
E
2''B 12
C
Benzolide maybe beneficial (4g)
Cyclization with hydroxyl 4''
to form chroman-4-one
results in loss of activity
I
8
13
12
D
11
7
1
3 OH
2''
5'' O 9 10
4
Br
3
''
1
''
21' O
OH OH
6
5
B
C
O
1''
O
OH
7
OH
O
2
O 910
1
1
3
OH
13
1
1'
Chroman-4-one exhibits
lower activity (4k) or
loss of activity (S9c)
4
Cl
4''
2''
3''
OH OH
2'
8
12
6
5
(
4e)
B
C
F
11
9
3
O
3'
O
OH
7
OH
O
1
0
OH OH 4
3''
3'
13
1
Cl
2
''
2'
2
''
O
7
O
1
2'
4''
3''
1'' 8
O 910
6
5
1'
3'
G
1
3
D
B
12
11
C
2
4'
1
2
O 1'' 8
1'
O
3 OH 5'
6
2
OH OH 4
B
C
1
1
Chiral center is still not essential to the activity (S7b
and S7d) and shorter linear chain is helpful (S7e).
5
2
HO
9
OH 4
3 OH
1
0OH
2
',3'-unsaturated ketone (4h) keeps the activity.
3'
O
OH
7
O
1
Carbonyl group is essential and
the reduction of ketone could
cause the loss of activity (S8c).
2'
1'
O
13
D
2
''
The substituents, which are migrated from 3'-
position to 2'-position, exhibit lower activity (S8b)
4
''
3
''
1''
E
O
2
8
12
6
5
B
C
5
''
11
9
3
OH
1
0
4
O
Cl
Cyclization with 2''-methylene to form
Carbonyl group is essential. The reduction of ketone
chroman-4-ones could retain bioactivity (4b).
could cause the loss of activity (S8e).
Halogen and cyano groups are benificial (Br>Cl>F>CN).
Replacement with methylene group or methyl formate causes
decreased activity (S10a).
Cyclization with hydroxyls to form seven-membered
ring causes complete activity loss (4i).
nd
Figure 5. SAR of 1 after the 2 round of optimization.
Figure 5 summarizes the full SAR at this second optimization step. Testing compounds 4a
(Figure 4A) and S7b, S7d (Table S3) showed that the chiral center in the chroman-4-one was
unnecessary. The results also supported the previous conclusion that shorter linear chains were
optimal (S7b, S7d, Table S3). Substituents of the D ring were preferred at the 2’-position
because S8a and S8b (Table S3) lacked activity. The lack of bioactivity of compounds S8c-S8e
(Table S3) indicated that the carbonyl group was also essential. The ranked contributions to
activity were Br>Cl>F>CN (4b, S11a-S11c, Figure 4A and Table S5). Using a 2,3-
dihydroquinolin-4(1H)-one to replace the chroman-4-one (4c and 4d) decreased the activity,
possibly because 2,3-dihydroquinolin-4(1H)-one, as a basic group, might neutralize a decrease in
pH . Chroman-4-one with a benzisoxazole ring (4f), lactone ring (4g) or 2',3'-unsaturated ketone
IB
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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
ring (4h) also contributed to improved activity in combination with the halogenation strategy,
implying that the chroman-4-one may not be essential.
Scheme 3. Synthetic route of the corresponding fragments of hydrogen bond mimic compoundsa.
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
R12 COOH
R12 COOC H
O
O
2
5
b
a
(C H O) PCH COOC H
+
R13
2
5
2
2
2
5
R12 R13
H
R13
H
8
2-84
78-80
+
R11
R11
O
O
C
OH
OH O
OH O
D
D
R11
R11
g
O
f
O
d
e
C
OMOM HO
OH
HO
OH
HO
OH
MOMO
OMOM
OMOM
Cl
68,69
MOMO
R7
,53-55,61
R7
11,64,67
Cl
70,71
Cl
72,73
5
e
O
O
OMOM
O
OH
c
f
g
E
B
E
B
HO
OMOM
O
R13
OMOM
O
O
OH
N
R12
75
74
14
i
OH O
OH O
O
O
D
O
OH
D
O
C
F
O
C
F
O
O
h
1
2
C
R
and
HO
C
E
O
B
HO
13 HO
OH
R
R7
HO
O
OH
HO
OH
OH
R7
Cl
Cl
105a
Cl
94
76
8
5a-93a,104a
85b-93b,104b
105b
O
OH
l
OH O
OH O
O
NH2
H3CO
m
H CO
Br
HO
3
j
k
C
D
D
C
NH
NH
HO
N
N
H
H
H CO
OCH3
3
H CO
3
98a
H CO
3
96
97
and
95
OH O
HO
O
O
OH
N
O
OH
N
O
N
O
O
i
I
C
F
n
o
I
N
HO
N
B
B
H
OH
Br
OH
O
OH
OH
98b
22
99
100
101
OR14
O
O
O
O
OH O
O
OH N
n
d
HO
HO
OH
HO
OH
HO
OH
HO
OH
Cl
Br
Br
06
Cl
1
6
3
1
02,103
OH O
O
O
K
K
1) p; 2) q
r
O
O
C
C
OH
HO
OH
108
87a,87b:
HO
OH
1
07
Br
3
7
R12 = CH3,
13
R =Cl,
R
13
= CH3
6
6
8,70,72: R11 = CH CH
2
3
7
R12 = CH CH CH , R = CH CH
11
88a,88b: R =Cl,
9,71,73: R = CH3
2
2
3
2
3
1
2
13
7
R12 = CH CH ,
R13 = CH CH
78,82: R = CH CH ,
R
= CH3
89a,89b: R =Cl,
2
3
2 3
2
3
79,83: R12 = CH CH CH , R = CH3
13
7
R12 = CH CH ,
2 3
R13 = CH3
R13 = CH3
R13 = CH3
R13 = CH3
R13 = CH3
90a,90b: R =Br,
2
2
3
80,84: R12 = CH CH ,
R13 = CH CH3
7
12
91a,91b: R =COOCH ,R = CH CH ,
2
3
2
3
2
3
7
R12 = CH CH ,
7
12
13
92a,92b: R =F,
85a,85b: R =CH , R =CH CH , R = CH3
2 3
3
2
3
7
R12 = CH CH ,
86a,86b: R =Cl, R12 = CH CH , R = CH3
7
13
93a,93b: R =CN,
2
3
2
3
7
R12 = CH3,
102: R14 = H
14
104a,104b: R =CH ,
103: R = CH3
3
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a
Reagents and conditions: (a) 65% NaH, THF, 0°C; (b) 2N KOH, 80 °C; (c) BF ·Et O, 70°C; (d) Acyl
3
2
chlorides, BF ·Et O, DCE, 50°C; (e) MOMCl, K CO , r.t; (f) (HCHO)n, diethylamine, EtOH, reflux; (g)
3
2
2
3
HCl/MeOH, reflux; (h) Zn powder, conc. HCl, MeOH, rt; (i) N,N,N',N'-tetramethylmethanediamine, DCM, rt;
(j) Ethyl Acetoacetate, NaBH CN, DCE, CH COOH, rt; (k) LiOH, THF/H O, rt; (l) HBr solution/CH COOH
4 3 2 3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(v/v=1:1), 140°C; (m) NH Br, Oxone, rt; (n) NH OH·HCl or NH OCH ·HCl, Na CO , MeOH, rt; (o) PPh ,
4
2
2
3
2
3
3
DDQ, anh. DCM, rt; (p) Ac O, CH COONa, reflux; (q) Na CO , H O, reflux; (r) NBS, THF, rt.
2
3
2
3
2
To synthesize the designed compounds in this section, all required corresponding fragments were
initially prepared as shown in Scheme 3. As illustrated in Scheme 3, intermediates 72 and 73
were prepared from 2-chlorobenzene-1,3,5-triol (61, Scheme 2) via F-C acylation catalyzed by
BF ·Et O (64 and 67), the alternative protection of MOM (68 and 69), cyclization with
3
2
paraformaldehyde and ethylenediamine, and finally, removal of the protective MOM group.
7
Through selective protection with MOM, when R was a methyl group (11), the MOM position
was changed due to different electronegativities. With various substituted ketones as starting
materials, intermediates 82-84 were prepared through the Horner-Wadsworth-Emmons
reaction28,29 and hydrolysis of the ester bond, followed by reactions with intermediates 5, 53-55
and 61 to gain chroman-4-ones (85a-93a, 85b-93b, 104a, 104b, 105a and 105b) with Lewis acid
BF ·Et O as a catalyst. The carbonyl of chroman-4-one (86a) was then reduced by Zn powder
3
2
3
0
and conc. HCl to attain 94. Intermediates 98a and 98b were made from 3,5-dimethoxyaniline
via the reaction steps of reductive amination (95), hydrolysis of the ester bond (96), F–C
acylation, removal of a methyl group (97) and a final substitution reaction. During the process of
F-C acylation, protonic acid (CH COOH, HBr (gas) in water) and Lewis acid (AlCl ) were each
3
3
investigated, yielding trace amounts of the anticipated product. Interestingly, HBr (gas) in
CH COOH provided an alternative approach to gaining the designed intermediates. Segments
3
1
01 (B and I ring cooperation) were synthesized via reaction steps of the hydroxyl oxime
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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
reaction (99), dehydration and cyclization.31,32 Intermediate 3 was treated with acetic anhydride,
anhydrous sodium acetate, followed by sodium carbonate hydrolysis to provide 107.33 The
critical C ring and K ring cooperation of 108 was then finally obtained with excellent yields
through selective bromination as described previously.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 4. Synthetic route of the corresponding fragments of hydrogen bond mimic compoundsa.
Br
Br
a
b
c
d
AcO
OAc
HO
OH
AcO
OAc
HO
OH
HO
OH
O
O
109
O
111
O
112
110
Br
O
HO
Cl
O
e
104a
G
C
C
OH
f
O
HO
HO
OH
B
H
Cl
O
O
113
4i
a
o
Reagents and conditions: (a) AlCl , Acyl chlorides, Chlorobenzene, 70 C; (b) Acetyl chloride, DIPEA, DCM,
3
o
rt; (c) NBS, AIBN, CCl , reflux; (d) H SO , MeOH, 70 C; (e) NCS, DCM, rt; (f) i) HCHO solution,
4
2
4
CH COOH, rt; ii) K CO , acetone, rt.
3
2
3
To prepare acylated 109 (Scheme 4), 1,3-dimethoxy-5-methylbenzene or 5-methylbenzene-1,3-
diol was studied for their selective F-C reactions. The results showed that the former F-C
acylation mainly occurred at the o-position of the methyl group; alternatively, the latter happened
at the p-position of the methyl group. In fact, we finally acetylated the diol group (110) to reduce
the electronegativity of benzene, followed by bromination (111),33 deacetylation (112), and
halogenation steps to provide 114. The designed compound 4i was finally gained through
reaction of 114 and 104a in the presence of HCHO/CH COOH and K CO in acetone. With
3
2
3
employment of the same conditions and with the corresponding fragments, all designed
compounds were synthesized (Scheme S1).
Scaffold-Hopping via Integration of Coumarin and Agrimophol Pharmacophores.
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Coumarin and its derivatives are attractive due to a variety of noncovalent bond interactions to
targeted proteins, i.e., π-π interactions, hydrophobicity, hydrogen bonding and van der Waals
3
4
35
36
37
38,39
forces, which confer antioxidant, anti-HIV, antibacterial, antivirus, anti-TB,
and other
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
biological activities. Coumarin derivatives were also reported as disruptors of pH homeostasis
IB
of Mtb.40 Taking advantage of pharmacophore displacement for drug lead identification, we
carried out a screening of coumarin analogs and agrimophol pharmacophores (Table S7).
Interestingly, the fragment F9, which is a 3-fluorinated coumarin, was able to decrease BCG’s
pH < 6.0 at an initial tested concentration of 12.5 μM. Upon the discovery by 1st round SAR,
IB
concluding that the shortest one of the 2-carbonyl aliphatic linear chains (C ring) exhibited the
most potent activity, the B ring of 3a was integrated with the F9 fragment to produce the
designed analog 5a and its analogs 5b, S13a, and S13b (Figure 6 and Table S8). Alternatively,
the B or C ring of 4b was integrated with the F9 fragment, resulting in 5c-5d and S13c-S13d
(Figure 6 and Table S9). Computational calculations indicated that these compounds 5a-5c and
S13a-S13b shared similar pKa values to natural 1 (Table 1, Figure S1).
O
O
OH
OH
O
O
OH
O
D
O
E
O
B
C
B
C
OH
OH
OH OH
OH OH
Br
4b
3a
O
O
OH
OH
O
O
OH
OH
O
O
OH
OH
O
C
OH
F
F
F
D
F
B
C
M
B
C
M
B
C
M
O
C
M
O
O
O
O
O
O
OH OH
OH OH
OH OH
HO
O
O
OH
O
O
O
OH
Br
O
5a
5b
5c
5d
BCG: 0.0975μ M (pHIB 6.00)
BCG: 0.19
μ M
μ M
(pHIB 5.97) BCG: 0.0975 (pHIB 6.00)
BCG: 12.5μ M (pHIB 6.29)
Figure 6. Selected results for the diphenylmethane scaffold through pharmacophore
displacement (the concentration indicated the minimum concentration able to decrease the pHIB
≤
6.0).
Table 1. Predicted LogP and pKa Values of Designed Compounds a.
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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
5
5
5
5
5
5
5
5
5
6
ID
1
Structure
pKaa
ID
5c
Structure
pKaa
O
OH
OH
O
O
OH
O
O
F
E
B
C
M
O
B
A
4.5±1.0
4.8±0.2
4.8±0.2
5.6±0.2
4.8±0.2
O
O
OH
OH OH
OH OH
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
OH
OH
O
OH
OH
F
F
B
C
M
O
E
O
5
a
S13c
S13d
C
M
O
5.6±0.2
6.1±0.2
O
O
OH OH
OH OH
Br
O
O
O
OH
OH
O
OH
OH
F
B
C
M
O
E
O
5
b
B
C
M
O
O
O
OH OH
O
OH OH
Br
O
O
OH
OH
F
S13a
B
C
M
O
O
OH OH
Br
a
The pKa values were all predicted by ACD/Percepta Platform software.20
At this third optimization step, eight compounds were designed (Tables S8-S9). Of note,
compounds 5a, 5b and 5c showed substantial potency in their ability to decrease the pH of
IB
BCG under acidic conditions (Figure 6). The 4-methyl group of coumarin (5a) was the most
favorable in comparison with 5b. The 3-fluorine of coumarin was also useful (5a-5c) in
achieving potency. The 8-acetyl group of coumarin also contributed substantially.
In this optimization step, the coumarin corresponding fragments were prepared by using
phloroglucinol as the starting material (Scheme 5). The Peckman reaction (a), F-C acetylation
reaction (b) and bromination reaction (c) were subsequently employed, as described in Scheme
5
. Using the method in Scheme 1, all designed compounds in Figure 6, Table S8 and Table S9
were prepared individually.
Scheme 5. Synthetic route of corresponding fragments a
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OH R20
R21
C
M
HO
HO
O
O
OH
OH R20
b
R21
a
O
120-123
C
M
HO
OH
HO
O
O
OH R20
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
c
R21
O
115-119
C
M
O
15: R20=CH3,
16: R20=CH3,
17: R20=CH CH ,
R21=H
R21=F
21=F
1
1
1
1
1
Br
1
24, 125
=F
21=F
20=CH3,
R
21
120: R
121: R20=CH CH ,
R
2
3
R
R21=F
2
3
18: R20=CH CH CH , R21=F
122: R20=CH CH CH ,
2
2
3
2
2
3
19: R20,R21=cyclohexane
24: R20=CH3,
23: R20,R21=cyclohexane
1
R21=H
125: R20=CH3,
R21=F
1
a
Reagents and conditions: (a) BF .Et O, EtOH, 85 C, 115) Ethyl acetoacetate; 116) Ethyl 2-fluoro-3-
3
2
oxobutanoate; 117) Ethyl 2-fluoro-3-oxopentanoate; 118) Ethyl 2-fluoro-3-oxohexanoate; 119) Ethyl 2-
oxocyclohexanecarboxylate; (b) AlCl , CH NO , 110  C; (c) NBS, DCM, rt.
3
3
2
The bactericidal activities against BCG of the most potent compounds 5a and 5c were compared
with natural 1 and other analogs 5b and S13b in a pH 4.5 environment. To compare the efficacy
at equivalent concentrations, we used the minimum concentration required to reduce the pH of
IB
BCG ≤ 6.0 (Figure 7) to determine the utility of the pH assay to predict antibacterial activity.
IB
We note that compound 5a, at a concentration of 0.195 µM, had equivalent bactericidal action as
natural 1 used at 6.25 µM. Compounds 5c and 5b also showed bactericidal action against BCG at
concentrations of 0.195 µM and 0.39 µM, respectively.
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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
Figure 7. Bactericidal activity of 1 and selected analogs. The relative survival of BCG was
measured when it was exposed to the indicated compounds at pH 4.5 for 2 days. The minimum
effective concentration able to reduce BCG’s pH ≤ 6.0 was used for the experiment, i.e., 1:
IB
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
.25 μM; 5a: 0.195 μM; 5b: 0.39 μM; S13b: 0.78 μM; 5c: 0.195 μM. Mean values ± SD of
triplicate samples are shown. The survival percentage is CFUCompound/CFUDMSO
.
Human Liver Microsome Stability, Hepatocyte Stability and Compound Solubility.
Screening the analogs for activity in disrupting pH revealed that compounds 3a, 4j, S11a, 4b,
IB
S11g, 5a, 5b and 5c exhibited superior activity. They were subjected to incubation with mouse
liver microsomes and human liver microsomes to assess their metabolic stability (Table 2). All
tested compounds were more stable when exposed to human liver microsomes than to mouse
liver microsomes. In human liver microsomes, the t1/2 values of coumarin-based 5a and 5b were
greater than 40 minutes, whereas that of natural 1 was 10.3 minutes; of note, that of 5a was 68.5
minutes. Furthermore, 5a was a substrate of supersome CYP3A4, with the observation of a
shortened t1/2 to 18.1 minutes, which increased to more than 90 minutes (t ) for other tested
1/2
supersomes CYP1A2, CYP2C9, CYP2C19, and CYP2D6 (Table 3). The supersome CYP3A4
can catalyze a variety of chemical reactions such as alkyl hydroxylation and O-demethylation.
Further testing indicated that the metabolic stability of compound 5a to human hepatocytes was
more than 500 minutes (Table 2). Because human hepatocytes contain both phase I and phase II
metabolic enzymes, these results strongly indicated that 5a would be metabolically stable in
humans. A glucuronidated metabolite of 5a was detectable by LC-MS, and its amount gradually
increased with extended incubation time (data not shown), indicating that 5a subsequently
reacted in phase II metabolization of human hepatocytes. The solubility of the compounds was
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5
6
7
8
9
measured from 10 mM (with the exception of S13b) or 1.25 mM (S13b) in DMSO stock added
to an acidic PBS buffer at pH 4.5. Compounds 5a and 5b possessed better solubility.
Table 2. Metabolic Stability in Human Liver Microsomes and Hepatocytes and Solubility
of the Potent Compounds.
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
T1/2 f (min)
aq sol.
μM)c
Compound
Structure
Human
(
MLMa
HLMb
Hepatocytes
O
O
OH
O
O
A
1
B
1.52
NDd
NDd
10.3
24.4
31.9
ND e
ND
15.45
10.59
ND
OH
OH OH
O
OH
OH O
3
a
B
C
O
OH
OH OH
O
O
OH
OH O
3
b
B
C
ND
OH
OH OH
O
O
OH
O
D
4
j
O
NDd
NDd
2.80
20.2
22.1
68.5
ND
ND
15.58
9.63
B
C
OH
OH OH
Br
O
OH
O
D
4
b
O
E
O
B
C
OH
OH OH
Br
O
OH
OH
F
B
C
M
O
5
a
>500
31.06
O
O
OH OH
O
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2
3
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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
O
O
OH
OH
F
B
C
M
O
5b
5.43
2.87
2.57
44.1
15.2
27.6
ND
ND
ND
26.54
BLQd
23.72
O
OH OH
O
O
O
OH
OH
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
B
C
M
O
S13b
O
OH OH
O
O
OH
OH
F
E
O
B
C
M
O
5
c
O
OH OH
O
a
b
c
MLM: Mouse liver microsome. HLM: Human liver microsome. Solubility was measured in
an acidic PBS buffer at pH 4.5, which was prepared with 200 mM sodium phosphate and 100
d
e
f
mM citric acid. BLQ means below the limit of quantitation. ND: not tested. Drug half-life.
Table 3. CYP Phenotyping Assay of Compound 5a in Human Liver Microsomes
T (min)
1
/2
Compound
Supersome
Supersome
Supersome
Supersome
Supersome
CYP1A2a
CYP2C9 a
CYP2C19 a
CYP2D6 a
CYP3A4 a
5
a
129
167
97.7
132
18.1
a
CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were all cytochrome P450 (CYP)
subtypes.
Toxicity Assays and Protein binding Rate Assay. Compounds 5a and 5c, which showed high
potency, were evaluated for their cytotoxicity to HepG2 cells in comparison with natural 1, and
4
1
42
with paclitaxel and rifampicin as controls representing a cytotoxic agent and a well-tolerated
antibiotic, respectively (Table 4). By determining the EC values of 1, 5a and 5c, the selectivity
5
0
indices (SIEC50) were calculated, which confirmed that 5a and 5c had a superior SIEC50 to natural
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1
. Meanwhile, the selectivity indices (SIMBC90) were also calculated with minimum bactericidal
concentration (MBC ) values of 1, 5a and 5c. Compound 5a exhibited a 45 times greater
9
0
SIMBC90 value to HepG2 cells than 1, suggesting that the planar structure of the coumarin ring
may help to reduce cytotoxicity. To test whether differences in cytotoxicity may be explained by
differential protein binding of the analogs compared with 1, we then tested the protein binding of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
, 5a and 5c with three concentrations (1 μM, 5 μM and 15 μM). The results showed that the
human plasma protein binding rates of 1, 5a and 5c were all greater than 99%, with no
differences between them and no concentration-dependent relationships (Table 4), indicating that
these compounds all exhibit strong protein binding capacity.
Table 4. The EC , IC , SI and protein-binding rate of measurements are shown.
5
0
50
EC50
IC50
(μM) b
0.0539
>50
MBC90
(μM) c
-
Protein binding
Compound
SIEC50d/ SIMBC90e
(
μM) a
rate (%) f
Paclitaxel
-
-
-
-
Rifampicin
-
-
-
1
0.9669
2.10
3.125
<0.78
3.125
2.17/0.67
381.42/>30
55.02/1.08
>99
>99
>99
5a
0.06502
0.0618
24.80
3.40
5
c
a
b
The EC value is derived from the activity of the compounds at reducing the pH ; The IC is
5
0
IB
50
defined as the concentration of compound that caused a 50% decrease in HepG2 cell growth
c
compared with the paclitaxel and rifampicin as controls. MBC is the minimum bactericidal
9
0
concentration with the ability to kill 90% of Mtb in vitro.43 d SIEC50 is the selectivity index
e
f
calculated as IC /EC . SI
is the selectivity index calculated as IC /MBC
Protein is
90.
5
0
50
MBC90
50
human plasma protein.
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In Vitro Antibacterial Activity Against Wild-type Mtb, and Clinical Multi- and Extensively
Drug-Resistant Mtb. Finally, the potencies of 5a and 5c to kill fully pathogenic Mtb were
assessed. Wild-type Mtb (H37Rv) was incubated at pH 4.5 with 5a, 5c, natural 1, and DMSO as
a control for 6 days at the indicated concentrations in Figure 8. All three active compounds
showed concentration-dependent killing activity by counting colony-forming units (CFU) of
H37Rv. Compound 5a was the most potent: it reduced CFU viability of Mtb by 1.5 logs at 0.78
µM, with up to a 3 log reduction when used at 3.125 µM (Figure 8A). In comparison, both 5c
and natural 1 achieved a maximum of 1 log reduction in CFU viability of H37Rv at the
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maximum tested concentration of 3.125 µM. Since targeting pH represents a novel therapeutic
IB
strategy compared with existing anti-tuberculosis drugs, we hypothesized that our bioactive
candidates would also possess bactericidal activity against drug-resistant clinical isolates of Mtb.
The antibacterial activities of 5a and 1 were then investigated against a multidrug-resistant
(MDR-Mtb) strain and an extensively-drug-resistant (XDR-Mtb) strain using 9,9-dimethyl-2-
nitro-7-propyl-10,11-dihydrofuro[2,3-f]pyrano[2,3-h]chromen-5(9H)-one
as
a
novel
fluorescence indicator44,45 (Figure 8B). Both 1 and 5a were active against MDR- and XDR-Mtb,
with MIC of 1.56 µM against MDR-Mtb and 6.25 µM against XDR-Mtb, respectively (Figures
90
8
B, 8C).
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0
Figure 8. Inhibitory activity of select compounds against Mtb-H37Rv and clinically isolated
MDR-Mtb and XDR-Mtb strains at pH=4.5. A) The CFU of wild-type H37Rv following
exposure to natural 1, 5a and 5c at the indicated concentrations in pH 4.5 buffer for 6 days.
Means ± SD of triplicate samples are shown. The inhibition rates of natural 1, 5a and 5c at
indicated
concentrations
with
9,9-dimethyl-2-nitro-7-propyl-10,11-dihydrofuro[2,3-
f]pyrano[2,3-h]chromen-5(9H)-one as a novel fluorescence indicator at pH 4.5 for 6 days against
B) MDR-Mtb strain 15042 and C) XDR-Mtb strain 16630. Each group was performed in
duplicate.
■
CONCLUSIONS
4
6
In summary, natural 1 was isolated and synthesized, and we verified its ability to disrupt the
pH homeostasis and inhibit the viability of Mtb. New diphenylmethane scaffold compounds,
IB
e.g., 3a, mimicked the ability of natural 1 in being able to decrease the pH values of both BCG
IB
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and Mtb (1 log reduction). Through a structure-hopping strategy and mimicking intramolecular
1
0
hydrogen bonds (I, II, III and IV) of natural 1, in combination with the calculation of pKa, novel
compounds 5a and 5c were synthesized integrating coumarin derivatives and the fully substituted
butan-1-one fragment (5a) or chroman-4-one fragment (5c) (Figure 9). These two compounds
0
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were two orders of magnitude more potent at reducing the pH of BCG compared with natural
IB
1
. Since there was a close correlation between potency in disrupting pHIB and antibacterial
efficacy for 5a, our data support that 1 and 5a are likely to function via the same pathway. Most
interestingly, compound 5a exhibited improved metabolic stability to human microsomes and
human hepatocytes and an acceptable selectivity index (>30, as calculated by MBC /IC ). In a
90
50
killing assay using wild-type Mtb and strain H37Rv, 5a at 0.78 μM resulted in a 1.5 log and at
1
0
3
.125 μM resulted in a 3.0 log reduction in CFU under a pH 4.5 environment, which is a two
10
log improvement of the mycobactericidal activity compared with natural 1. Compound 5a also
1
0
exhibited bactericidal activity to clinical isolates of MDR- and XDR-Mtb. These findings
together suggest a new approach for the development of novel anti-TB drugs through various
concepts of medicinal chemistry that work via disruption of pHIB homeostasis (Figure 9).
4
7
Furthermore, since an acidic environment is associated with multidrug tolerance in Mtb , these
agents may also play a role in shortening TB treatment regimens.
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0
Figure 9. The discovery that 5a and 5c derived from 1 via exploration of a diphenylmethane
scaffold-hopping strategy and pharmacophore displacement strategy could disrupt the pHIB
homeostasis and inhibit the viability of Mtb.
■
EXPERIMENTAL SECTION
Chemistry. Unless otherwise noted, commercially available materials were used without further
purification. All NMR experiments were carried out on a Bruker Advance 400 MHz
spectrometer using DMSO-d , methanol-d , acetone-d and CDCl as solvents. Chemical shifts
6
4
6
3
are given in ppm (δ) relative to the residual solvent and coupling constants (J) in Hz. Multiplicity
is tabulated as s for singlet, d for doublet, t for triplet, q for quadruplet, and m for multiplet.
Melting points were determined without correction with a Yanaco micro-melting point
apparatus. UPLC-MS analysis was performed on a Thermo Finnigan LCQ Advantage mass
spectrometer equipped with an Agilent pump, an Agilent detector, an Agilent liquid handler, and
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a fluent splitter. The column is a Kromasil C18 column (4.6 μm, 4.6 mm × 50 mm). The eluent
was a mixture of acetonitrile and water containing 0.05% HCOOH with a linear gradient ranging
from 5:95 (v/v) to 95:5 (v/v) of acetonitrile-H O within 5 minutes at a 0.3 mL/minute flow rate
2
0
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2
3
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0
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2
3
4
5
6
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8
9
0
for analysis. The UV detection was carried out at a UV wavelength of 254 nm. Five percent of
the eluent was split into the MS system. Mass spectra were recorded in either positive or
negative ion mode using electrospray ionization (ESI). High resolution LC-MS (HRMS) was
carried out by Agilent LC/MSD TOF using an Agilent ZORBAX SB-C18 column (rapid
resolution, 3.5 μm, 2.1 mm × 30 mm) at a flow rate of 0.40 mL/min. The solvent was
methanol/water = 75/25 (v/v) containing 5 mmol/L ammonium formate. The ion source was
electrospray ionization (ESI). The column chromatography was performed with silica gel 60
(200-300 mesh) from Qingdao Haiyang Chemical Factory. The tested compounds were purified
≥
95%, as characterized by HPLC under UV 254 nm wavelength, NMR, melting point, and
HRMS.
The details on the synthesis of all chemical entities described in this paper are in the Supporting
Information.
General Procedure For the Synthesis of the Final Compounds.
Method one: To a solution of the two corresponding fragments (0.1 mmol) in acetic acid (1.0
mL) was added 37% formaldehyde aqueous solution (0.5 mL) at room temperature. The mixture
was stirred at room temperature for 8 h, and then saturated NaHCO aqueous solution was added
3
to pH=7.0. The solution was extracted with ethyl acetate (5.0 mL), and the organic layer was
evaporated in vacuo. The compound was obtained and characterized after purification by silica
gel column chromatography (ethyl acetate/petroleum ether).
Method two: To a solution of the two corresponding fragments (0.1 mmol) in DCM (3.0 mL)
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was added TsCl (0.1 mmol), aldehyde (0.5 mmol) and anhydrous sodium sulfate (200 mg). The
mixture was refluxed for 8 h. The insoluble substance was filtered off, and the filtrate was
evaporated in vacuo. The compound was obtained and characterized after purification by silica
gel column chromatography (ethyl acetate/petroleum ether). Method 3: To a solution of the two
1
1
1
1
1
1
1
1
1
1
2
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5
5
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0
corresponding fragments (0.1 mmol) in dry dioxane (2.0 mL) was added ZnCl (0.2 mmol). The
2
mixture was refluxed for 12 h and then concentrated in vacuo. The compound was obtained and
characterized after purification by silica gel column chromatography (ethyl acetate/petroleum
ether).
Method three: To a solution of the two corresponding intermediates (0.1 mmol) in dry dioxane
(2.0 mL) was added ZnCl (0.2 mmol). The mixture was reflux for 12 h and then concentrated in
2
vacuo. The corresponding compounds were obtained and characterized after purification by
silica gel column chromatography.
Synthesis of 1-(3-(3-butyryl-2,6-dihydroxy-4-methoxy-5-methylbenzyl)-2,4,6-trihydroxy-5-
methylphenyl)-2-methylbutan-1-one (2). Method one. Yellow solid. Yield 48%. mp 58-60 C.
1
H NMR (300 MHz, CDCl ) δ 15.52 (s, 1H), 9.58 (s, 1H), 9.36 (s, 1H), 3.83 (s, 2H), 3.74-
3
3.80 (m, 1H), 3.71 (s, 3H), 3.07 (t, J = 7.2 Hz, 2H), 2.10 (s, 3H), 2.08 (s, 3H), 1.80-1.92 (m,
1
H), 1.67-1.77 (m, 2H), 1.37-1.48 (m, 1H), 1.17 (d, J = 7.2 Hz, 3H), 0.98 (t, J = 7.2 Hz, 3H),
1
3
0
.92 (t, J = 7.2 Hz, 3H). C NMR (100 MHz, CDCl ) δ 210.9, 206.9, 161.8, 160.3, 160.2,
3
159.6, 112.1, 109.5, 107.8, 105.9, 103.5, 101.8, 61.5, 45.8, 44.3, 26.9, 18.2, 16.7, 16.2, 13.9,
+
1
2.0, 9.1, 7.5. HRMS calcd for C H O [M+H] : 461.2170; found: 461.2175.
2
5
33
8
Synthesis of 3-(3-butyryl-2,6-dihydroxy-4-methoxy-5-methylbenzyl)-2,4,6-trihydroxy-5-
1
methyl benzaldehyde (3a). Method one. Yellow solid. Yield 61%. mp > 210 C. H NMR (400
MHz, CDCl ) δ 15.56 (s, 1H), 13.78 (s, 1H), 10.11 (s, 1H), 9.72 (s, 1H), 9.06 (s, 1H), 3.80 (s,
3
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