Indolyl Azaspiroketal Mannich Bases Are Potent Antimycobacterial Agents with Selective Membrane Permeabilizing Effects and in Vivo Activity

Article abstract of DOI:10.1021/acs.jmedchem.8b00777

The inclusion of an azaspiroketal Mannich base in the membrane targeting antitubercular 6-methoxy-1-n-octyl-1H-indole scaffold resulted in analogs with improved selectivity and submicromolar activity against Mycobacterium tuberculosis H37Rv. The potency enhancing properties of the spiro-fused ring motif was affirmed by SAR and validated in a mouse model of tuberculosis. As expected for membrane inserting agents, the indolyl azaspiroketal Mannich bases perturbed phospholipid vesicles, permeabilized bacterial cells, and induced the mycobacterial cell envelope stress reporter promoter piniBAC. Surprisingly, their membrane disruptive effects did not appear to be associated with bacterial membrane depolarization. This profile was not uniquely associated with azaspiroketal Mannich bases but was characteristic of indolyl Mannich bases as a class. Whereas resistant mycobacteria could not be isolated for a less potent indolyl Mannich base, the more potent azaspiroketal analog displayed low spontaneous resistance mutation frequency of 10^-8/CFU. This may indicate involvement of an additional envelope-related target in its mechanism of action.

Full text of DOI:10.1021/acs.jmedchem.8b00777

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Article
Indolyl Azaspiroketal Mannich Bases Are Potent Anti-mycobacterial Agents
with Selective Membrane Permeabilizing Effects and In Vivo Activity
Samuel Agyei Nyantakyi, Ming Li, Pooja Gopal, Matthew Zimmerman,
Veronique Dartois, Martin Gengenbacher, Thomas Dick, and Mei-Lin Go
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00777 • Publication Date (Web): 12 Jun 2018
Downloaded from http://pubs.acs.org on June 13, 2018
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Indolyl Azaspiroketal Mannich Bases Are Potent Anti-mycobacterial Agents with
Selective Membrane Permeabilizing Effects and In Vivo Activity
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# +
+
§
Samuel Agyei Nyantakyi,
Ming Li, ^ Pooja Gopal, ^ Matthew Zimmerman, Véronique
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Dartois, § Martin Gengenbacher, § Thomas Dick * $ § and MeiꢀLin Go, * #
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# Department of Pharmacy, 18 Science Drive 4, National University of Singapore, Singapore
117543; ^ Department of Medicine, 14 Medical Drive, National University of Singapore,
Singapore 117599; $ Department of Microbiology and Immunology, 5 Science Drive 2, National
§
University of Singapore, Singapore 117545; Public Health Research Institute, New Jersey
Medical School, Rutgers, The State University of New Jersey, 225, Warren Street, Newark, New
Jersey, USA 07 103ꢀ2714.
+ Joint first authors
Abstract
The inclusion of an azaspiroketal Mannich base in the membrane targeting antiꢀtubercular 6ꢀ
methoxyꢀ1ꢀnꢀoctylꢀ1Hꢀindole scaffold resulted in analogs with improved selectivity and
submicromolar activity against Mycobacterium tuberculosis H37Rv. The potency enhancing
properties of the spiroꢀring fused motif was affirmed by SAR and validated in a mouse model of
tuberculosis. As expected for membrane inserting agents, the indolyl azaspiroketal Mannich
bases perturbed phospholipid vesicles, permeabilized bacterial cells and induced the
mycobacterial cell envelope stress reporter promoter piniBAC. Surprisingly, their membrane
disruptive effects did not appear to be associated with bacterial membrane depolarization. This
profile was not uniquely associated with azaspiroketal Mannich bases but was characteristic of
indolyl Mannich bases as a class. Whereas resistant mycobacteria could not be isolated for a
less potent indolyl Mannich base, the more potent azaspiroketal analog displayed low
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spontaneous resistance mutation frequency of 10ꢀ8/CFU. This may indicate involvement of an
additional envelopeꢀrelated target in its mechanism of action.
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INTRODUCTION
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1
Tuberculosis (TB) has been a scourge for most of history.
In spite of advances in our
understanding of the pathogenic organism Mycobacterium tuberculosis (Mtb), TB remains
frustratingly difficult to treat and continues to extract a high toll of human lives. In 2016, TB
surpassed HIVꢀAIDS and malaria as the leading cause of death due to a single infectious agent.
2
The past decade has witnessed continuing resistance to first line antiꢀTB drugs isoniazid and
rifampicin (multiꢀdrug resistance), additional resistance to fluoroquinolones and secondꢀline
injectables (extensive drug resistance), and more recently, cases of programmatically incurable
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TB where treatment regimens constructed with available drugs completely fail.
Most drugs
used today were discovered decades ago and the global TB drug pipeline, notwithstanding
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recent additions like bedaquiline, remains thin. While the urgency for new drugs cannot be
overemphasized, a more compelling need is for agents that kill both replicating and
phenotypically drug tolerant nonreplicating populations of mycobacteria. Disrupting the
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mycobacterial membrane has been posited as a viable means of achieving these objectives.
First, the loss of an architecturally intact and functional membrane is fatal to both growing and
quiescent bacteria. 5 Second, the ensuing perturbation of multiple membraneꢀembedded targets
would further delay resistance development among surviving organisms. 5 The feasibility of this
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approach is corroborated by membraneꢀactive small molecules (verapamil,
boromycin,
amphiphilic xanthones, 8 propyltriphenylphosphonium phenothiazine conjugates, 9 1ꢀoctylindolyl
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Mannich bases
) that have antimycobacterial activity. Involvement of the mycobacterial
membrane was inferred from the ability of these compounds to permeabilize mycobacterial
7, 8, 10
6, 7
7, 8
membranes,
disrupt transmembrane potential
and/or deplete intracellular ATP.
Tellingly, conjugation of the membrane targeting triphenylphosphonium (TPP) moiety to
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antimycobacterial phenothiazines led to significant improvements in activity, likely due to
enhanced localization within the mycobacterial membrane.9 Relatably, grafting TPP onto an
inactive indole scaffold resulted in mycobactericidal cationic amphiphilic TPP indoles that rapidly
depolarized the membrane of M. bovis BCG. 11 1ꢀOctylindolyl Mannich bases embedded with a
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cationic amphiphilic motif were similarly mycobactericidal and additionally, more potent and
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selective than the TPP indoles.
Among the more potent indolyl Mannich bases, compound
1 (8ꢀ[(6ꢀmethoxyꢀ1ꢀoctylꢀ1Hꢀindolylꢀ3ꢀyl)methyl]ꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4,5]decane) stood out as
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the only member with an azaspiroketal side chain (Figure 1).
We noted that a structurally
4, 12
similar motif was present in the preclinical TB candidate BTZ043 (2, Figure 1).
Mechanistically, the azaspiroketal has not been implicated in the potent activity of BTZ043
which was attributed to inhibition of the oxidoreductase DprE1, a key enzyme involved in
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mycobacterial cell wall synthesis.
The contribution of the azaspiroketal to the antiꢀTB
activity of BTZ043 remains elusive as structural variations have yielded mixed results. Notably,
the antimycobacterial activity of 3 which does not possess a spirocyclic side chain, was
comparable to BTZ043 (Figure 1) 15 whereas replacing the ketal oxygens in BTZ043 with sulfur
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resulted in a more active analog 4 (Figure 1).
Spirocyclic motifs are widely perceived to
possess attractive attributes from a drug design perspective due to their unique dimensionality
17
and complexity.
Evidence from literature has shown that the inclusion of spirocycles in
molecules led to tighter and more selective binding to desired targets, improved solubility and
18,19
greater metabolic stability.
Viewed in this context and as part of our efforts to optimize the
antimycobacterial activity of indolyl Mannich bases, we explored the azaspirocyclic motif with
two goals in mind: First, to assess its potential as a potencyꢀenhancing chemotype, and second,
to determine if it is associated with distinctive mechanistic effects that set it apart from other
Mannich bases.
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Figure 1: Structures of indolyl Mannich base 1, BTZ043 (2) and its benzothiazinone congeners
3 and 4
Chemistry
Three series of indolyl Mannich bases were synthesized to interrogate the structureꢀactivity
contribution of the spirocyclic motif. In Series 1, diverse azaspirocycles were introduced as the
basic moiety at position 3 of the 1ꢀnꢀoctylꢀ6ꢀmethoxyindolyl scaffold. We then sought to optimize
activity of the more potent Series 1 members by replacing nꢀoctyl with ring bearing side chains
(Series 2), and introducing alternative substituents (nitro, cyano, propoxy, isopropoxy,
benzyloxy) in place of methoxy (Series 3). Also prepared were 1ꢀoctylindoles in which the
azaspirocycle was not part of the Mannich base motif but attached to indole via an alkoxy linker
(Series 4).
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Series 1ꢀ3 were synthesized by methods previously reported for indolyl Mannich bases.
Scheme 1 outlines the synthesis of Series 1 which involves reacting 6ꢀmethoxyꢀ1Hꢀindole with
1ꢀbromooctane in the presence of sodium hydride to give the 1ꢀ(nꢀoctyl) analog 5, followed by
the insertion of the basic 3ꢀsubstituted aminomethyl side chain by two approaches. In
the first, the indoleꢀ3ꢀcarbaldehyde 6 was prepared by a VilsmeierꢀHaack reaction and then
subjected to reductive amination to give compounds 21, 22, 28 – 32, 35. In the second
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approach, 5 was reacted with an amine and formaldehyde in the presence of an acid catalyst
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(acetic acid or zinc chloride) to give the remaining Series 1 compounds.
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Scheme 1: Reagents and conditions: (i) NaH, CH3(CH2)6CH2Br, anhydrous DMF, 0 °C to RT, 2
– 8 h (ii) POCl3, DMF, 0 °C – 40 °C, 2 h (iii) CH3COOH, appropriate amines, Na(AcO)3BH,
anhydrous THF, RT, 24 – 48 h (iv) CH2O (aq., 36%), appropriate amines, CH3COOH, RT or
ZnCl2, EtOH, RT, 3 – 24 h.
Most of the amines indicated in Scheme 1 were commercially available but those required for 1,
7-16 were synthesized. These were the azaspirocycles 1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane (1a),
(S)ꢀ2ꢀmethylꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane
(7a),
(R)ꢀ2ꢀmethylꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]ꢀ
decane (8a), 2,3ꢀdimethylꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane (9a), 1ꢀoxaꢀ4ꢀthiaꢀ8ꢀazaspiroꢀ
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[4.5]decane (10a), 1,4ꢀdithiaꢀ8ꢀazaspiro[4.5]decane (11a), 1,5ꢀdioxaꢀ9ꢀazaspiro[5.5]undecane
(12a), 1ꢀoxaꢀ5ꢀthiaꢀ9ꢀazaspiro[5.5]undecane (13a), 1,5ꢀdithiaꢀ9ꢀazaspiro[5.5]undecane (14a), 1ꢀ
oxaꢀ4ꢀthiaꢀ8ꢀazaspiro[4.6]undecane (15a) and 1ꢀoxaꢀ5ꢀthiaꢀ9ꢀazaspiro[5.6]dodecane (16a).
These amines were prepared by reacting piperidinꢀ4ꢀone or azepanꢀ4ꢀone with the relevant
alkanediols, alkanedithiols or mercaptoalkanols in the presence of catalytic amounts of pꢀ
toluenesulfonic acid.
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In Series 2, we replaced the nꢀoctyl side chain of 1, 12 and 6ꢀmethoxyꢀ1ꢀoctylꢀ3ꢀ(piperidinꢀ1ꢀ
ylmethyl)ꢀ1Hꢀindole (52) with various ringꢀbearing side chains, namely 3ꢀphenoxypropyl,
fluoro/methoxy/methylꢀsubstituted
3ꢀphenoxypropyl,
4ꢀphenoxybutyl,
3ꢀbenzyloxypropyl,
cinnamyl, 3ꢀphenylpropyl and 4ꢀphenylbutyl. Of these side chains, phenoxypropyl bromide (53)
and 1ꢀ(3ꢀbromopropoxy)ꢀ4ꢀmethoxybenzene (54) were prepared by reacting phenol / 4ꢀ
methoxyphenol with 1,3ꢀdibromopropane in the presence of potassium carbonate in acetonitrile
(Supporting Information, Scheme S1). Scheme 2 outlines the synthesis of Series 2 which
involved firstly, Nꢀalkylation of 6ꢀmethoxyꢀ1Hꢀindole as described in Scheme 1 to give the 1ꢀring
substituted 6ꢀmethoxyindoles (41-51), followed by the Mannich reaction with bases 1a, 12a and
piperidine to give the desired compounds (41a,b,c – 50a,b,c; 51a,b). The 1ꢀoctylindolyl
Mannich base 52 was synthesized by a similar method. 10
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Scheme 2: Reagents and conditions: (i) NaH, appropriate nꢀoctyl, phenalkyl, phenoxyalkyl,
benzyloxyalkyl or cinnamyl bromide, anhydrous DMF, 0 °C to RT, 2 – 8 h (ii) CH2O (aq., 36%),
amines (1a, 12a or piperidine), CH3COOH, RT or ZnCl2, EtOH, RT, 3 – 24 h.
In Series 3, other functionalities (nitro, cyano, propoxy, isopropoxy, benzyloxy) were introduced
in place of methoxy on the indole ring while retaining nꢀoctyl and selected Mannich bases at
positions 1 and 3 respectively. The 6ꢀisopropoxy (55a), 6ꢀpropoxy (55b) and 6ꢀbenzyoxyindoles
(55c) were synthesized from 6ꢀhydroxyꢀ1Hꢀindole and the corresponding alkyl/benzyl halide in
the presence of an inorganic base (cesium carbonate Cs2CO3 or potassium carbonate K2CO3)
in DMF. The regioisomeric nitroindoles and 6ꢀcyanoindoles were purchased. The substituted
indoles were then alkylated with 1ꢀbromooctane and reacted via a Mannich reaction to give the
final compounds 57a-h, 58a-e and 59a-c (Scheme 3).
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Scheme 3: Reagents and conditions: (i) 1ꢀiodopropane; 2ꢀiodopropane; or benzyl bromide,
K2CO3 or Cs2CO3, anhydrous DMF, 80 ºC, 3 – 4 h (ii) NaH, CH3(CH2)6CH2Br, anhydrous DMF, 0
°C to RT, 2 – 8 h (iii) CH2O (aq., 36%), amines (1a, 12a or piperidine), CH3COOH, RT or ZnCl2,
EtOH, RT, 3 – 24 h.
To obtain Series 4, we first reacted regioisomeric 4ꢀ, 5ꢀ, 6ꢀ and 7ꢀhydroxyꢀ1Hꢀindoles with 1ꢀ
bromoꢀ3ꢀchloropropane or 1ꢀbromoꢀ2ꢀchloroethane in the presence of K2CO3 in ethanol to
obtain the chloropropoxyindole (60a-d) or 5ꢀ(2ꢀchloroethoxy)ꢀ1Hꢀindole (60e). Displacement of
the terminal chlorine with iodine followed by reaction with amines azepane, 1,5ꢀdioxaꢀ9ꢀazaspiro
[5.5]undecane (12a), piperidine, (S)ꢀ2ꢀmethylꢀ1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane (7a), and
pyrrolidine in the presence of K2CO3 or direct reaction of 60a-e with the appropriate amine and
Cs2CO3 gave 61a-b, 62a-c, 63, 64a-d, 65a-e. The latter were then alkylated as before to give
66a-b, 67a-c, 68, 69a-d, 70a-e.
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Scheme 4: Reagents and conditions: (i) 1ꢀBromoꢀ3ꢀchloropropane or 1ꢀbromoꢀ2ꢀchloroethane,
K2CO3, ethanol, reflux, 3 h (ii) KI, K2CO3, appropriate amine, MeCN, reflux, 24 – 48 h; or
Cs2CO3, appropriate amine, MeCN, reflux, 24 – 48 h (iii) NaH, CH3(CH2)6CH2Br, anhydrous
DMF, RT, 12 – 24 h.
RESULTS AND DISCUSSION
Series 1 compounds with azaspiroketal and isosterically related sidechains have
submicromolar potencies and outstanding selectivity
The minimum inhibitory concentrations (MIC50, MIC90) of Series 1 were determined by turbidity
measurements on M. bovis BCG, an attenuated tubercule bacillus and mycobacterial model
organism (Table 1). The more potent analogs (MIC50 < 5 ꢁM) were assessed on mammalian
African Green Monkey kidney epithelial Vero cells for selective cytotoxicities. Also assessed
were the hemolytic potential of some analogs on human red blood cells (RBCs). This was in
view of the cationic amphiphilic nature of these compounds which would predispose them to
nonꢀselective membrane disruptive effects.
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Table 1: Antimycobacterial activity (M. bovis BCG), mammalian Vero cell cytotoxicity (IC50) and
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hemolytic activity (HC50, human RBC) of Series 1 compounds.
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No
R
MIC (BCG) (ꢁM)a
Vero IC50
(ꢁM)b
46
SIc
23
68
HC50
(ꢁM)d
75
LogPe
5.01
MIC50
2
MIC90
4
1
7
8
0.5
0.6
0.9
0.8
34
37
51
75
5.33
62
5.33
9
0.7
0.6
0.5
0.3
0.4
0.4
1.6
0.8
0.8
0.8
0.8
0.8
34
64
49
107
>400
97
76
5.65
5.73
6.46
5.12
5.84
6.56
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12
13
14
>300
>300
64
>200
29
39
98
>300
>300
152
380
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0.7
1.2
35
50
>300
5.70
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6
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9
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0.5
17
17
1
34
68
132
NDf
NDf
5.95
4.47
4.75
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24
42
NDf
NDf
NDf
NDf
19
20
1
1
2
2
44
31
44
31
>300
>300
6.93
7.25
21
22
13
13
18
25
NDf
NDf
NDf
NDf
NDf
NDf
5.17
5.58
23
24
2
4
31
16
88
4.07
4.85
15
46
NDf
NDf
NDf
25
1.8
6.3
>500
>278
>300
4.16
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NDf
NDf
NDf
3.74
5
6
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9
27
28
2.3
2
4.4
4
14
10
6
5
69
33
7.03
5.46
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28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
29
30
1.6
1.1
2.3
1.9
20
10
13
9
NDf
19
5.88
6.09
31
32
4
6
24
21
6
NDf
NDf
6.13
5.31
1.8
4.6
12
33
34
35
36
3.6
15
6.3
24
22
NDf
NDf
31
6
207
NDf
NDf
109
4.83
4.63
4.92
5.96
NDf
NDf
8
21
50
3.8
7.6
37
17
30
NDf
NDf
NDf
3.70
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2
3
4
5
6
7
8
9
38
39
40
2
6
8
4
44
22
>300
NDf
6.07
4.40
3.18
15
22
NDf
NDf
NDf
NDf
NDf
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Minimum Inhibitory Concentrations required to reduce growth by 50% (MIC50) or 90% (MIC90)
b
compared to untreated controls. Average of 2 or more determinations. Concentration required
to reduce growth of Vero cells by 50% compared to untreated controls. Average of 2 or more
c
d
determinations. Selective Index = IC50 Vero /MIC50 BCG.
Concentration required to hemolyze
e
50% of human RBCs, compared to 2% Triton Xꢀ100 (100% hemolysis). Log P values were
f
determined on ChemDraw Professional Version 15.1.0.144. ND = Not Determined
Structurally diverse Mannich bases were explored at R in Series 1 (Table 1). These were (i)
azaspiroketals (7-9) and congeners in which one or both ketal oxygen atoms were replaced by
sulfur (10-16); (ii) azaspirocycles without the ketal moiety (19-27); (iii) secondary amines
substituted with fused/bridged bicyclic rings (28-31), the spirocycle 1ꢀoxaspiro[4.4]nonane (32)
and the spiroketal 1,4ꢀdioxaspiro[4.5]decane (33); (iv) azabicycles (35-37) and (v) substituted
azetidines 38,39 and piperidine 40. Of these, only the azaspiroketals and their sulfur isosteres
(7-16, MIC50 0.3ꢀ0.7 ꢁM) surpassed the original lead 1 (MIC50 2 ꢁM). These significant gains in
potency which also led to better selective cytotoxicities, were achieved by modifying the ketal or
piperidine ring of 1 in the following ways: (i) methyl or dimethyl substitution of the ketal ring; (ii)
replacing one or both ketal oxygen atoms with isosteric sulfur to give 10 (MIC50 0.6 ꢁM) and 11
(MIC50 0.5 ꢁM); (iii) expanding the 5ꢀmembered ketal bearing ring to a 6ꢀmembered homolog
(12, MIC50 0.3 ꢁM); (iv) submitting 12 to oxygenꢀsulfur isosteric substitution to give 13 and 14
(MIC50 0.4 ꢁM for both) and (v) homologation of the piperidine ring in 11 and 13 to the 7ꢀ
membered azepanyl homologs 15 (MIC50 0.7 ꢁM) and 16 (MIC50 0.5 ꢁM) respectively.
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7
8
9
Interestingly, these modifications resulted in compounds that were largely equipotent. We then
asked if the narrow variation in MICs could be due to hydrolysis of the spirocyclic side chain to a
common potent intermediate. Hydrolysis would yield the piperidinone 17 or azepanone 18 and if
the hypothesis holds, these compounds should be as active as their spirocyclic precursors. That
both 17 and 18 had only moderate activities (MIC50 17 ꢁM) led us to conclude otherwise.
The remaining compounds (19-40) in Series 1 were less potent than 1 but provided useful
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
insight into the structural requirements for activity.
First, the diminished activities of
azaspirocycles that were not ketals (19-27) emphasized the importance of the intact
azaspiroketal motif. Tellingly, the nonꢀketal azaspirocycles 24 and 25 displayed mediocre
activity (MIC50 15 ꢁM, 1.8 ꢁM), in spite of being regioisomers of the azaspiroketals 1 and 12
respectively. Second, positioning the basic nitrogen outside the spirocycle or bicycle resulted in
analogs (28-33) that were moderately potent (MIC50 1.1ꢀ4 ꢁM) but with strikingly poor selective
cytotoxicities (SI 5ꢀ13). The comparison of 1 (MIC50 2 ꢁM, SI =23) with 33 (MIC50 3.6 ꢁM, SI =6)
was particularly compelling in this regard. As before, hydrolysis of the spiroketal side chain in 33
was dismissed in view of the limited activity of the Nꢀcyclohexanone analog 34 (MIC50 15 ꢁM).
Third, there was no apparent advantage in deploying rigid scaffolds like those in the azabicycles
35-37 or conformationally more flexible azacycles such as 38-40.
We also collated the log P values of the Series 1 analogs. Lipophilicities varied over a wide 104
range, from 3.2 (40) to 7.3 (20) (Table 1). A cursory inspection showed that the least active
compounds (MIC50 > 10 ꢁM) had low log P values (3.2 – 5.6), an indication that activity may be
driven by lipophilicity. Indeed, we found a significant inverse relationship beween MIC50 and
log P (Spearman ρ correlation coefficient ꢀ0.567, p = 0.01, 2ꢀtailed), althought the potent
azaspiroketal Mannich bases (7-16), with an average log P of 5.8, were only moderately
lipophilic.
In all, these results pointed to considerable tolerance for structurally diverse Mannich bases at
R, most of which have low micromolar activities (MIC50 1ꢀ10 ꢁM). However, only azaspiroketals
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2
3
4
and their sulfur isosteres (7-16) were uniquely associated with
highly selective and
5
6
submicromolar potencies.
7
8
9
Series 2 analogs with ring bearing side chains at the indole N were less potent than n-
octyl counterparts.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Series 2 was designed to interrogate substitution at the indole N. Here, ringꢀbearing
phenoxyalkyl, phenylalkyl and phenylalkenyl side chains were introduced in place of nꢀoctyl. As
shown in Table 2, the R1 substitution was explored in three 6ꢀmethoxyindolyl Mannich bases,
namely the early hit 1, the potent submicromolar hit 12 and a previously reported
piperidinylmethyl analog 52. 10
Table 2: Antimycobacterial activity (M. bovis BCG), mammalian Vero cell cytotoxicity (IC50) and
hemolytic activity (HC50, human RBC) of Series 2 compounds.
MIC (BCG) (ꢁM)a
Vero
HC50
LogP d
b
No
R1
R2
IC50
(ꢁM)c
MIC50
MIC90
41a
3
10
NDe
NDe
3.87
41b
41c
42a
1.3
30
5
3.1
50
11
NDe
NDe
NDe
>300
NDe
NDe
3.98
4.13
4.03
O
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4
5
6
7
8
9
42b
42c
43a
1.5
20
2.6
NDe
NDe
NDe
>300
NDe
NDe
4.14
4.29
4.36
43
11
4.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NDe
NDe
NDe
>300
NDe
NDe
4.46
4.62
3.75
O
43b
43c
44a
1.3
19
6
2.6
41
13
44b
44c
45a
1.5
34
3.5
>50
11
46
>300
NDe
NDe
3.85
4.00
4.85
NDe
NDe
4.6
45b
45c
46a
1.7
18
20
2.7
25
41
NDe
NDe
NDe
>300
NDe
NDe
4.95
5.10
4.33
46b
46c
47a
11
16
16
25
41
40
NDe
NDe
NDe
NDe
NDe
NDe
4.43
4.58
3.78
47b
47c
2.0
46
6
54
NDe
NDe
3.89
4.04
>50
NDe
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4
48a
6
18
35
NDe
4.53
5
6
7
8
9
48b
48c
49a
2.6
36
9.8
>50
17
NDe
43
NDe
NDe
NDe
4.64
4.79
4.41
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7.4
31
49b
49c
50a
4.1
18
15
9.3
39
31
NDe
NDe
NDe
NDe
NDe
NDe
4.51
4.66
4.35
50b
50c
51a
4.8
31
9
NDe
NDe
NDe
NDe
NDe
NDe
4.46
4.61
4.95
49
4.9
11.5
51b
1
1.6
2
3.1
4
21
46
>300
75
5.05
5.01
12
52
0.3
7
0.8
13
29
64
5.12
5.27
ꢀ C8H17ꢀn
NDe
NDe
a-d
As described in footnote of Table 1. e ND = Not Determined.
The results largely recapitulated the activity advantage of the R1 nꢀoctyl side chain that was
observed in a related series where R1 was varied from ethyl (ꢀC2H5) to dodecyl (ꢀC12H25) .10
Here, none of the compounds with ring bearing R1, for the same Mannich base R2, were more
potent than their nꢀoctyl counterparts. Tellingly, the ring bearing analogs were also less lipophilic
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3
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5
6
7
8
9
than their nꢀoctyl counterparts with the same R2 group. However, unlike Series 1, Spearman
correlation analysis did not reveal any significant relationship between MIC50 and log P in this
series. However, it is still likely that the favored status of the nꢀoctyl side chain is due to its
lipophilic and less bulky features. As to whether there was an optimal ring bearing R1, we found
that this was largely determined by R2. Of the three R2 side chains explored, there was an
unambiguous preference for the 6/6 spiroꢀring fused 1,5ꢀdioxaꢀ9ꢀazaspiro[5.5]undecane (41b-
51b), followed by its 6/5 homolog 1,4ꢀdioxaꢀ8ꢀazaspiro[4.5]decane (41a-51a) and lastly the
monocyclic piperidine (41c-50c). For the 6/5 and 6/6 azaspiroketals, there was a preference for
R1 side chains that were phenoxypropyl (41a,b), substituted phenoxypropyl (42a,b-45a,b) and
phenylbutyl (51a,b). Benzyloxypropyl at R1 was favored only when paired with 1,5ꢀdioxaꢀ9ꢀ
azaspiro[5.5]undecane at R2 (47b).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The activity advantage of replacing 6-methoxy with other ring substituents in Series 3
was strongly dependent on the nature of the Mannich base
Series 3 was designed to explore the replacement of 6ꢀmethoxy with other groups while
retaining the nꢀoctyl chain and Mannich base (1,5ꢀdioxaꢀ9ꢀazaspiro[5.5]undecanylmethyl, 1,4ꢀ
dioxaꢀ8ꢀazaspiro[4.5]decanylmethyl, piperidinylmethyl) of Series 1 and 2 respectively (Table 3) .
The electron donating methoxy was replaced by electron withdrawing groups (nitro, cyano) and
sterically bulkier/ more lipophilic alkoxy groups (nꢀpropoxy, isopropoxy, benzyloxy). To
determine if there was a preference for specific positions on the indole ring, we prepared nitro
regioisomers of the 1,5ꢀdioxaꢀ9ꢀazaspiro[5.5]undecanyl analog 12 (Table 3). When greater
potency was found for the 6ꢀnitro regioisomer (57c, MIC50 0.3 ꢁM), subsequent substitutions
were limited to position 6. Here, we found that 6ꢀcyano (57e), 6ꢀpropoxy (57f), and 6ꢀ
isopropoxy (57g) analogs were as potent (MIC50 0.5ꢀ0.7 ꢁM) as the 6ꢀnitro 57c. Only the
insertion of the bulkier and more lipophilic benzyloxy at position 6 was not favored (57h, MIC50
2.2 ꢁM). Interestingly, none of the compounds were more potent than the 6ꢀmethoxy analog 12.
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5
6
7
8
9
In contrast, selective replacement of 6ꢀmethoxy in 1 and 52 resulted in more potent analogs.
These were the 6ꢀnitro 58a (MIC50 0.8 ꢁM) and 6ꢀcyano 59b (MIC50 2 ꢁM) which were more
potent than 1 and 52 respectively. Other substitutions yielded compounds (58b-e, 59a,c) that
were comparable to 1 and 52. Thus, R3 substitution benefited the less potent analogs (1, 52) to
a greater exent than the more potent 12. Taken together, two structureꢀactivity trends were
evident: First, there was no preference for electron donating or withdrawing R3 groups but size
was a limiting factor. Notwithstanding, lipophilicity was not found to significantly influence
activity. Second, the optimal R3 in this series was conspicously influenced by the Mannich base
at R2, reinforcing the SAR trend observed in Series 2.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3: Antimycobacterial activity (M. bovis BCG), mammalian Vero cell cytotoxicity (IC50) and
hemolytic activity (HC50, human RBC) of Series 3 compounds.
No
R2
R3
MIC (ꢁM)a
MIC50 MIC90
Vero IC50
(ꢁM)b
NDe
NDe
28
HC50
(ꢁM)c
NDe
NDe
>300
>300
>300
27
LogP d
57a
4ꢀNO2
5ꢀNO2
6ꢀNO2
7ꢀNO2
6ꢀCN
10
7
25
5.13
4.92
5.87
6.06
5.53
7.14
6.98
57b
57c
57d
57e
57f
18
0.3
1.3
0.5
0.8
3.1
1.2
1.2
1.6
49
22
6ꢀC3H7ꢀn 0.7
6ꢀC3H7ꢀi 0.6
29
57g
20
29
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2
3
4
5
6
7
8
9
57h
12
6ꢀBzO
6ꢀMeO
6ꢀNO2
6ꢀCN
2.2
0.3
0.8
2.5
3
3.1
0.8
1.6
5
27
>300
NDe
>300
>300
31
7.42
6.12
5.67
5.33
6.94
6.78
7.21
5.92
6.35
6.01
7.46
6.60
NDe
62
58a
58b
58c
58d
58e
1
59
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6ꢀC3H7ꢀn
6ꢀC3H7ꢀi
6ꢀBzO
6ꢀMeO
6ꢀNO2
6ꢀCN
5
29
3
6
41
56
4
5.5
4
36
>300
NDe
NDe
NDe
NDe
NDe
2
NDe
23
59a
59b
59c
4
5
2
6
22
6ꢀC3H7ꢀi
6ꢀMeO
4
6
26
52
7
13
NDe
a-c
d
As described in footnote of Table 1. Log P values were determined on ACD Labs
Physchem History Version 12.5. e ND = Not Determined.
Non-Mannich base Series 4 analogs have modest antimycobacterial activities
The indole scaffold in Series 4 comprised an nꢀoctyl side chain at position 1 and a basic
spirocyclic or nonꢀspirocyclic moiety attached to the benzenoid carbons of indole via an alkoxy
linker. In a significant departure from the preceding series, these compounds retained the
cationic amphiphilic signature but were not Mannich bases. Interestingly, this alteration resulted
in analogs with mediocre activity (Table 4). Even analogs bearing the highly favored 6/6 spiro
ring fused 1,5ꢀdioxaꢀ9ꢀazaspiro[5.5]undecane ring (66a, 66b: MIC50 6 ꢁM, 14 ꢁM), were no
more potent than their nonꢀspirocyclic piperidinyl counterparts (67a-c: MIC50 5ꢀ13 uM). Clearly
the privileged status of the azaspiroketal motif that was so pronounced in the indolyl Mannich
bases of Series 1 did not apply to this scaffold. Here, the most potent members were 70b
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2
3
4
5
6
7
8
9
(MIC50 1.9 ꢁM) and 70e (MIC50 2 ꢁM), both of which have azepanylalkoxy side chains.
Collectively these results led us to conclude that the topography of the cationic amphiphilic
indole was pivotal for activity. To this end, the location of the azaspiroketal moiety was decisive
and reconfiguring it to a nonꢀMannich base was disadvantageous.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4: MIC50 / MIC90 values of Series 4 on M. bovis BCG
No
R
Position on ring
MIC (ꢁM)a
MIC50
LogPb
MIC90
31
66a
66b
5
6
14
5.28
5.28
5.6
28
67a
67b
67c
5
6
7
13
13
5
40
25
13
5.43
5.43
5.43
6
11
25
68
5.49
69a
69b
69c
69d
70a
70bc
70cd
70d
4
5
6
7
4
5
6
7
18
31
20
10
9.5
1.9
3.9
5
36
48
42
22
22
8
5.02
5.02
5.02
5.02
5.85
5.85
5.85
5.85
12
11
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2
3
4
70e
5
2
6
5.74
5
6
7
a
b
As described in footnote of Table 1.
Log P values were determined on ChemDraw
8
9
Professional Version 15.1.0.144. c Vero cell IC50 34 ꢁM. d Vero cell IC50 33 ꢁM.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Antimycobacterial profiles of azaspiroketals (10-14) with submicromolar potencies
Encouraged by the potent and selective antimycobacterial activities of the azaspiroketals 10-14,
we proceeded to investigate their antimycobacterial profiles in greater detail. As shown in Table
5, these compounds retained submicromolar MICs against pathogenic Mtb H37Rv. Both 12 and
13 were cidal against Mtb and achieved 1000ꢀfold kill at 3.2 ꢁM (4 x MIC90). Together with
remaining compounds, they were cidal against M. bovis BCG, with MBC99.9 equivalent to 1ꢀ2x
MIC90 BCG. We also assessed the ability of 12 and 13 to eradicate nonꢀgrowing, phenotyoically
drug tolerant organisms by assessing their cidality on Mtb cultures grown under nutrient
deprived conditions (Loebel model). Both compounds were cidal in this model, with LCC99 of 18
ꢁM for 12 and LCC99.9 < 18 ꢁM for 13 (Supporting Information, Figure S1).
Table 5: Antimycobacterial profiles of Compounds 1, 10-14
M.bovis
Mtb H37Rv (ꢁM)
BCG
(ꢁM)
MIC50 (ꢁM) (M.bovis BCG) in
No
R a
7H9 Media b
Standard Without
With
10%
FBS
1.1
b
b
c
c
MIC50
MIC90
2.3
MBC99.9 MBC99.9 Medium
Glycerol
d
1
1.2
0.6
ND
ND
3ꢀ6
1.5
1.6
10
0.95
0.8ꢀ1.6 0.36
0.37
0.32
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2
3
4
5
6
7
8
9
11
12
13
14
0.7
0.44
0.5
1.1
0.76
0.8
ND
3.2
3.2
ND
0.8ꢀ1.6 0.29
0.8ꢀ1.6 0.30
0.4ꢀ1.6 0.26
0.8ꢀ1.6 0.28
0.27
0.35
0.41
0.43
0.28
0.33
0.6
10
11
12
13
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0.5
0.78
0.48
a
Refer to general structure given in Table 1; b Average of 2 or more separate determinations.
c MBC99.9 : Minimum bactericidal concentration required to kill 99.9% of bacteria. Average of 2 or
more separate determinations. MBC99.9 of 1, 10, 11, 14 on Mtb H37Rv were not determined
(ND).
d Standard 7H9 medium contains glycerol and does not contain FBS
The antimycobacterial activities of 12, 13 and other analogs (1, 10, 11) were not dependent on
media composition. MICs were unchanged in the absence of glycerol or in 7H9 medium
containing serum. We also determined the spontaneous resistance mutation frequencies of M.
bovis BCG and Mtb H37Rv to 12. A low spontaneous mutation frequency (10ꢀ8/CFU) was
obtained on Mtb H37Rv exposed to 12 at 4 x and 8 x MIC90. The Mtb H37Rv mutants were
confirmed to be resistant to 12, displaying on average 10ꢀfold shifts in MIC50 values (based on
analysis of 6 resistant strains). This is in contrast to the indolyl Mannich base 3ꢀ(azepanꢀ1ꢀ
ylmethyl)ꢀ4ꢀfluoroꢀ1ꢀoctylꢀ1H indole, 71 (Supporting Information, Figure S2) which we had
10
reported earlier.
For compound 71 which was not an azaspiroketal Mannich base, the
mutation frequency was < 10ꢀ9/CFU for M. bovis BCG.
Here, we confirmed in sideꢀbyꢀside
10
experiments that the spontaneous resistance mutation frequency of 71 was < 10ꢀ9/CFU for Mtb
H37Rv as well.
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The azaspiroketal Mannich bases 10-13 disrupted the melting profiles of
dimyristoylphosphatidylglycerol (DMPG) liposomal vesicles
9
Phosphatidylglycerols are negatively charged phospholipids that are abundant in bacterial
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20,21
plasma membranes but almost absent from membranes derived from eukaryotic cells.
Previously, we employed DMPG liposomal vesicles as surrogates of bacterial membranes and
found that indolyl Mannich bases which altered the melting profiles of DMPG vesicles
(monitored by differential scanning calorimetry) also permeabilized mycobacterial membranes
as seen from greater propidium iodide (PI) uptake by mycobacterial cultures.10 Conversely,
antimycobacterial indolylalkyltriphenylphosphonium analogs that did not disrupt the thermotropic
11
profiles of DMPG vesicles failed to increase PI uptake into mycobacteria.
Figure 2: Representative calorimetric curves, in heating mode, of DMPG vesicles in the
absence and presence (1 in 10 parts DMPG) of test compounds (A) 10-13, and (B) 70b. The
melting transition Tm (oC) and molar enthalpy ꢀH (in kJ/mol) for each treatment condition are
indicated. Control DMPG vesicles showed a main Tm (22.3oC) and a shoulder at 23.05 oC. This
appearance has been noted by others. 22
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Here, we found that 10-13 caused pronounced changes in the melting profile of DMPG vesicles
(Figure 2). Their presence caused a downward shift (≈ 4 o) in the phase transition temperature
7
8
9
Tm of the phospholipid and a pronounced 6ꢀ9 fold reduction in the calorimetric enthalpy (ꢀH) of
the gel to liquid transition. Collectively these changes pointed to weakened interactions within
the lipid bilayer, conceivably due to the insertion of these compounds within the hydrophobic
core of the bilayer. They also closely mimicked those reported earlier for DMPG vesicles
containing 1, determined under similar conditions. 10
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Intriguingly, the nonꢀMannich base 70b of Series 4 had significantly diminished effects on the
melting profile of the DMPG vesicles, namely a minimal (1o) shift in Tm and a reduction of ꢀH by
only half (Figure 2B). Clearly, 70b did not disrupt the DMPG vesicles to the same extent as 12.
It would seem that relocating the azaspiroketal in 70b so that it is no longer part of a Mannich
base had seriously hampered its interaction with the lipid bilayer.
The azaspiroketal Mannich bases 12 and 13 induced permeabilization of mycobacteria
before onset of cell death.
In view of the perturbative effects of 10-13 on DMPG vesicles, we posited that they should
permeabilize mycobacterial cells. Hence, changes in the permeability of M. bovis BCG cultures
treated with representative members 12 and 13 (4x MIC90) were monitored over time (up to
48h) using the nucleic acid dyes SYTO 9 and propidium iodide (PI). Similar experiments were
carried out with the less potent analog 1 and isoniazid. Briefly, SYTO 9 is taken up by all cells
and emits a green fluorescence whereas PI is selectively concentrated in cells with
compromised membranes where it emits a red fluorescence. A decrease in the green/red ratio
with time would signify progessive permeabilization of the mycobacterial cultures. The ratios
were normalized against values obtained from untreated cultures (no permeabilization) and
SDSꢀtreated cultures (complete permeabilization) to give the % permeabilization. To confirm
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that increased permeability was not an epiphenomenon of cell death, we monitored the viability
of the treated cultures by plating and colony counting over the same time period.
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8
9
Figure 3A shows the timeꢀkill profiles of 12, 13 and isoniazid at 6 h, 12 h, 24 h and 48 h. The
onset of cidal activity was more rapid for isoniazid but by 48 h, all three compounds had killed
the mycobacterial cultures by more than 100ꢀfold. The timeꢀkill profile of 1 was determined
separately and here we found that cultures treated with 1 were still viable at 48h (Figure 3B).
A comparison of the time kill profiles with the loss in membrane permeability at the various time
points showed that 12 and 13 increased the permeability of bacteria before the onset of massive
cell death. This was observed early for 13 (12 h) and later (24 h) for 12 (Figure 3C). 13 induced
noticeably less permeabilization at 24 h than 12 but by 48 h, both compounds had
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permeabilized the cultures to the same extent. The slower acting 1 had a similar timeꢀkill
/permeability profile as 12, except that permeabilization did not increase at 48 h (Figure 3D).
These differences notwithstanding, the overall profile was one in which increased permeability
was incurred early and before the onset of significant cell death, suggesting that increased
permeability may have caused cell death. In the case of isoniazid, whose mode of action does
not directly involve membrane disruption, both events – cell death and permeabilization – were
aligned as would be expected if cell death was causal to the loss of the permeability barrier.
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Figure 3: Timeꢀkill kinetics (A,B) and membrane permeabilization (C,D) of M. bovis BCG
cultures by test compounds (1,12,13) and isoniazid (INH). Mid log phase cultures were treated
with (A) 12, 13 and (B) 1 over 48h at 4x MIC90 for the determination of timeꢀkill profiles. Samples
were concurrently withdrawn at the indicated time points for the determination of green/red
fluorescence ratios, normalized to drug free (DF) inoculum at the same time point, to give %
permeabilization induced by (C) 12, 13 and (D) 1. Dashed lines in (A,B) represent the limit of
detection. Experiments were performed 3 times independently. Mean and SD from
representative experiments are shown
Compounds 12 and 13 induce the mycobacterial promoter piniBAC
The promoter of the mycobacterial iniBAC operon is upregulated under conditions of cell
23. 24
envelope stress such as those encountered when cell wall biosynthesis is disrupted
or
10
when the mycobacterial membrane is permeabilized.
To further verify the membrane
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disruptive effects of the azaspiro ketal Mannich bases, we monitored the fluorescent signal
emitted by cultures of M. bovis BCG expressing Red Fluorescent Protein (RFP) under control of
piniBAC promoter, that had been treated with various concentrations (0.4 ꢀ100 ꢁM) of 1, 12, 13
and the positive control isoniazid. Upregulation of transcriptional activity would increase the
fluorescent signal of the RFP. Here we found that 12 and 13 increased the transcriptional
activity of the piniBAC in a concentration dependent manner (Figure 4). The piniBAC activation
profiles of 1, 12 and 13 were broadly similar to that of isoniazid. In all, the ability of 1, 12 and 13
to perturb phospholipid vesicles, permeabilize mycobacterial cultures and upregulate cell
envelope stress reporter genes were in accord with their membrane disruptive properties.
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Figure 4: Concentration dependent induction of piniBAC promoter activity by 1, 12, 13 and
isoniazid (INH) in a recombinant strain of M. bovis BCGꢀpiniBACꢀRFP after 24 h incubation.
Experiments were performed in biological replicates, data shown here is from a representative
experiment.
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Compounds 12 and 13 did not depolarize mycobacterial membranes.
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We expected the permeabilizing effects of the azaspiroketals on mycobacterial cultures to be
accompanied by a loss in membrane potential. Hence, we examined the ability of 12 and 13 to
dissipate the membrane potential of M. bovis BCG cultures over time (minutes to days) using
the fluorescent membrane permeable dye 3,3ꢀdiethyloxacarbocyanine iodide (DiOC2). Briefly,
DiOC2 emits red fluorescence in cells with polarized membranes and green fluorescence in cells
that are depolarized. A fall in the red/green fluorescence ratio of DiOC2 would indicate a loss in
the membrane potential. Here, we monitored time dependent changes in the DiOC2 ratios of M.
bovis BCG cultures treated with 1, 12, 13, the membrane depolarizer carbonyl cyanide mꢀ
chlorophenylhydrazine (CCCP) and rifampicin.
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As shown in Figure 5, the changes in the DiOC2 ratios of CCCP and rifampicinꢀtreated
mycobacteria reflected their roles as positive and negative controls respectively. In cultures
treated with 12 and 13, the DiOC2 ratios were comparable to drugꢀfree controls over the short
time intervals (Figure 5A,B). Only at Days 3ꢀ5 did the ratios declined, by which time the losses
would be attributed to the dying cultures. A similar observation was made for 1 (Supporting
Information, Figure S3). Thus the azaspiroketal Mannich bases had the surprising effect of not
depolarizing mycobacterial membranes in spite of their ability to induce permeabilization.
There was however precedent to the separation of these closely related membrane effects.
First, several antimycobactericidal indolylpentyltriphenylphosphonium analogs were found to
9
rapidly depolarize M. bovis BCG cultures without inducing membrane permeabilization.
Second, Okano and coworkers reported that peripheral modifications of vancomycin resulted in
a potent analog that increased permeability of Enterococcus faecium without inducing
membrane depolarization or cell lysis.25 The authors posited that the compound in question had
a “specific” mode of action that could have led to membrane perturbation.
We had earlier reported that the indolyl Mannich base 71 (Supporting Iinformation, Figure S2)
induced both permeabilization and depolarization of live M. bovis BCG cultures.10 In view of the
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