Tryptamine derivatives disarm colistin resistance in polymyxin-resistant gram-negative bacteria

Article abstract of DOI:10.1016/j.bmc.2019.03.019

The last three decades have seen a dwindling number of novel antibiotic classes approved for clinical use and a concurrent increase in levels of antibiotic resistance, necessitating alternative methods to combat the rise of multi-drug resistant bacteria. A promising strategy employs antibiotic adjuvants, non-toxic molecules that disarm antibiotic resistance. When co-dosed with antibiotics, these compounds restore antibiotic efficacy in drug-resistant strains. Herein we identify derivatives of tryptamine, a ubiquitous biochemical scaffold containing an indole ring system, capable of disarming colistin resistance in the Gram-negative bacterial pathogens Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli while having no inherent bacterial toxicity. Resistance was overcome in strains carrying endogenous chromosomally-encoded colistin resistance machinery, as well as resistance conferred by the mobile colistin resistance-1 (mcr-1) plasmid-borne gene. These compounds restore a colistin minimum inhibitory concentration (MIC) below the Clinical & Laboratory Sciences Institute (CLSI) breakpoint in all resistant strains.

Full text of DOI:10.1016/j.bmc.2019.03.019

Accepted Manuscript
Tryptamine Derivatives Disarm Colistin Resistance in Polymyxin-Resistant
Gram-negative Bacteria
William T. Barker, Courtney E. Chandler, Roberta J. Melander, Robert K. Ernst,
Christian Melander
PII:
S0968-0896(19)30208-1
https://doi.org/10.1016/j.bmc.2019.03.019
BMC 14811
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 February 2019
6 March 2019
7 March 2019
Please cite this article as: Barker, W.T., Chandler, C.E., Melander, R.J., Ernst, R.K., Melander, C., Tryptamine
Derivatives Disarm Colistin Resistance in Polymyxin-Resistant Gram-negative Bacteria, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.03.019
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Tryptamine Derivatives Disarm Colistin Resistance in Polymyxin-Resistant Gram-negative
Bacteria
William T. Barkera, Courtney E. Chandlerb, Roberta J. Melandera, Robert K. Ernstb, and
Christian Melandera*
aDepartment of Chemistry & Biochemistry, University of Notre Dame, Notre Dame, IN, 46556
USA
bDepartment of Microbial Pathogenesis, University of Maryland-Baltimore, Baltimore, MD
21201 USA
*to whom correspondence should be addressed: cmelande@nd.edu
Abstract
The last three decades have seen a dwindling number of novel antibiotic classes approved
for clinical use and a concurrent increase in levels of antibiotic resistance, necessitating
alternative methods to combat the rise of multi-drug resistant bacteria. A promising strategy
employs antibiotic adjuvants, non-toxic molecules that disarm antibiotic resistance. When co-
dosed with antibiotics, these compounds restore antibiotic efficacy in drug-resistant strains.
Herein we identify derivatives of tryptamine, a ubiquitous biochemical scaffold containing an
indole ring system, capable of disarming colistin resistance in the Gram-negative bacterial
pathogens Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli while having
no inherent bacterial toxicity. Resistance was overcome in strains carrying endogenous
chromosomally-encoded colistin resistance machinery, as well as resistance conferred by the
mobile colistin resistance – 1 (mcr-1) plasmid-borne gene. These compounds restore a colistin
minimum inhibitory concentration (MIC) below the Clinical Laboratory Sciences Institute
(CLSI) breakpoint in all resistant strains.
1. Introduction
The proliferation of antibiotic resistant bacteria threatens a collapse of modern medicine.
As multi-drug resistant (MDR) bacterial strains continue to grow in both strength and number
1

while the antibiotic clinical arsenal remains stagnate, a “Post-Antibiotic World” has become
increasingly tangible. Antibiotic resistant infections currently claim more than 700,000 lives
world-wide each year.1 These infections are most often caused by one or more of the ESKAPE
pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa, and Enterobacter species). Efforts to combat these six
agents have been met with limited success, as only two novel antibiotic classes, lipopeptides and
oxazolidinones, have been deployed clinically in the last two decades. Both are active only
against Gram-positive bacteria, and offer a transient solution as bacterial resistance to each has
been documented.2 Given the lack of treatment options to combat MDR Gram-negative bacteria,
colistin (polymyxin E), a decades-old cationic polypeptide, has reemerged as salvage therapy for
MDR Gram-negative bacterial infections despite causing severe nephrotoxicity.3 This “last
resort” antibiotic is not exempt from resistance development, as numerous primary clinical
isolates have shown resistance that far exceeds the Clinical Laboratory Standards Institute
(CLSI) breakpoint for safe and effective treatment.4
Colistin elicits bacterial cell lysis by displacing divalent cations chelated by phosphate
moieties on lipid A, a component of lipopolysaccharide (LPS), which destabilizes the outer
membrane leading to envelope rupture and cell death.5 Bacteria become resistant to colistin by
modifying lipid A in a number of ways, including modification of the phosphate group with
positively charged residues. Addition of aminoarabinose, galactosamine, and
phosphoethanolamine residues to the terminal phosphate moieties of lipid A, the membrane
anchor of lipopolysaccharide (LPS) has been documented.6 These modifications all impart
decreased electrostatic attraction toward the polycationic polymyxin, leading to drug resistance.
This resistance mechanism is typically evolved under intense selection pressure, comes at a
2

fitness cost, and is controlled and encoded by endogenous genomic material.7 The latter of the
three modifications, however, can also be acquired from the mobile colistin resistance – 1 (mcr-
1) or related genes. These compact, plasmid-borne transcripts can be rapidly disseminated
amongst pathogenic bacteria.8 While mcr-1 was first identified in China, several additional mcr
gene variants (mcr-1-8) have since been found in bacteria across the globe.9
Antibiotic adjuvant therapy offers an alternative to novel antibiotic discovery by targeting
non-essential bacterial systems and restoring susceptibility to a given antibiotic in a concurrent
treatment strategy. Our research group has demonstrated diverse modulation of bacterial
behavior using compounds derived from and inspired by marine natural products. Among these
active compounds, a 2-aminoimidazole (2-AI) or 2-aminobenzimidazole (2-ABI) moiety has
been a key component of the adjuvant scaffold in previous reports.10, 11 Herein, we expand the
breadth of active scaffolds and report that tryptamine, a well-established biogenic amine and
component of the amino acid tryptophan, serves as a synthetic platform for novel, non-toxic
small molecules that successfully disarm colistin resistance, and report a lead molecule active
against multiple colistin-resistant (colR) strains.
2. Results and Discussion
In a previous report from our group, a comprehensive screening of our in-house library
identified that compound 1 (Figure 1) disarms colistin resistance in Gram-negative strains
carrying the mcr-1 gene.12 When dosed at 60 µM, 1 returned up to a 32-fold reduction in the
colistin minimum inhibitory concentration (MIC) against five engineered mcr-1 colR Gram-
negative strains.13 This compound was one of six tryptamine derivatives (1-6, Figure 1) of the
natural product oroidin first prepared in a previous report from our group aimed toward
discovery of anti-biofilm agents against P. aeruginosa.14 Interestingly, biofilm dispersion or
3

inhibition activity was not noted for any of these six molecules. When we screened the remaining
five compounds from this original study (2-6) for synergy with colistin in four colR Gram-
negative strains, two that harbor chromosomally encoded resistance (A. baumannii 4106, K.
pneumoniae B9), and two transfected with the mcr-1 gene contained in a plasmid vector (A.
baumannii ATCC 17978+mcr-1, and E. coli ATCC 25922+mcr-1) (Table 1), we found consensus
activity in the molecule subset. Of these, compound 2 was consistently twice as active as 1 and
returned MICs below the CLSI breakpoint (4 µg/mL) and thus became our lead molecule and the
subject of a structure activity relationship (SAR) study.
Figure 1. Structures of compounds 1-6 and structural segmentation.
Table 1. MIC (µg/mL (fold reduction)) of colistin with in-house library adjuvants dosed
at 60 µM.
A. baumannii
A. baumannii
E. coli ATCC
K. pneumoniae B9
4106
ATCC 17978+mcr-1
25922+mcr-1
No compound
1 (19.3 µg/mL)
2 (24.7 µg/mL)
3 (19.9 µg/mL)
4 (20.8 µg/mL)
5 (16.1 µg/mL)
6 (15.2 µg/mL)
1024 µg/mL
4 (256)
1024 µg/mL
1 (1024)
0.5 (2048)
8 (128)
16 µg/mL
0.5 (32)
0.5 (32)
1 (16)
8 µg/mL
1 (8)
2 (512)
1 (8)
16 (64)
4 (2)
32 (32)
256 (4)
4 (4)
4 (2)
512 (2)
1024 (0)
1024 (0)
16 (0)
8 (0)
512 (2)
8 (2)
8 (0)
With lead compound 2 in hand, we envisioned an SAR strategy that focused on dividing
the molecule along the amide bond and launching two separate diversification campaigns, one
focused on the indole head group and carbon linker, and the other on the brominated carboxy-
pyrrole tail (Figure 1). As each of the thirty-three analogs were synthesized, their activity with
4

colistin was determined in the same four Gram-negative colR strains used previously (Table 2).
Interestingly, all analogs synthesized in this study showed no antimicrobial activity in
monotherapy (MIC = >200 µM) and were initially tested at 60 µM when co-dosed with colistin.
We synthesized a panel of analogs with a variety of changes to the head group scaffold via
condensing 7 with commercially available amines under basic conditions (Scheme 1).14 The
two-carbon linker of 2 was first both shortened and lengthened using 1H-indole-3-methanamine
and homotryptamine as reactive amines to afford compounds 8 and 9 respectively, both of which
showed increased activity over 2 in several strains. Reactions with histamine, benzylamine, 2-
pyridineethanamine, and 2-aminoindane yielded compounds 10, 11, 12, and 13, respectively, all
of which were inactive. We moved the tail group from the 3- position to the 2- position of the
indole ring (14) via reaction with 1H-indole-2-methanamine,and moved it to the 6- position (15)
using 1H-indole-6-methanamine as the amine source. Both 14 and 15 were roughly equipotent
with 2. Using this same methodology, we then employed both 5-hydroxytryptamine and 5-
methoxytryptamine to afford the corresponding indole ring substitution in final products 16 and
17, which were both upwards of eight-fold less active than 2.
Scheme 1. Reaction scheme and structures of compounds 8 through 17. (a): Br2, CHCl3,
2h, RT. (b): RCH2NH2, K2CO3, DMF, 18h, RT.
5

Further modifications to the head group required preparation of derivatized amines
(Scheme 2). We first sought to substitute benzothiophene for indole. Starting from commercially
available benzo[b]thiophene-3-acetonitrile, reduction with SmI2 gave the corresponding
ethylamine 18,15 which was then condensed with 7 to give 19, a product with greatly reduced (32
fold) activity compared to 2. To access additional substitutions of the indole ring, synthesis of
substituted tryptamine precursors was necessary due to either lack of commercial availability or
cost. Briefly, a nitrovinyl group was appended to the 3-position of the appropriately substituted
indole via an addition-elimination reaction with 1-(dimethylamino)-2-nitroethylene (DMANE) in
trifluoroacetic acid and dichloromethane. The nitroalkenes were then reduced with lithium
aluminum hydride at room temperature to give the corresponding tryptamine derivatives.16
Starting from 6-fluoroindole and 7-methylindole, this route gave both 6-fluorotryptamine (22)
and 7-methyltryptamine (23), which were each condensed with 7 to afford 24 and 25 in three
steps overall. This same route also proved useful to access N-alkylated analogs of 2. N-methyl,
N-butyl, and N-benzyl indole (26a-c) were each accessed by N-alkylating with the appropriate
alkyl halide, and were then converted to the nitroalkene intermediate (27a-c) and finally reduced
to the tryptamine derivative (28a-c). Each was then condensed with 7 to afford 29a-c.
Table 2. Initial screen of head-group substituted analogs dosed at 60 µM with colistin
given as MIC (µg/mL (fold reduction)).
A. baumannii
A. baumannii
E. coli ATCC
K. pneumoniae B9
4106
ATCC 17978+mcr-1
25922+mcr-1
No compound
1024 µg/mL
0.5 (2048)
2 (512)
128 (8)
512 (2)
1024 (0)
128 (8)
0.5 (2048)
4 (256)
1024 µg/mL
0.5 (2048)
0.25 (4096)
1024 (0)
512 (2)
512 (2)
1024 (0)
0.25 (4096)
0.5 (2048)
16 µg/mL
0.5 (32)
0.5 (32)
16 (0)
16 (0)
16 (0)
16 (0)
0.5 (32)
0.5 (32)
8 µg/mL
0.5 (16)
0.5 (16)
8 (0)
8 (0)
8 (0)
8 (0)
0.5 (16)
1 (8)
8 (23.8 µg/mL)
9 (25.5 µg/mL)
10 (21.7 µg/mL)
11 (21.5 µg/mL)
12 (22.4 µg/mL)
13 (23.0 µg/mL)
14 (23.8 µg/mL)
15 (23.8 µg/mL)
6

16 (25.6 µg/mL)
17 (26.5 µg/mL)
19 (25.7 µg/mL)
24 (25.7 µg/mL)
25 (25.5 µg/mL)
29a (25.5 µg/mL)
29b (28.0 µg/mL)
29c (30.1 µg/mL)
256 (4)
8 (128)
32 (32)
2 (512)
4 (256)
256 (4)
1024 (0)
1024 (0)
128 (8)
8 (128)
64 (16)
16 (64)
2 (512)
1024 (0)
1024 (0)
1024 (0)
4 (4)
2 (8)
2 (8)
0.5 (32)
0.5 (32)
16 (0)
16 (0)
16 (0)
2 (4)
4 (2)
2 (4)
1 (8)
1 (8)
8 (0)
8 (0)
8 (0)
From the biological data, we see that in general, an indole ring is required for activity
though its orientation with respect to the aliphatic linker is flexible, as is the case for the overall
length of the aliphatic linker itself. Substitutions about the indole ring are well tolerated in some
cases but do not return increases in activity over the original lead 2, while indole N-alkylation
renders a complete loss of activity.
Scheme 2. Reaction scheme and structures of compounds 18 through 29c; (a): SmI2,
TEA, H2O, THF; (b): K2CO3, DMF, 18h, RT (c): NaH, DMF, 0C, 30 min, then R-X,
18h, RT; (d): DMANE, TFA, DCM; (e): LAH, THF.
Turning our attention to the pyrrole tail moiety, tryptamine was either directly reacted
with commercially available acid chlorides, or coupled with commercially available carboxylic
7

acids through activation with PyBOP, to access compounds 30-39 (Scheme 3, biological data
given in Table 3). These compounds offer diversification of the 4,5-dibromopyrrole with
multiple five and six membered heterocycles with varying halide substituents, and revealed that
alteration of the 4,5-dibromopyrrole moiety is deleterious for activity with few exceptions.
Isolated activity was seen with 37 in K. pneumoniae B9, returning a colistin MIC of 1 µg/mL
(1024 fold reduction) when dosed at 60 µM, while remaining inactive in other Gram-negative
strains.
Scheme 3. Reaction scheme and structures of compounds 30 through 39; (a): RCOCl,
TEA, DMF; (b): RCOOH, TEA, PyBOP, DMF.
Mirroring efforts towards the indole N-alkylated analogs 29a-c, we accessed N-alkylated
derivatives of the 4,5-dibromopyrrole moiety, namely N-methyl, N-butyl, and N-benzyl analogs
of 7. Each of these derivatives was condensed with tryptamine to afford compounds 41a-c
(Scheme 4). We observed diminished biological activity with increased steric bulk. To further
explore the impact that halogenation patterns of the pyrrole tail had upon activity, the 4,5-
diiodopyrrole analog 44 was synthesized from 7 (Scheme 4). Iodination of the 2-trichloroacetyl
8

pyrrole using molecular iodine and silver trifluoroacetate generated the 4,5-diiodinated pyrrole
along with the 4-iodopyrrole as a side product as an inseparable mixture.17 Condensation of this
mixture with tryptamine gave both the 4,5-diiodoinated product 44, as well as the 4-iodinated 45,
which were readily isolated on silica gel. Both 44 and 45 were most effective in K. pneumoniae
B9 and returned a 2048 fold reduction in colistin MIC when dosed at 60 µM.
Table 3. Colistin MIC (µg/mL (fold reduction)) of colR strains co-challenged with tail-group
substituted and shortened analogs of 2 dosed at 60 µM.
A. baumannii
A. baumannii
E. coli ATCC
K. pneumoniae B9
4106
ATCC 17978+mcr-1
25922+mcr-1
1024 µg/mL
1024 µg/mL
16 µg/mL
8 µg/mL
No compound
30 (16.2 µg/mL)
31 (17.3 µg/mL)
32 (24.7 µg/mL)
33 (25.7 µg/mL)
34 (20.0 µg/mL)
35 (21.0 µg/mL)
36 (25.4 µg/mL)
37 (20.7 µg/mL)
38 (20.7 µg/mL)
39 (15.9 µg/mL)
41a (25.5 µg/mL)
41b (28.0 µg/mL)
41c (30.1 µg/mL)
44 (30.3 µg/mL)
45 (22.8 µg/mL)
46 (20.7 µg/mL)
47 (29.5 µg/mL)
128 (8)
64 (16)
16 (64)
32 (32)
64 (16)
16 (64)
128 (8)
64 (16)
256 (4)
128 (8)
32 (32)
64 (16)
128 (8)
8 (128)
16 (64)
32 (32)
1024 (0)
1024 (0)
128 (8)
2 (512)
2 (512)
16 (64)
8 (128)
8 (128)
1 (1024)
128 (8)
1024 (0)
4 (256)
64 (16)
64 (16)
0.5 (2048)
0.5 (2048)
32 (32)
1024 (0)
16 (0)
8 (2)
1 (16)
1 (16)
8 (2)
4 (4)
16 (0)
8 (2)
16 (0)
16 (0)
1 (16)
8 (2)
8 (0)
4 (2)
4 (2)
4 (2)
8 (0)
4 (2)
8 (0)
4 (2)
8 (0)
8 (0)
4 (2)
8 (0)
8 (0)
2 (4)
4 (2)
8 (0)
8 (0)
8 (2)
1 (16)
1 (16)
16 (0)
16 (0)
Preliminary screening showed promising results for compounds 8, 37, and 44 (MIC fold-
reduction of >512 in any tested strain, Tables 2 and 3) and thus we elected to combine successful
alterations on both sides of the amide bond (Scheme 4). Coupling 1H-indole-3-methanamine
with 6-bromopicolinic acid delivered 46, a shortened analog of 37. We also generated 47, the
analogous shortened derivative of 44 via condensation of 1H-indole-3-methanamine with the
9

corresponding 4,5-diiodopyrrole to afford 47. Compound 46 had modest activity (32-fold MIC
colistin reduction) against two strains (A. baumannii 4106, K. pneumoniae B9), while compound
47 was completely inactive (Table 3). All analogs having with equal or greater activity
compared to 2 in any of the four test strains were then taken forward for further testing. These
criteria eliminated twenty-three analogs and identified a cohort of nine molecules of interest (8,
9, 14, 15, 24, 25, 37, 44, and 45.)
Scheme 4. Reaction scheme and structures of compounds 41a through 47; (a): NaH,
DMF, 30 mins, 0C, then R-X, RT, 18h; (b): tryptamine, K2CO3, DMF, 18h; (c): I2,
AgCOCF3, CHCl3; (d): 6-bromopicolinic acid, PyBOP, TEA, DMF; (e): 42, K2CO3,
DMF.
These nine compounds were then subjected to further screening with nine additional
bacterial strains: five primary clinical isolates that harbor chromosomally-encoded colistin
resistance (A. baumannii 3941, A. baumannii 3942, A. baumannii 4112, K. pneumoniae A5, and
K. pneumoniae C3) as well as the mcr-1 parent strains without plasmid (A. baumannii ATCC
17978, and E. coli ATCC 25922) and two other colistin sensitive strains (A. baumannii 5075 and
K. pneumoniae ATCC BAA 2146). These results are tabulated in Table 4.
Table 4. MIC (µg/mL (fold reduction)) of colistin with adjuvants dosed at 60 µM against
nine Gram-negative strains.
10

2
8
9
14
15
24
25
37
44
45
(24.7
(23.8
(25.5
(23.8
(23.8
(25.7
(25.5
(20.7
(30.3
(22.8
Colistin
MIC
µg/mL) µg/mL) µg/mL) µg/mL) µg/mL) µg/mL) µg/mL) µg/mL) µg/mL) µg/mL)
512
µg/mL
512
µg/mL
0.5
(1024)
0.25
(2048)
2 (256)
1 (512)
2 (512)
2 (256) 2 (256) 4 (128) 1 (512) 2 (256)
2 (256) 2 (256) 4 (128) 1 (512) 2 (256)
64 (8)
64 (8)
4 (128)
8 (64)
A. baumannii 3941
A. baumannii 3942
A. baumannii 4112
K. pneumoniae A5
K. pneumoniae C3
A. baumannii 5075
4 (128) 16 (32)
1024
µg/mL
0.5
(2048)
4 (256) 2 (512) 4 (256) 2 (512) 4 (256) 128 (8) 8 (128) 32 (32)
1024
1024
µg/mL
512
µg/mL
4 (256) 2 (512) 4 (256) 2 (512) 4 (256) 4 (256) 4 (256)
4 (256) 32 (32)
(0)
0.5
(1024)
0.25
(2048)
0.5
(1024)
0.25
(2048)
1 (512) 1 (512) 1 (512) 1 (512) 1 (512) 4 (128)
1
0.5 (2) 0.25 (4) 0.25 (4) 0.5 (2)
0.25 (4) 0.25 (4) 0.25 (4) 0.5 (2)
0.5 (2)
0.5 (2)
0.5 (2)
1 (0)
0.5 (2)
0.5 (2)
0.5 (2)
0.5 (2)
0.5 (2)
0.5 (2)
0.5 (2)
0.5 (2)
1 (0)
0.5 (2)
0.5 (2)
0.5 (2)
0.5 (2)
1(0)
µg/mL
1
A. baumannii
0.5 (2)
0.5 (2)
0.5 (2)
0.5 (2)
0.5 (2)
0.5 (2)
ATCC 17978parent
µg/mL
1
E. coli ATCC
0.25 (4) 0.25 (4) 0.5 (2)
0.5 (2) 1 (0) 0.5 (2)
0.5 (2)
1 (0)
25922parent
µg/mL
1
K. pneumoniae
ATCC 2146NDM-1
µg/mL
This second round of screening established that all molecules were efficacious in colR
primary clinical isolates of A. baumannii and K. pneumoniae, and identified 8 as the most active
colistin modulator, in some cases returning a 2048-fold reduction in colistin MIC and a four-fold
increase in activity compared to the original lead molecule 2. A subsequent dose-response study
with 8 (Table 5) showed concentration-dependent activity in two Gram-negative clinical isolates
(A. baumannii 3942, K. pneumoniae C3). To explore potential aggregate effects, this same
experiment was repeated supplementing Cation-Adjusted Mueller Hinton Broth (CAMHB) with
0.001% Triton X100. No significant changes in colistin MIC were observed in the presence of
detergent (Table S1, Supporting Information). Additionally, growth curves were constructed for
A. baumannii 3942 treated with 60 µM 8, (Figure S1) which showed that 8 did not perturb
bacterial proliferation. To identify changes in membrane permeability, the BacLight cell viability
assay was used. This duplex colorimetric fluorescence screen offers quantized detection of
potential increases in cell permeability caused by xenobiotics. The ratio of nucleic-acid bound
11

STYO-9 (green-fluorescent), which readily crosses healthy cellular membranes, to the red-
fluorescence of propidium iodide, which only enters cells with compromised cellular envelopes,
is determined by scanning the distinct emission wavelengths of the two dyes. In endotoxin-free
water dosed with 60 µM 8, membrane permeability was increased by a factor of 1.9 in A.
baumannii 3942 and of 1.5 in K. pneumoniae C3 compared to untreated controls. Conversely, no
significant change in cell permeability was detected in either strain treated with the inactive
compound 29b at the same concentration. In a separate assay, neither 8 nor 29b lysed red blood
cells (<1%) when dosed in phosphate-buffered saline (PBS) at 100 µM.
Table 5. Colistin MIC (µg/mL (fold-reduction) of A. baumannii 3942 and K. pneumoniae C3
treated with various concentrations of 8.
A. baumannii 3942
0.25 (2048)
1 (512)
K. pneumoniae C3
0.25 (2048)
1 (512)
60 µM 8 (23.8 µg/mL)
50 µM 8 (19.8 µg/mL)
40 µM 8 (15.9 µg/mL)
30 µM 8 (11.9 µg/mL)
20 µM 8 (7.9 µg/mL)
15 µM 8 (6.0 µg/mL)
10 µM 8 (4.0 µg/mL)
5 µM 8 (2.0 µg/mL)
2 (256)
4 (128)
32 (16)
64 (8)
256 (2)
512 (0)
2 (256)
4 (128)
4 (128)
8 (64)
16 (32)
128 (4)
In conclusion, we have presented the synthesis of thirty-three analogs of a tryptamine-
based lead-molecule previously observed to suppress colistin resistance. This diverse panel
allowed for preliminary relationships between structure and activity to be uncovered indicating
that there are rigid constraints on the scaffold to retain suppression of colistin resistance. The
indole head group is necessary for activity, and neither substitution about the ring nor N-
alkylation enhance efficacy. We further saw that deviation from the 4,5-dibromopyrrole to other
five and six membered heterocycles greatly reduced activity. These molecules are non-toxic to
bacteria, each having an MIC >200 µM in all strains that were studied. Based on activity, this
panel of thirty-three analogs was narrowed to nine compounds for further screening against an
12

additional nine bacterial strains. This second round of screening revealed that while they had
unremarkable activity in colistin-sensitive strains, they were effective in both colR primary
clinical isolates of A. baumannii and K. pneumoniae, as well as strains transfected with the mcr-1
gene, and returned sub-breakpoint colistin MICs, in some cases as low as 0.25 µg/mL.
In the end, shortening the aliphatic linker by one carbon accessed compound 8 with four-
fold greater activity than the parent molecule. In subsequent studies we observed dose-dependent
activity of 8, and when given as monotherapy, it caused no abnormalities in bacterial growth and
did not lyse red blood cells. Currently, the mechanism-of-action of this family of compounds is
unknown. Previous reports from our research group have disclosed colistin adjuvants containing
a 2-AI heterocycle reversed lipid A modification leading to re-sensitization to colisin. These
tryptamine-based adjuvants, however, do not reverse lipid A modification, thus appear to act via
a novel mechanism. In all, this study identified several scaffolds that can be tasked in future
probes of colistin resistance pathways and potentially new leads for further SAR studies while
concurrent in vivo investigations are pursued.
3. Experimental Methods
General Biological Assay Methods: All antibiotics, media, and other biological reagents were
purchased from commercial sources and used without further purification. Compounds
synthesized in this study were dosed from DMSO stocks.
Single Compound MIC Determination via Broth Microdilution: This procedure follows the
guidelines set by the Clinical Laboratory Sciences Institute.4 Bacteria were cultured for 4 to 6
hours in Cation-Adjusted Mueller-Hinton Broth (CAMHB) and subcultured to 5 x105 CFU/mL
in fresh CAMHB. For each compound to be tested, a 1 mL aliquot of subculture was taken and
dosed with compound to a final concentration of 200 µM. Samples were then dispensed (200 μL)
13

into the first row of a 96-well microtiter plate in which subsequent wells were prefilled with 100
μL of subculture. 100 µL of dosed subculture was then serially diluted a total of 6 times in each
subsequent row of the plate save for the last row which served as a control. Plates were then
sealed and incubated stationary at 37°C. After 18 hours, the plates were removed and MIC
values were recorded. All compounds tested had a purity of >95%.
Antibiotic Resensitization MIC Determination via Broth Microdilution: This procedure was
adapted from the guidelines set by the Clinical Laboratory Sciences Institute.4 Bacteria were
cultured for 4 to 6 hours in Cation-Adjusted Mueller-Hinton Broth (CAMHB) and subcultured to
5 x105 CFU/mL in fresh CAMHB. For each compound to be tested, a 5 mL aliquot of subculture
was taken and dosed with adjuvant to a set concentration. A 1 mL aliquot of each dosed
subculture was collected and dosed with antibiotic to a set concentration. Co-dosed aliquots were
then dispensed (200 μL) into the first row of a 96-well microtiter plate in which subsequent wells
were prefilled with 100 μL of the corresponding dosed subculture. 100 µL of co-dosed
subculture was then serially diluted a total of 6 times in each subsequent row of the plate save the
last row as a control to afford serial dilution of the antibiotic while holding a constant
concentration of adjuvant. Plates were then sealed and incubated stationary at 37°C. After 18
hours, the plates were removed and MIC values were recorded. All compounds tested had a
purity of >95%.
Bacterial Growth Curve: Bacteria were cultured overnight in CAMHB and subcultured to 5 ×
105 CFU/mL in fresh CAMHB. The subculture was then transferred to culture tubes in 5 mL
aliquots, which were dosed with adjuvant to a set concentration, save one aliquot as a control.
All subcultures were then incubated at 37°C with shaking. At 2, 4, 6, 8, and 24 hours, 100 µL
samples of each culture was serially diluted in 900 µL aliquots of CAMHB for a total of 5 to 7
14

times. 100 µL of each dilution point were plated on nutrient agar and incubated stationary
overnight. The total number of bacterial colonies on each plate was recorded.
BacLight Cell Permeability Assay: Bacteria were cultured overnight in CAMHB at 37C and
diluted 1:10 in fresh CAMHB and grown for an additional 4 hours to an OD600 of ~0.3. Cultures
were centrifuged at 10,000 g for 15 mins, supernatants were discarded, and cell pellets were
washed once with sterile water and resuspended in 1/10th the original volume in sterile water or
water dosed with compounds. Suspensions were incubated for 1 hour at 37C with shaking and
centrifuged at 10,000 g for 15 mins, washed with sterile water and resuspended in sterile water
supplemented with 1:1 SYTO-9 and propidium iodide (3 µL/mL, from Invitrogen BacLight Kit).
100 µL of each test condition were added to a 96 well plate which was covered and allowed to
stand at room temperature. After 15 mins, the plate was read at 530 nm and 645 nm (excitation
485 nm). The ratio of green to red fluorescence was calculated as a percentage of the control.
Ovine Erythrocyte Lysis Assay: Hemolysis assays were performed on mechanically
difibrinated sheep blood (Hemostat Labs: DSB100). Difibrinated blood (1.5 mL) was placed into
a microcentrifuge tube and centrifuged for 10 min at 10,000 rpm. The supernatant was then
removed and then the cells were resuspended in 1 mL of phosphate-buffered saline (PBS). The
suspension was centrifuged, the supernatant was removed and cells were resuspended two
additional times in PBS. The final cell suspension was then diluted 10-fold in PBS dosed with
test compounds from DMSO stock solutions. DMSO was used as a negative control and a zero
hemolysis marker. Triton X100 (1%) was used as a positive control serving as the 100% lysis
marker. Samples were incubated at 37C with shaking. After one hour, the samples were
centrifuged for 10 min at 10,000 rpm. The supernatant was diluted 1:40 in distilled water. The
absorbance of the supernatant at 540 nm was then measured with a UV spectrometer.
15

General Synthetic Methods: All reagents used for chemical synthesis were purchased from
commercial sources and used without further purification. Flash chromatography was performed
using 60Å mesh silica gel (Sorbtech, Norcross, GA). Both 1H (300 and 400 MHz) and 13C NMR
(75 MHz and 100 Mhz) spectra were collected on Varian Mercury spectrometers at 25C.
Ultraviolet spectra were obtained from a Genesys 10 Scanning UV/Vis spectrophotometer (max
in nm). Infrared spectra were obtained on a solid-phase FT-IR-4100 spectrophotometer (max in
cm-1). High-resolution mass spectra were obtained by the NCSU Molecular Education,
Technology, and Research Innovation Center (METRIC). All NMR spectra are available in this
article’s supplemental information document. The purity of all reported compounds was
determined to be greater than or equal to 95% both by NMR spectra and subsequent liquid
chromatography UV/Vis trace analysis.
General Procedure for Condensing Primary Amines with 2,2,2-trichloroacetylpyrroles:
This method was adapted from methods described by Richards et al.14 One equivalent of the
appropriate 2,2,2-trichloroacetylpyrrole was combined with 2 equivalents of the appropriate
primary amine along with 3 equivalents of anhydrous potassium carbonate and dissolved in 4
mLs of anhydrous dimethylformamide (DMF) under argon and stirred for 18 hours. The reaction
was poured into 75 mLs EtOAc and 25 mLs of deionized water, the aqueous layer was discarded
and the organic was further washed thrice with water, twice with 25 mLs 1N HCl, and once with
25 mLs brine. The organic layer was then dried with anhydrous magnesium sulfate and
evaporated under reduced pressure. The crude solid was purified by flash chromatography (5-
20% EtOAc/Hexanes) to yield the final products.
General Procedure for Synthesis of 3-(2-nitrovinyl) indoles: This procedure was adapted
16
from the methods described by Holloway. The corresponding indole (0.9 equiv) was dissolved
16

in 4 mLs anhydrous dichloromethane (DCM) under argon and stirred for 15 mins at RT. In a
separate flask, N,N-dimethyl-2-nitroethen-1-amine (DMANE) (1 equiv) was dissolved in 3 mLs
trifluoroacetic acid (TFA) and stirred for 15 mins at RT. The solution of indole in DCM was
added dropwise to the stirring solution of TFA and stirred for an additional 30 mins at RT. The
reaction was quenched with the addition of water (10 mLs) and was poured into 25 mLs water
and extracted three times with 25 mLs DCM. The organic layers were combined and dried with
anhydrous magnesium sulfate and evaporated under reduced pressure. The resulted crude solid
was recrystallized from chloroform to give the products with no further purification necessary.
General Procedure for N-alkylation of Indole: Indole (1 g, 8.54 mmols) was dissolved in
15mLs DMF and cooled to 0C under argon. Sodium hydride (410 mg, 10.2 mmols) was added
in portions to the solution which was then stirred at 0C for 30 mins. Two equivalents of the
appropriate alkyl halide were then added to the solution which was allowed to warm to RT for 18
hours. The reaction was poured into 75 mLs EtOAc and 25 mLs of deionized water, the aqueous
layer was discarded and the organic was further washed thrice with water, twice with 25mLs 1N
HCl, and once with 25 mLs brine. The organic layer was then dried with anhydrous magnesium
sulfate and evaporated under reduced pressure. The crude solid was purified by flash
chromatography (5-20% EtOAc/Hexanes) to yield the final products.
General Procedure for the Reduction of N-alkylated 3-nitrovinyl Indoles: This procedure
was adapted from the methods described by Muratore.18 The corresponding N-alkylated 3-
nitrovinyl indole was dissolved in 15 mLs anhydrous tetrahydrofuran (THF) and cooled to -78C
for 20 mins. A 2.4M solution (5 mLs) of lithium aluminum hydride (LAH) in THF) was added
dropwise to the stirring solution which was then allowed to come to room temperature for 18
hours. The stirring solution was then cooled to 0C and the following solutions were added
17

dropwise in sequence: 0.86 mL of water, 0.86 mL of 15% aq. NaOH, and finally, 2.2 mL of
deionized water. After 30 mins, magnesium sulfate was added to the slurry which was
subsequently filtered through a pad of celite and washed twice with 20 mLs DCM. The organics
were evaporated under reduced pressure and the crude amine was taken to further
transformations with no further purification.
General Procedure for the Coupling of Carboxylic Acids to Primary Amines using PyBOP:
The corresponding carboxylic acid (1 equiv), benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP) (1 equiv), and triethylamine (TEA) (3 equiv) were dissolved in
anhydrous DMF under argon and stirred for 15 mins at RT. The appropriate primary amine (2
equiv) was then added in one portion and the reaction was allowed to stir for 18 hours at RT and
then poured into 75 mLs EtOAc and 25 mLs deionized water. The aqueous layer was discarded
and the organic layer was washed twice with water (25 mLs) and once with brine (25 mLs). The
organic layer was dried with magnesium sulfate and evaporated under reduced pressure. The
crude solids were purified by flash chromatography (5-20% EtOAc/Hexanes) to afford pure
products.
N-(2-(1H-indol-3-yl)ethyl)-4,5-dichloro-1H-pyrrole-2-carboxamide (1): Compound was
synthesized using the methods previously reported by Richards et al. Spectral data were
consistent with previous reports.14
N-(2-(1H-indol-3-yl)ethyl)-4,5-dibromo-1H-pyrrole-2-carboxamide (2): Compound was
synthesized using the methods previously reported by Richards et al. Spectral data were
consistent with previous reports.14
18

N-(2-(1H-indol-3-yl)ethyl)-4-bromo-1H-pyrrole-2-carboxamide
(3):
Compound
was
synthesized using the methods previously reported by Richards et al. Spectral data were
consistent with previous reports.14
N-(2-(1H-indol-3-yl)ethyl)-4-bromo-1-methyl-1H-pyrrole-2-carboxamide (4): Compound
was synthesized using the methods previously reported by Richards et al. Spectral data were
consistent with previous reports.14
N-(2-(1H-indol-3-yl)ethyl)-1-methyl-1H-pyrrole-2-carboxamide (5):
Compound
was
synthesized using the methods previously reported by Richards et al. Spectral data were
consistent with previous reports.14
N-(2-(1H-indol-3-yl)ethyl)-1H-pyrrole-2-carboxamide (6): Compound was synthesized using
the methods previously reported by Richards et al. Spectral data were consistent with previous
reports.14
2,2,2-trichloro-1-(4,5-dibromo-1H-pyrrol-2-yl)ethan-1-one (7): Compound was synthesized
using the methods previously reported by Richards et al. Spectral data were consistent with
previous reports.14
N-((1H-indol-3-yl)methyl)-4,5-dibromo-1H-pyrrole-2-carboxamide (8): Compound was
synthesized using the general procedure for condensing primary amines with 2,2,2-
trichloroacetyl pyrroles with (1H-indol-3-yl)methanamine (323 mg, 2.14 mmols) and 7 (396 mg,
1
1.07 mmols) as condensation partners to afford 8 as a tan solid (372 mg, 77%). H NMR (400
MHz, DMSO-d6) δ 12.69 (s, 1H), 10.94 (s, 1H), 8.41 (s, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.36 (d, J
= 8.1 Hz, 1H), 7.28 (s, 1H), 7.07 (t, J = 7.5 Hz, 1H), 7.01 – 6.88 (m, 2H), 4.57 (d, J = 5.5 Hz,
2H); 13C NMR (101 MHz, dmso) δ 158.54, 136.20, 128.26, 126.46, 123.82, 121.20, 118.81,
118.56, 112.71, 112.25, 111.44, 104.21, 97.86, 33.91 ppm; UV (λmax nm) 288; IR vmax (cm-1)
19

3392, 1504, 804, 749, 492; HRMS (ESI) calcd for C14H11Br2N3O [M+H]- 393.9196, found
393.9192.
4,5-dibromo-N-(2-(1-butyl-1H-indol-3-yl)ethyl)-1H-pyrrole-2-carboxamide (9): Compound
was synthesized using the general procedure for condensing primary amines with 2,2,2-
trichloroacetyl pyrroles with (1H-indol-3-yl)propylamine (150 mg, 0.86 mmols) and 7 (287 mg,
1
0.78 mmols) as condensation partners to afford 9 as a tan solid (186 mg, 38%). H NMR (400
MHz, DMSO-d6) δ 12.70 (s, 1H), 8.26 (s, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H),
7.16 (s, 1H), 7.14 – 7.07 (m, 1H), 7.05 – 6.96 (m, 1H), 6.92 (s, 1H), 4.07 (t, J = 6.5 Hz, 2H), 3.62
– 3.39 (m, 2H), 2.91 (t, J = 6.9 Hz, 2H), 1.82 – 1.49 (m, 2H), 1.19 (dd, J = 14.7, 7.3 Hz, 2H),
0.84 (t, J = 7.2 Hz, 3H); 13C NMR (101 MHz, acetone) δ 160.09, 137.48, 129.43, 129.00, 126.97,
122.00, 119.62, 119.26, 113.08, 113.05, 112.35, 110.34, 99.44, 46.24, 40.79, 33.20, 26.21, 20.74,
14.01 ppm; UV (λmax nm) 296; IR vmax (cm-1) 3117, 1611, 1557, 742, 492; HRMS (ESI) calcd for
C19H21Br2N3O [M+H]- 463.9978, found 463.9976.
N-(2-(1H-imidazol-4-yl)ethyl)-4,5-dibromo-1H-pyrrole-2-carboxamide (10): Compound was
synthesized using the methods previously reported by Richards et al. Spectral data were
consistent with previous reports.14
N-benzyl-4,5-dibromo-1H-pyrrole-2-carboxamide (11): Compound was synthesized using the
general procedure for condensing primary amines with 2,2,2-trichloroacetyl pyrroles with
benzylamine (89 µL, 0.81 mmols) and 7 (300 mg, 0.81 mmols) as condensation partners to
afford 11 as a white solid (162 mg, 56%). 1H NMR (300 MHz, DMSO-d6) δ 12.73 (s, 1H), 8.68
13
(s, 1H), 7.40 – 7.17 (m, 5H), 6.98 (d, J = 2.8 Hz, 1H), 4.42 (d, J = 5.8 Hz, 2H); C NMR (75
MHz, dmso) δ 158.93, 139.51, 128.36, 128.06, 127.21, 126.86, 112.73, 104.78, 97.92, 41.99
20

ppm; UV (λmax nm) 286; IR vmax (cm-1) 3187, 1602, 1558, 1232, 711; HRMS (ESI) calcd for
C12H10Br2N2O [M+H]- 354.9087, found 354.9088.
4,5-dibromo-N-(2-(pyridin-2-yl)ethyl)-1H-pyrrole-2-carboxamide (12): Compound was
synthesized using the general procedure for condensing primary amines with 2,2,2-
trichloroacetyl pyrroles with 2-(pyridin-2-yl)ethan-1-amine (97 µL, 0.81 mmols) and 7 (300 mg,
0.81 mmols) as condensation partners to afford 12 as a white solid (97 mg, 32%). 1H NMR (300
MHz, DMSO-d6) δ 12.67 (s, 1H), 8.50 (d, J = 6.4 Hz, 1H), 8.23 (t, J = 5.4 Hz, 1H), 7.69 (td, J =
7.6, 1.9 Hz, 1H), 7.23 (dd, J = 14.0, 7.2 Hz, 2H), 6.87 (s, 1H), 3.56 (q, J = 6.7 Hz, 2H), 2.95 (t, J
13
= 7.3 Hz, 2H); C NMR (101 MHz, dmso) δ 159.05, 158.91, 149.11, 136.55, 128.25, 123.23,
121.60, 112.49, 104.52, 97.84, 38.65, 37.50 ppm; UV (λmax nm) 288; IR vmax (cm-1) 3108, 1626,
1566, 1327, 765; HRMS (ESI) calcd for C12H11Br2N3O [M+H]- 369.9196, found 369.9197.
4,5-dibromo-N-(2,3-dihydro-1H-inden-2-yl)-1H-pyrrole-2-carboxamide (13): Compound
was synthesized using the general procedure for condensing primary amines with 2,2,2-
trichloroacetyl pyrroles with 2-aminoindane (211 µL, 1.62 mmols) and 7 (300 mg, 0.81 mmols)
1
as condensation partners to afford 13 as a gray solid (102 mg, 33%). H NMR (300 MHz,
DMSO-d6) δ 12.68 (s, 1H), 8.29 (d, J = 6.2 Hz, 1H), 7.20 (d, J = 20.0 Hz, 4H), 6.99 (s, 1H), 4.85
13
– 4.38 (m, 1H), 3.22 (dd, J = 16.3, 7.3 Hz, 2H), 2.88 (dd, J = 16.0, 5.5 Hz, 2H); C NMR (101
MHz, dmso) δ 158.80, 141.22, 128.21, 126.46, 124.52, 112.97, 104.50, 97.84, 50.12, 39.07 ppm;
UV (λmax nm) 288; IR vmax (cm-1) 3133, 1635, 1503, 811, 740; HRMS (ESI) calcd for
C14H12Br2N2O [M+H]- 380.9243, found 380.9245.
N-((1H-indol-2-yl)methyl)-4,5-dibromo-1H-pyrrole-2-carboxamide (14): Compound was
synthesized using the general procedure for condensing primary amines with 2,2,2-
trichloroacetyl pyrroles with (1H-indol-2-yl)methanamine (237 mg, 1.62 mmols) and 7 (300
21

1
mg, 0.81 mmols) as condensation partners to afford 14 as a yellow solid (239 mg, 74%). H
NMR (300 MHz, DMSO-d6) δ 12.76 (s, 1H), 10.96 (s, 1H), 8.64 (s, 1H), 7.45 (d, J = 7.7 Hz,
1H), 7.33 (d, J = 8.2 Hz, 1H), 7.07 – 6.99 (m, 2H), 6.98 – 6.89 (m, 1H), 6.28 (s, 1H), 4.57 (s,
2H); 13C NMR (101 MHz, dmso) δ 158.98, 137.01, 136.18, 128.08, 127.98, 120.72, 119.60,
118.92, 113.08, 111.14, 104.82, 99.20, 98.01, 36.25 ppm; UV (λmax nm) 294; IR vmax (cm-1)
3314, 1685, 1521, 1229, 684; HRMS (ESI) calcd for C15H14ClN3O [M+H]- 393.9196, found
393.9198.
N-((1H-indol-6-yl)methyl)-4,5-dibromo-1H-pyrrole-2-carboxamide (15): Compound was
synthesized using the general procedure for condensing primary amines with 2,2,2-
trichloroacetyl pyrroles with (1H-indol-6-yl)methanamine (236 mg, 1.62 mmols) and 7 (300 mg,
0.81 mmols) as condensation partners to afford 14 as a white solid (268 mg, 83%). 1H NMR (300
MHz, DMSO-d6) δ 12.73 (s, 1H), 11.01 (s, 1H), 8.66 (s, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.30 (s,
2H), 7.00 (s, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.38 (s, 1H), 4.51 (d, J = 5.9 Hz, 2H); 13C NMR (101
MHz, dmso) δ 158.85, 136.06, 132.19, 128.30, 126.64, 125.30, 119.90, 118.85, 112.73, 110.00,
104.68, 100.94, 97.97, 42.54 ppm; UV (λmax nm) 292; IR vmax (cm-1) 3403, 1637, 1512, 867, 769;
HRMS (ESI) calcd for C15H14ClN3O [M+H]- 393.9196, found 393.9198.
4,5-dibromo-N-(2-(5-hydroxy-1H-indol-3-yl)ethyl)-1H-pyrrole-2-carboxamide
(16):
Compound was synthesized using the general procedure for condensing primary amines with
2,2,2-trichloroacetyl pyrroles with one exception: final product was purified by flash
chromatograpy (0.5 – 5% MeOH-NH3/DCM) in lieu of EtOAc/Hexanes. 5-hydroxytryptamine
(117 mg, 0.54 mmols) and 7 (200 mg, 0.54 mmols) were condensed to afford 16 as a tan solid
(48 mg, 21%). 1H NMR (400 MHz, DMSO-d6) δ 12.68 (s, 1H), 10.50 (s, 1H), 8.61 (s, 1H), 8.25
(s, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.06 (s, 1H), 6.93 (s, 1H), 6.87 (s, 1H), 6.61 (d, J = 8.6 Hz, 1H),
22

13
3.53 – 3.42 (m, 2H), 2.82 (t, J = 7.0 Hz, 2H); C NMR (101 MHz, dmso) δ 158.86, 150.24,
130.85, 128.44, 127.93, 123.16, 112.46, 111.73, 111.35, 110.79, 104.40, 102.29, 97.83, 39.44,
25.46 ppm; UV (λmax nm) 296; IR vmax (cm-1) 3161, 1569, 1178, 829, 614; HRMS (ESI) calcd for
C15H13Br2N3O2 [M+H]- 423.9301, found 423.9304.
4,5-dibromo-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-1H-pyrrole-2-carboxamide
(17):
Compound was synthesized using the general procedure for condensing primary amines with
2,2,2-trichloroacetyl pyrroles with 5-methoxytryptamine (318 mg, 1.62 mmols) and 7 (300 mg,
0.81 mmols) as condensation partners to afford 14 as a pink solid (207 mg, 58%). 1H NMR (300
MHz, DMSO-d6) δ 12.69 (s, 1H), 10.66 (s, 1H), 8.26 (s, 1H), 7.22 (d, J = 8.6 Hz, 1H), 7.12 (s,
1H), 7.04 (s, 1H), 6.91 (s, 1H), 6.71 (d, J = 8.7 Hz, 1H), 3.53 – 3.39 (m, 2H), 2.87 (t, J = 7.1 Hz,
2H); 13C NMR (101 MHz, dmso) δ 158.94, 153.04, 131.43, 128.46, 127.65, 123.43, 112.50,
112.09, 111.69, 111.18, 104.42, 100.16, 97.85, 55.28, 39.72, 25.35 ppm; UV (λmax nm) 296; IR
vmax (cm-1) 3340, 1606, 1467, 1167, 923; HRMS (ESI) calcd for C16H15Br2N3O2 [M+H]-
437.9458, found 437.9460.
2-(benzo[b]thiophen-3-yl)ethan-1-amine (18): Compound was synthesized using the methods
previously reported by Szostak. Spectral data were consistent with previous reports.15
N-(2-(benzo[b]thiophen-3-yl)ethyl)-4,5-dibromo-1H-pyrrole-2-carboxamide
(19):
Compound was synthesized using the general procedure for condensing primary amines with
2,2,2-trichloroacetyl pyrroles with 18 (100 mg, 0.56 mmols) and 7 (209 mg, 0.56 mmols) as
1
condensation partners to afford 19 as a yellow solid (105 mg, 44%). H NMR (300 MHz,
DMSO-d6) δ 12.71 (s, 1H), 8.35 (s, 1H), 7.95 (dd, J = 16.0, 7.2 Hz, 2H), 7.48 (s, 1H), 7.45 –
13
7.33 (m, 2H), 6.89 (s, 1H), 3.54 (q, J = 6.8 Hz, 2H), 3.05 (t, J = 7.2 Hz, 2H); C NMR (101
MHz, dmso) δ 159.03, 139.73, 138.81, 133.73, 128.24, 124.35, 124.11, 122.98, 122.86, 121.77,
23

112.52, 104.59, 97.85, 38.50, 28.34 ppm; UV (λmax nm) 286; IR vmax (cm-1) 3410, 3117, 2921,
1629, 739; HRMS (ESI) calcd for C15H12Br2N2OS [M+H]- 424.8964, found 424.8969.
(E)-6-fluoro-3-(2-nitrovinyl)-1H-indole (20): Compound was synthesized using the methods
previously reported by Muratore. Spectral data were consistent with previous reports.18
(E)-7-methyl-3-(2-nitrovinyl)-1H-indole (21): Compound was synthesized using the general
procedure for synthesis of 3-(2-nitrovinyl) indoles with 7-methyl indole (800 mg, 6.09 mmols) as
the starting material to afford 21 as an orange solid (730 mg, 59%). 1H NMR (400 MHz, DMSO-
d6) δ 12.22 (s, 1H), 8.35 (d, J = 13.4 Hz, 1H), 8.19 (d, J = 2.7 Hz, 1H), 7.93 (d, J = 13.4 Hz, 1H),
13
7.68 (d, J = 7.8 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 7.00 (d, J = 7.1 Hz, 1H), 2.44 (s, 3H); C
NMR (101 MHz, dmso) δ 137.17, 135.99, 134.82, 131.02, 124.53, 123.99, 122.21, 122.09,
117.98, 108.68, 16.62 ppm; UV (λmax nm) 396; IR vmax (cm-1) 3104, 1609, 1521, 1201, 793;
HRMS (ESI) calcd for C11H10N2O2 [M+H]- 201.0669, found 201.0669.
2-(6-fluoro-1H-indol-3-yl)ethan-1-amine (22): Compound was synthesized using the methods
previously reported by Muratore and taken to the next reaction without further purification.18
2-(7-methyl-1H-indol-3-yl)ethan-1-amine (23): Compound was synthesized using the methods
previously reported by Blough and taken on to the next reaction without further purification.19
4,5-dibromo-N-(2-(6-fluoro-1H-indol-3-yl)ethyl)-1H-pyrrole-2-carboxamide
(24):
Compound was synthesized using the general procedure for condensing primary amines with
2,2,2-trichloroacetyl pyrroles with 22 (204 mg, 1.14 mmols) and 7 (300 mg, 0.81 mmols) as
1
condensation partners to afford 24 as a brown solid (129 mg, 37%). H NMR (300 MHz,
Chloroform-d) δ 11.25 (s, 1H), 8.11 (s, 1H), 7.50 (dd, J = 8.6, 5.3 Hz, 1H), 7.09 – 7.00 (m, 2H),
6.90 (td, J = 9.1, 2.1 Hz, 1H), 6.37 (s, 1H), 5.97 (s, 1H), 3.76 (q, J = 6.4 Hz, 2H), 3.03 (t, J = 6.5
Hz, 2H); 13C NMR (101 MHz, cd3od) δ 161.64, 159.89, 137.95, 128.85, 125.40, 123.79, 120.13,
24

114.21, 113.48, 108.09, 105.88, 99.94, 98.19, 41.34, 26.33 ppm; UV (λmax nm) 288; IR vmax (cm-
1) 2924, 1613, 1556, 1454, 799; HRMS (ESI) calcd for C15H12Br2FN3O [M+H]- 425.9258, found
425.9262.
4,5-dibromo-N-(2-(7-methyl-1H-indol-3-yl)ethyl)-1H-pyrrole-2-carboxamide
(25):
Compound was synthesized using the general procedure for condensing primary amines with
2,2,2-trichloroacetyl pyrroles with 23 (201 mg, 1.14 mmols) and 7 (300 mg, 0.81 mmols) as
condensation partners to afford 25 as a tan solid (141 mg, 41%). 1H NMR (400 MHz, DMSO-d6)
δ 12.70 (s, 1H), 10.79 (s, 1H), 8.27 (s, 1H), 7.40 (d, J = 7.4 Hz, 1H), 7.15 (s, 1H), 6.93 (s, 1H),
6.88 (t, J = 9.1 Hz, 2H), 3.50 (q, J = 6.3 Hz, 2H), 2.91 (t, J = 7.2 Hz, 2H), 2.44 (s, 3H); 13C NMR
(101 MHz, dmso) δ 158.85, 135.79, 128.42, 126.92, 122.39, 121.45, 120.46, 118.49, 115.90,
112.43, 112.19, 104.34, 97.79, 39.57, 25.42, 16.79 ppm; UV (λmax nm) 286; IR vmax (cm-1) 3392,
3085, 1559, 777, 448; HRMS (ESI) calcd for C16H15Br2N3O [M+H]- 421.9509, found 421.9514.
1-methyl-1H-indole (26a): Compound was synthesized via the general procedure for N-
alkylation of indole with methyl iodide as the electrophile. Spectral data were consistent with
previous reports.20
1-butyl-1H-indole (26b): Compound was synthesized via the general procedure for N-alkylation
of indole with butyl iodide (4.84 mLs, 42.7 mmols) as the electrophile to afford 26b as a yellow
oil (1.36 g, 92%). 1H NMR (400 MHz, Chloroform-d) δ 7.90 (d, J = 8.5 Hz, 1H), 7.57 (d, J = 8.2
Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.37 (t, J = 7.4 Hz, 1H), 7.28 (d, J = 3.1 Hz, 1H), 6.74 (d, J =
3.1 Hz, 1H), 4.27 (t, J = 7.1 Hz, 2H), 2.01 (p, J = 7.2 Hz, 2H), 1.59 – 1.51 (m, 2H), 1.17 (t, J =
7.4 Hz, 3H); 13C NMR (101 MHz, cdcl3) δ 136.08, 128.71, 127.79, 121.34, 120.99, 119.22,
109.44, 100.92, 46.06, 32.37, 20.22, 13.75 ppm; UV (λmax nm) 298; IR vmax (cm-1) 2928, 1510,
1313, 734, 425; HRMS (ESI) calcd for C12H15N [M+H]+ 174.1277, found 174.1277.
25

1-benzyl-1H-indole (26c): Compound was synthesized using the methods previously reported
by Yu. Spectral data were consistent with previous reports.21
(E)-1-methyl-3-(2-nitrovinyl)-1H-indole (27a): Compound was synthesized using the general
procedure for synthesis of 3-(2-nitrovinyl) indoles with 26a (536 mg, 4.08 mmols) as the starting
material to afford 27a as an orange solid (115 mg, 14%). 1H NMR (400 MHz, DMSO-d6) δ 8.34
(d, J = 13.4 Hz, 1H), 8.16 (s, 1H), 8.03 – 7.89 (m, 2H), 7.56 (d, J = 8.0 Hz, 1H), 7.33 (t, J = 7.6
13
Hz, 1H), 7.27 (t, J = 7.5 Hz, 1H), 3.84 (s, 3H); C NMR (101 MHz, dmso) δ 139.48, 138.27,
134.20, 131.00, 125.06, 123.42, 122.24, 120.69, 111.26, 107.25, 33.33 ppm; UV (λmax nm) 394;
IR vmax (cm-1) 2918, 1614, 1296, 948, 740; HRMS (ESI) calcd for C11H10N2O2 [M+H]- 201.0669,
found 201.0672
(E)-1-butyl-3-(2-nitrovinyl)-1H-indole (27b): Compound was synthesized using the general
procedure for synthesis of 3-(2-nitrovinyl) indoles with 26b (1.21 g, 6.98 mmols) as the starting
material to afford 27b as a brown oil (545 mg, 32%). 1H NMR (400 MHz, Chloroform-d) δ 8.26
(d, J = 14.6 Hz, 1H), 7.78 – 7.71 (m, 2H), 7.55 (s, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.40 – 7.35 (m,
1H), 7.35 – 7.29 (m, 1H), 4.14 (t, J = 7.1 Hz, 2H), 1.84 (dq, J = 12.5, 5.7, 5.0 Hz, 2H), 1.37 (h, J
13
= 7.3 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H); C NMR (101 MHz, cdcl3) δ 137.74, 136.29, 133.63,
131.51, 125.51, 123.67, 122.32, 120.58, 110.83, 107.91, 46.75, 31.67, 19.96, 13.54 ppm; UV
(λmax nm) 394; IR vmax (cm-1) 2929, 1615, 1294, 1174, 740; HRMS (ESI) calcd for C14H16N2O2
[M+H]+ 245.1284, found 245.1285.
(E)-1-benzyl-3-(2-nitrovinyl)-1H-indole (27c): Compound was synthesized using the general
procedure for synthesis of 3-(2-nitrovinyl) indoles with 26c (770 mg, 3.71 mmols) as the starting
1
material to afford 27c as an orange solid (390 mg, 38%). H NMR (400 MHz, Chloroform-d) δ
8.23 (d, J = 13.4 Hz, 1H), 7.75 (d, J = 13.3 Hz, 2H), 7.53 (s, 1H), 7.37 (td, J = 14.0, 12.6, 9.3 Hz,
26

5H), 7.19 (d, J = 6.0 Hz, 2H), 5.33 (s, 2H); 13C NMR (101 MHz, cdcl3) δ 137.99, 136.27, 135.38,
133.43, 132.14, 129.11, 128.39, 127.17, 125.68, 124.04, 122.63, 120.72, 111.19, 108.55, 50.73
ppm; UV (λmax nm) 394; IR vmax (cm-1) 3105, 1616, 1488, 1247, 701; HRMS (ESI) calcd for
C17H14N2O2 [M+H]- 277.0982, found 277.0982.
(E)-7-methyl-3-(2-nitrovinyl)-1H-indole (28a): Compound was synthesized using the general
procedure for the reduction of N-alkylated 3-nitrovinyl indoles and taken to the next reaction
without further purification.
2-(1-butyl-1H-indol-3-yl)ethan-1-amine (28b): Compound was synthesized using the general
procedure for the reduction of N-alkylated 3-nitrovinyl indoles and taken to the next reaction
without further purification.
2-(1-benzyl-1H-indol-3-yl)ethan-1-amine (28c): Compound was synthesized using the general
procedure for the reduction of N-alkylated 3-nitrovinyl indoles and taken to the next reaction
without further purification.
4,5-dibromo-N-(2-(1-methyl-1H-indol-3-yl)ethyl)-1H-pyrrole-2-carboxamide
(29a):
Compound was synthesized using the general procedure for condensing primary amines with
2,2,2-trichloroacetyl pyrroles with 28a (330 mg, 1.89 mmols) and 7 (701 mg, 1.89 mmols) as
1
condensation partners to afford 29a as a white solid (476 mg, 59%). H NMR (400 MHz,
DMSO-d6) δ 12.75 (s, 1H), 8.33 (s, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.21
– 7.07 (m, 2H), 7.08 – 6.91 (m, 2H), 3.70 (s, 3H), 3.58 – 3.44 (m, 2H), 2.94 (t, J = 6.4 Hz, 2H);
13C NMR (101 MHz, dmso) δ 158.99, 136.70, 128.45, 127.62, 127.13, 121.17, 118.59, 118.43,
112.57, 111.22, 109.60, 104.48, 97.92, 39.77, 32.26, 25.28 ppm; UV (λmax nm) 298; IR vmax (cm-
1) 3113, 1639, 1516, 824, 736; HRMS (ESI) calcd for C16H15Br2N3O [M+H]- 421.9509, found
421.9521.
27

4,5-dibromo-N-(2-(1-butyl-1H-indol-3-yl)ethyl)-1H-pyrrole-2-carboxamide
(29b):
Compound was synthesized using the general procedure for condensing primary amines with
2,2,2-trichloroacetyl pyrroles with 28b (204 mg, 0.95 mmols) and 7 (350 mg, 0.95 mmols) as
1
condensation partners to afford 29b as a tan solid (168 mg, 38%). H NMR (400 MHz, DMSO-
d6) δ 12.70 (s, 1H), 8.26 (s, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.16 (s, 1H),
7.14 – 7.07 (m, 1H), 7.05 – 6.96 (m, 1H), 6.92 (s, 1H), 4.07 (t, J = 6.5 Hz, 2H), 3.62 – 3.39 (m,
2H), 2.91 (t, J = 6.9 Hz, 2H), 1.82 – 1.49 (m, 2H), 1.19 (dd, J = 14.7, 7.3 Hz, 2H), 0.84 (t, J = 7.2
Hz, 3H); 13C NMR (101 MHz, acetone) δ 160.09, 137.48, 129.43, 129.00, 126.97, 122.00,
119.62, 119.26, 113.08, 113.05, 112.35, 110.34, 99.44, 46.24, 40.79, 33.20, 26.21, 20.74, 14.01
ppm; UV (λmax nm) 296; IR vmax (cm-1) 3111, 1638, 1506, 745, 494; HRMS (ESI) calcd for
C19H21Br2N3O [M+H]- 463.9978, found 463.9976.
N-(2-(1-benzyl-1H-indol-3-yl)ethyl)-4,5-dibromo-1H-pyrrole-2-carboxamide
(29c):
Compound was synthesized using the general procedure for condensing primary amines with
2,2,2-trichloroacetyl pyrroles with 28c (400 mg, 1.59 mmols) and 7 (300 mg, 0.81 mmols) as
1
condensation partners to afford 29c as a tan solid (117 mg, 15%). H NMR (300 MHz, DMSO-
d6) δ 12.69 (s, 1H), 8.25 (s, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.32 (s, 1H),
7.24 (t, J = 8.0 Hz, 3H), 7.16 (d, J = 7.7 Hz, 2H), 7.12 – 7.05 (m, 1H), 7.03 – 6.97 (m, 1H), 6.90
(s, 1H), 5.35 (s, 2H), 3.54 – 3.46 (m, 2H), 2.92 (t, J = 7.3 Hz, 2H); 13C NMR (101 MHz, dmso) δ
158.91, 138.40, 136.10, 128.51, 128.40, 127.86, 127.26, 127.04, 126.75, 121.28, 118.74, 118.63,
112.44, 111.71, 110.11, 104.43, 97.84, 48.94, 39.43, 25.16 ppm; UV (λmax nm) 290; IR vmax (cm-
1) 3109, 1638, 824, 746, 490; HRMS (ESI) calcd for C22H19Br2N3O [M+H]- 497.9822, found
497.9821.
28

N-(2-(1H-indol-3-yl)ethyl)thiophene-2-carboxamide (30): Tryptamine (152 mg, 0.984 mmols)
and TEA (0.13 mLs, 0.984 mmols) were dissolved in 3 mLs anhydrous DCM and stirred for 10
mins at RT. To the stirring solution was added thiophene-2-carbonyl chloride (100 µL, 0.984
mmols) dropwise over five minutes. The reaction was stirred for an additional hour at RT and
then poured into 75 mLs EtOAc and 25 mLs of deionized water, the aqueous layer was discarded
and the organic was further washed thrice with water, twice with 25mLs 1N HCl, and once with
25 mLs brine. The organic layer was then dried with anhydrous magnesium sulfate and
evaporated under reduced pressure. The crude solid was purified by flash chromatography (5-
20% EtOAc/Hexanes) to afford 30 as a brown solid (192 mg, 80%).1H NMR (300 MHz, DMSO-
d6) δ 10.83 (s, 1H), 8.65 (s, 1H), 7.74 (d, J = 4.0 Hz, 2H), 7.58 (d, J = 7.8 Hz, 1H), 7.34 (d, J =
8.0 Hz, 1H), 7.18 (s, 1H), 7.16 – 7.11 (m, 1H), 7.07 (t, J = 7.4 Hz, 1H), 6.98 (t, J = 7.3 Hz, 1H),
13
3.57 – 3.47 (q, 2H), 2.94 (t, J = 7.5 Hz, 2H); C NMR (101 MHz, cd3od) δ 164.44, 140.40,
138.12, 131.36, 129.42, 128.78, 128.68, 123.41, 122.31, 119.60, 119.34, 113.30, 112.21, 42.03,
26.38 ppm; UV (λmax nm) 292; IR vmax (cm-1) 3401, 1551, 741, 717, 504; HRMS (ESI) calcd for
C15H14N2OS [M+H]+ 271.0899, found 271.0899.
N-(2-(1H-indol-3-yl)ethyl)-4-chloro-1H-pyrrole-2-carboxamide (31): Compound was
synthesized using the general procedure for condensing primary amines with 2,2,2-
trichloroacetyl pyrroles with tryptamine (81 mg, 0.41 mmols) and 2,2,2-trichloro-1-(4-chloro-
1H-pyrrol-2-yl)ethan-1-one (50 mg, 0.20 mmols) as condensation partners to afford 31 as a tan
solid (46 mg, 79%). 1H NMR (300 MHz, DMSO-d6) δ 11.76 (s, 1H), 10.82 (s, 1H), 8.24 (s, 1H),
7.58 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.17 (s, 1H), 7.07 (t, J = 7.5 Hz, 1H), 7.03 –
13
6.92 (m, 2H), 6.77 (s, 1H), 3.49 (q, J = 6.8 Hz, 2H), 2.91 (t, J = 7.3 Hz, 2H); C NMR (101
29