Broad activity of diphenyleneiodonium analogues against Mycobacterium tuberculosis, malaria parasites and bacterial pathogens

Article abstract of DOI:10.1016/j.ejmech.2017.10.010

In this study, a structure-activity relationship (SAR) compound series based on the NDH-2 inhibitor diphenyleneiodonium (DPI) was synthesised. Compounds were evaluated primarily for in vitro efficacy against Gram-positive and Gram-negative bacteria, commonly responsible for nosocomial and community acquired infections. In addition, we also assessed the activity of these compounds against Mycobacterium tuberculosis (Tuberculosis) and Plasmodium spp. (Malaria). This led to the discovery of highly potent compounds active against bacterial pathogens and malaria parasites in the low nanomolar range, several of which were significantly less toxic to mammalian cells.

Full text of DOI:10.1016/j.ejmech.2017.10.010

Accepted Manuscript
Broad activity of diphenyleneiodonium analogues against Mycobacterium
tuberculosis, malaria parasites and bacterial pathogens
Nghi Nguyen, Danny W. Wilson, Gayathri Nagalingam, James A. Triccas, Elena K.
Schneider, Jian Li, Tony Velkov, Jonathan Baell
PII:
S0223-5234(17)30802-4
10.1016/j.ejmech.2017.10.010
EJMECH 9802
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 June 2017
Revised Date: 7 September 2017
Accepted Date: 4 October 2017
Please cite this article as: N. Nguyen, D.W. Wilson, G. Nagalingam, J.A. Triccas, E.K. Schneider,
J. Li, T. Velkov, J. Baell, Broad activity of diphenyleneiodonium analogues against Mycobacterium
tuberculosis, malaria parasites and bacterial pathogens, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.10.010.
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ACCEPTED MANUSCRIPT
Graphical abstract

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Broad activity of diphenyleneiodonium analogues against Mycobacterium tuberculosis,
malaria parasites and bacterial pathogens
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Nghi Nguyen,a¥ Danny W. Wilson,b¥ Gayathri Nagalingam,c James A. Triccas,c Elena K.
Schneider,a Jian Li,d Tony Velkov,a* Jonathan Baella*
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Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences Monash University, VIC,
Australia, 3052a; Research Centre for Infectious Diseases, School of Biological Sciences,
University of Adelaide, Adelaide 5005, Australiab; Discipline of Biological Sciences, Priority
Research Centre in Reproductive Biology, Faculty of Science and IT, University of
Newcastle, University Drive, Callaghan NSW, 2308, Australiab; Microbial Pathogenesis and
Immunity Group, Discipline of Infectious Diseases and Immunology, Sydney Medical
School, University of Sydney, Sydney, NSW, Australiac; Monash Biomedicine Discovery
Institute, Department of Microbiology, Monash University, VIC, 3800, Australiad
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* Corresponding Authors:
Dr Jonathan Baell, Phone: +61 3 99039044
E-mail : Jonathan.Baell@monash.edu
OR
Dr Tony Velkov, Phone: +61 3 99039539
E-mail: Tony.Velkov@monash.edu
¥These authors contributed equally
Key words: diphenyleneiodonium, type II NADH-quinone oxidoreductase, Gram-negative,
Plasmodium, malaria.

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Abbreviations: DPI, diphenyleneiodonium; NADH, nicotinamide adenine dinucleotide
reduced; NDH-2, type II NADH-quinone oxidoreductases
Abstract
In this study, a structure-activity relationship (SAR) compound series based on the NDH-2
inhibitor diphenyleneiodonium (DPI) was synthesised. Compounds were evaluated primarily
for in vitro efficacy against Gram-positive and Gram-negative bacteria, commonly
responsible for nosocomial and community acquired infections. In addition, we also assessed
the activity of these compounds against Mycobacterium tuberculosis (Tuberculosis) and
Plasmodium spp. (Malaria). This led to the discovery of highly potent compounds active
against bacterial pathogens and malaria parasites in the low nanomolar range, several of
which had favourable toxicity profiles against mammalian cells.
1.
Introduction
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Antibiotic resistance has evolved into a serious global health concern [1, 2]. In the
United States over 23,000 people die each year due to infections with antibiotic-resistant
bacteria. Notwithstanding the human cost, antibiotic resistance is also a massive economic
burden which has been estimated to cost as much as $20 billion USD in excess healthcare
expenses, with associated lost productivity estimated to be as high as $35 billion USD/year
[2]. Sadly, the ‘magic bullet’ antimicrobial therapies we have gratuitously used over the past
decades are rapidly losing their calibre. Modern healthcare over the last century has been
founded on the basis that bacterial infections can be effectively treated using antimicrobial
drugs. In a world without effective antibiotics, modern medical procedures that we take for
granted, such as chemotherapy or simple surgery, will have increasing risk due to the threat
of untreatable bacterial infections. Once again common bacterial infections will more than
often result in death. Medicine is clearly entering a critical period, if bacteria continue
developing resistance to multiple antibiotics at the present rate, and at the same time the
pipeline continues to dry up, there could be catastrophic costs to healthcare and society [3].

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In the developing world, infectious diseases caused by microbial pathogens remain a
major disease burden with malaria (Plasmodium spp.; 429,000 deaths in 2015)[4] and TB
(Mycobacterium tuberculosis; 1,800,000 deaths in 2015)[5] contributing to >2 million deaths
every year globally [6]. Multi-drug resistant malaria parasites, particularly P. falciparum
which causes the greatest burden of mortality, and M. tuberculosis are spreading in the Asia-
Pacific region [7, 8], reducing the effectiveness of current front-line drugs and increasing
potentially deadly treatment failures.
There is an urgent unmet medical need to discover new scaffolds with superior
activity against these problematic human pathogens. Enzymes involved in energy metabolism
are emerging as very important novel drug targets for anti-infective drug development [9-15].
Encouragingly, respiratory chain inhibitors appear to be the Achilles’ heels of dormant non-
replicating cells (‘persisters’), that are often refractory to antibiotics and difficult to treat [16].
Instead of the multi-subunit complex I respiratory enzyme found in mammalian cells,
protozoa, bacteria and plants possess a single sub-unit non-proton pumping, rotenone
insensitive alternative complex I [10, 14, 17, 18]. This type II NADH-menaquinone
oxidoreductase (NDH-2) contains a single non-covalently bound flavin adenine dinucleotide
(FAD) cofactor and catalyzes the oxidation of NADH with menaquinone [19]. The absence
of NDH-2 in the respiratory chain of mammalian mitochondria makes it a very attractive
target for antibiotic drug development.
Diphenyleneiodonium (DPI) is known to be a potent, yet undeveloped, inhibitor of
NDH-2 [15, 20]. A few very early reports investigated the potential of iodonium compounds,
including DPI, as skin antiseptics against Gram-negative bacteria [21-23]. In the present
study we have synthesized a series of novel DPI analogues and evaluated their in vitro
effectiveness against infectious human pathogens that rely on NDH-2 for their energy needs,
namely Gram-positive and Gram-negative bacteria commonly responsible for nosocomial

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and community acquired infections and Mycobacterium tuberculosis. In addition, we
explored the sensitivity of Plasmodium spp. malaria parasites, which have a type II NADH-
menaquinone oxidoreductase of unclear function, to the DPI analogues. The potential of these
compounds as antimicrobial leads with a novel mode of action are discussed.
2.
2.1 Chemistry
The synthetic route to access the diphenyleneiodonium core typically involves 3 steps
Results
as described in the literature (Scheme 1)[24]. The biphenyl amine 3 is classically formed in
good to excellent yield using a Suzuki-coupling reaction between an appropriately substituted
2-iodoaniline 1 and an appropriately substituted phenylboronic acid 2. The biphenyl amine 3
was subsequently transformed into the biphenyl iodide 4 through diazotisation in Sandmeyer
reaction, followed by oxidation of the biphenyl iodide 4 utilising m-CPBA under acidic
conditions to afford the corresponding cyclic diphenyleneiodonium compound 5 (Scheme 1).
The desired final products were obtained as precipitates from the reaction mixture and were
easily isolated in high purity and acceptable yields. This procedure was successfully adopted
for the synthesis of all novel DPI analogues reported herein as shown in Table 1.

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Scheme 1. Synthesis of Cyclic Diphenyleniodonium Trifluoromethanesulfonate 5a aReagents
.
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and conditions: a) Dioxane/H2O (9:1), K2CO3, TBAB, Pd(dppf)Cl2, 130 oC, 1 h; b) 1. THF, 4 M HCl, 0 oC, aq.
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NaNO2, 20 min. 2. aq. KI, 0 oC 10 min, r.t, 1 h; c) CH2Cl2, m-CPBA, TfOH, r.t, 1 h.
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Table 1
Structures of DPI analogues synthesised and numbering system adopted
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Compound
5o
R
Compound
R
Compound
R
Compound
5aa
R
H
5-F, 5’-F
5n
5m
5l
3-F
5i
4-F
5d
5c
5-F
5z
5y
5x
5-F, 5’-Cl
5-Cl, 2’-Cl
5-F, 2’Cl
3-Cl
3-CN
5h
5g
4-Cl
4-CN
5-Cl
5k
5j
3-OMe
3-Me
5f
4-OMe
4-Me
5b
5a
5-OMe
5-Me
5w
5v
5u
5-Me, 2’-Cl
5-Cl, 2-Cl
5-Cl, 2-F
5e
5p
4,5-benzo
5q
5-CF3
5t
5-Cl, 2-OMe

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5r
5-OCF3
5s
5-Cl, 2-Me
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Note: The ring numbering system used herein is only for readers’ convenience (not IUPAC nomenclature).
The compounds were made iteratively in two series. The first series comprises 5o, 5a, 5b, 5c,
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5d, 5e, 5f, 5g, 5h, 5i, 5j, 5k, 5l, 5m, 5n, 5p and will be referred to hereon as DPI series 1,
while DPI series 2 comprises 5q, 5r, 5s, 5t, 5u, 5v, 5w, 5y, 5x, 5z, 5aa.
2.2 Gram-positive and -negative bacteria MIC determination
MIC determinations for DPI series 1 against several problematic Gram-positive and -
negative bacterial species revealed very potent activity for several analogues. In support of
our recent report [25], DPI itself (5o) displayed good activity against Pseudomonas
aeuruginosa (MICs generally 2.3-75 µM) and Acinetobacter baumannii (MICs 0.58-19 µM).
Activity was slightly weaker against Klebsiella pneumoniae (MICs 37 and 75 µM),
vancomycin-resistant Enterococcus faecium (MICs 37 µM) but better against drug resistant
strains of Staphylococcus aureus (MICs 4.7 and 19 µM) (Table 2).
Several analogues had very similar profiles to DPI, these being 5a, 5h, 5i, 5l, 5n and
5p, indicating that activity was not greatly influence by installation of a 5-Me, 4-Cl, 4-F, 3-
CN, 3-F or a fused phenyl ring. One analogue lost significant activity in a broad spectrum
sense, this being 5b, suggesting that the electron donating methoxy group in the 5-position is
particularly disfavoured. There were six other analogues (5e, 5f, 5g, 5j and 5k) that were
broadly similar in profile to DPI with the exception of selectively weaker activity against
certain strains, an example being 5f (4-OMe) and 5k (3-OMe) with strikingly weaker activity
against Pa QLD PSA (70 µM), Pa M146201 and Ab07AC-366 (Table 2). In contrast, 5c
displayed exquisitely potent activity across a broad spectrum of strains, with potency greater
than the detectable limit for 11 bacterial strains.

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The stability of the DPI analogues were investigated by incubating of analogue (5j) in
bacterial culture media under standard assay conditions (37 °C, 200 µL Cation-Adjusted
Mueller-Hinton Broth, 96-well polypropylene microtitre plates) and tested the resulting
chemical species of the compound by LCMS in the next day. The LCMS analysis showed
that the compound was stable under this condition.
Further, the impact of the counter anion towards the biological activity was
investigated. A small selected set of chloride counter ion analogues were synthesised, where
the chloride counter ion replaces the triflate counter ion. These chloride containing
compounds were tested against a selected Gram-negative bacteria (Pa ATCC 27853). The
results indicate that there is no notable anion linked trend in these results, hence there appears
to be no strong link between the biological activity and the presence/absence of triflate anion.
Table 2
Gram-positive and -negative bacteria MIC determination
MIC Activity µM (SI)
5o
5a
5b
5c
5d
5e
5f
5g
PMB
75
(0.39)
9.3
18
(0.14)
4.5
>70
(>0.55)
>70
<0.27
(<2.4)
0.54
2.2
(2.1)
4.5
>73
(>0.52)
36
>70
(>0.29)
70
71
(0.32)
18
Pa ATCC 27853
Pa QLD PSA 1
Pa #912
0.77
0.77
1.5
(3.1)
4.7
(6.3)
2.3
(13)
4.7
(6.3)
9.3
(3.1)
9.3
(3.1)
4.7
(6.3)
2.3
(13)
4.7
(6.3)
19
(1.6)
0.58
(50)
37
(0.78)
37
(0.78)
(0.58)
1.1
(2.3)
0.57
(4.6)
1.1
(2.3)
2.3
(1.2)
2.3
(1.2)
2.3
(1.2)
1.1
(2.3)
2.3
(1.2)
2.2
(>0.55)
70
(0.55)
70
(0.55)
70
(0.55)
>70
(>0.55)
35
(1.1)
70
(0.55)
35
(1.1)
17
(2.2)
70
(0.55)
35
(1.1)
>70
(>0.55)
70
(1.2)
(1.0)
4.5
(1.0)
9.0
(4.2)
9.0
(4.2)
9.0
(4.2)
36
(1.0)
18
(2.2)
9.0
(4.2)
9.0
(4.2)
9.0
(4.2)
18
(2.2)
1.1
(33)
72
(0.52)
72
(0.29)
8.7
2.3)
18
(1.2)
35
(0.58)
70
(0.29)
35
(0.58)
35
(0.58)
18
(1.2)
8.7
(2.3)
70
(0.29)
4.4
(4.6)
70
(0.29)
70
(0.13)
8.8
(2.5)
4.4
(5.1)
8.8
(2.5)
8.8
(2.5)
18
(0.13)
18
(0.13)
4.4
(5.1)
2.2
(10)
71
(0.32)
2.2
(10)
35
(0.63)
71
<0.27
(<2.4)
<0.27
(<2.4)
<0.27
(<2.4)
0.54
(1.0)
<0.28
(<16)
<0.28
(<16)
1.1
(4.1)
0.56
(8.2)
<0.28
(<16)
0.56
(8.2)
0.56
(8.2)
1.1
Pa 19147nm
25
Pa 18878B klon 1
Pa M146201
>25
3.1
(1.2)
<0.27
(<2.4)
<0.27
(<2.4)
<0.27
(<2.4)
<0.27
(<2.4)
<0.27
(<2.4)
<0.27
(<2.4)
4.3
Ab ATCC 19606
Ab 246-01-C
0.77
0.19
0.38
98
Ab ATCC 17978
Ab ATCC 19606 col10
Ab 07AC-336
(1.2)
0.57
(4.6)
18
(0.14)
36
(4.1)
<0.28
(<16)
4.5
(1.0)
9.0
6.2
Ab ATCC 17978 col10
Kp ATCC 13883
Kp M320445
12
(0.15)
8.6
(0.08)
12
(0.07)
(0.55)
(0.51)
(0.52)
(0.29)
(0.32)
0.38

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75
72
>70
8.6
9.0
>72
>70
>71
Kp #1
(0.78)
75
(0.78)
37
(0.78)
37
(0.78)
37
(0.04)
>72
(>0.04)
>72
(>0.04)
18
(0.14)
>72
(>0.55)
>70
(>0.55)
>70
(>0.55)
70
(0.55)
>70
(0.08)
8.6
(0.08)
8.6
(0.08)
0.54
(1.2)
0.54
(1.2)
2.2
(0.51)
9.0
(0.51)
9.0
(0.51)
1.1
(4.2)
1.1
(>0.52)
>72
(>0.52)
72
(0.52)
72
(0.52)
>72
(>0.29)
>70
(>0.29)
>70
(>0.29)
>70
(>0.29)
>70
(>0.32)
>71
(>0.32)
71
(0.32)
71
(0.32)
>71
98
>25
12
Kp 224-11-C
Kp 248-33-D
Ec N2381
1.5
Ec N4149
(0.78)
37
(>0.04)
18
(>0.55)
>70
(4.2)
4.5
(>0.52)
72
(>0.29)
>70
(>0.32)
71
1.5
Ec N11281
(0.78)
4.7
(6.3)
4.7
(6.3)
19
(1.6)
19
(0.14)
2.3
(1.2)
36
(0.07)
36
(0.07)
36
(>0.55)
8.7
(4.4)
35
(1.1)
35
(1.1)
35
(0.31)
<0.27
(<2.4)
1.1
(0.61)
2.3
(0.29)
1.1
(0.61)
(1.0)
0.56
(8.2)
2.2
(2.1)
2.2
(2.1)
2.2
(2.1)
(0.52)
4.5
(8.3)
9.0
(4.2)
18
(2.2)
18
(>0.29)
4.4
(4.6)
18
(1.5)
35
(0.58)
35
(0.32)
1.1
(20)
1.1
(20)
8.8
(2.5)
8.8
1.5
VRE ATCC 700221
MRSA ATCC 43300
VISA ATCC 700698
VRSA ATCC 700699
>25
>25
>25
>25
(1.6)
(0.07)
(1.1)
(2.2)
(0.58)
(2.5)
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Table 2. (continued)
MIC Activity µM (SI)
5h
8.7
(1.9)
8.7
(1.9)
4.3
(3.7)
4.3
(3.7)
2.2
5i
18
(0.67)
18
(0.67)
2.2
(5.3)
1.1
(11)
2.2
5j
72
(0.05)
>72
(>0.05)
4.5
(0.81)
4.5
(0.81)
4.5
5k
>70
(>0.11)
70
(0.11)
>70
(>0.11)
8.7
(0.81)
18
5l
35
(0.09)
8.8
(0.37)
4.4
(0.75)
2.2
(1.5)
4.4
5m
8.7
(0.13)
2.2
(0.51)
1.1
(1.0)
0.54
(2.0)
0.54
(2.0)
2.2
5n
8.9
(0.66)
8.9
(0.66)
1.1
(5.3)
0.56
(11)
1.1
5p
8.4
(0.09)
4.2
(0.17)
2.1
(0.34)
2.1
(0.34)
2.1
PMB
0.77
0.77
1.5
Pa ATCC 27853
Pa QLD PSA 1
Pa #912
Pa 19147nm
25
Pa 18878B klon 1
Pa M146201
(7.5)
4.3
(3.7)
4.3
(5.3)
4.5
(2.7)
4.5
(0.81)
36
(0.11)
9.1
(0.39)
70
(0.11)
18
(0.75)
18
(0.19)
18
(5.3)
2.2
(2.6)
4.5
(0.34)
8.4
(0.09)
4.2
>25
3.1
(0.51)
2.2
Ab ATCC 19606
Ab 246-01-C
(3.7)
4.3
(3.7)
2.2
(7.5)
2.2
(7.5)
8.7
(1.9)
0.5
(30)
8.7
(1.9)
35
(0.47)
35
(2.7)
4.5
(2.7)
2.2
(5.3)
2.2
(5.3)
9.1
(1.3)
0.56
(2.0)
18
(0.67)
18
(0.67)
36
(0.33)
(0.41)
18
(0.21)
4.5
(0.81)
4.5
(0.81)
18
(0.21)
1.1
(3.2)
72
(0.05)
36
(0.11)
72
(0.39)
35
(0.21)
18
(0.39)
2.2
(3.1)
>70
(>0.11)
2.2
(3.1)
>70
(>0.11)
70
(0.11)
>70
(0.19)
18
(0.19)
4.4
(0.75)
2.2
(1.5)
>71
(>0.05)
2.2
(1.5)
18
(0.19)
35
(0.09)
>71
(0.51)
2.2
(0.51)
0.54
(2.0)
1.1
(1.0)
2.2
(0.51)
<0.27
(<4.1)
8.7
(0.13)
8.7
(0.13)
17
(0.06)
(2.6)
4.5
(2.6)
1.1
(5.3)
2.2
(2.6)
8.9
(0.66)
0.56
(11)
36
(0.16)
36
(0.16)
72
(0.08)
(0.17)
4.2
(0.17)
1.1
(0.69)
1.1
(0.69)
8.4
(0.09)
0.52
(1.4)
8.4
(0.09)
17
(0.04)
34
(0.02)
0.77
0.19
0.38
98
Ab ATCC 17978
Ab ATCC 19606 col10
Ab 07AC-336
Ab ATCC 17978 col10
Kp ATCC 13883
Kp M320445
6.2
12
12
0.38
98
Kp #1
(0.47)
(0.05)
(>0.11)
(>0.05)
35
36
72
>70
>71
17
72
34
Kp 224-11-C
(0.47)
17
(0.93)
(0.33)
18
(0.67)
(0.05)
72
(0.05)
(>0.11)
>70
(>0.11)
(>0.05)
>71
(>0.05)
(0.06)
17
(0.06)
(0.08)
36
(0.16)
(0.02)
34
(0.02)
>25
12
Kp 248-33-D
17
18
72
35
71
8.7
18
8.4
Ec N2381
(0.93)
17
(0.67)
36
(0.05)
72
(0.21)
>70
(0.05)
71
(0.13)
8.7
(0.08)
18
(0.09)
34
1.5
Ec N4149
(0.93)
(0.33)
(0.05)
(>0.11)
(0.05)
(0.13)
(0.08)
(0.02)
1.5
17
18
36
>70
71
4.3
18
17
Ec N11281
(0.93)
8.7
(0.67)
4.5
(0.11)
4.5
(>0.11)
4.4
(0.05)
2.2
(0.26)
1.1
(0.08)
2.2
(0.04)
1.1
1.5
VRE ATCC 700221
MRSA ATCC 43300
VISA ATCC 700698
(1.9)
2.2
(7.5)
4.3
(3.7)
(2.7)
4.5
(2.7)
18
(0.67)
(0.81)
4.5
(0.81)
9.1
(0.41)
(1.6)
4.4
(1.6)
8.7
(0.78)
(1.5)
4.4
(0.75)
8.8
(0.37)
(1.0)
2.2
(0.51)
2.2
(0.51)
(2.6)
2.2
(2.6)
8.9
(0.66)
(0.69)
2.1
(0.34)
4.2
(0.17)
>25
>25
>25

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4.3
9.1
9.05
8.7
8.8
2.2
8.9
4.2
VRSA ATCC 700699
(3.7)
(1.3)
(0.398)
(0.78)
(0.37)
(0.51)
(0.66)
(0.17)
>25
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(SI) = selectivity index relative to HEPG2 cells and was calculated by CC50 (1%FBS)/MIC.
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2.3 Inhibition of Mycobacterium tuberculosis growth
Shown in Table 3 are the results of testing DPI series 1 compounds and DPI series 2
compounds against M. tuberculosis. It can be seen in Table 3 that all compounds displayed
anti-mycobacterial activity and that the weakest compound, 5b, nicely matched the
observation that this compound was also the weakest in the bacterial panel. Further, 5e, 5f, 5j
and 5k are also common to the groupings of compounds that are slightly less potent than DPI.
The observation that 5c was very potent in the bacterial panel but also amongst the
most potent compounds against M. tuberculosis led us to synthesise a set of second
generation analogues based around the structure of 5c. This compound contains a 5-chloro
substituent and so our DPI series 2 focuses on analogues with a halogen in a 5-position.
Gratifyingly, as show in Table 3, these compounds were uniformly extremely potent. In
particular, 5s, with 5-Cl, 2-Me substitution, was extremely potent, and 3-fold more potent
than 5o (DPI itself), with an MIC of 0.13 µM.
Table 3
Inhibition of Mycobacterium tuberculosis growth
DPI Series 1
MIC µM (SI)
0.30 (96)
0.14 (19)
4.4 (8.8)
DPI Series 2
MIC µM (SI) (SI)a
0.26 (0.09) (0.36)a
0.25 (0.21) (0.99)a
0.13 (0.74) (2.8)a
0.51 (0.31) (0.44)a
0.27 (3.4) (10)a
5o
(5a
5b
5c
5q
5r
5s
0.28 (2.4)
0.29 (16)
0.57 (66)
1.1 (19)
5t
5d
5e
5u
5v
5w
0.50 (1.8) (3.7)a
5f
0.27 (0.22) (1.8)a

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5g
0.29 (78)
0.54 (30)
0.29 (41)
0.57 (6.4)
0.28 (24)
0.29 (12)
0.28 (3.9)
0.29 (20)
0.52 (2.4)
0.30
5x
0.27 (0.19) (1.4)a
0.26 (0.32) (2.1)a
1.0 (0.38) (0.80)a
0.28 (0.32) (0.58)a
5h
5y
5i
5z
5j
5aa
5k
5l
5m
5n
5p
Rifampicin
0.61
161
162
163
(SI) = selectivity index relative to HEPG2 cells and was calculated by CC50 (1%FBS)/MIC.
(SI)a = selectivity index relative to HEPG2 cells and was calculated by CC50 (10%FBS)/MIC.
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2.4 In vitro activity of DPI against Plasmodium spp.
DPI has been shown to inhibit P. falciparum growth in vitro, however, the reported
IC50 values and the proposed mechanism of action vary [26, 27] . In this study, the growth
inhibitory IC50 for D10 (chloroquine sensitive; 0.13 µM) parasites after 90 hours of treatment
(early ring stage to late trophozoite stage next cycle) was 3.3-fold higher than that achieved
for the CS2 chloroquine resistant line (0.04 µM; Table 4 and Suppl. Fig. 1A), indicating that
DPI is inhibitory to both chloroquine resistant and sensitive strains in vitro. The zoonotic
human pathogen P. knowlesi YH1[28], an emerging pathogen in Southeast Asia and a
laboratory adapted model for the major human pathogen P. vivax, had an IC50 5.6 fold higher
than that of the chloroquine sensitive D10 line at 0.74 µM (Table 4 and Suppl. Fig. 1A).
Whether this represents a difference in parasite sensitivities, or is due to the shorter lifecycle
of P. knowlesi (around 32 hours in these assays) and therefore reduced exposure time is not
clear.
The in vitro growth inhibitory activity after 90 hours of treatment of the 26 DPI
analogues was tested against the D10 chloroquine sensitive line (Table 5, Suppl. Fig. 1B and

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1C). The activity of the analogues ranged from >3-fold decrease in growth inhibitory IC50
compared to DPI (to below 0.045 µM), to a loss of growth inhibition up to a concentration of
1 µM. Six of the 26 analogues had a greater than 2-fold increase in growth inhibitory activity.
P. knowlesi growth inhibition was also tested against three analogues which displayed
increased potency against P. falciparum. Analogues 5c (P.k. YH1 0.08 µM versus D10 0.05
µM, p>0.054, Table 5), 5d (P.k. YH1 0.08 µM versus D10 0.05 µM, p>0.032) and 5g (P.k.
YH1 0.11 µM versus D10 0.04 µM, p>0.0016), showed significantly improved potency over
DPI and inhibited P. knowlesi growth to a similar extent to P. falciparum.
Next we attempted to make the D10 chloroquine sensitive line resistant to DPI. After
6 days of treatment with 0.2 µM DPI, the DPI concentration was raised to 0.4 µM for a
further 6 days. The DPI concentration was then reduced back to 0.2 µM and parasites grown
continuously under drug pressure. After a further 18 days of culture, viable parasites were
visible and maintained under drug pressure until a stable population could be obtained (called
D10-DPIR). A sub culture of DPI selected parasites was made and the drug pressure was
removed to test whether a tolerant, rather than resistant, parasite population had been selected
for (line with DPI drug pressure removed for between 2-5 weeks is called D10-DPIoff). There
was no statistical difference between the growth inhibitory IC50 for the D10-DPIr line (0.36
µM) and the D10-DPIoff line (0.46 µM; p=0.264). In contrast, there was a ~2-fold difference
between both the D10-DPIr (p=0.011) and D10-DPIoff (p=0.005) lines compared to D10 (0.19
µM, Fig. 2) in parallel experiments. Sequence comparison between the D10-PfPHG parental,
D10-DPIr and D10-DPIoff lines showed no mutations in PfNDH2, suggesting it is unlikely
that mutations in PfNDH2 are causing the reduced sensitivity to DPI. The sequence of Type
II Pf dihydroorotate dehydrogenase, reported as an alternative target of PfNDH2 inhibitors
[27], was also found to have no mutations associated with reduced sensitivity to DPI. It
remains to be determined the mechanism by which resistance to DPI occurs in these

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parasites. Attempts to select drug resistant parasites at a DPI concentration of 0.8 µM
between days 6 and 12 were unsuccessful.
Changes in sensitivity of the D10-DPIR line to more potent DPI analogues 5q, 5c, 5d
and 5g were assessed by comparing the growth inhibitory IC50 between the resistant and
parental lines (Table 5). The D10-DPIR line was significantly more tolerant of analogue 5g
(D10-DPIR 0.09 µM versus D10 0.04 µM, p=0.003), but not of 5c (D10-DPIR 0.07 µM versus
D10 0.05 µM, p=0.165), 5d (D10-DPIR 0.08 µM versus D10 0.05 µM, p=0.0567) and 5q
(D10-DPIR 0.14 µM versus D10 0.13 µM, p=0.091). These data indicate that selection for a
DPI resistant line did not necessarily confer resistance to analogues of DPI.
In general, the potencies of DPI analogues in series 1 for bacterial species and M.
tuberculosis (e.g increased potency for 5c; reduced potency for 5b) were reflected in the
relative potency for P. falciparum. However, some discrepancies were observed with 5g and
5l, both potent inhibitors of P. falciparum that were very poor inhibitors of bacteria. This
result suggests that the hydrophilic electron withdrawing groups at the 3- and 4-position are
particularly disfavoured against bacterial isolates. In terms of series 2 compounds, there were
broad similarities between the IC50 for P. falciparum and M. tuberculosis (R2 0.27, p=0.101),
with the exception of 5u and 5v which were inhibitory to M. tuberculosis but not P.
falciparum growth, suggesting that 2 electron withdrawing substituents at the 2- and 5-
position on the same phenyl ring are not tolerated for P. falciparum growth inhibition. When
5u and 5v were removed from the comparison, the correlation between growth inhibitory
IC50s for P. falciparum and M. tuberculosis improved significantly (R2 0.455, p=0.046),
confirming the broad similarity of drug potency for series 2 compounds against these
important human pathogens.

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Table 4
In vitro activity of DPI against P. falciparum and P. knowlesi malaria
Parasite line
IC50-90 hour
(µM)
Pf D10 (chloroquine sensitive)
Pf CS2 (chloroquine resistant)
P. knowlesi YH1
0.13
0.04
0.74
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Table 5
In vitro activity of DPI analogues against P. falciparum and P. knowlesi malaria
DPI
IC50 µM (SI)
Pf D10
IC50 µM (SI)
Pf D10 r
IC50 µM (SI)
Pk YH1
DPI
IC50 µM (SI) IC50 µM (SI)
(SI)a
(SI)a
Series 1
Series 2
Pf D10
Pf D10r
5o
5a
5b
5c
5d
5e
5f
0.13 (221)
0.07 (37)
5q
5r
5s
0.13 (0.18)
(0.72)a
0.14 (0.18)
(0.70)a
0.13 (0.39)
(1.9)a
0.17 (225)
0.05 (13)
0.11 (0.82)
(3.1)a
0.07 (8.9)
0.08 (61)
0.08 (8.5)
0.08 (59)
5t
0.13 (1.2)
(1.75)a
0.05 (96)
5u
5v
5w
5x
5y
5z
5aa
>1 (>0.93)
(>2.7)a
0.08 (500)
0.10 (213)
0.04 (531)
0.08 (194)
0.10 (125)
0.23 (15.8)
>1 (>0.89)
(>1.9)a
0.11 (0.63)
(5.1)a
5g
5h
5i
0.09 (256)
0.11 (232)
0.19 (0.28)
(2.1)a
0.36 (0.23)
(1.5)a
>1 (>0.39)
(0.83)a
5j
0.84 (0.11)
(0.19)a
5l
0.05 (62)
0.07 (15)
5m

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5n
0.06 (105)
0.12 (5.8)
0.03
5p
Chloroquine
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(SI) = selectivity index relative to HEPG2 cells and was calculated by CC50 (1% FBS)/IC50.
(SI)a = selectivity index relative to HEPG2 cells and was calculated by CC50 (10% FBS)/MIC.
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Fig. 1A
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Fig. 1B
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Fig. 1C

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Supplementary Fig 1: P. falciparum growth inhibitory activity of DPI and analogues in vitro. (A) Dose
response curves demonstrate that DPI is inhibitory to P. falciparum growth in vitro, with an IC50 <0.2 µM after
90 hours of parasite treatment for both chloroquine sensitive (D10) and chloroquine resistant (CS2) lines.
Analogues of DPI in many cases demonstrated improved growth inhibitory activity against D10 parasites with
(B) 11 out of 15 analogues for series 1, and (C) 1 out of 11 analogues for series 2 demonstrating >2 fold
reduction in growth inhibitory IC50.
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Fig 2. Parasite line selected for resistance to DPI shows reduced drug sensitivity. D10 parasites selected for
resistance to DPI during continuous culture (D10-DPIR) exhibited ~2 fold reduction in sensitivity to DPI, even
after extended removal of drug pressure (D10-DPIoff, 2-5 weeks without drug pressure) when compared to D10
parental parasites.
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2.5 Cytotoxicity assay

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In parallel with the above assays, we assessed both series of DPI analogues for
cytotoxicity against HEPG2 cells. As shown in Table 6, DPI series 1 displayed a wide range
of cytotoxicity in 1% FBS, from as low as 0.66 µM for 5c to as high as 38 µM for 5b. Ten of
the 15 series 1 compounds had improved antimalarial activity in the low nM range (Table. 5;
5a, 5d, 5e, 5f, 5g, 5h, 5i, 5k, 5l, 5m) whilst maintaining a CC50 above 1 µM. For M.
tuberculosis (Table. 4), 8 out of 15 compounds met this criteria (5a, 5d, 5g, 5i, 5k, 5l, 5m,
5n), confirming that improved DPI analogue activity against broad pathogens is not
absolutely at the expense of mammalian cell toxicity. As seen for compound 5c from which
series 2 was derived, analogues with a halogen in a 5-position were significantly more
cytotoxic (Table 6). However, this is at conditions artificially low in serum concentration
(1%). When retested in the presence of 10% FBS, cytotoxicity decreased up to 8-fold and
toxicity would decrease further in vivo where serum concentration is 100%. Nevertheless,
cytotoxicity, particularly for the series 2 compounds, is a clear potential liability and will
need to be addressed in future DPI analogue optimisation studies.
Table 6
Cytotoxicity Assay
CC50[µM]
1%FBS
29.2
CC50[µM]
1%FBS
DPI Series 1
10%FBS
DPI Series 2
10%FBS
5o
5a
5b
5c
5d
5e
5f
5q
5r
5s
0.02361
0.05026
0.09295
0.1504
0.9303
0.8938
0.0601
0.05238
0.0947
0.2508
0.3488
0.2242
2.724
2.6
38.3
5t
0.66
5u
5v
5w
5x
4.6
37.5
1.848
20.2
0.4863
0.3864
5g
22.3

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5h
5i
5y
16.1
11.9
3.6
0.08457
0.3915
0.5436
0.8331
0.161
5z
5j
5aa
0.08862
5k
5l
6.8
3.3
5m
5n
5p
1.1
5.9
0.72
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3.
Discussion
There has been a steady decline in the number of FDA approved antibiotics, with only
10 antibiotics considered “New Molecular Entities” being approved by the FDA from 2004 to
2012 [29, 30]. This is dwarfed in comparison to the 20 new classes of antibiotics developed
between 1930 and 1962, and the 30 new antibiotics approved between 1983 and 1992 [29].
There were no new classes of antimicrobials discovered between 1968 and 2000, and the two
novel classes discovered in 2000 and 2003 (daptomycin and linezolid) are only effective
against Gram-positive bacteria [29]. The lack of new antibiotics renders physicians impotent
to treat emerging resistance to existing antibiotics. Globally, spreading resistance to
antimalarial and anti-tubercular drugs is of urgent concern [7, 8], with increasing treatment
failures and the potential for increased mortality in the years ahead unless alternative
treatments become available. The present report helps address this urgent need for new anti-
infectives by evaluating the in vitro antibacterial and antimalarial activity of a novel series of
DPI compounds.
In view of the increasing incidence of MDR pathogens there is an urgent need for new
antibiotics with novel modes of action. Agents that selectively target respiratory enzymes that
are unique to the pathogen such as NDH-2 in the electron transport chain of bacteria and

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certain parasites will offer a superior approach for treating persistent infections, greatly
reduced treatment periods and provide a high selectivity for the pathogen versus the host. DPI
has been show to inactivate flavin enzymes via a radical reaction mechanism which leads to
the covalent modification of the reduced flavin cofactor [31-33]. The reduced flavin transfers
an electron to DPI, generating semiquinone and a diphenyliodyl radical. The free radical
fragments to give iodobenzene and a phenyl radical, the latter undergoes recombination with
the flavin semiquinone to form various phenyl adducts [32]. We have previously shown that
DPI inhibits NDH-2 activity in isolated Escherichia coli membranes [34]. Coincidently, we
also demonstrated that a secondary mode of action of the polymyxin lipopeptide antibiotics
against Gram-negative bacteria involves the inhibition of NDH-2 activity [34].
The unique structure of the Gram-negative cell wall provides an often impermeable
barrier to antibiotics, particularly hydrophobic compounds [35]. A major advantage of diaryl-
iodonium compounds is their amphipathic character which allows them to easily cross even
the most formidable membrane barrier such as the Gram-negative cell wall. Despite their
apparent attractiveness as anti-infective agents, iodonium compounds have undergone limited
drug development.
A few very early reports investigated the potential of iodonium compounds including
DPI as skin antiseptics against Gram-negative bacteria [21-23]. The compounds per se or as
emulsions with pine oils showed bactericidal activity. The MIC values reported against both
Gram-positive and Gram-negative bacteria obtained in the present study were much lower
( 10-fold) compared to those of early reports, suggesting DPI is much more active than
previously thought [36-38]. This discrepancy may have arisen due to the fact these authors
employed DPI dissolved in aqueous solutions without any co-solvent and then attempted to
estimate the concentration using the dipicrylamine chemical reactivity technique [36-38].

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Acute toxicity studies with dogs (received an intraperitonal dose of 70 mg/kg) and
monkeys (received intraperitonal doses of doses of 30 and 50 mg/kg) indicated DPI has some
toxic effects on the central nervous system and skeletal muscle at high concentrations [39,
40]. DPI has also been shown to act as a primary eye irritant and a moderate irritant via
dermal exposure in humans [41, 42]. In oral exposure studies with dogs at levels of 100
mg/kg, principle toxicity included frequent vomiting, reduced cardiac efficiency and
electrolyte imbalance [43, 44]. DPI has also been reported to induce hypoglycaemia in rats at
high concentrations, which can be minimized by fortifying the drinking water with glucose
[45]. The LD50 of DPI administered orally to rats is 60 mg/kg. Based on these animal
toxicity studies it appears that DPI related toxicity occurs at very high concentrations well
beyond the very low µM levels required to kill the tested bacterial species in vitro. This offers
some assurance of safety and a high degree of pathogen versus host selectivity, however, this
will also be dependent upon the pharmacokinetics of DPI. Encouragingly, tissue distribution
studies in rats that were administered [125I]-DPI revealed a predominant localization of
radioactivity in the liver, kidneys, heart and adipose tissue [46].
From a medicinal chemistry perspective, an iodonium chemotype would usually be
viewed with great caution. However, data achieved in this study which shows analogues can
be synthesised with improved pathogen killing activity that maintain desirably low
mammalian cell toxicity suggests such a compound could be considered worthy of further
elaboration to investigate structure-activity relationships. For the first time, we have reported
here a detailed assessment of the SAR across a range of microorganisms for a novel set of
DPI analogues. We show that some of our novel DPI analogues are much more potent than
DPI itself with IC50 values in the low nanomolar range. However, some compounds,
particularly those with a halogen in a 5-position in series 2, are relatively cytotoxic to

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mammalian cells and need to be further modified to determine if cytotoxicity can be reduced
while potency against bacterial and parasite pathogens is maintained.
While DPI is one of the less cytotoxic compounds against HEPG2 cells, it is one of
the most potently cytotoxic compounds against THP1 cells. Therefore there seems to be
something of a disconnect between our observation of in vitro cytotoxicity and the historical
use of DPI in vivo with an apparently acceptable, albeit moderate, toxicity profile. For this
reason, while cytotoxicity-associated liability would necessarily require monitoring, it is
possible that the extreme potency of some of our DPI analogues could find an application in
certain antibacterial settings after further improvements in the cytotoxicity profile. Whether
sufficiently divergent SAR can be established to build on the antibacterial potency we have
achieved and minimise mammalian cytotoxicity for further development of this class for in
vivo use will be the focus of future research.
4. Conclusion
A series of DPI analogues were synthesised and subsequently assessed for their
biological activity. Several of these compounds exhibited high potency, with low nanomolar
activity against problematic Gram-negative and Gram-positive bacteria, Mycobacterium
tuberculosis and Plasmodium spp., protozoan parasites.
5. Experimental
5.1 Chemistry
All non-aqueous reactions were performed under an atmosphere of nitrogen, unless otherwise
specified. Commercially available reagents were used without further purification. Thin-layer
chromatography (TLC) was performed on silica gel 60F254 pre-coated aluminum sheets (0.25
mm, Merck). Automated flash column chromatography was performed with a Biotage Isolera

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One instrument using Biotage SNAP cartridges packed with silica (KP-SilTM). Nuclear
Magnetic Resonance (NMR) spectra were recorded at 400.13 Hz on an Avance III Nanobay
400 MHz Bruker spectrometer coupled to the BACS 60 automatic sample changer. Proton
resonances are annotated as: chemical shift (δ), multiplicity (s, singlet; d, doublet; m,
multiplet), coupling constant (J, Hz), and the number of protons. Mass spectrometry was
performed with an Agilent 6224 TOF LC/MS coupled to an Agilent 1290 Infinity (Agilent,
Palo Alto, CA). All data were acquired and reference mass corrected via a dual-spray
electrospray ionisation (ESI) source. Analytical HPLC was acquired on an Agilent 1260
Infinity analytical HPLC coupled with a G1322A degasser, G1312B binary pump, G1367E
high-performance autosampler, and G4212B diode array detector. Conditions were as
follows: Zorbax Eclipse Plus C18 rapid resolution column (4.6 × 100 mm) with UV detection
at 254 and 214 nm, 30 °C; the sample was eluted using a gradient of 5−100% solvent B in
solvent A, where solvent A was 0.1% aq. TFA and solvent B was 0.1% TFA in CH3CN
(5−100% B [9 min], 100% B [1min]; 0.5 mL/min).
5.1.1 General Method A: Preparation of Biphenylamine Derivatives 3a-3aa.
Substituted 2-iodoaniline 1 (1.0 eq.), substituted phenylboronic acid 2 (1.2 eq.), K2CO3 (3.0
eq.), tetrabutylammonium bromide (0.1 eq.), PdCl2(dppf) (0.1 eq.) and dioxane/H2O (9:1)
(0.5 M) were added to a 10 mL microwave-vial. The vial was sealed with a cap and placed in
a Cem Discover-microwave cavity. After irradiation at 130 °C for 1 h and subsequent
cooling, the solvent was removed in vacuo. The residue was taken up into EtOAc (30 mL)
and washed once with water and brine. The organic layer was dried over MgSO4, filtered, and
concentrated. The crude product was purified by flash column chromatography using 0-10%
EtOAc/petroleum benzine to give the biphenylamine product 3a-3aa.

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5.1.2 The following compounds have all been reported previously and their H spectra data
showed good agreement with the literature data:
2'-Methyl-[1,1'-biphenyl]-2-amine (3a)[47], 2'-Methoxy-[1,1'-biphenyl]-2-amine (3b)[48], 2'
Fluoro-[1,1'-biphenyl]-2-amine (3c)[49], 2'-Chloro-[1,1'-biphenyl]-2-amine (3d)[50], 3'-
Methyl-[1,1'-biphenyl]-2-amine (3e)[51], 3'-Methoxy-[1,1'-biphenyl]-2-amine (3f)[51], 3'-
Fluoro-[1,1'-biphenyl]-2-amine (3h)[51], 3'-Chloro-[1,1'-biphenyl]-2-amine (3i)[51], 4'-
Methyl-[1,1'-biphenyl]-2-amine (3j)[51], 4'-Methoxy-[1,1'-biphenyl]-2-amine (3k)[51], 4'-
Fluoro-[1,1'-biphenyl]-2-amine (3m)[51], 4'-Chloro-[1,1'-biphenyl]-2-amine (3n)[51], [1,1'-
Biphenyl]-2-amine (3o)[52], 2-(Naphthalen-2-yl)aniline (3p)[53],
biphenyl]-2-yl)-2,2,2-trifluoroethan-1-one (3r)[54].
(2'-Amino-[1,1'-
5.1.3 2'-Amino-[1,1'-biphenyl]-3-carbonitrile (3g). Light yellow solid (0.29 g, 65% yield).
1H NMR (400 MHz, CDCl3) δ 7.81 (ddd, J = 2.2, 1.7, 0.8 Hz, 1H), 7.78 – 7.72 (m, 1H), 7.68
– 7.63 (m, 1H), 7.57 (td, J = 7.7, 0.5 Hz, 1H), 7.24 (ddd, J = 8.0, 7.4, 1.6 Hz, 1H), 7.12 (dd, J
= 7.6, 1.4 Hz, 1H), 6.90 (td, J = 7.5, 1.1 Hz, 1H), 6.85 (dd, J = 8.0, 0.8 Hz, 1H), 4.19 (br s,
2H). 13C NMR (101 MHz, CDCl3) δ 143.42, 140.94, 133.64, 132.64, 130.72, 130.31, 129.68,
129.48, 125.02, 118.95, 118.72, 116.04, 112.95. LCMS Rt 3.21 min, m/z 195.1 [M + H]+.
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5.1.4 2'-Amino-[1,1'-biphenyl]-4-carbonitrile (3l). Brown oil (0.27 g, 61% yield). H NMR
(400 MHz, CDCl3) δ 7.75 (dq, J = 5.4, 1.7 Hz, 2H), 7.65 – 7.61 (m, 2H), 7.26 (d, J = 7.7 Hz,
1H), 7.15 (d, J = 7.6 Hz, 1H), 6.93 (t, J = 7.5 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 4.41 (s, 2H).
13C NMR (101 MHz, CDCl3) δ 144.68, 143.40, 132.71, 130.33, 129.93, 129.72, 125.55,
119.10, 118.94, 116.19, 110.96. LCMS Rt 3.22 min, m/z 195.1 [M + H]+.

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5.1.5 2'-(Trifluoromethyl)-[1,1'-biphenyl]-2-amine (3q). Dark brown oil (0.39 g, 74% yield).
1H NMR (400 MHz, CDCl3) δ 7.47 – 7.34 (m, 4H), 7.25 (td, J = 7.8, 1.6 Hz, 1H), 7.13 (dd, J
13
= 7.5, 1.4 Hz, 1H), 6.92 (t, J = 8.8 Hz, 2H), 4.42 (s, 2H). C NMR (101 MHz, CDCl3) δ
147.05, 144.04, 132.84, 132.52, 130.97, 129.25, 127.39, 122.54, 121.83, 121.53, 119.26,
118.50, 115.80. LCMS Rt 3.39 min, m/z 239.3 [M + H]+.
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5.1.6 2'-Chloro-5'-methyl-[1,1'-biphenyl]-2-amine (3s). Brown solid (0.39 g, 79% yield). H
NMR (400 MHz, CDCl3) δ 7.40 (d, J = 8.1 Hz, 1H), 7.24 (ddd, J = 8.0, 7.4, 1.6 Hz, 1H), 7.20
– 7.11 (m, 2H), 7.08 (dd, J = 7.5, 1.5 Hz, 1H), 6.91 – 6.82 (m, 2H), 3.72 (s, 2H), 2.37 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 143.87, 137.70, 137.24, 132.61, 130.81, 130.49, 129.93,
129.69, 129.15, 125.66, 118.46, 115.63, 20.94. LCMS Rt 3.41 min, m/z 218.1 [M + H]+.
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5.1.7 2'-Chloro-5'-methoxy-[1,1'-biphenyl]-2-amine (3t). Brown oil (0.46 g, 87% yield). H
NMR (400 MHz, CDCl3) δ 7.46 – 7.35 (m, 1H), 7.26 – 7.19 (m, 1H), 7.09 (dd, J = 7.5, 1.5
13
Hz, 1H), 6.95 – 6.78 (m, 4H), 3.83 (s, 3H), 3.62 (s, 2H). C NMR (101 MHz, CDCl3) δ
158.70, 143.81, 138.86, 130.69, 130.41, 129.30, 125.55, 125.25, 118.45, 116.77, 115.69,
115.36, 55.73. LCMS Rt 3.35 min, m/z 234.1 [M + H]+.
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5.1.8 2',5'-Dichloro-[1,1'-biphenyl]-2-amine (3u). Colourless oil (0.43 g, 80% yield). H
NMR (400 MHz, CDCl3) δ 7.46 (d, J = 8.6 Hz, 1H), 7.38 (s, 1H), 7.32 (dd, J = 8.5, 2.6 Hz,
1H), 7.29 – 7.24 (m, 1H), 7.07 (dd, J = 7.6, 1.5 Hz, 1H), 6.89 (ddd, J = 10.7, 8.5, 4.6 Hz, 2H),
4.11 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 143.72, 139.70, 133.05, 132.46, 131.93, 131.12,
130.36, 129.70, 129.22, 124.17, 118.58, 115.83. LCMS Rt 3.49 min, m/z 238.0 [M + H]+.

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5.1.9 2'-Chloro-5'-fluoro-[1,1'-biphenyl]-2-amine (3v). Yellow oil (0.38 g, 76% yield). H
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NMR (400 MHz, CDCl3) δ H NMR (400 MHz, CDCl3) δ 7.46 – 7.35 (m, 3H), 7.34 – 7.28
(m, 1H), 7.16 (td, J = 8.2, 6.3 Hz, 1H), 6.63 (dd, J = 11.1, 8.5 Hz, 2H), 5.07 – 3.98 (m, 2H).
13C NMR (101 MHz, CDCl3) δ 162.72, 160.26, 143.69, 139.91, 139.83, 131.31, 131.23,
130.32, 129.64, 129.02, 124.38, 118.98, 118.76, 118.53, 116.36, 116.13, 115.82. LCMS Rt
3.88 min, m/z 222.1 [M + H]+.
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5.1.10 2'-Chloro-3-methyl-[1,1'-biphenyl]-2-amine (3w). Brown oil (0.32 g, 79% yield). H
NMR (400 MHz, CDCl3) δ 7.59 – 7.48 (m, 1H), 7.36 (q, J = 2.9 Hz, 3H), 7.16 (dd, J = 7.4,
0.6 Hz, 1H), 6.98 (dd, J = 7.5, 1.3 Hz, 1H), 6.85 (t, J = 7.5 Hz, 1H), 4.03 (s, 2H), 2.30 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 142.05, 138.37, 134.14, 132.11, 130.36, 130.06, 129.13,
128.28, 127.33, 125.13, 122.59, 117.96, 17.95. LCMS Rt 3.44 min, m/z 218.1 [M + H]+.
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5.1.11 2'-Chloro-3-fluoro-[1,1'-biphenyl]-2-amine (3x). Brown oil (0.31 g, 69% yield). H
NMR (400 MHz, CDCl3) δ 7.57 – 7.50 (m, 1H), 7.38 – 7.32 (m, 3H), 7.15 (dd, J = 7.4, 0.8
Hz, 1H), 6.97 (dd, J = 7.6, 1.1 Hz, 1H), 6.84 (t, J = 7.5 Hz, 1H), 3.87 (s, 2H). 13C NMR (101
MHz, CDCl3) δ 161.82, 159.40, 145.77, 145.72, 134.89, 134.44, 130.50, 130.32, 129.74,
129.63, 128.64, 128.24, 114.18, 110.99, 110.97, 105.30, 105.07. LCMS Rt 3.40 min, m/z
222.1 [M + H]+.
5.1.12 2',6-Difluoro-[1,1'-biphenyl]-2-amine (3y). Brown solid (0.36 g, 85% yield). 1H NMR
(400 MHz, CDCl3) δ 7.46 – 7.37 (m, 2H), 7.27 – 7.14 (m, 3H), 6.66 – 6.59 (m, 2H), 4.12 (s,
2H). 13C NMR (101 MHz, CDCl3) δ 162.23, 161.67, 159.80, 159.20, 146.27, 146.22, 132.47,
132.44, 130.38, 130.30, 130.06, 129.95, 124.65, 124.61, 120.03, 119.86, 116.47, 116.25,
110.99, 110.97, 109.29, 109.09, 105.16, 104.93. LCMS Rt 3.28 min, m/z 206.1 [M + H]+.

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5.1.13 2'-Chloro-6-fluoro-[1,1'-biphenyl]-2-amine (3z).Brown oil (0.27 g, 77% yield). H
NMR (400 MHz, CDCl3) δ 7.58 – 7.50 (m, 1H), 7.37 (t, J = 4.0 Hz, 3H), 7.07 (ddd, J = 10.8,
8.1, 1.3 Hz, 1H), 6.89 (d, J = 7.3 Hz, 1H), 6.80 (td, J = 7.9, 5.2 Hz, 1H), 3.56 (s, 2H). 13C
NMR (101 MHz, CDCl3) δ 153.01, 150.63, 136.86, 136.83, 133.89, 132.84, 132.72, 131.89,
130.16, 129.53, 127.41, 127.31, 127.27, 125.81, 125.78, 117.67, 117.59, 114.83, 114.64.
LCMS Rt 3.41 min, m/z 222.1 [M + H]+.
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5.1.14 2',3-Dichloro-[1,1'-biphenyl]-2-amine (3aa). Brown solid (0.28 g, 75% yield). H
NMR (400 MHz, CDCl3) δ 7.45 (d, J = 8.6 Hz, 1H), 7.39 (d, J = 2.5 Hz, 1H), 7.34 – 7.23 (m,
13
2H), 7.08 (dd, J = 7.6, 1.5 Hz, 1H), 6.91 (dd, J = 13.8, 7.5 Hz, 2H), 4.48 (s, 2H). C NMR
(101 MHz, CDCl3) δ 143.73, 139.69, 133.02, 132.43, 131.91, 131.10, 130.34, 129.68,
129.19, 124.12, 118.53, 115.80. LCMS Rt 3.47 min, m/z 238.0 [M + H]+.
5.2.1 General Method B: Synthesis of Cyclic Diphenyleniodonium Trifluoromethanesulfonate
Derivatives 5a-5aa.
The preparation was performed according to the literature procedure.[24] To a stirred
solution of biphenylamine 3a-3aa (1.0 eq.) in THF (0.1 M) was added 4 M HCl (3 mL), and
the solution was cooled in an ice water bath. A solution of NaNO2 (1.2 eq.) in H2O (3 mL)
was added dropwise. After 20 min, a solution of KI (2.5 eq.) in H2O (3 mL) was added, and
stirred for 10 min in an ice water bath. Then the solution was slowly warmed to room
temperature and stirred for 1 h before an aqueous solution of 20% Na2S2O3 was added until
the colour of the mixture didn’t change. The phases were separated, and the aqueous phase
extracted with EtOAc (15 mL x 3). Then the combined organic layers were washed with H2O
and brine, dried over MgSO4, and concentrated. The residue was purified by flash column

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chromatography using 0-5% EtOAc/petroleum benzine to give the biphenyliodide 4. These
compounds were used directly in the next step.
To a stirred solution of biphenyliodide 4 (1.0 eq.) in anhydrous CH2Cl2 (0.2 M) was added m-
CPBA (75%, 1.5 eq.), TfOH (0.003 eq.). The solution was stirred for 1 h at room
temperature. The solvent was removed by rotary evaporation and Et2O (2 mL) was added to
the remained solid. The mixture was stirred for 20 min, and then filtered. The obtained solid
was washed with Et2O (3x), dried in a vacuum oven to afford the desired cyclic
diphenyleniodonium trifluoromethanesulfonate 5a-5aa.
1
5.2.2 The following compounds have all been reported previously and their H spectra data
showed good agreement with the literature data:
1-Methyldibenzo[b,d]iodol-5-ium
Methoxydibenzo[b,d]iodol-5-ium
Methoxydibenzo[b,d]iodol-5-ium
Cyanodibenzo[b,d]iodol-5-ium
Chlorodibenzo[b,d]iodol-5-ium
trifluoromethanesulfonate
trifluoromethanesulfonate
trifluoromethanesulfonate
trifluoromethanesulfonate
trifluoromethanesulfonate
(5a)[55],
(5k)[24]
(5k)[24],
(5l)[24],
(5m)[55],
2-
3-
3-
3-
3-
Fluorodibenzo[b,d]iodol-5-ium trifluoromethanesulfonate (5n)[55], Dibenzo[b,d]iodol-5-ium
trifluoromethanesulfonate
(5o)[24],
Benzo[b]naphtho[1,2-d]iodol-11-ium
trifluoromethanesulfonate (5p)[24]
5.2.3 1-Methoxydibenzo[b,d]iodol-5-ium trifluoromethanesulfonate (5b). Off-white solid
(0.05 g, 22% yield). 1H NMR (400 MHz, DMSO) δ 8.83 (dd, J = 8.1, 1.4 Hz, 1H), 8.28 (dd, J
= 8.2, 1.1 Hz, 1H), 7.91 – 7.88 (m, 1H), 7.86 – 7.82 (m, 1H), 7.74 – 7.60 (m, 2H), 7.58 – 7.50
(m, 1H), 4.11 (s, 3H). 13C NMR (101 MHz, DMSO) δ 160.26, 141.91, 131.48, 131.30,
130.96, 130.76, 130.34, 130.07, 122.81, 122.49, 120.56, 114.16, 57.10. LCMS Rt 2.89 min,

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m/z 309.0 [M - OTf]+. HRMS (ESI) calcd for C13H10IO [M- OTf]+, 308.9771, found
308.9771. HPLC purity >95%, Rt 4.59 min.
5.2.4 1-Chlorodibenzo[b,d]iodol-5-ium trifluoromethanesulfonate (5c). Colourless solid (0.3
1
g, 87% yield). H NMR (400 MHz, DMSO) δ 9.14 (d, J = 7.6 Hz, 1H), 8.30 (t, J = 7.6 Hz,
2H), 7.98 (d, J = 7.8 Hz, 1H), 7.91 (t, J = 7.4 Hz, 1H), 7.80 (t, J = 7.3 Hz, 1H), 7.66 (t, J =
8.0 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 140.74, 137.85, 134.34, 133.35, 131.92, 131.21,
131.09, 131.01, 130.92, 130.38, 123.50, 121.66. LCMS Rt 2.99 min, m/z 312.9 [M - OTf]+.
HRMS (ESI) calcd for C12H7ClI [M- OTf]+, 312.9275, found 312.9276. HPLC purity >95%,
Rt 4.86 min.
5.2.5 1-Fluorodibenzo[b,d]iodol-5-ium trifluoromethanesulfonate (5d). Colourless solid (0.2
g, 66% yield). 1H NMR (400 MHz, DMSO) δ 8.42 (d, J = 7.9 Hz, 1H), 8.25 (dd, J = 8.2, 0.7
Hz, 1H), 8.10 (dd, J = 8.0, 0.7 Hz, 1H), 7.89 (t, J = 7.6 Hz, 1H), 7.83 – 7.68 (m, 3H). 13C
NMR (101 MHz, DMSO) δ 160.05, 139.50, 139.45, 132.09, 132.00, 131.59, 131.54, 131.08,
130.51, 130.29, 130.12, 127.41, 122.03, 121.28, 118.89, 118.68. LCMS rt 2.81 min, m/z
297.0 [M - OTf]+. HRMS (ESI) calcd for C12H7FI [M- OTf]+, 296.9571, found 296.9570.
HPLC purity >95%, Rt 4.53 min.
5.2.6 2-Methyldibenzo[b,d]iodol-5-ium trifluromethanesulfonate (5e). Off-white solid (0.2 g,
71% yield). 1H NMR (400 MHz, DMSO) δ 8.46 (d, J = 7.5 Hz, 1H), 8.34 (s, 1H), 8.21 (d, J =
7.9 Hz, 1H), 8.07 (d, J = 8.2 Hz, 1H), 7.86 (t, J = 7.0 Hz, 1H), 7.71 (t, J = 7.4 Hz, 1H), 7.55
(d, J = 8.2 Hz, 1H), 2.52 (s, 3H). 13C NMR (101 MHz, DMSO) δ 142.19, 142.15, 141.43,
132.53, 131.48, 131.17, 131.08, 130.62, 127.82, 127.37, 122.16, 118.42, 21.22. LCMS rt 2.88

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min, m/z 293.0 [M - OTf]+. HRMS (ESI) calcd for C13H10I [M- OTf]+, 292.9822, found
292.9821. HPLC purity >95%, Rt 4.61 min.
5.2.7 2-Cyanodibenzo[b,d]iodol-5-ium trifluoromethanesulfonate (5g). Off-white solid (0.04
g, 39% yield). 1H NMR (400 MHz, DMSO) δ 9.10 (s, 1H), 8.63 (s, 1H), 8.41 (s, 1H), 8.19 (d,
J = 46.2 Hz, 2H), 7.86 (d, J = 51.6 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 143.53, 140.76,
133.69, 132.51, 132.25, 131.37, 131.01, 128.10, 127.31, 123.01, 118.26, 114.26. LCMS rt
2.70 min, m/z 303.9 [M - OTf]+. HRMS (ESI) calcd for C13H7IN [M- OTf]+, 303.9618, found
303.9618. HPLC purity >95%, Rt 4.14 min.
5.2.8 2-Chlorodibenzo[b,d]iodol-5-ium trifluoromethanesulfonate (5h). Off-white solid (0.13
g, 69% yield). 1H NMR (400 MHz, DMSO) δ 8.68 (d, J = 2.2 Hz, 1H), 8.61 – 8.57 (m, 1H),
8.21 (dd, J = 15.0, 8.3 Hz, 2H), 7.88 (t, J = 7.2 Hz, 1H), 7.81 – 7.72 (m, 2H). 13C NMR (101
MHz, DMSO) δ 144.32, 141.02, 136.87, 132.53, 132.18, 131.26, 131.12, 131.03, 128.03,
127.17, 122.70, 119.99. LCMS rt 2.91 min, m/z 312.9 [M - OTf]+. HRMS (ESI) calcd for
C12H7ClI [M- OTf]+, 312.9275, found 312.9276. HPLC purity >95%, Rt 4.82 min.
5.2.9 2-Fluorodibenzo[b,d]iodol-5-ium trifluoromethanesulfonate (5i). Off-white solid (0.07
1
g, 56% yield). H NMR (400 MHz, DMSO) δ 8.52 (ddd, J = 12.7, 8.9, 2.0 Hz, 2H), 8.27 –
13
8.19 (m, 2H), 7.92 – 7.84 (m, 1H), 7.78 – 7.71 (m, 1H), 7.62 (td, J = 8.8, 2.8 Hz, 1H). C
NMR (101 MHz, DMSO) δ 165.72, 163.79, 163.26, 144.88, 144.79, 141.28, 141.25 (JCF
=
3.1 Hz), 133.02, 132.93, 132.13, 131.26, 131.08, 127.98, 122.70, 119.10, 118.86, 116.05,
116.03, 114.59, 114.34. LCMS rt 2.81 min, m/z 297.0 [M - OTf]+. HRMS (ESI) calcd for
C12H7FI [M-OTf]+, 296.9571, found 296.9570. HPLC purity >95%, Rt 4.39 min.