Substituted Carbazoles-A New Class of Anthelmintic Agent

Article abstract of DOI:10.1071/CH16169

A series of novel carbazoles were synthesized based on structural modifications to lead carbazole 1 (EC100≤2.5M against Haemonchus contortus in vitro), which was revealed in a small molecule screening program as a potentially promising platform for the development of new anthelmintic drugs. Subsequently, analogues 19, 21, 41, 42 (EC100≤ 1.25M, all), and 39 (EC100≤0.625M) were demonstrated to exhibit enhanced in vitro anthelmintic activity over the lead structure, with compound 39 also being shown to be active in vivo against Heligmosomoides polygyrus.

Full text of DOI:10.1071/CH16169

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CSIRO PUBLISHING
Aust. J. Chem.
Full Paper
http://dx.doi.org/10.1071/CH16169
Substituted Carbazoles – A New Class
of Anthelmintic Agent
A D
,
A
A
B
David Rennison,
Stephanie M. Gueret, Olivia Laita, Ross J. Bland,
B
C
A D
,
Ian A. Sutherland, Ian K. Boddy, and Margaret A. Brimble
A
School of Chemical Sciences, University of Auckland, Auckland, New Zealand.
AgResearch Limited, Hopkirk Institute, Corner University Avenue and Library Road,
Palmerston North, New Zealand.
B
C
ParaCo Technologies Limited, Hamilton, New Zealand.
D
Corresponding authors. Email: d.rennison@auckland.ac.nz; m.brimble@auckland.ac.nz
A series of novel carbazoles were synthesized based on structural modifications to lead carbazole 1 (EC100 ¼ 2.5 mM
against Haemonchus contortus in vitro), which was revealed in a small molecule screening program as a potentially
promising platform for the development of new anthelmintic drugs. Subsequently, analogues 19, 21, 41, 42 (EC100
1.25 mM, all), and 39 (EC100 ¼ 0.625 mM) were demonstrated to exhibit enhanced in vitro anthelmintic activity over the
lead structure, with compound 39 also being shown to be active in vivo against Heligmosomoides polygyrus.
¼
Manuscript received: 18 March 2016.
Manuscript accepted: 28 April 2016.
Published online: 30 May 2016.
Introduction
of new anti-parasitic agents, including anthelmintics, to ensure
the long-term sustainability of pastoral agriculture.[11,12]
Parasites are responsible for a variety of diseases detrimental to
both human and animal health. In livestock, parasitic infections
can lead to substantial decreases in productivity,[1] and in
extreme cases animal death. Helminths, in particular gastroin-
testinal nematodes or roundworms, are generally considered to
be the most economically significant parasites affecting sheep
and cattle, worldwide.[1] Effective control of these parasites is
essential not only for animal welfare, but also in enabling
intensive livestock agriculture to produce food and fibre at
practical economic cost.
Results and Discussion
Synthesis
A small molecule screening program revealed carbazole 1
(Fig. 1) to display modest, yet encouraging, anthelmintic
activity (against H. contortus) in vitro. An extended series of
carbazoles were synthesized, based on rational modifications to
the lead structure, in an endeavour to improve potency.
Helminth control relies almost exclusively on the regular
application of anti-parasitic agents (e.g. anthelmintics) to
the animal in an effort to minimize worm populations and
therefore maintain animal performance.[2] The sustained and
widespread application of such agents has inadvertently led to
the manifestation of organisms impervious to current treat-
ments.[3] Resistance to the three major anthelmintic drug
classes, namely, benzimidazoles,[4] imidazothiazoles,[5] and
macrocyclic lactones,[6] is now widespread in small ruminants
throughout the world,[7] and is likewise becoming an escala-
ting problem in cattle.[8] In terms of new product development,
with the exception of the cyclodepsipeptides,[9] represented
amongst others by emodepside, and the amino-acetonitrile
derivatives or AADs,[10] which include the broad-spectrum
anthelmintic monepantel (2), no novel anthelmintic class has
reached the market in the past 30 years. Despite these
advances, history suggests resistance will slowly develop
following a few years of use; and in some cases misuse.
Accordingly, there is an ongoing need for the development
Compounds 3–18 were prepared through the reductive
amination of aldehyde 51, using the appropriate amine in
combination with sodium triacetoxyborohydride and acetic
acid. Compounds 32 and 33 were synthesized in a similar
fashion from carbazoles 52 and 53, respectively (Scheme 1,
Tables 1–3). Rieche formylation[13] of carbazoles 48 and 49,
using dichloromethyl methyl ether and titanium(IV) chloride, led
to aldehydes 51 and 52, respectively. Vilsmeier–Haack formy-
lation[14] of carbazole 50 gave aldehyde 53. Precursors 48, 49,
and 50 were accessed through the alkylation of carbazole 47,
using either ethyl bromide, benzyl bromide, or ethyl bromoa-
cetate, respectively. Acid-promoted hydrolysis of ethyl ester 11
revealed carboxylic acid 12, whereas the amination of carbazole
11 using methylamine gave amide 13. Oxidative debenzyl-
ation[15] of carbazole 32, using potassium tert-butoxide and
dimethyl sulfoxide in the presence of oxygen, provided carba-
zole 31, with subsequent alkylation, using 2-(dimethylamino)
ethyl chloride and sodium hydride, leading to amine 37. Treat-
ment of ethyl ester 33 with either aqueous hydrochloric acid or
Journal compilation Ó CSIRO 2016
www.publish.csiro.au/journals/ajc

B
D. Rennison et al.
F
CN
SCF3
N
H
4
H
N
3
O
2
CN
O
CF3
2
1
N
1
Fig. 1. Structures of lead carbazole 1 and monepantel (2).
NR1R2
O
b
c
N
R
N
R
N
R
3-18, 32-33
4 R ꢀ Et, R1 ꢀ CH2CH2Ph, R2 ꢀ H
21 R ꢀ Et, R1 ꢀ CH2CH2Ph, R2 ꢀ Me
47 R ꢀ H
48 R ꢀ Et
51 R ꢀ Et
d
a
a
a
52 R ꢀ CH2Ph
53 R ꢀ CH2CO2Et
11 R1 ꢀ CH2CO2Et
12 R1 ꢀ CH2CO2H
47 R ꢀ H
e
f
49 R ꢀ CH2Ph
R ꢀ Et, R2 ꢀ H
11 R1 ꢀ CH2CO2Et
13 R1 ꢀ CH2CONHMe
47 R ꢀ H
50 R ꢀ CH2CO2Et
32 R ꢀ CH2Ph
31 R ꢀ H
g
h
i
33 R ꢀ CH2CO2Et
34 R ꢀ CH2CO2H
33 R ꢀ CH2CO2Et
R1 ꢀ CH2CH2C6H4pF, R2 ꢀ H
35 R ꢀ CH2CONHMe
33 R ꢀ CH2CO2Et
36 R ꢀ CH2CH2OH
j
31 R ꢀ H
k
37 R ꢀ CH2CH2NMe2
Scheme 1. Reagents and conditions: (a) NaH, EtBr, DMF, rt, 18 h, 76 % (to give 48) or NaH, PhCH2Br, DMF, rt, 18 h, 75 % (to give 49) or NaH,
BrCH2CO2Et, DMF, rt, 18 h, 60 % (to give 50); (b) 48, Cl2CHOMe, TiCl4, DCM, ꢀ788C, 4 h, 89 % (to give 51) or 49, Cl2CHOMe, TiCl4, DCM,
ꢀ788C, 4 h, 83 % (to give 52) or 50, POCl3, DMF, 1008C, 2 h, 55 % (to give 53); (c) RNH2, NaBH(OAc)3, AcOH, THF, rt, 18 h (3–18 and 32–33, see
Tables 1, 2 and 3); (d) 4, aq. formaldehyde, NaBH(OAc)3, AcOH, THF, rt, 18 h, 75 %; (e) 6 M HCl(aq), reflux, 10 min, 55 %; (f) MeNH2, EtOH, rt,
18 h, 89 %; (g) t-BuOK, O2, THF-DMSO (4 : 1), rt, 1 h, 69 %; (h) 6 M HCl(aq), reflux, 10 min, 60 %; (i) MeNH2, EtOH, rt, 18 h, 84 %; (j) LiAlH4, THF,
rt, 18 h, 77 %; (k) NaH, ClCH2CH2NMe2.HCl, DMF, rt, 18 h, 70 %.
ethanolic methylamine afforded carboxylic acid 34 and amide
35, respectively. Reduction of ester 33 using lithium alumi-
nium hydride led to alcohol 36. Reductive amination of
carbazole 4 using aqueous formaldehyde gave tertiary amine
derivative 21.
Compounds 19, 22–26, 29, and 30 were prepared from
common aldehyde precursor 51 (Scheme 2). Treatment of
aldehyde 51 with hydroxylamine led to oxime 56, with subse-
quent O-benzylation affording carbazole 25. Reduction of
oxime 56 using lithium aluminium hydride gave amine 57,
which was then acylated to reveal amide 22. Alternatively,
reduction of aldehyde 51 using sodium borohydride afforded
alcohol 60, which was then alkylated using benzyl bromide to
give ether 26. Nitrile 59, prepared over two-steps via oxime 56,
was converted into amidine 24 through reaction with phenethy-
lamine under Pinner-type conditions. Oxidation of aldehyde 51
using potassium permanganate led to carboxylic acid 58, which
was then converted into amide 23 via the corresponding acid
chloride. b-Nitrovinyl derivative 54 was accessed through the
Henry reaction of nitromethane and aldehyde 51, with lithium
aluminium hydride induced reduction leading to amine 55;
which then underwent reductive amination in the presence of
benzaldehyde to afford carbazole 19. Carbazole 29 was prepared
over two-steps from aldehyde 51 using Wittig chemistry
(to reveal alkene 30), followed by palladium(0)-catalyzed
hydrogenation.
Compounds 20, 27, and 28 were accessed from common
aniline precursor 62, which was prepared via the sequential
nitration and reduction of carbazole 48 (Scheme 3). Reductive
amination of aniline 62 with hydrocinnamaldehyde, under
conditions similar to those described previously, gave carbazole
20. Acylation of aniline 62 with cinnamoyl chloride afforded
cinnamide 28, which was subsequently hydrogenated over
palladium(0) to reveal saturated amide 27.

Substituted Carbazoles
C
Table 1. Anthelmintic activity of compounds 3–16
Table 3. Anthelmintic activity of analogues 31–37
NHR
F
N
N
H
Compound
R
Yield [%] Log PA EC100B [mM]
N
R
3
CH2CH2C6H4pCl
CH2CH2Ph
2-Pyridinylmethyl
2-Furanylmethyl
2-Thienylmethyl
CH2CH2CMe3
CH2CH2OH
CH2CH2OMe
CH2CO2Et
65
64
64
69
71
73
70
39
73
–
5.34
4.78
3.73
3.26
4.62
4.81
2.25
2.61
2.78
2.17
1.76
1.80
2.77
3.10
4.94
3.00
2.5
2.5
5
4
5
Compound
R
Yield [%]
Log PA
EC100B [mM]
6
2.5
5
7
31
H
–
75
69
–
4.36
6.33
4.47
3.87
3.45
4.08
4.60
4.94
3.00
2.5
10
8
1.25
32
CH2Ph
9
10
33
CH2CO2Et
CH2CO2H
CH2CONHMe
CH2CH2OH
CH2CH2NMe2
–
10
10
10
.10
.10
.10
.10
5
34
5
11
35
–
5
12
CH2CO2H
36
–
2.5b
2.5
2.5
0.0125
13
CH2CONHMe
CH2CH2NHAc
CH2CH2NMe2
CH2CH3
–
37
–
14
62
65
68
–
1
–
15
Monepantel
–
–
16
5
ALog P (in silico, ChemBioDraw Ultra version 12).
BAgainst H. contortus in vitro.
1
–
2.5
0.0125
Monepantel
–
–
ALog P (in silico, ChemBioDraw Ultra version 12).
BAgainst H. contortus in vitro.
by tin(II) chloride induced reduction, led to hydrazine 64, which
was then reacted with cyclohexanone in the presence of acetic
acid[17] to afford tetrahydrocarbazole 65. Catalytic dehydroge-
nation[17] of carbazole 65, using palladium(0) at elevated
temperature, gave carbazole 66. Methyl ester 66 was reduced
using lithium aluminium hydride to give alcohol 67, which was
then oxidized using pyridinium chlorochromate to reveal alde-
hyde 68. Alkylation of carbazole 68 using ethyl bromide gave
carbazole 69. Finally, reductive amination of aldehyde 69 and
2-(p-fluorophenyl)ethylamine, in the presence of triacetoxy-
borohydride and acetic acid, led to target carbazole 38.
Compound 39, a second regioisomeric example of carbazole
1, was built from commercially available 4-chloro-3-
nitrobenzaldehyde (70) over 4 steps; via a modified Cadogan
cyclization[16,18] (Scheme 5). Suzuki coupling[19] of phenyl-
boronic acid and aldehyde 70, in the presence of tetrakis
(triphenylphosphine)palladium(0), led to 2-nitrobiphenyl
derivative 71. Cyclization of compound 71 was achieved via a
reductive deoxygenation mechanism,[20] using triphenylpho-
sphine at elevated temperature, to give carbazole 72. Alkylation
of carbazole 72 furnished compound 73. Reductive amination of
73 with 2-(p-fluorophenyl)ethylamine, as described for carba-
zole 38, afforded carbazole 39.
In similar fashion, compound 40, a third regioisomeric
analogue of carbazole 1, was prepared over 5 steps from com-
mercially available 1-bromo-2-nitrobenzene (74) (Scheme 6).
Sequential Suzuki coupling[21] of 2-methylphenylboronic acid
and aryl bromide 74 (to give 75), followed by reductive cycliza-
tion,[20] gave carbazole 76. Oxidation of compound 76 using
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) under UV
irradiation revealed aldehyde 77, with subsequent ethylation
leading to carbazole 78. Finally, reductive amination of aldehyde
78 with 2-(p-fluorophenyl)ethylamine, as detailed previously,
furnished carbazole 40.
Table 2. Anthelmintic activity of compounds 17–30
X
N
Compound
X
Yield [%] Log PA EC100B [mM]
17
CH2NHCH2
68
64
–
4.64
5.20
4.78
5.13
5.16
4.02
4.36
4.88
5.33
4.72
4.37
4.35
6.55
6.23
4.94
3.00
2.5
18
CH2NHCH2CH2CH2
CH2CH2NHCH2
NHCH2CH2CH2
CH2N(Me)CH2CH2
CH2NHC(=O)CH2
C(=O)NHCH2CH2
C(=NH)NHCH2CH2
C=NOCH2
2.5
19
1.25
20
–
.10
1.25
.10
21
–
22
–
23
–
.10
.10
.10
.10
.10
.10
.10
.10
24
–
25
–
26
CH2OCH2
–
27
NHC(=O)CH2CH2
NHC(=O)CH=CH
CH2CH2CH2CH2
CH=CHCH2CH2
–
–
28
–
29
–
30
–
1
–
2.5
0.0125
Monepantel
–
–
ALog P (in silico, ChemBioDraw Ultra version 12).
BAgainst H. contortus in vitro.
Compound 38, a regioisomer of lead carbazole 1, was
prepared over 8 steps from commercially available 2-anthranillic
acid methyl ester (63); using a modified Fischer–Borsche
approach[16] (Scheme 4). Diazotization of aniline 63, followed
Compounds 41–43 were prepared from common aldehyde
precursor 51 over 2 steps (Scheme 7). Reaction of carbazole 51

D
D. Rennison et al.
Ph
H
N
O
Ph
Ph
N
H
N
19
29
22
N
N
a, b, c
d, e, f
q, r
O
O
Ph
Ph
o, p
N
H
O
N
g, h, i
51
N
N
26
23
d, n
j, k, l, m
Ph
NH
N
H
NOCH2Ph
.HCl
N
N
25
24
Scheme 2. Reagents and conditions: (a) MeNO2, NH4OAc, AcOH, 808C, 2 h (to give 54); (b) LiAlH4, THF, rt, 18 h, 61 % (over 2 steps to give 55);
(c) PhCHO, NaH(OAc)3, AcOH, THF, rt, 18 h, 67 %; (d) NH2OH.HCl, NaOH(aq), EtOH, rt, 18 h, 83 % (to give 56); (e) LiAlH4, THF, rt, 18 h, 60 % (to give
57); (f) PhCH2COCl, Et3N, DMAP, DCM, rt, 18 h, 82 %; (g) KMnO4, acetone, rt, 3 h, 86 % (to give 58); (h) SOCl2, reflux, 3 h; (i) PhCH2CH2NH2, Et3N,
DMAP, DCM, rt, 18 h, 82 % (over 2 steps); (j) NH2OH.HCl, Py, rt, 18 h; (k) Ac2O, reflux, 6 h, 61 % (to give 59 over 2 steps); (l) 4 M HCl/1,4-dioxane, EtOH,
rt, 48 h; (m) PhCH2CH2NH2, EtOH, reflux, 3 days, 15 % (over 2 steps); (n) NaH, PhCH2Br, DMF, rt, 18 h, 86 %; (o) NaBH4, EtOH-THF (1 : 1), rt, 3 h, 96 %
(to give 60); (p) NaH, PhCH2Br, DMF, rt, 18 h, 59 %; (q) PhCH2CH2CH2PPh3Br, n-BuLi, THF, ꢀ788C to rt, 2 h, 55 % (to give 30); (r) H2, Pd/C, EtOAc, rt,
18 h, 100 %.
NH2
NHR
NO2
a
b
c or d
N
N
N
N
48
61
62
20 R ꢀ CH2CH2CH2Ph
28 R ꢀ COCH ꢀ CHPh
27 R ꢀ COCH2CH2Ph
e
Scheme 3. Reagents and conditions: (a) HNO3(aq), ClCH2CH2Cl, 108C, 1h, 81%; (b) SnCl2ꢁ2H2O, EtOH, reflux, 18 h, 86 %; (c) PhCH2CH2CHO,
NaH(OAc)3, AcOH, THF, rt, 18 h, 50% (to give 20) or (d) PhCH=CHCOCl, Et3N, DMAP, DCM, rt, 18 h, 88 % (to give 28); (e) H2, Pd/C, EtOAc, rt, 18 h, 86%.
with the appropriate lithiated pyridine, generated from the
corresponding bromopyridine using n-butyllithium, gave
pyridinylmethanols 79–81. Reduction of compounds 79–81,
using platinum(IV) oxide in the presence of hydrochloric
acid, led directly to dehydroxylated piperidines 41–43; whereas
performing the same reaction in glacial acid afforded
piperidinylmethanols 44–46.
compound being the concentration at which 100% of the
nematodes present were killed) (Tables 1–5). To elucidate
which regions of the lead structure were potentially involved in
binding to the alleged macromolecular target, a comprehensive
structure–activity study was undertaken. In a rational, stepwise
fashion, each functional group was now modified, masked, or
removed to determine its importance/contribution towards
biological activity.
Lipophilic substituents have the capacity to interact with
hydrophobic regions of a binding domain in several different
ways. Although recognised as being weaker than both hydrogen
bonds and ion-pair interactions, given that such moieties can
Biological Evaluation
Compounds 3–46 were screened in vitro for activity against the
helminth Haemonchus contortus; where lead carbazole 1 was
previously revealed to have an EC100 of 2.5 mM (the EC100 of a

Substituted Carbazoles
E
c
a, b
d
NH2
NHNH2
N
H
N
H
MeO2C
MeO2C
63
64
65
66
CO2Me
CO2Me
F
e
f, g
h
N
N
H
N
67
69
38
N
O
OH
H
Scheme 4. Reagents and conditions: (a) NaNO2, HCl(aq), H2O, ,108C, 0.5 h; (b) SnCl2ꢁH2O, HCl(aq), H2O; (c) cyclohexanone,
AcOH, reflux, 3 h, 40 % (over 3 steps); (d) Pd/C, 2608C, 2 h; (e) LiAlH4, THF, rt, 1 h, 56 % (over 2 steps); (f) PCC, DCM, rt, 1.5 h, 76 %
(to give 68); (g) NaH, EtBr, DMF, rt, 18 h, 90 %; (h) p-FC6H4CH2CH2NH2, NaBH(OAc)3, AcOH, THF, rt, 18 h, 70 %.
Cl
H
N
O
a, b
c, d
F
O
O2N
N
H
N
70
72
39
Scheme 5. Reagents and conditions: (a) PhB(OH)2, Pd(Ph3P)4, K2CO3, PhMe, 1008C, 18 h (to give 71); (b) Ph3P, DMA, 1508C, 18 h,
71 % (over 2 steps); (c) NaH, EtBr, DMF, rt, 18 h, 89 % (to give 73); (d) p-FC6H4CH2CH2NH2, NaBH(OAc)3, AcOH, THF, rt, 18 h, 70 %.
H
O
N
F
Br
a, b
c, d
e
NO2
N
H
N
N
74
76
78
40
Scheme 6. Reagents and conditions: (a) 2-Methylphenylboronic acid, Pd(Ph3P)4, Na2CO3, DME-EtOH, reflux, 18 h, 94 % (to give 75); (b) Ph3P,
DMA, 1508C, 18 h, 91 %; (c) DDQ, 1,4-dioxane-H2O, AcOH, 1108C, mn (150 W), 5.5 h, 21 % (to give 77); (d) NaH, EtBr, DMF, rt, 18 h, 93 %;
˚
(e) p-FC6H4CH2CH2NH2, MgSO4, DCM, rt, 18 h then AcOH, 4A MS, 508C, 16 h then NaBH4, MeOH, rt, 18 h, 63 %.
HO
R
X
X
Y
Y
O
Z
Z
a
b or c
N
N
N
51
79 X ꢀ N, Y ꢀ Z ꢀ CH
80 X ꢀ Z ꢀ CH, Y ꢀ N
81 X ꢀ Y ꢀ CH, Z ꢀ N
41 X ꢀ NH, Y = Z ꢀ CH2
42 X ꢀ Z = CH2, Y ꢀ NH
43 X ꢀ Y = CH2, Z ꢀ NH
R ꢀ H
44 X ꢀ NH, Y = Z ꢀ CH2
45 X ꢀ Z ꢀ CH2, Y ꢀ NH
46 X ꢀ Y ꢀ CH2, Z ꢀ NH
R ꢀ OH
Scheme 7. Reagents and conditions: (a) 2-bromopyridine, n-BuLi, Et2O, ꢀ788C, 4 h, 84 % (to give 79) or 3-bromopyridine, n-BuLi,
THF, ꢀ788C, 4 h, 69 % (to give 80) or 4-bromopyridine (prepared from 4-bromopyridine hydrochloride and K2CO3), n-BuLi, THF,
ꢀ788C, 4 h, 64 % (to give 81); (b) 79, H2, PtO2, HCl(aq), EtOH, 18 h, 100 % (to give 41) or 80, H2, PtO2, HCl(aq), EtOH, 18 h, 91 %
(to give 42) or 81, H2, PtO2, HCl(aq), EtOH, 18 h, 90 % (to give 43); (c) 79, H2, PtO2, AcOH, 18 h, 51 % (to give 44) or 80, H2, PtO2,
AcOH, 18 h, 64 % (to give 45) or 81, H2, PtO2, AcOH, 18 h, 67 % (to give 46).

F
D. Rennison et al.
Table 4. Anthelmintic activity of analogues 38–40
Table 5. Anthelmintic activity of analogues 41–46
F
R
X
Y
Z
4
3
N
H
N
2
N
1
Compound
R
X
Y
Z
Log PA
EC100B [mM]
41
H
NH
CH2
CH2
NH
CH2
CH2
–
CH2
NH
CH2
CH2
NH
CH2
–
CH2
CH2
NH
CH2
CH2
NH
–
3.76
3.77
3.70
2.94
2.73
2.68
4.94
3.00
1.25
1.25
2.5
Compound
Ring substituent position
Log PA
EC100B [mM]
42
H
43
H
38
1
2
4
3
–
4.94
4.94
4.94
4.94
3.00
10
44
OH
OH
OH
–
10
39
0.62C
1.25
2.5
45
.10
.10
2.5
40
46
1
1
Monepantel
0.0125
Monepantel
–
–
–
–
0.0125
ALog P (in silico, ChemBioDraw Ultra version 12).
BAgainst H. contortus in vitro.
CLowest dose tested.
ALog P (in silico, ChemBioDraw Ultra version 12).
BAgainst H. contortus in vitro.
often be present in sizeable numbers, the resulting cumulative
effect can contribute significant binding. Aryl–aryl interactions
are of particular importance, for instance the interactions
between aromatic ligands and aryl-containing amino acids such
as phenylalanine, tyrosine, tryptophan, and histidine.[22] The
introduction of substituents or insertion of heteroatoms into
aromatic rings can have a pronounced influence on both the
strength and geometry of aryl–aryl interactions. As a rule, the
stacking arrangements of electron-rich and electron-poor
aromatic rings profit from charge transfer. In similar fashion,
stacking between electron-deficient rings is generally preferred
over the stacking of electron-rich ones.[23] To probe the impor-
tance of the aryl ring substituent on the activity of lead carbazole
1, chlorophenyl derivative 3 was prepared and subsequently
demonstrated to be equipotent to its fluorinated counterpart.
Unsubstituted carbazole 4 was likewise shown to be of similar
activity to halogenated carbazoles 1 and 3. It is known that the
insertion of a heteroatom into an aryl ring produces an electron-
deficient system, which in turn can improve aryl–aryl stacking
interactions with aromatic amino acid side-chains. Of the
heteroaromatic derivatives prepared, furan 6 was revealed to
be comparable to carbazole 1, whereas pyridine 5 and thiophene
7 were found to be marginally less active; linker shortened
analogues 6–8 were prepared on synthetic grounds (and can be
directly compared with carbazole 17). As an example of a non-
aromatic hydrophobic substituent (hence removing the capacity
of the side-chain group to form p–p interactions), tert-butyl
derivative 8 was demonstrated to be of similar potency to its aryl
counterparts. With regards to the overall lipophilicity of com-
pounds 3–8, as determined by their in silico generated log
P values, no apparent trends were observed in this particular
instance (Table 1).
functional groups in both lead compounds and drugs, they are
still relatively prevalent. Again, they have the capacity to form
hydrogen bonds, either as the free acid or as the ionized
carboxylate ion. In addition, carboxylate ions can form strong
ionic interactions with charged aminium and guanidinium ions
(e.g. lysine and arginine residues, respectively). Alcohol 9 was
subsequently shown to be significantly less active than carba-
zole 1, as were methyl ether 10, ethyl ester 11, carboxylic acid
12, and amide regioisomers 13 and 14. On the basis of these
findings it would appear that no significant hydrogen bonding
interaction was attained through the incorporation of these
specific polar moieties into this particular region of the mole-
cule. This is not to say that the putative binding pocket does not
possess the capacity to form hydrogen bonds, but that the
intermolecular distances, geometry, and directionality involved
may not have been optimal to allow for a favourable interaction.
With regards to overall lipophilicity, a potential correlation
between the activity of compounds 9–14 and their lower log
P values could also be put forward as a further reason for their
inferior performance in vitro (compared with their more lipo-
philic counterparts, compounds 3–8, Table 1).
Amines also have the potential to form hydrogen bonds, the
specific nature of which is largely determined by whether the
amine is in an ionized or non-ionized form; which itself is
dictated by the pH of the physiological environment. When
ionized, the capacity of an amine to operate as a hydrogen bond
acceptor is lost; however, on the other hand, protonated amines
can now act as much stronger hydrogen bond donors. Moreover,
aminium ions have the potential to form strong ionic interactions
with charged carboxylate-containing amino acids such as aspar-
tate and glutamate. A subtle recovery in activity was noted for
amine 15 (versus other examples bearing polar functional
groups within the series, namely compounds 9–14). p-Cation
interactions are also known to play an important role in the
binding of aminium groups to aromatic or heteroaromatic
moieties.[24] It is recognised that methyl groups interact favour-
ably with the p-face of aromatic rings when bound to an
electronegative atom. Given that a positively charged nitrogen
is a particularly strong electron-withdrawing species, the
Next, a selection of carbazoles bearing polar functional
groups were investigated. Hydrogen bonds are acknowledged
as being one of the most important specific interactions in the
biological recognition process. Among others, alcohols,
ketones, amides, ethers, and esters can all act as hydrogen bond
acceptors, and in the case of alcohols and amides, hydrogen
bond donors. While carboxylic acids are less common

Substituted Carbazoles
G
interaction between alkylammonium ions and aromatic rings
can lead to even stronger alkyl–aryl interactions. Carbazole 16,
an example bearing no terminal group, only the linker, was
found to be of moderate activity.
Table 6. In vivo evaluation of compounds 1 and 39
Compound
Concentration [mM]
Average adult nematodesA
1
100
39.8 ꢂ 4.0
14.9 ꢂ 4.4
32.4 ꢂ 5.5
In parallel to the preparation of compounds 3–16, carbazoles
17 and 18 were designed to probe the importance of linker length
on anthelmintic activity (Table 2). Homologues 17 and 18,
incorporating linkers of 3 and 5 atoms, respectively (while
retaining the position of the linker nitrogen), were demonstrated
to be of similar activity to carbazole 4 (4 atom linker example).
This would appear to suggest that there is a degree of tolerance in
terms of the postulated binding of the linker amine in combina-
tion with that of the carbazole core and the side-chain aromatic
moiety. With regards to carbazole 17, as a by-product of
removing a methylene unit from the side-chain, the basicity of
the amine was concurrently lowered due to the increasing
influence of the neighbouring electron-withdrawing phenyl
group (based on in silico pKa values of 8.7 and 9.3 for com-
pounds 17 and 1, respectively; ACD laboratories version 12).
For compounds 1, 4, and 17–19 it should be noted that at pH 7.4
and below the proportion of ionized amine is calculated to
be . 95 %; thus the linker amine has the capacity to act as both a
hydrogen bond donor and/or form strong ion-pair interactions.
Next, the position of the nitrogen atom within the linker was
investigated through the preparation of carbazoles 19 and 20.
Moving the linker nitrogen one atom further away from the
carbazole core, as found in analogue 19, led to an improvement
in activity, suggesting that a more favourable interaction was
attained as a consequence. Conversely, carbazole 20 was found
to be inactive, most likely due to the amine character of the
molecule being removed to reveal a non-basic aniline deriva-
tive. Gratifyingly, tertiary amine 21 was demonstrated to be
more potent than its secondary amine counterpart, carbazole 4.
Potential explanations for this observation may relate to those
outlined earlier for compound 15. Replacing the amine linker of
carbazole 4 with an amide, through regioisomers 22 and 23,
again likely reiterated the importance of having a basic group in
this region of the molecule. This was possibly further reinforced
by the fact that both oxime 25 and ether 26 were similarly devoid
of all activity. Curiously amidine 24, prepared as a linker
example incorporating a non-amine basic moiety, was demon-
strated to be inactive. Finally, carbazoles 29 and 30, possessing
linkers solely constituted of carbon atoms, were also confirmed
to be inactive, as were cinnamides 27 and 28.
39
100
DMSO
Control
AAgainst H. polygyrus.
structure. It could be postulated that the spatial orientation of
both the linker amine and carbazole core of carbazoles 1, 39, and
40 is more favourable, in terms of binding to the target, than it is
in example 38.
In a final effort to further optimize the spatial positioning of
the linker amine with respect to the carbazole core, piperidine
derivatives 41–43 were prepared (Table 5). Gratifyingly, com-
pounds 41 and 42 were demonstrated to be more active than
carbazole 1, while compound 43 was found to be equipotent.
Precursors 44–46 were revealed to be inactive, suggesting that
the presence of a hydroxy group in this position led to an
unfavourable interaction with the binding domain.
Under these assay conditions, the in vitro EC100 for mon-
epantel (2), the active ingredient in Zolvix, a commercially
available broad spectrum anthelmintic for the treatment of
gastrointestinal roundworms in sheep, was found to be
0.0125 ppm (against H. contortus).
Based on their performance in vitro, compounds 1 and 39
were next screened for anthelmintic activity against the
helminth H. polygyrus, in mice (Table 6). Whereas lead carba-
zole 1 was demonstrated to be inactive in vivo, encouragingly,
compound 39 was found to have an EC50 in the region of 50 mM.
Conclusion
In summary, carbazole 1 underwent an array of structural
modifications to reveal compound 39, which exhibited a mini-
mum 4-fold improvement in anthelmintic activity (in vitro) over
the lead structure. Although monepantel (2) was shown to be
around 50 times more potent against H. contortus in vitro, the
fact that compound 39 displayed activity in vivo (against
H. polygyrus) suggests that carbazoles remain a genuine plat-
form for further development in the search for a new class of
anthelmintic agent.
Derivatization of the carbazole ring nitrogen subsequently
revealed both benzyl and ethoxycarbonylmethyl substituted
analogues, compounds 32 and 33 respectively, to be weakly
active. A slight recovery in activity was noted for carboxylic
acid derivative 34 and amide 35, while alcohol 36 and amine 37,
along with unsubstituted carbazole 31, were found to be equi-
potent to lead carbazole 1 (Table 3). In the absence of any clear
trend in terms of molecular interaction (other than possibly
steric intolerance), the overall lipophilicity of the molecule may
be the dominant parameter in this instance; it could be postulated
that the optimal log P within this series of compounds is between
4–5 (based on in silico log P values; ChemBioDraw Ultra
version 12).
Next, through the preparation of regioisomeric carbazoles
38–40, our focus moved onto determining the importance of the
location of the side-chain (Table 4). Incorporation of the
substituent at the 1-position of the carbazole ring resulted in a
marked loss in activity. Conversely, analogues 39 and 40 were
revealed to be of greater anthelmintic activity than the lead
Experimental
Synthesis
General Experimental Methods
All reagents were used as supplied unless stated otherwise.
Analytical thin-layer chromatography (TLC) was carried out on
pre-coated silica gel plates (Merck/UV254) and products were
visualized by UV fluorescence and/or staining. Flash chroma-
tography was performed using silica gel (Riedel-de Hae¨n,
particle size 0.032–0.063 mm). NMR spectra were recorded
on a Bruker AVANCE 300 (1H, 300 MHz) spectrometer at
298 K. For 1H NMR data, chemical shifts are described in
parts per million (ppm) relative to tetramethylsilane (d 0.00)
and are reported consecutively as position (dH), relative
integral, multiplicity (s ¼ singlet, bs ¼ broad singlet, d ¼ doublet,
t ¼ triplet, q ¼ quartet, m ¼ multiplet), coupling constant
(J, Hz), and assignment. Mass spectra were recorded on a
VG-70SE mass spectrometer (EI, CI, and FAB). The purity of
all target compounds was assigned using reverse phase-HPLC

H
D. Rennison et al.
(Dionex P680 system using a Phenomenex Gemini C18-Si
column, 150 mm ꢃ 4.6 mm, 5 mm) – eluted using a gradient of
100 : 0 % A/B to 5 : 95 % A/B over 15 min at 1 mL minꢀ1; where
solvent A was water (þ0.1 % trifluoroacetic acid) and solvent B
was CH3CN (þ0.1 % trifluoroacetic acid); with detection at 210,
254, and 280 nm.
(15 mL) was purged with nitrogen, followed by the addition of
tetrakis(triphenylphosphine)palladium(0) (125 mg, 0.11 mmol).
The mixture was heated at 1008C overnight. The reaction was
allowed to cool, diluted with diethyl ether, and washed with
water, and then brine, dried over anhydrous magnesium sulfate,
and the solvent removed under vacuum to afford 71 as a pale
yellow solid (2.5 g), which was used without further purifica-
tion. Spectroscopic data was in agreement with that reported in
the literature.[19]
9-Ethyl-N-[2-(4-fluorophenyl)ethyl)-9H-carbazole-2-
methanamine (39)
A solution of 73 (160 mg, 0.72 mmol), 2-(4-fluorophenyl)
ethylamine (100 mg, 0.72 mmol), and glacial acetic acid (1 drop,
cat.) in anhydrous tetrahydrofuran (5 mL) was stirred at room
temperature for 1 h. Sodium triacetoxyborohydride (150 mg,
0.72 mmol) was added in a single portion and the mixture stirred
for a further 18 h. The reaction mixture was diluted with ethyl
acetate and washed with a saturated aqueous solution of sodium
hydrogen carbonate. The combined organic phases were washed
with water, and then brine, dried over anhydrous magnesium
sulfate, and the solvent removed under vacuum. Purification by
column chromatography (ethyl acetate) afforded 39 as a pale
orange oil (175 mg, 0.51 mmol, 70 %). dH (300 MHz, CDCl3)
1.38 (3H, t, J 7.2, NCH2CH3), 2.80 (2H, m), 2.94 (2H, m), 3.98
(2H, s, CH2NH), 4.31 (2H, q, J 7.2, NCH2CH3), 6.91–6.98 (2H,
m, ArH), 7.09–7.22 (4H, m, ArH), 7.31–7.45 (3H, m, ArH), 8.01
(1H, d, J 7.8, ArH), 8.05 (1H, d, J 7.8, ArH). dC (75 MHz,
CDCl3) 13.7 (CH3), 35.5 (CH2), 37.4 (CH2), 50.5 (CH2), 54.5
(CH2), 107.7 (CH), 108.4 (CH), 115.1 (CH, d, JCF 20.3), 118.7
(CH), 119.1 (CH), 120.1 (CH), 120.2 (CH), 121.9 (C), 122.8 (C),
125.3 (CH), 130.0 (CH, d, JCF 7.5), 135.7 (C, d, JCF 3.0), 138.0
(C), 140.1 (C), 140.2 (C), 161.4 (C, d, JCF 242). m/z (ESI) 347
([M þ H]þ, 90 %), 208 (100).
Biological Evaluation
Compounds (3–46) were screened for anti-parasitic activity
against the helminth Haemonchus contortus using an EC100
assay (the EC100 of a compound being the concentration at
which 100% of the nematodes present were killed). Hae-
monchus contortus were recovered from faeces using literature
methods.[27]
A centrifuge tube containing eggs was shaken well and a
100 mL aliquot of the egg solution taken; the eggs were counted
using the McMaster Chamber in accordance with the manufac-
turer’s instructions. Distilled water was either added or removed
(by centrifuging and removing the appropriate quantity of
water) to obtain an egg solution with a concentration of 100
eggs per 100 mL. Working stock solutions of each compound
(100 mM) were prepared by dissolving and/or diluting each
compound in DMSO. Additional dilutions were performed with
DMSO, as required.
The compounds were assayed using 96 well Nunc tissue
culture plates. Agar (Merck-101614) was prepared as a 2 %
solution and then heated by microwave before cooling to
,458C. A phosphate buffered saline (PBS, 0.85 %) solution
was prepared by dissolving one PBS tablet (Sigma P4417) in
125 mL of distilled water. Earle’s balanced salt solution (1X)
was prepared from 10X Earle’s balanced salt solution (Sigma
E7510). A yeast solution (1 %) was prepared from 0.25 g of
yeast extract (Sigma Y-1000), 22.5 mL of 0.85 % PBS solution,
and 2.5 mL of Earle’s balanced salt solution (1X).
Larval development assay (LDA) media was prepared by
mixing 15 mL of a 0.015 % solution of lyophilised E. coli (strain
W (ATCC) 9637; Sigma Ec9637), 15 mL of a 1 % yeast solution,
and 45 mL of a 5 mg mLꢀ1 solution of amphotericin B (Sigma
A-9528) in distilled water, and either used immediately or stored
overnight at 48C.
A solution of test compound, or water as a negative control
(2 mL), was added to each well, followed by agar (100 mL). The
agar was allowed to set at room temperature, and then a solution
of nematode eggs (60 mL; 100 eggs per 100 mL) and LDA media
(40 mL) were added to each well. The plates were incubated for
up to 10 days at 258C in a plastic container, with a lid covering
,70 % of the opening. The larvae were aerated by blowing air
over the plate following 24 h, and thereafter on every third day
until the plates were evaluated.
Compounds 1 and 39 were also screened for anti-parasitic
activity against the helminth Heligmosomoides polygyrus, in
mice. Mice were infected with 100 Heligmosomoides polygyrus
L3 larvae by oral gavage. Approximately 10 days later, infection
was confirmed by faecal egg count. The infected mice were
dosed with either compound 1 or 39 at 100 mM, or a DMSO
control, by oral gavage based on bodyweight. After 7 days, the
mice were killed and their intestines removed. The contents of
the small intestines were flushed out with 5 mL of water into a
Petri-dish (using a syringe). Adult worms were identified and
counted using a dissecting microscope.
9-Ethyl-9H-carbazole-2-carboxaldehyde (73)[25]
To a solution of 72 (0.51 g, 2.61 mmol) in anhydrous
dimethylformamide (10 mL) at 08C under nitrogen was carefully
added sodium hydride (130 mg, 3.26 mmol, 60 % w/w in oil) in a
single portion. After 0.5h ethyl bromide (0.22 mL, 2.87 mmol)
was added and the mixture was stirred at room temperature
overnight. The reaction mixture was poured onto ice and
extracted with ethyl acetate. The combined organic phases were
washed with water, and then brine, dried over anhydrous
magnesium sulfate, and the solvent removed under vacuum.
Purification by column chromatography (hexane/ethyl acetate,
20 : 1) afforded 73 as a pale yellow solid (0.52 g, 2.33 mmol,
89 %). Spectroscopic data was in agreement with that reported in
the literature.[25]
9H-Carbazole-2-carboxaldehyde (72)[26]
A solution of 71 (2.5 g) and triphenylphosphine (14.2 g,
54 mmol) in dimethylacetamide (20 mL) was heated at 1508C
overnight. The reaction was allowed to cool, diluted with ethyl
acetate, washed with water, and then brine, dried over anhydrous
magnesium sulfate, and the solvent removed under vacuum.
Purification by column chromatography (hexane/ethyl acetate,
7 : 1 then 3 : 1) afforded 72 as a light brown solid (1.50 g,
7.7 mmol, 71 % over 2 steps). Spectroscopic data was in agree-
ment with that reported in the literature.[26]
4-Formyl-2-nitrobiphenyl (71)[19]
A solution of 4-chloro-3-nitrobenzaldehyde (70) (2.0 g,
10.8 mmol), phenylboronic acid (1.43 g, 11.8 mmol), and aque-
ous potassium carbonate (11 mL, 21.6 mmol, 2 M) in toluene

Substituted Carbazoles
I
Supplementary Material
[11] R. B. Besier, Trends Parasitol. 2007, 23, 21. doi:10.1016/J.PT.2006.
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[12] T. G. Geary, N. C. Sangster, D. P. Thompson, Vet. Parasitol. 1999, 84,
275. doi:10.1016/S0304-4017(99)00042-4
Experimental methods and compound characterization are
available on the Journal’s website.
[13] A. Rieche, H. Gross, E. Ho¨ft, Org. Synth. 1967, 47, 1. doi:10.15227/
ORGSYN.047.0001
Acknowledgements
The authors thank ParaCo Technologies Limited, New Zealand (whose
interests in this study are protected under US patent application 61/468, 317)
for their financial support.
[14] A. Vilsmeier, A. Haack, Ber. Dtsch. Chem. Ges. 1927, 60, 119.
[15] A. A. Haddach, A. Kellemanb, M. V. Deaton-Rewolinskia, Tetrahe-
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[16] H.-J. Kno¨lker, K. R. Reddy, Chem. Rev. 2002, 102, 4303. doi:10.1021/
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