Design and synthesis of 1,3,5-trisubstituted 1,2,4-triazoles as CYP enzyme inhibitors

Article abstract of DOI:10.1016/j.tetlet.2011.09.049

A series of 1,3,5-trisubstituted 1,2,4-triazoles was designed and synthesized as potential inhibitors of ste-roidogenic CYP enzymes. The 1,2,4-triazole is part of the core structure fixing the geometry of the substances. A pyridine moiety was introduced as heme-binder. The target compounds were synthesized in two to four steps using silver carbonate mediated ring closure and Suzuki cross coupling reaction as key synthetic transformations. Biological testing of the synthesized compounds for the inhibition of the most important steroidogenic CYPs revealed compounds 29a and 30 as moderate inhibitors of aldo-sterone synthase (CYP11B2).

Full text of DOI:10.1016/j.tetlet.2011.09.049

Tetrahedron Letters 52 (2011) 6372–6375
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Tetrahedron Letters
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Design and synthesis of 1,3,5-trisubstituted 1,2,4-triazoles as CYP
enzyme inhibitors
Yaseen A. Al-Soud a, ,à, Michael Heydel a, , Rolf W. Hartmann a,b,
⇑
a Pharmaceutical and Medicinal Chemistry, Saarland University, P.O. Box 15 11 50, D-66041 Saarbrücken, Germany
b Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, D-66123 Saarbrücken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 June 2011
Revised 5 September 2011
Accepted 12 September 2011
Available online 17 September 2011
A series of 1,3,5-trisubstituted 1,2,4-triazoles was designed and synthesized as potential inhibitors of ste-
roidogenic CYP enzymes. The 1,2,4-triazole is part of the core structure fixing the geometry of the sub-
stances. A pyridine moiety was introduced as heme-binder. The target compounds were synthesized in
two to four steps using silver carbonate mediated ring closure and Suzuki cross coupling reaction as
key synthetic transformations. Biological testing of the synthesized compounds for the inhibition of
the most important steroidogenic CYPs revealed compounds 29a and 30 as moderate inhibitors of aldo-
sterone synthase (CYP11B2).
Keywords:
1,2,4-Triazoles
CYP inhibitors
Steroidogenic CYP enzymes
CYP11B1/2
Ó 2011 Elsevier Ltd. All rights reserved.
1,2,4-Triazoles are a class of heterocycles which are very impor-
tant in organic synthesis. However, in nature 1,2,4-triazoles are
uncommon and only a few examples have been described so far.
The first report of an occurrence of 1,2,4-triazole in nature dates
and fungi. The molecular target is the cytochrome P450 (CYP) en-
zyme CYP51A1 (lanosterol-14
-demethylase).12,13
Further examples of CYP enzyme inhibitors containing 1,2,4-tri-
azole are letrozole 714 and anastrozole 8.15 Both compounds are
potent inhibitors of aromatase CYP19. These substances are de-
picted in Figure 2.
a
to the year 1985 when L-1,2,4-triazole-3-alanine 1 (Fig. 1) was iso-
lated from a Streptomyces sp. strain.1 The compound acts as a his-
tidine antagonist.2,3 Further (and only) examples of natural
products containing 1,2,4-triazole are the antibiotic Essramycin
In all of the above mentioned CYP inhibitors the 1,2,4-triazole
moiety is responsible for the interaction of the compound with
the heme of the CYP enzyme.16 Our group has been working for
more than 20 years on steroidogenic CYP enzymes and is highly
experienced in the development of potent and selective inhibitors
of CYP11B1,17a,b CYP11B2,18a–h CYP17,19a–i and CYP19.20a–f In this
Letter we report on the design of new inhibitors of steroidogenic
CYP enzymes which bear 1,2,4-triazole as part of the core struc-
ture. By adding nitrogen containing heterocycles as heme-binders,
such as pyridine, inhibitors of steroidogenic CYP enzymes should
be developed. The synthesized compounds were tested in our
established assay system in order to evaluate their potency on
the most important steroidogenic CYP enzymes CYP11B1,
CYP11B2, CYP17, and CYP19.
The design strategy starts from 1,2,4-triazole as the central ele-
ment of the core structure. We know from previously described
CYP enzyme inhibitors that the triazole scaffold is well tolerated
and is suitable as drug component. Compared to a benzene core
a triazole moiety possesses a higher hydrophilicity resulting in a
better solubility of the inhibitors. Furthermore, its basicity is signif-
icantly lower compared to an imidazole core. A second reason for
choosing a 1,2,4-triazole as central core is the characteristic geom-
etry of the resulting inhibitors. As we do not want the triazole to
24 and 1-(b- -ribofuranosyl)-1,2,4-triazole 35 isolated from the
D
sea urchin Glyptocidaris crenularis. Both compounds were pub-
lished very recently as natural products but the latter one is al-
ready known in the literature as a synthetic compound.6
Medicinal chemists involved in drug discovery also paid a lot of
attention on 1,2,4-triazoles. In the past years many triazole con-
taining drugs were discovered. The most important application of
1,2,4-triazoles is as biocides but also as drugs for usage for instance
in cancer or immunosuppressed patients for prophylaxis and treat-
ment of life-threatening invasive fungal infections.7 Figure 1 illus-
trates posaconazole8,9 4, fluconazole10 5, and triadimenol11 6 as
examples for antifungal drugs (4, 5) or fungicide (6), respectively.
All of these compounds inhibit the biosynthesis of ergosterol
which is important for the growth of the cell membrane in yeast
⇑
Corresponding author. Tel.: +49 681 302 70300; fax: +49 681 302 70308.
E-mail address: rwh@mx.uni-saarland.de (R.W. Hartmann).
These authors contributed equally to this work.
Address: Department of Chemistry, College of Science, University of Al al-Bayt,
à
P.O. Box 1240, 25115 Al-Mafraq, Jordan.
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.09.049

Y. A. Al-Soud et al. / Tetrahedron Letters 52 (2011) 6372–6375
6373
Figure 3. General structures of the synthesized compounds.
Figure 1. Natural products and antifungal drugs bearing a 1,2,4-triazole moiety.
Figure 2. Aromatase inhibitors letrozole 7 and anastrozole 8.
Scheme 1. Reagents and conditions: (a) toluene, rt, 12 h; (b) NCS, DMS, CH2Cl2, 0 °C
to ꢀ78 °C to rt, 46–49% (over two steps); (c) 13, TEA, CH3CN, rt, 12 h; (d) Ag2CO3,
CH3CN, rt, 2 h, 48–77% (over two steps).
act as heme binder it needs to be shielded by appropriate substit-
uents. The interaction with the heme shall be achieved by a pyridyl
substituent which is connected to the 5-position of the triazole via
a short variable (n = 0, 1) alkyl or phenyl linker. In the 3-position
either a phenyl group or a 3- or 4-methoxyphenyl group (R1 = H,
OMe) is linked to the triazole. The methoxy group (R1) is supposed
to act as H-bond acceptor in a similar way as the natural steroidal
substrates. The resulting 3-aryl-1,2,4-triazole core structure mim-
ics the A- and B-ring of the natural steroidal substrate. The 1-posi-
tion of the triazole is furthermore substituted by a methyl, phenyl
or, benzyl group (R2). The increasing bulkiness of those substitu-
ents could give insights into the shape of the active site. We know
from previous SAR in other inhibitor classes that for instance a
benzyl group may exploit a subpocket present in CYP11Bs, thus
increasing the selectivity toward other steroidogenic CYP enzy-
mes.18e The resulting general structures of the desired inhibitors
are visualized in Figure 3.
For compounds with R2 = Ph the synthesis starts from benzalde-
hyde 9a or 4-methoxybenzaldehyde 9b, respectively, which is con-
densed with phenylhydrazine 10 to give hydrazones 11a,b which
were used in the next step without further purification
(Scheme 1).21 Hydrazones 11 are converted to the corresponding
hydrazonyl chlorides 12 by the reaction with n-chlorosuccinimide
in the presence of dimethylsulfide. The chlorine was displaced by
primary amines 13 following the approach by Buzykin to give
the triazene intermediates 14a–e22,23 which were not isolated
and immediately cyclized to the final products 15. The desired
1,3,5-substituted 1,2,4-triazoles 15a–e were obtained after cycliza-
tion of intermediates 14 mediated by silver carbonate.21
formed N0-benzyl benzohydrazonamide 18 with substituted het-
eroaryl acid chlorides 19 (Scheme 2). The intermediate benzohy-
drazonamide 19 was synthesized from ethyl benzimidate 16
which was reacted with benzyl hydrazine 17. This reaction se-
quence afforded compounds 20a–c. Removal of the bromine sub-
stituent of 20a using palladium and hydrazine yielded 20d. A
Suzuki cross coupling reaction of 20a with (3-methoxyphenyl)
boronic acid under Pd catalysis and microwave irradiation led to
compound 20e.
The synthesis of 1-methyl substituted 1,2,4-triazoles (R2 = Me)
is outlined in Scheme 3. Ethyl 4-methoxybenzimidate 21a was re-
acted with 5-bromonicotinoyl chloride to give intermediate 23
which was used in the next step without further purification and
was subsequently cyclized under mild conditions with meth-
ylhydrazine via an Einhorn–Brunner reaction24 yielding compound
24.
The preparation of compounds 29a,b and 30 proceeded in a
similar manner as for compound 24 (Scheme 4). After cyclization
the intermediates 27a,b were reacted with either 3-pyridine boro-
nic acid 28a or 4-pyridine boronic acid 28b in a Pd-catalyzed Suzu-
ki cross coupling reaction to afford the desired target substances
29a,b and 30.
The 14 synthesized 1,3,5-trisubstituted 1,2,4-triazoles 15a–e,
20a–e, 24, 29a,b and 30 were tested in our established screening
system with regard to their ability to inhibit the most important
human steroidogenic CYP enzymes CYP11B1, CYP11B2, CYP17,
and CYP19. The assays were performed as described previously
for CYP11B1,17a CYP11B2,17a CYP17,25 and CYP19.25b,26 After the
The 1,2,4-triazoles with benzyl substituent in 1-position
(R2 = Bn) 20 were readily prepared via ring closure of the initially

6374
Y. A. Al-Soud et al. / Tetrahedron Letters 52 (2011) 6372–6375
Scheme 2. Reagents and conditions: (a) EtOH, 30–40 °C, 4 h, 48%; (b) 19, toluene,
110–120 °C, 10 h, 45–80%; (c) Pd/C, N2H4ꢁHCl, NaOH, toluene, 25 °C, 24 h, 57%; (d)
(3-methoxy-phenyl)boronic acid, NaHCO3, Pd(PPh3)4, DMF, MWI, 15 min, 50%.
Scheme 4. Reagents and conditions: (a) TEA, CH2Cl2, 30–40 °C, 6 h; (b) MeNHNH2,
CH2Cl2, 30–40 °C, 4 h, 52% (over two steps); (c) 28a or 28b, NaHCO3, Pd(PPh3)4,
DMF, MWI, 15 min, 41–83%.
Table 1
Inhibition of human steroidogenic CYP enzymes in vitro
a
Compound
IC50
(l
M)
CYP11B1b
CYP11B2b
CYP17c
CYP19d
15a
15b
15c
15d
15e
20a
20b
20c
20d
20e
24
ni
ni
ni
ni
1.00
ni
ni
ni
1.23
ni
ni
ni
ni
ni
3.20
4.37
ni
ni
2.11
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
1.62
ni
ni
ni
ni
ni
ni
ni
Scheme 3. Reagents and conditions: (a) TEA, CH2Cl2, 30–40 °C, 6 h; (b) MeNHNH2,
CH2Cl2, 30–40 °C, 4 h, 35% (over two steps).
initial screening of all compounds at a single concentration (data
not shown) the IC50 values of all active compounds were deter-
mined in order to demonstrate the dose dependency of the enzyme
inhibition. The obtained IC50 values are summarized in Table 1.
Some compounds showed an inhibition of steroidogenic CYP
enzymes. Thus compound 20b could be identified as a weak inhib-
itor of aromatase (CYP19) having an IC50 value of 1.62 lM whereas
all other tested compounds showed no activity toward this en-
zyme. None of the tested compounds showed an inhibition of
ni
4.76
ni
ni
0.65
ni
29a
29b
30
0.66
0.52
a
Mean values of at least two experiments, standard deviation less than 25%.
Chinese hamster lung fibroblasts expressing stably either human CYP11B1 or
b
CYP11B2; substrate [3H]-labeled 11-deoxycorticosterone, 100 nM; reference com-
17a-hydroxylase/17,20-lyase (CYP17).
pound: rac-fadrozole IC50 B1 = 10 nM, IC50 B2 = 1 nM.
c
Regarding the CYP enzymes responsible for corticoid formation
E. coli expressing human CYP17; substrate progesterone, 25 lM; reference
(CYP11B) some compounds with weak to moderate potency to-
ward CYP11B1 and CYP11B2 could be identified. Most of the active
compounds show IC50 values in the low micromolar range. Two
compounds, 29a and 30, inhibit CYP11B2 with IC50 values in the
submicromolar range. The IC50 values for the inhibition of CYP11B1
and CYP11B2 by 30 were determined to be 663 nM for CYP11B1
and 515 nM for CYP11B2, thus the compound is a moderate inhib-
itor but shows no selectivity. This result is not very surprising as
these two enzymes share 93% of their protein sequence thus exhib-
iting a high homology.27 The big challenge in drug development is
to create inhibitors that are able to discriminate between CYP11B1
and CYP11B2. The IC50 value of compound 29a for the inhibition of
compound: abiraterone IC50 = 72 nM.
d
Human placental CYP19; substrate androstenedione, 500 nM; reference com-
M.
pound: letrozole 7 IC50 = 36 nM. ni = no inhibition, IC50 value higher than 10
l
CYP11B2 is 650 nM. Interestingly, this compound inhibits CYP11B2
seven-fold stronger than CYP11B1 (IC50 = 4.76 M). Both com-
pounds, 29a and 30, differ only in the position of the terminal
methoxy- and 3-pyridine substituents. So we could show that in
this compound class a slight variation in the molecular geometry
could turn an unselective inhibitor into a selective one.
l
Data show that the nature of residue R2 is important for the
inhibitory activity. Only in the case of R2 = Me or Bn active

Y. A. Al-Soud et al. / Tetrahedron Letters 52 (2011) 6372–6375
13. Nebert, D. W.; Russel, D. W. Lancet 2002, 360, 1155–1162.
6375
compounds were obtained. Phenyl substituted compounds turned
out to be inactive. An explanation may be the steric hindrance of
the phenyl group. Compound 15e having an additional CH2 spacer
connecting core structure and the heme-binding pyridine results in
a more flexible structure and thus a moderate inhibition of
CYP11Bs could be observed. The position of the nitrogen in the
heme-binding pyridine is also important. 4-pyridine as present in
15e seems to enhance the inhibition of CYP11B1 whereas a 3-pyr-
idine is more favorable to inhibition of CYP11B2.
In this project we synthesized 14 compounds and screened
them for the inhibition of steroidogenic CYP enzymes. Two inhibi-
tors of CYP11B with moderate potency could be identified. With
this work we were able to demonstrate that 1,2,4-triazole is a suit-
able core structure for inhibitors of steroidogenic CYP enzymes.
The identified unselective CYP11B inhibitor 30 blocks the corticoid
biosynthesis with IC50 values in the nanomolar range. In compari-
son to 30, compound 29a possesses a slightly decreased activity to-
ward CYP11B2 but shows some selectivity toward the highly
homologous CYP11B1. The molecular scaffolds of 29a and 30 can
therefore be used as lead structures for further inhibitor design.
By combination of the scaffolds and additional introduction of
appropriate substituents at the 1,2,4-triazole core it should be pos-
sible to obtain inhibitors with enhanced activity toward CYP11B2
and selectivity toward CYP11B1.
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Acknowledgments
Y.A.S. is grateful to the Alexander von Humboldt-Foundation for
a scholarship. We thank Professor Hermans (University of Maas-
tricht) for providing us with V79MZh11B1 cells. Finally, we want
to thank Ms. Gertrud Schmitt and Ms. Viktoria Kraemer for per-
forming the biological assays.
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