Identification and development of novel inhibitors of toxoplasma gondii enoyl reductase

Article abstract of DOI:10.1021/jm9017724

Toxoplasmosis causes significant morbidity and mortality, and yet available medicines are limited by toxicities and hypersensitivity. Because improved medicines are needed urgently, rational approaches were used to identify novel lead compounds effective against Toxoplasma gondii enoyl reductase (TgENR), a type II fatty acid synthase enzyme essential in parasites but not present in animals. Fifty-three compounds, including three classes that inhibit ENRs, were tested. Six compounds have antiparasite MIC₉₀s ≤ 6 μM without toxicity to host cells, three compounds have IC₉₀s < 45 nM against recombinant TgENR, and two protect mice. To further understand the mode of inhibition, the cocrystal structure of one of the most promising candidate compounds in complex with TgENR has been determined to 2.7 A?. The crystal structure reveals that the aliphatic side chain of compound 19 occupies, as predicted, space made available by replacement of a bulky hydrophobic residue in homologous bacterial ENRs by Ala in TgENR. This provides a paradigm, conceptual foundation, reagents, and lead compounds for future rational development and discovery of improved inhibitors of T. gondii.

Full text of DOI:10.1021/jm9017724

NIH Public Access
Author Manuscript
J Med Chem. Author manuscript; available in PMC 2011 September 9.
Published in final edited form as:
J Med Chem. 2010 September 9; 53(17): 6287–6300. doi:10.1021/jm9017724.
Identification and Development of Novel Inhibitors of
Toxoplasma gondii Enoyl Reductase
‡,+
†,+
#,+
^,+
Suresh K. Tipparaju , Stephen P. Muench , Ernest J. Mui , Sergey N. Ruzheinikov
,
§
#
#
§
∫
Jeffrey Z. Lu , Samuel L. Hutson , Michael J. Kirisits , Sean T. Prigge , Craig W. Roberts ,
Fiona L. Henriquez , Alan P. Kozikowski , David W. Rice , and Rima L. McLeod
∫
‡
†
#,*
‡ Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University
of Illinois at Chicago, Chicago, Illinois, USA
† Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK
# Department of Ophthalmology and Visual Sciences, Pediatrics (Infectious Diseases),
Committees on Genetics, Immunology and Molecular Medicine, Institute of Genomics and
Systems Biology, and The College, The University of Chicago, Chicago, Illinois, USA
^ Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK
§ Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of
Public Health, Baltimore, Maryland, USA
∫ Department of Immunology and Strathclyde Institute of Pharmacy and Biomedical Sciences,
University of Strathclyde, Glasgow, Scotland, UK
Abstract
Toxoplasmosis causes significant morbidity and mortality and yet available medicines are limited
by toxicities and hypersensitivity. Since improved medicines are needed urgently, rational
approaches were used to identify novel lead compounds effective against Toxoplasma gondii
enoyl reductase (TgENR), a type II fatty acid synthase enzyme essential in parasites but not
present in animals. Fifty-three compounds, including three classes that inhibit ENRs, were tested.
Six compounds have anti-parasite MIC s ≤6μM without toxicity to host cells, three compounds
90
have IC s <45nM against recombinant TgENR and two protect mice. To further understand the
90
mode of inhibition, the co-crystal structure of one of the most promising candidate compounds in
complex with TgENR has been determined to 2.7Å. The crystal structure reveals that the aliphatic
side chain of compound 19 occupies, as predicted, space made available by replacement of a bulky
hydrophobic residue in homologous bacterial ENRs by Ala in TgENR. This provides a paradigm,
conceptual foundation, reagents, and lead compounds for future rational development and
discovery of improved inhibitors of T. gondii.
Introduction
Toxoplasmosis causes substantial morbidity and mortality, especially in persons who are
congenitally infected or immune-compromised and this parasite is the most frequent
1–4
infectious cause of uveitis.
Toxoplasma gondii (T. gondii, Tga) is acquired as a
*To whom correspondence should be addressed. d; rmcleod@midway.uchicago.edu.
+
These authors contributed equally to this work.
The PDB ID for TgENR co-crystallized with compound 19 is 3NJ8.
Supporting Information Available
Table S1 contains HPLC purity analysis data of the tested TgENR inhibitors.

Tipparaju et al.
Page 2
sporozoite from oocysts formed in cats or bradyzoites from cysts in meat. In humans, this
parasite has a simple life cycle consisting of two stages; tachyzoites and bradyzoites. The
former are a rapidly growing, obligate intracellular forms of T. gondii present when parasites
are first acquired in acute infections. T. gondii then develops into slowly growing, encysted,
latent bradyzoites, sequestered within cysts inside cells, with a competent host immune
response. When a cyst ruptures, stage transition from latent bradyzoites back to rapidly
growing tachyzoites occurs, causing destruction of surrounding tissue.
Reasons for recrudescence of eye disease have not been completely defined but is a lifelong
problem in individuals infected congenitally as well as some of those whose infection is
3,4
acquired after birth. This is an especially pressing problem in Brazil, as 80% of the
population is infected with particularly pathogenic parasite strains, with a high incidence
beginning in childhood. In some regions of Brazil, 20% of these individuals and 50% of
those over 50 years old have eye disease. In immunocompromised persons such as those
with AIDS, disease due to recrudescence (especially in the brain) is frequent, occurring in
50% of those with AIDS whose HIV infection remains untreated. Life threatening
toxoplasmosis occurs in those immunocompromised by malignancies, organ transplantations
and autoimmune disease with associated treatments. Rarely, there is significant organ
damage in those without known immune compromise. An epidemic of multivisceral, lethal
disease caused by a hypervirulent strain of parasite was reported recently in Guyana, making
this emerging infection potentially even more problematic with globalization of food
supplies. This parasite can easily contaminate food supplies or the environment and is a
potential bioterrorism pathogen. There have been several recent epidemics associated with
contaminated water supplies.
Consequences of chronic infections present in ~30% of the population (~2 billion people)
worldwide, throughout their lifetimes, are not thoroughly characterized. Recently, memory
impairment was reported in healthy, young to middle aged professionals in association with
this infection and presence of a susceptibility allele of a gene encoding an enzyme that
degrades dopamine, catechol O-methyl transferase (COMT) (Yolken et al. 2009 Personal
Communication). There is also a higher prevalence of antibody to T. gondii in those with
cryptogenic epilepsy and schizophrenia, although cause and effect between T. gondii
infection and these neurologic observations has not yet been proven.
1–4
There are only a few medicines that restrict growth of tachyzoites,
and use of these
medicines is associated with significant incidences of hypersensitivity (up to 25%) and
5
toxicity. No medicines eliminate encysted, latent bradyzoites. Better approaches to treat
this disease are greatly needed including medicines that eliminate active parasites causing
disease and means to eliminate latent parasites.
6–15
16
Recent work by our group,
and a recent report by others, provide the foundation for
the present work to develop a new class of medicines to better treat toxoplasmosis.
Specifically, the prokaryotic-like type II fatty acid biosynthetic (fas) pathway in T. gondii is
a validated molecular target in tachyzoites it is essential for parasite survival in vitro and in
16
vivo. In particular, the enoyl reductase (ENR) enzyme, which catalyses the last reductive
step of the type II fatty acid synthesis pathway, is present in all T. gondii life cycle stages
11
except microgametes and ENRs in other organisms have been shown to be the target for a
wide range of potent inhibitors. Importantly, compounds which inhibit type II fatty acid
synthesis (including triclosan and a number of newly designed and synthesized compounds)
not only inhibit T. gondii tachyzoite growth but are effective against other apicomplexan
a
Abbreviations: T. gondii and Tg, Toxoplasma gondii; ENR, enoyl reductase; COMT, catechol O-methyl transferase; HLM, Human
liver microsomes.
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Tipparaju et al.
Page 3
,7,16–20
parasites, such as the hepatic stage of Plasmodia
which causes malaria. However,
while triclosan is a potent inhibitor of the enzyme, its poor solubility makes it unsuitable as a
potential therapeutic. This prompted the investigation into different triclosan scaffolds as
potential therapeutic agents for toxoplasmosis, with improved activity, as well as
physicochemical properties and toxicity profiles.
This report details activity of a panel of 53 compounds (Figure 1) based on previously
identified ENR inhibitors, with two of these compounds (compounds 2 and 19) showing low
toxicity and activity in the low nM range. A promising adaptation to the triclosan scaffold is
the addition of an n-propyl group at the 4-position, exploits an increase in space in the
parasitic ENR binding pocket. In order to validate this, a co-crystal structure of one of the
most promising inhibitors (compound 19) in complex with TgENR, was determined to 2.7Å.
This structure revealed that these inhibitors utilize the extra space within the binding pocket
of the parasitic ENR family. The data presented herein provides insights into lead
compounds which form the basis for future development of highly active inhibitors of ENR
with potential for progression to medicines that effectively treat toxoplasmosis and related
apicomplexan diseases.
Results
Synthesis of Inhibitors
Synthesis of compounds tested is shown in Figure 2 (Synthetic Schemes). The numbers,
molecular weights, ClogPs and structures of compounds are shown in Figures 1 and 2. The
requisite diphenyl ethers were synthesized either by nucleophilic aromatic substitution
(Method A) or through Cu catalyzed coupling reactions (Method B). The choice of the
method was dependent on the product desired and the choice of the starting phenol. In each
case, the methoxy diarylether precursors so obtained were demethylated to phenols using
boron tribromide (Scheme 1). The aryl boronic acid 20 was prepared by lithiation of 1-(3-
chlorophenoxy)-2-methoxy-4-propylbenzene followed by quenching with trimethyl borate
followed by acidic hydrolysis. The diarylether derivatives 2, 4, 6, 8, 9–12, 14–19, 21–24, 29,
21
22
31, and the 2-pyridone derivatives 38–44 were previously published by us. Amides 7, 25
and 27 were prepared from their corresponding aniline precursors using standard amide
bond forming procedures (Scheme 2). Pivaloyl ester 3 was prepared from triclosan (Scheme
23
2). Aminopyridines 52 and 53 were synthesized from 3-(6-aminopyridin-3-yl)-acrylic acid
according to Scheme 3. Benzimidazolones 46–48 were prepared using standard procedures
as depicted in Scheme 3. The HPLC and high resolution mass spectroscopy were employed
to determine the purity of the tested compounds. Purity was between 96 and 100% as shown
in Table S1.
Inhibition of T. gondii tachyzoites in vitro
A summary of ability of each compound to inhibit the parasite’s growth by 50% (MIC )
50
and 90% (MIC ) and effect on growth and survival of nonconfluent cultures of the same
90
fibroblasts host cells in the parasite inhibition assay, as an indication of toxicity, are in
Figure 1.
Analysis of the 53 compounds identified 6 compounds that robustly inhibited parasites
(~60–95% inhibition of parasite growth) with less than 20% toxicity to host cells with
MIC s <6μM (Figure 1). An additional six compounds had modest inhibitory effect on
90
parasites (~20% to 60% inhibition of parasite growth) and an additional three had ~20–40%
inhibition of host cell uptake of tritiated thymidine (Figure 1). The next six compounds had
less than 40% inhibitory effect on uptake of uracil by parasites or were very toxic to host
cells precluding interpretation of parasite inhibition assays. The last compounds shown had
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Tipparaju et al.
Page 4
minimal inhibitory effect and/or harmed host cell growth substantially (Figure 1). These data
showing IC and IC are in Figure 1. To illustrate these results in specific experiments
50
90
with more detail, a representative experiment with compounds 25, which was toxic for
fibroblasts, and 19, which was efficacious and not toxic are shown in Figure 3. Of the initial
53 compounds tested, compounds that displayed the greatest effect and least toxicity were 2,
3, 19, and 39.
Inhibition of TgENR activity
Compounds that inhibited the growth of T. gondii in culture (Figure 1, shaded regions) were
initially tested for inhibition of TgENR enzymatic activity at three concentrations (0.2, 2,
and 20μM). Compounds which displayed significant inhibitory activity at 2μM were assayed
in triplicate at ten concentrations to determine IC values as summarized in Figure 1 and
50
shown in detail in Figure 3. This assay uses 20nM TgENR, preventing the accurate
measurement of IC values below this concentration. Seven compounds (including
50
triclosan) are listed as having IC values below 20nM. Compound 39 turned out to be a
50
poor inhibitor of TgENR and hence appears to have an off target effect on parasites and
requires further investigation. This result indicates that this compound may be of interest for
further development but does not target ENR specifically.
+
Co-crystallization and structure solution of TgENR in complex with NAD and compound
19
In order to gain insights into the mode of binding for compound 19 which is very active
against the ENR enzyme (IC <20nM; Figure 3 bottom panel) and the parasite in tissue
50
culture, structural studies were conducted. TgENR was co-crystallized in the presence of
+
NAD and compound 19 with the subsequent crystals diffracting to beyond 2.7 Å (Data are
in Table 1). The refined structure showed clear and continuous density for the bound
inhibitor allowing for its unambiguous placement within the structure (Figure 5a). The
superposition of TgENR in complex with compound 19 and triclosan shows that the mode
of binding for both inhibitors is similar with the aromatic rings of both compounds adopting
+
the same position, with ring A in both cases forming stacking interactions with the NAD
co-factor. The additional bulk of the alkyl substituent of compound 19, when compared to
triclosan, occupies the space made available by replacement of a bulky hydrophobic residue
common to the bacterial enzyme family (Met206 in E. coli) by alanine (Ala247 in TgENR)
which is conserved in the apicomplexan family (Figure 5c). Comparison of the triclosan and
compound 19 TgENR complex also reveals that there is little change in the position of the
mainchain atoms with an overall Cα r.m.s.d value of 0.3 Å (Figure 5b). However, the side
chain of Phe 243 does move significantly by ~1.5Å about its Cβ atom to accommodate the
extra bulk of compound 19 compared to triclosan, without significant change to its
mainchain position (Figure 5b, c). As such elaborating the nature of the substituent on the
phenoxy ring of the candidate compound exploits the increase in space which is a conserved
feature of the apicomplexan ENR family, without perturbing the overall fold of the binding
pocket.
Inhibition of T. gondii tachyzoites in vivo
Effect of Compounds 2 and 19 on T. gondii in vivo are in Figure 4. Representative
experiment of two replicate studies, indicate that compounds 2 and 19 can reduce parasite
burden in mice (P<0.05).
Cytochrome P450 inhibition and human liver microsomal stability
2 and 19 were tested for in vitro inhibition of recombinant CYP450 isozymes 2C9, 2D6 and
3A4. At 10μM, 2 and 19 showed strong inhibition of CYP2C9 (both >85%) and relatively
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Tipparaju et al.
Page 5
low inhibition of CYP2D6 and CYP3A4 (both <22%). Further, metabolic stabilities in
pooled human liver microsomes (HLM) indicated that they were moderately metabolized in
HLM, and remaining parent drugs after incubation for 60 minutes were 51.23% and 45.14%,
respectively. Intrinsic clearances, metabolic rates, of 2 and 19 in HLM were slower than the
control drug verapamil.
Discussion
Triclosan is a broad spectrum antibacterial agent incorporated into a wide range of consumer
24–26
products.
coli
It inhibits the ENR (FabI) enzyme in a number of microorganisms like E.
,27,28
29
30
P. aeruginosa and S. aureus. Significant variation in the degree of inhibition of
different pathogens by triclosan has been observed
28–30
suggesting that it should be possible
to modify the compound to improve its efficacy against a particular organism through
systematic structure-based as well as ligand-based drug design methods to improve
properties such as potency, solubility and toxicity.
The crystal structure of T. gondii and P. falciparum ENR in complex with triclosan shows
the binding geometry of triclosan to be similar to that seen with other organisms. Thus the
+
phenoxy ‘ring A’ (Figure 1 and 2) of triclosan and the nicotinamide ring of NAD engage in
π-stacking interactions, while the phenolic hydroxyl group on ring A (Figure 1) is involved
in a hydrogen bond with a conserved Tyr (OH), and the ether linkage has two hydrogen
+
bonds, one to the hydroxyl group of Tyr, and the other to the ribose unit of the NAD .
7
Encouraged by our earlier results on triclosan activity against TgENR, we tested a number
of newly synthesized triclosan derivatives in addition to those available to us from our
21,22
previous work.
In addition, we designed and tested compounds based on diazaborine
and aminopyridine inhibitor families shown to inhibit the enzyme with the goal of attaining
improved inhibitors.
Several of the triclosan derivatives identified were active against parasites at low
micromolar levels with sub-micromolar inhibitory activity toward TgENR (Figures 1 and 3).
In general, presence of a strong electron withdrawing group on the B ring contributed to
improved activity while the presence of an electron donating group reduced the activities of
these compounds (compare compounds 2 vs 1, 8 vs 10, 4 vs 10. Freundlich and co-workers
31
showed that compound 2 has an IC of 0.2μM against P. falciparum ENR. This
50
compound is highly active against TgENR as well, which is not surprising as the active sites
of these two enzymes share significant homology. Unlike the observation of Sivaraman et
,32,33
al
in the case of TgENR, deletion of chlorine atoms on the B ring of triclosan (as in
compound 4) does not improve activity toward both TgENR and the parasite, suggesting
that the presence of a more lipophilic group would favor the improvement in activity.
However, modification of the B ring to a biphenyl to increase its hydrophobicity did not
+
improve activity (compound 6). This is not surprising since this group faces the NAD co-
factor and would cause a large degree of steric hindrance accounting for the large increase in
IC value (>2000 nM). When the B ring of triclosan is replaced by a hetero-aromatic ring
50
resulted in compounds 17 and 18 with compound 17, the pyridine analog of compound 4
showing some activity. However, compound 18, the corresponding pyrazine analog had no
activity. Since compound 18 is smaller than triclosan this lack of activity is unlikely to be
caused simply through steric hindrance within the binding pocket highlighting the
importance of presentation of an appropriate electronic distribution in this ring to allow for
optimal interaction with the active site. Moreover, the pyrazine may be attracted towards the
+
NAD cofactor, destabilizing both the rings A and B, driven in part by the formation of a
+
hydrogen bond between the backbone oxygen of NAD and nitrogen of compound 18.
Further studies are required to test this hypothesis. Although of the 11 compounds reflecting
changes focused on the B ring (2, 4, 8–10, 14–18) only compound 2 displays a comparable
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Tipparaju et al.
Page 6
efficacy to triclosan. These observations, taken together open up additional avenues for
further development of a new class of heteroaryl analogs of triclosan. However, caution
+
must be taken to not disturb the important interaction the A ring makes with the NAD
cofactor through changes in the B ring.
The phenolic hydroxyl group on ring A is considered to be critical to triclosan’s activity, as
it is involved in hydrogen bonding to a conserved Tyr residue in the active site and forming
+
packing interactions with the NAD cofactor. This has been observed by us in our earlier
10
work, and is also evident by the inactivity of compound 5. The significant change in
potency between compounds 4 (IC >20<200 nM) and 5 (IC >20000 nM), can only be
50
50
attributed to a change of the OH group to NH . In light of this observation, it is interesting to
2
note that 3 shows improved activity on the parasites over triclosan itself. Since the pivalate
group would produce severe steric clashes within the binding pocket (as highlighted by the
increased IC value) we suspect that this heightened activity may be due to the enhanced
50
ability of this pro-drug form to enter parasites, whereby the ester is hydrolyzed. Similarly,
while the antiparasitic activities of compounds 4 and 13 were comparable, there is
significant difference in their TgENR activity indicating that a tertiary butyl group is indeed
too bulky at this position. The differences in the antiparasitic activity and TgENR inhibitory
activity observed in some of the analogs is not surprising and could be attributed to off
target effects and problems with delivery, particularly with delivery of an inhibitory
compound into the apicoplast that has 4 surrounding membranes and in which ENR resides.
We intend to investigate this finding further.
Diazaborines are known to inhibit ENR through covalent bond formation between the 2′-
+
hydroxyl of the nicotinamide ribose unit of the NAD molecule and the boron atom of the
34
drug. Consequently, we believed that it would be valuable to incorporate this type of
functionality into our triclosan based ENR inhibitors, and thus we prepared the boronic acid
analog 20. This compound also proved to be a very effective inhibitor of TgENR, although
further studies are needed to determine whether a covalent bond has been formed with the
nicotinamide ribose. Other attempts to vary the meta position of the B ring with H-bond
donors and acceptors did not give encouraging results (compounds 29, 31, and 33).
23
Although the indole aminopyridine compound 50 was very effective against S. aureus and
E. coli ENR, it shows only moderate activity toward TgENR. We synthesized several
aminopyridine mimics of this compound, namely 50, 52 and 53, in order to determine
whether this series had any special merits that might warrant further investigation.
Unfortunately, none of these showed any promising activity, this may in part be due to the
steric hindrance which can be brought about by Phe243 (TgENR numbering) in the binding
15
pocket as previously reported.
When the A ring was modified by replacing the chlorine atom at the para position by an
hydrophilic OH group, the activity decreased (compounds 11 and 12). These changes on
ring A may be brought about by the disturbance of the stacking interactions with the NAD
+
cofactor which are critical for inhibitor binding. Furthermore, this inactivity could also be
influenced by the lower lipophilicity of these compounds rather than the electron-donor
effect of the hydroxyl group. To test this hypothesis, we attempted to append a long
hydrophobic alkyl chain at the para position. This can be accommodated since the bacterial
and plant ENR family contain a bulky hydrophobic residue (Met, Leu or Ile) close to the
ring A of triclosan, whereas the ENR of apicomplexan species such as T. gondii and
Plasmodium have a fully conserved alanine residue. This sequence change produces an
increase in space at the base of the binding pocket and reduces the van der Waals packing
interactions with the triclosan based inhibitors in comparison to other members of the ENR
15
family. When we synthesized a number of triclosan derivatives bearing an n-propyl group
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Tipparaju et al.
Page 7
at the 4-position which were predicted to be optimal length to fit within the binding site,
these inhibitors showed, in general, better or similar activities than the triclosan scaffold
(compounds 19–24), with no detrimental effect of this extension to their IC values with
50
derivatives that bear electron withdrawing groups on the B ring (compounds 19, 21–24)
producing among the most active compounds identified to date.
To further investigate the mode of binding of the most promising inhibitor which contains
an n-propyl group at the 4 position, TgENR was co-crystallized in the presence of
+
compound 19 and NAD . The structure reveals that compound 19 binds in very similar
35–45
mode to triclosan (Figure 5c).
A structural comparison of the complex with that of the
+
E. coli ENR/NAD /triclosan enzyme reveals that the additional bulk of the alkyl substituent
of compound 19 compared to triclosan does occupy the space made available by the
replacement of a bulky hydrophobic residue of the E. coli enzyme Met by alanine in TgENR
within the substrate binding pocket. Moreover, the structure shows that n-propyl is not the
most optimal group as there is still some additional space at the base of the pocket which
may be further utilized through further modifications. The only significant difference in side
chain positions between the compound 19 and triclosan complex is a small change in the
position of Phe243 to accommodate the larger bulk of compound 19 (Figure 5c). Presence of
an n-propyl group on the 4-position clearly does not adversely affect the activity of these
inhibitors (compare compounds 2 and 22), hence this side chain could be further modified to
modulate both physicochemical properties and activity of these compounds. Moreover,
despite triclosan and compound 19 showing similar solubility, as shown by their ClogP,
ALOGpS, and tPSA values (Table 2), there is more scope for improvement through
modifications of compound 19, (in particular the additional n-propyl group) when compared
to triclosan. Since the ability to bind the triclosan analogues with the extension on the n-
propyl group is only achieved within the parasitic ENR it may be possible to develop an
inhibitor which is specific for the parasitic and not bacterial family. Although the addition of
the n-propyl side chain maximizes packing interactions within the active site, these
interactions are not fully exploited and could explain why the n-propyl group did not
enhance activity. The additional n-propyl group does however produce a compound which is
more tailored towards the apicomplexan and not bacterial ENR family. Moreover, further
development of this inhibitor family is feasible particularly with the crystal structure
showing there is still additional space at the base of the inhibitor binding pocket which could
be further modified to improve binding.
By maintaining the A ring of these compounds which is involved in packing interactions
+
with the NAD , and changing the B ring, it is clear that subtle variations in the MIC90 value
can be achieved. Since these changes are small it may be that they affect the packing of the
B ring within the active site or change its electrostatic properties without destabilizing the
+
interaction with the A ring and NAD such that the mode of binding will be very similar to
triclosan. Among these, inhibitor 19, bearing a 4-cyano group on the B ring seemed to be
most promising with the least toxicity to host cells. Interestingly, compound 24 with a 4-
amino functionality on the B ring also showed reasonable activity albeit lower than
compound 19. This lower activity is caused through the removal of the chlorine and
replacement of CN with NH on ring B, this again affects the properties of the B ring whilst
2
+
allowing the A ring to stack onto the NAD cofactor such that the IC value is still
50
comparable with triclosan. From a medicinal chemistry perspective, triclosan with two
chlorine atoms at the 2′ and 4′ positions on the ring B presents little opportunity to
incorporate any polar susbstitutions in order to reduce the lipophilicity of the drug. The
rationale behind introduction of functionalities such as CN or NO on ring B is to create an
2
opportunity to modify these groups in future rounds of ligand optimization to improve the
physicochemical properties of these compounds. However, increased electron density on the
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Tipparaju et al.
Page 8
A ring (a difference between compound 19 vs. 30, and 32) which enhances ability of
+
compound 19 to π-stack with the nicotinamide ring of NAD did not improve activity.
As a final proof of concept for the promising activity of compounds 2 and 19, they were
tested in an animal model for Toxoplasma infection with both protecting mice against
parasite burden in vivo with an efficacy approximately 5 times higher than triclosan itself
(Figure 4). In addition, a further study into the metabolism of these derivatives was
performed. Both compounds 2 and 19 were tested for in vitro inhibition of recombinant
CYP450 isozymes 2C9, 2D6 and 3A4. At 10μM, 2 and 19 showed strong inhibition of
CYP2C9 (both >85%) and relatively low inhibition of CYP2D6 and CYP3A4 (both <22%).
Further metabolic stabilities in pooled HLM indicated that they were moderately
metabolized in HLM, and remaining parent drugs after incubation for 60 minutes were
51.23% and 45.14%, respectively. Intrinsic clearances and metabolic rates of 2 and 19 in
HLM were slower than the control drug verapamil. Interestingly, when certain of these
compounds recently were used to test their efficacy against Bacillus anthracis some were
21,22
noted to be effective.
The scavenging of fatty acids which abrogates the lethal effect of
the elimination of Type 2 FAS with Staphlococus aureus in mice is not seen with T. gondii.
46
10,47
Triclosan was noted to be safe in mice in earlier studies.
Conclusions
In all a series of fifty-three compounds were tested for their activity against T. gondii, with
six compounds having anti-parasite MIC s ≤6μM without toxicity to host cells. Three
90
compounds had IC s <45nM against recombinant TgENR and of the 44 triclosan analogs
90
tested, 6 showed better activity than triclosan. Moreover, those with the best toxicological
profiles were tested in mice (compounds 2 and 19), and were found to be several times more
active than triclosan itself in protecting mice. To investigate if, as predicted, the larger bulk
of a subset of these compounds exploits the increase in space in the parasitic enzymes, a co-
+
crystal structure of compound 19 in complex with TgENR, and NAD was solved. This
structure clearly revealed that the compound’s aliphatic side chain does, as predicted,
occupy space made available in the parasitic ENR family and reveals that further
modifications may be possible. Moreover compound 19 with its adaptations on both the A
and B ring is more amenable to chemical modifications which could improve both potency
and solubility.
This work provides a foundation for discovery of medicines to treat toxoplasmosis by
targeting ENR and for a program which includes a structure based program to develop new
medicines against ENR. This work has produced new insights into the efficacy of a range of
inhibitor scaffolds. The co-crystal structures proved useful in understanding structure
activity relationships. In particular the co-crystal structure of compound 19 has revealed that
a subset of these compounds exploit the increase in space within the parasitic ENR family
which these inhibitors target. This strategy can now be utilized in future medicinal chemistry
efforts to develop compounds that inhibit TgENR with increased avidity along with
modifications which will increase bioavailability.
Experimental Section
Chemistry
1
13
Synthesis of Inhibitors— H NMR and C NMR spectra were recorded on Bruker
spectrometer with TMS as an internal standard. Standard abbreviation indicating multiplicity
was used as follows: s = singlet, d = doublet, t = triplet, q = quadruplet, quin = quintuplet, m
= multiplet and br = broad. HRMS experiment was performed on Q–TOF–2TM
(Micromass). The progress of all reactions was monitored by TLC on precoated silica gel
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Tipparaju et al.
Page 9
plates (Merck Silica Gel 60 F254). Preparative TLC was performed with Analtech 1000–
mm silica gel GF plates. Column chromatography was performed using Merck silica gel
(40–60 mesh).
Compounds triclosan, 5, 36, 49, 54 and 37 were commercially available and have been
23
purchased. Compounds 50 and 51 were synthesized according to the reported procedures.
21
The new diarylether analogs of triclosan were synthesized by Schemes 1 and 2. The
synthesis of aminopyridine compounds 53 and 52, as well as benzimidazole-2-one
derivatives 46, 47, and 48 were carried out according to the Scheme 3. All the compounds
21,22
not illustrated in the synthetic schemes were reported earlier.
Descriptions of synthetic
experimental details and analytical data of compounds in Figure 1 is as follows:
General Methods
Method A—The aryl halide (1 mmol), phenol (1 mmol), and K CO or Cs CO (2–4
2
3
2
3
mmol) in DMSO (1.5 mL) were heated to 100 °C under nitrogen until the reaction was
shown to be complete by TLC (8–12 h). After cooling to rt, the reaction mixture was diluted
with ethyl acetate, and washed with 5% aqueous NaOH solution. The aqueous layer was
further extracted with ethyl acetate, and the combined organic layers were washed with
brine. The organic layer was dried over Na SO , and concentrated under vacuum to give the
2
4
crude product, which was subsequently purified by flash chromatography on silica gel.
Method B—To the phenol (1 mmol) dissolved in DMF (1.75 mL) was added KOtBu (1.1
mmol) in one portion and the mixture was heated at 45 °C under mild vacuum. After 2 h, the
reaction mixture was cooled to rt. Aryl halide (1 mmol) and (CuOTf) . Ph (0.05 mmol) were
2
added and the mixture was heated to reflux at 145 °C for 16–20 h. After cooling to rt, the
reaction mixture was diluted with ethyl acetate, filtered over Celite, and extracted with ethyl
acetate. The extract was washed with 5% aqueous NaOH solution. The aqueous layer was
then extracted with ethyl acetate, and the combined organic layers were washed with brine.
The organic layer was dried over Na SO , and concentrated under vacuum to give the crude
2
4
product, which was subsequently purified by flash chromatography on silica gel.
Method C—In each case, the methoxy diarylether derivatives obtained by the above
methods were converted to the corresponding phenols using boron tribromide according to
the following procedure: A solution of boron tribromide (2–8 mmol of 1.0 M solution in
dichloromethane) was added to a solution of the corresponding methoxy diphenylether
analog (1 mmol) in dry dichloromethane (4 mL) maintained at −78 °C under nitrogen. The
reaction mixture was stirred at the same temperature (1 h), and then at rt (3–8 h) while
monitoring by TLC. Following completion, the reaction was quenched with methanol at −78
°C and concentrated under vacuum. The concentrate was further dissolved in ethyl acetate,
washed with 10% aqueous sodium bicarbonate solution, and the organic layer was separated
and washed with water and then with brine. The aqueous layer was extracted twice with
ethyl acetate. The combined organic layers were then dried over Na SO , concentrated
2
4
under vacuum, and the crude product was purified by flash chromatography on silica gel.
2-(3-Chlorophenoxy)-5-propylphenol (33)—To the 2-methoxy-4-propyl-phenol (1 g,
6.0 mmol) dissolved in DMF (11 mL) was added KOtBu (0.81 g, 7.2 mmol) in one portion
and the mixture was heated at 45 °C under vacuum. After 2 h, the reaction mixture was
cooled to room temperature. 1-chloro-3-iodobenzene (1.72 g, 7.2 mmol), and (CuOTf) . Ph
2
(0.31 g, 0.6 mmol) were added, and the mixture was heated to reflux at 145 °C for 18 h
(Method B above). Usual workup and purification by flash chromatography (SiO , 10%
2
EtOAc/Hexanes) gave analytically pure 1-(3-chlorophenoxy)-2-methoxy-4-propylbenzene
1
as a colorless oil (62%). H NMR (400 MHz, CDCl ): δ = 1.00 (t, J = 7.3 Hz, 3H), 1.70
3
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(quin, J = 7.5 Hz, 2H), 2.62 (t, J = 7.5 Hz, 2H), 3.83 (s, 3H), 6.79 (dd, J = 8.0, 1.7 Hz, 1H),
6.85–6.83 (m, 2H), 6.91 (t, J = 2.1 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 7.00 (dd, J = 7.9, 1.0
13
Hz, 1H), 7.21 ppm (t, J = 8.1 Hz, 2H); C NMR (100 MHz, CDCl ): δ = 13.5, 24.3, 37.6,
3
55.5, 112.8, 114.3, 116.3, 120.6, 121.4, 121.7, 129.8, 134.4, 140.3, 141.2, 151.0, 159.0 ppm.
A mixture of above intermediate 1-(3-chlorophenoxy)-2-methoxy-4-propylbenzene (0.22
g, 0.79 mmol) and BBr (1M, 2.5 mL, 2.5 mmol) were subjected to the general
3
demethylation procedure outlined in method C above. The crude product was purified by
flash chromatography (SiO , 10% EtOAc/Hexanes) to give analytically pure 33 as a
2
1
colorless oil (62%). H NMR (400 MHz, CDCl ): δ = 0.99 (t, J = 7.3 Hz, 3H), 1.67 (quin, J
3
= 7.5 Hz, 2H), 2.58 (t, J = 7.5 Hz, 2H), 5.43 (br s, 1H), 6.72 (d, J = 8.1 Hz, 2H), 6.86 (d, J =
8.2 Hz, 2H), 6.92–6.89 (m, 2H), 7.02 (s, 1H), 7.09 (d, J = 8.0 Hz, 1H), 7.26 ppm (t, J = 8.1
13
Hz, 1H); C NMR (100 MHz, CDCl ): δ = 13.5, 24.1, 37.2, 115.1, 116.2, 117.3, 119.2,
3
120.5, 122.9, 130.2, 134.8, 139.9, 140.3, 146.9, 157.8 ppm. HRMS (ESI-positive): calcd for
+
C H ClO ([M–H] ): 261.06878, found: 261.0693.
15 15
2
3-Chloro-4-(2-hydroxy-6-methoxy-4-propylphenoxy)benzonitrile (30)—2,6-
Dimethoxy-4-propylphenol (1.0 g, 5.1 mmol), 3-chloro-4-fluoro-benzonitrile (0.79 g, 5.1
mmol) and K CO (1.41 g, 10.2 mmol) in DMSO (8 mL) were heated to 100 °C under
2
3
nitrogen for 12 h (Method A above). Usual workup followed by purification of the crude
reaction mixture by column chromatography (SiO , 10% EtOAc/Hexanes) afforded the
2
1
compound 3-chloro-4-(2,6-dimethoxy-4-propylphenoxy)benzonitrile (1.57 g, 93%). H
NMR (400 MHz, CDCl ): δ = 1.00 (t, J = 7.3 Hz, 3H), 1.70 (quin, J = 7.5 Hz, 2H), 2.62 (t, J
3
= 7.5 Hz, 2H), 3.78 (s, 6H), 6.49 (s, 2H), 6.62 (d, J = 8.6 Hz, 1H), 7.38 (dd, J = 8.6, 1.8 Hz,
13
1H), 7.73 ppm (d, J = 1.8 Hz, 1H); C NMR (100 MHz, CDCl ): δ = 13.4, 24.2, 38.2, 55.8,
3
105.0, 114.5, 117.6, 122.8, 128.3, 131.5, 133.4, 141.4, 151.9, 157.6 ppm. MS (ESI) m/z:
+
354.1 [M + Na]
A mixture of above intermediate 3-chloro-4-(2,6-dimethoxy-4-
propylphenoxy)benzonitrile (0.29 g, 0.86 mmol) and BBr (1M, 3.4 mL, 3.4 mmol) were
3
subjected to the general demethylation procedure outlined in method C above. The reaction
resulted in two products (30, 20% yield) and (26, 60% yield) that were separated by flash
chromatography (SiO , 15% EtOAc/Hexanes) to give analytically pure samplesas white
2
solids.
1
3-Chloro-4-(2-hydroxy-6-methoxy-4-propylphenoxy)benzonitrile (30)— H NMR
(400 MHz, CDCl ): δ = 0.99 (t, J = 7.3 Hz, 3H), 1.68 (quin, J = 7.5 Hz, 2H), 2.57 (t, J = 7.5
3
Hz, 2H), 3.73 (s, 3H), 5.67 (s, 1H), 6.40 (s, 1H), 6.54 (s, 1H), 6.73 (d, J = 8.6 Hz, 1H), 7.40
13
(d, J = 8.6 Hz, 1H), 7.70 ppm (s, 1H); C NMR (100 MHz, CDCl ): δ = 13.4, 23.9, 37.8,
3
55.6, 104.4, 105.8, 108.9, 115.0, 117.2, 123.0, 126.8, 131.6, 133.5, 141.8, 148.3, 151.2,
+
156.8 ppm. HRMS (ESI-positive): calcd for C H ClNO ([M–H] ): 316.0746, found:
17 16
3
316.0755.
1
3-Chloro-4-(2,6-dihydroxy-4-propylphenoxy)benzonitrile (26)— H NMR (400
MHz, CDCl ): δ = 0.98 (t, J = 7.3 Hz, 3H), 1.66 (quin, J = 7.5 Hz, 2H), 2.53 (t, J = 7.5 Hz,
3
2H), 5.04 (s, 2H), 6.47 (s, 2H), 6.85 (d, J = 8.6 Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.77 ppm
13
(d, J = 1.9 Hz, 1H); C NMR (100 MHz, CDCl ): δ = 13.4, 23.7, 37.4, 106.7, 108.8, 115.1,
3
117.0, 123.2, 126.0, 131.9, 133.8, 142.3, 147.7, 156.1 ppm. HRMS (ESI-positive): calcd for
+
C H ClO ([M–H] ): 302.05894, found: 302.0606.
16 14
3
4-(2,6-Dihydroxy-4-propylphenoxy)benzonitrile (32)—2,6-Dimethoxy-4-
propylphenol (0.63 g, 3.2 mmol), 4-fluoro-benzonitrile (0.39 g, 3.2 mmol) and Cs CO (2.1
2
3
g, 6.4 mmol) in DMSO (9 mL) were reacted according to the Method A above. Usual
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Tipparaju et al.
Page 11
workup followed by purification of the crude reaction mixture by column chromatography
(SiO , 10% EtOAc/Hexanes) afforded the compound 4-(2,6-dimethoxy-4-
2
propylphenoxy)benzonitrile (0.81 g, 86%). A mixture of this intermediate compound (0.20
g, 0.67 mmol) and BBr (1M, 3.4 mL, 3.4 mmol) were subjected to the general
3
demethylation procedure outlined in Method C above. The crude product was purified by
flash chromatography (SiO , 10% EtOAc/Hexanes) to give analytically pure 32 as a
2
1
colorless oil (74%). H NMR (400 MHz, (CD ) SO): δ = 0.90 (t, J = 7.2 Hz, 3H), 1.55
3 2
(quin, J = 7.2 Hz, 2H), 2.40 (t, J = 7.2 Hz, 2H), 6.27 (s, 2H), 6.91 (d, J = 8.8 Hz, 2H), 7.73
13
(d, J = 8.8 Hz, 2H), 9.41 ppm (s, 2H); C NMR (100 MHz, (CD ) SO): δ = 14.1, 24.2,
3 2
37.6, 103.7, 107.9, 116.3, 119.6, 127.0, 134.4, 140.5, 150.7, 162.3 ppm. HRMS (ESI-
+
positive): calcd for C H NO ([M–H] ): 268.09792, found: 268.0988.
16 15
3
3-Chloro-4-(2,6-dihydroxy-4-propylphenoxy)benzoic acid (34)—A mixture of the
nitrile 3-chloro-4-(2,6-dimethoxy-4-propylphenoxy)benzonitrile (0.60 g, 1.8 mmol)
whose synthesis is described in the preparation of 30 above and 25% aqueous NaOH (0.9
mL) in ethanol (5 mL) was refluxed with stirring (20 h). After cooling, the reaction mixture
was acidified with dilute hydrochloric acid. The precipitate was filtered, dried, and purified
by column chromatography (SiO , 6% MeOH/CHCl ) to yield the 3-chloro-4-(2,6-
2
3
1
dimethoxy-4-propylphenoxy)benzoic acid (0.51 g, 80%) as a white solid. H NMR (400
MHz, (CD ) SO): δ = 0.94 (t, J = 7.2 Hz, 3H), 1.66 (quin, J = 7.6 Hz, 2H), 2.58 (t, J = 7.6
3 2
Hz, 2H), 3.38 (br s, 1H), 3.71 (s, 6H), 6.53 (d, J = 8.8 Hz, 1H), 6.67 (s, 2H), 7.76 (d, J = 8.8
13
Hz, 1H), 7.97 ppm (s, 1H); C NMR (100 MHz, (CD ) SO): δ = 14.2, 24.5, 38.2, 56.4,
3 2
105.9, 114.1, 121.0, 125.4, 128.3, 130.3, 131.6, 141.7, 152.3, 157.4, 166.3 ppm. MS (ESI)
+
m/z: 372.1 [M + Na] A mixture of this intermediate (0.40 g, 1.14 mmol) and BBr (1M, 6.8
3
mL, 6.8 mmol) were subjected to the general demethylation procedure outlined in Method C
above. The crude product was purified by flash chromatography (SiO , 5% MeOH/
2
1
Chloroform) to give analytically pure 34 as a white solid (76%). H NMR (400 MHz,
(CD ) SO): δ = 0.91 (t, J = 7.2 Hz, 3H), 1.56 (quin, J = 7.2 Hz, 2H), 2.41 (t, J = 7.6 Hz,
3 2
2H), 6.28 (s, 2H), 6.61 (d, J = 8.4 Hz, 1H), 7.77 (dd, J = 8.6, 1.6 Hz, 1H), 7.95 (d, J = 2.0
13
Hz, 1H), 9.45 ppm (br s, 1H); C NMR (100 MHz, CDCl ): δ = 14.1, 24.2, 37.6, 107.9,
3
114.6, 121.3, 125.0, 127.2, 130.1, 131.4, 140.6, 150.6, 157.8, 166.4 ppm. HRMS (ESI-
+
positive): calcd for C H ClO ([M–H] ): 321.0535, found: 321.0545.
16 15
5
3-(2-Hydroxy-4-propylphenoxy)phenyl boronic acid (20)—1-(3-chlorophenoxy)-2-
methoxy-4-propylbenzene (0.83 g, 3.0 mmol), whose synthesis is described in the
preparation of 33, was dissolved in 20 mL of anhydrous THF and treated with n-BuLi (1.6
M in hexanes, 3.8 mL, 6 mmol) at −78 °C for 2.5 h under Ar. Trimethyl borate (3.1 g, 30
mmol) was added drop wise to this solution at −78 °C and the reaction mixture was stirred
for 3 h. The reaction was quenched by careful addition of 14 mL of 2N HCl at −78 °C and
stirred at room temperature for 18 h. The solvent was evaporated and the concentrate was
further dissolved in ethyl acetate, washed with 10% aqueous sodium bicarbonate solution,
and the organic layer was separated and washed with water and brine. The aqueous layer
was extracted twice with ethyl acetate. The combined organic layers were then dried over
Na SO , concentrated under vacuum, and the crude product was purified by flash
2
4
chromatography on silica gel (20% EtOAc/Hexanes) to result in 3-(2-methoxy-4-
1
propylphenoxy)phenyl boronic acid (0.65 g, 76%) as a grey solid. H NMR (400 MHz,
(CD ) SO): δ = 0.91 (t, J = 7.3 Hz, 3H), 1.62 (quin, J = 7.4 Hz, 2H), 2.56 (t, J = 7.4 Hz,
3 2
2H), 3.35 (s, 3H), 6.36 (d, J = 8.2 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.93 (d, J = 8.0 Hz,
13
1H), 7.00–6.98 (m, 2H), 7.16 (t, J = 8.0 Hz, 1H), 8.38 ppm (s, 2H); C NMR (100 MHz,
(CD ) SO): δ = 13.7, 24.2, 37.2, 55.7, 112.6, 120.8, 121.7, 121.8, 128.5, 130.1, 135.7,
3 2
139.9, 142.0, 151.2, 160.6 ppm. A mixture of this boronic acid intermediate (0.34 g, 1.2
mmol) and BBr (1M, 6.0 mL, 6.0 mmol) were subjected to the general demethylation
3
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Tipparaju et al.
Page 12
procedure outlined in Method C above. The crude product was purified by flash
chromatography (SiO , 2% MeOH/chloroform) to give analytically pure 20 as a grey solid
2
1
(72%). H NMR (400 MHz, (CD ) SO): δ = 0.89 (t, J = 7.1 Hz, 3H), 1.57 (quin, J = 7.2 Hz,
3 2
2H), 2.48 (t, J = 7.1 Hz, 2H), 6.65 (d, J = 7.4 Hz, 1H), 6.82–6.78 (m, 3H), 6.91 (d, J = 8.0
13
Hz, 1H), 7.03 (d, J = 7.6 Hz, 1H), 7.29 ppm (t, J = 8.1 Hz, 1H); C NMR (100 MHz,
(CD ) SO): δ = 13.7, 24.0, 36.9, 114.5, 115.6, 117.3, 119.7, 121.5, 122.2, 130.9, 133.7,
3 2
139.4, 140.4, 149.1, 159.4 ppm.
4-(2,6-Dihydroxy-4-propylphenoxy)benzamide (28)—A mixture of the (2,6-
dimethoxy-4-propylphenoxy)benzonitrile (0.60 g, 1.8 mmol) whose synthesis is described in
the preparation of 32 above, H O (38%, 1.5 mL, 12.4 mmol) and 3N NaOH (0.3 mL) in
2
2
ethanol (10 mL) was refluxed with stirring (20 h). After cooling, the reaction mixture was
acidified with dilute hydrochloric acid. The precipitate was filtered, dried to obtain 4-(2,6-
dimethoxy-4-propylphenoxy)benzamide as a white solid. A mixture of this intermediate
compound (0.15 g, 0.48 mmol) and BBr (1M, 3.4 mL, 3.4 mmol) were subjected to the
3
general demethylation procedure outlined in Method C above. The crude product was
purified by flash chromatography (SiO , 10% MeOH/chloroform). The product was
2
recrystallized from MeOH/chloroform to give analytically pure 28 as white prisms
1
(74%). H NMR (400 MHz, (CD ) SO): δ = 0.91 (t, J = 7.2 Hz, 3H), 1.55 (quin, J = 7.2 Hz,
3 2
2H), 2.40 (t, J = 7.2 Hz, 2H), 6.26 (s, 2H), 6.78 (d, J = 8.4 Hz, 2H), 7.18 (br s, 1H), 7.80–
13
7.78 (m, 3H), 9.28 ppm (s, 2H); C NMR (100 MHz, (CD ) SO): δ = 14.2, 24.3, 37.6,
3 2
107.9, 114.6, 127.5, 129.5, 140.0, 150.9, 161.1, 167.9 ppm.
5-Propyl-2-(4-(2H-tetrazol-5-yl)phenoxy)-3-hydroxyphenol (35)—A mixture of the
4-(2,6-dimethoxy-4-propylphenoxy)benzamide (0.18 g, 0.6 mmol) (from above), SiCl
4
(0.20 g, 1.2 mmol) and NaN (0.15 g, 2.3 mmol) was refluxed at 90 °C in CH CN (0.5 mL)
3
3
for 6 h under Ar. After cooling, the reaction mixture was diluted with EtOAc, washed with
saturated sodium bicarbonate solution, the organic layer was separated and washed with
water followed by brine. The aqueous layer was extracted twice with ethyl acetate. The
combined organic layers were then dried over Na SO , concentrated under vacuum, and the
2
4
crude product was purified by flash chromatography on silica gel (8% MeOH/chloroform)
to result in 5-(4-(2,6-dimethoxy-4-propylphenoxy)phenyl)-2H-tetrazole (0.18 g, 97%) as
1
a grey solid. H NMR (400 MHz, CDCl ): δ = 0.96 (t, J = 7.2 Hz, 3H), 1.66 (quin, J = 7.4
3
Hz, 2H), 2.57 (t, J = 7.6 Hz, 2H), 3.72 (s, 6H), 6.45 (s, 2H), 6.88 (d, J = 8.1 Hz, 2H), 8.01
13
(d, J = 8.2 Hz, 2H), 12.56 ppm (br s, 1H); C NMR (100 MHz, (CD ) SO): δ = 13.9, 24.2,
3 2
37.9, 55.9, 105.5, 114.9, 121.1, 128.2, 128.5, 140.6, 152.4 ppm. A mixture of this
intermediate compound (0.17 g, 0.50 mmol) and BBr (1M, 4.0 mL, 4.0 mmol) were
3
subjected to the general demethylation procedure outlined in Method C above. The crude
product was purified by flash chromatography (SiO , 10% MeOH/chloroform) to give
2
1
analytically pure 35 as off-white solid (78%). H NMR (400 MHz, (CD ) SO): δ = 0.91 (t, J
3 2
= 7.2 Hz, 3H), 1.56 (quin, J = 7.2 Hz, 2H), 2.40 (t, J = 7.6 Hz, 2H), 6.28 (s, 2H), 6.97 (d, J =
13
8.0 Hz, 2H), 7.94 (d, J = 8.4 Hz, 2H), 9.34 ppm (s, 2H); C NMR (100 MHz, (CD ) SO): δ
3 2
= 14.2, 24.3, 37.6, 107.9, 116.1, 127.4, 128.9, 140.1, 150.9, 161.0 ppm. HRMS (ESI-
+
positive): calcd for C H N O ([M+H] ): 313.1295, found: 313.1296.
16 16
4 3
Hexa-2,4-dienoic acid (4-(4-chloro-2-hydroxyphenoxy)-phenyl)amide (25)—A
21
mixture of 4-(4-chloro-2-methoxyphenoxy)phenylamine (0.19 g, 0.8 mmol), EDAC·HCl
(0.15 g, 0.8 mmol) and HOBt (0.1 g, 0.8 mmol) in 8 mL DMF was stirred for 10 minutes at
rt under Ar. To this, a mixture of sorbic acid (0.09 g, 0.8 mmol) and triethylamine (0.08 g,
0.8 mmol) in 2 mL of DMF was added at rt and the reaction mixture was stirred at rt. After
16 h, the reaction mixture was diluted with EtOAc, washed with saturated sodium
bicarbonate solution, the organic layer was separated and washed with water followed by
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Tipparaju et al.
Page 13
brine. The aqueous layer was extracted twice with ethyl acetate. The combined organic
layers were then dried over Na SO , concentrated under vacuum, and the crude product was
2
4
purified by flash chromatography on silica gel (3% MeOH/chloroform) to result in
hexa-2,4-dienoic acid (4-(4-chloro-2-methoxyphenoxy)-phenyl)amide (0.18 g, 67%) as a
1
grey solid. H NMR (300 MHz, CDCl ): δ = 1.87 (d, J = 7.1 Hz, 3H), 3.85 (s, 3H), 5.89 (d,
3
J = 19.7 Hz, 1H), 6.20 (dd, J = 19.7, 6.5 Hz, 2H), 7.03–6.89 (m, 5H), 7.42–7.32 (m, 2H),
7.53–7.50 ppm (m, 1H). A mixture of this intermediate compound (0.18 g, 0.50 mmol) and
BBr (1M, 1.5 mL, 1.5 mmol) were subjected to the general demethylation procedure
3
outlined in Method C above. The crude product was purified by flash chromatography
1
(SiO , 2% MeOH/chloroform) to give analytically pure 25 as a brown gel (76%). H NMR
2
(300 MHz, CDCl ): δ = 1.88 (d, J = 6.9 Hz, 3H), 6.18–5.74 (m, 2H), 6.22–6.16 (m, 2H),
3
13
6.80–6.77 (m, 2H), 7.07–6.98 (m, 3H), 7.54 ppm (d, J = 11.2 Hz, 2H); C NMR (100 MHz,
CD OD): δ = 17.2, 116.7, 117.1, 117.2, 119.3, 121.0, 121.4, 121.5, 121.6, 129.0, 129.7,
3
+
137.9, 141.7, 149.7, 154.0 ppm. HRMS (ESI-positive): calcd for C H ClNO ([M+H] ):
18 16
3
330.0891, found: 330.0883.
Hexa-2,4-dienoic acid (4-(2,4-dichlorophenoxy)-3-hydroxyphenyl)amide (27)—
A mixture of 2,4-dichlorophenol (1.0 g, 6.3 mmol), KOtBu (0.83 g, 7.5 mmol), 2-iodo-5-
nitroanisole (1.9 g, 6.9 mmol), and (CuOTf) PhCH (0.16 g, 0.3 mmol) in DMF (11 mL)
2
3
were reacted according to the Method B above. Usual workup and purification by flash
chromatography (SiO , 5% EtOAc/Hexanes) gave analytically pure 4-(2,4-
2
1
dichlorophenoxy)-3-methoxynitrobenzene as a brown oil (62%). H NMR (400 MHz,
(CD ) SO): δ = 4.14 (s, 3H), 6.36 (d, J = 4.8 Hz, 1H), 7.13 (d, J = 5.2 Hz, 1H), 7.46 (dd, J =
3 2
8.8, 2.4 Hz, 1H), 7.61 (dd, J = 4.2, 2.4 Hz, 1H), 7.69 (d, J = 2.4 Hz, 1H), 8.09 (d, J = 8.4 Hz,
13
2H); C NMR (100 MHz, (CD ) SO): δ = 57.5, 105.9, 108.7, 117.3, 117.6, 118.1, 122.2,
3 2
125.7, 129.5, 129.7, 130.7, 140.2, 150.2 ppm. A suspension of the above compound (2.0 g,
6.5 mmol), and 10% Pd/C (0.32 g) in EtOH (25 mL) was stirred under hydrogen atmosphere
at 40 PSI at room temperature for 16 h. The catalyst was removed by filtration through a pad
of Celite, and the residue was thoroughly washed with EtOAc. The solvent was evaporated
and purification by flash chromatography (SiO , 20% EtOAc/Hexanes) gave the compound
2
1
4-(2,4-dichlorophenoxy)-3-methoxyaniline (0.7 g, 38%). H NMR (400 MHz, CDCl ): δ =
3
3.48 (br s, 2H), 3.69 (s, 3H), 6.27 (dd, J = 4.2, 2.4 Hz, 1H), 6.36 (d, J = 2.4 Hz, 1H), 6.61 (d,
J = 8.8 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 7.06 (dd, J = 4.4, 2.3 Hz, 1H), 7.42 ppm (d, J =
13
2.4 Hz, 1H); C NMR (100 MHz, CDCl ): δ 55.4, 100.2, 106.7, 116.2, 122.4, 122.9, 126.1,
3
127.1, 129.5, 144.7, 151.7, 153.2 ppm. A mixture of this aniline intermediate (0.15 g, 0.5
mmol), EDAC·HCl (0.10 g, 0.5 mmol) and HOBt (0.07 g, 0.5 mmol) in 6 mL DMF was
stirred for 10 minutes at rt under Ar. To this, a mixture of sorbic acid (0.06 g, 0.5 mmol) and
triethylamine (0.08 g, 0.8 mmol) in 2 mL of DMF was added at rt and the reaction mixture
was stirred at rt. After 16 h, the reaction mixture was diluted with EtOAc, washed with
saturated sodium bicarbonate solution, the organic layer was separated and washed with
water followed by brine. The aqueous layer was extracted twice with ethyl acetate. The
combined organic layers were then dried over Na SO , concentrated under vacuum, and the
2
4
crude product was purified by flash chromatography on silica gel (3% MeOH/chloroform)
to result in hexa-2,4-dienoic acid (4-(2,4-dichlorophenoxy)-3-methoxyphenyl)amide
1
(0.18 g, 93%) as brown liquid. H NMR (400 MHz, (CD ) SO): δ = 1.84 (d, J = 6.2 Hz,
3 2
3H), 3.34 (s, 3H), 6.35–6.09 (m, 3H), 6.64 (d, J = 8.6 Hz, 1H), 7.04 (d, J = 8.6 Hz, 1H),
13
7.28–7.15 (m, 3H), 7.65 (d, J = 8.6 Hz, 2H), 10.15 ppm (s, 1H); C NMR (100 MHz,
(CD ) SO): δ = 18.8, 56.0, 105.1, 112.0, 117.5, 122.2, 122.9, 123.2, 126.4, 128.7, 130.1,
3 2
+
130.3, 138.3, 138.5, 141.4, 151.2, 153.2, 164.4 ppm. MS (ESI) m/z: 402.1 [M + H] A
mixture of this intermediate compound (0.12 g, 0.33 mmol) and BBr (1M, 1.6 mL, 1.6
3
mmol) were subjected to the general demethylation procedure outlined in Method C above.
The crude product was purified by flash chromatography (SiO , 3% MeOH/chloroform) to
2
1
give analytically pure 27 as a brown oil (76%). H NMR (400 MHz, (CD ) SO): δ = 1.83
3 2
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Tipparaju et al.
Page 14
(d, J = 6.2 Hz, 3H), 6.33–6.09 (m, 2H), 6.64 (d, J = 8.6 Hz, 1H), 6.96 (d, J = 8.6 Hz, 1H),
7.06 (d, J = 8.4 Hz, 1H), 7.19–7.13 (m, 1H), 7.28 (d, J = 8.6 Hz, 1H), 7.55 (s, 1H), 7.65 (s,
13
1H), 10.02 ppm (s, 1H); C NMR (100 MHz, (CD ) SO): δ = 18.8, 108.7, 111.0, 117.5,
3 2
122.4, 122.8, 123.4, 126.1, 128.6, 129.9, 130.3, 137.4, 137.9, 138.2, 141.2, 149.4, 153.3,
164.2 ppm.
5-Chloro-2-phenoxy-N-pivaloyl aniline (13)—A suspension of commercially available
5-chloro-2-phenoxyaniline (0.13 g, 0.61 mmol) and Et N (0.15 g, 1.5 mmol) in 2 mL
3
methylene chloride at 0 °C was treated with pivaloyl chloride (0.07 g, 0.61 mmol), and the
reaction mixture was stirred for 3h. The reaction mixture was diluted with ethyl acetate,
extracted and the combined organic layers were washed with brine. Purification by flash
1
chromatography (SiO , 5% EtOAc/Hexanes) gave analytically product. H NMR (400 MHz,
2
CD OD): δ = 1.14 (s, 9H), 6.97 (t, J = 8.5 Hz, 3H), 7.12 (t, J = 7.4 Hz, 3H), 7.18 (dd, J =
3
13
4.4, 2.4 Hz, 3H), 7.35 ppm (t, J = 8.2 Hz, 3H); C NMR (100 MHz, CD OD): δ = 25.7,
3
38.8, 99.6, 116.5, 120.3, 122.9, 123.6, 124.8, 128.4, 129.3, 130.4, 145.9, 156.4, 177.7 ppm.
+
HRMS (ESI-positive): calcd for C H ClNO ([M+H] ): 304.1099, found: 304.1098.
17 18
2
2-Amino-N-(5-chloro-2-phenoxyphenyl)nicotinamide (7)—A mixture of 2-
aminonicotinic acid (0.27 g, 1.9 mmol), EDAC·HCl (0.38 g, 1.9 mmol) and HOBt (0.26 g,
1.9 mmol) in 15 mL DMF was stirred for 10 minutes at rt under Ar. To this, a mixture of
commercially available 5-chloro-2-phenoxyaniline (0.43 g, 1.9 mmol) and triethylamine (0.2
g, 1.9 mmol) in 5 mL of DMF was added at rt and the reaction mixture was stirred at rt.
After 16 h, DMF was evaporated, the reaction mixture was diluted with EtOAc, washed with
saturated sodium bicarbonate solution. The organic layer was separated and washed with
water followed by brine. The aqueous layer was extracted twice with ethyl acetate. The
combined organic layers were then dried over Na SO , concentrated under vacuum, and the
2
4
crude product was purified by flash chromatography on silica gel (1% MeOH/chloroform)
1
to result in 7. H NMR (400 MHz, CDCl ): δ = 6.46 (br s, 2H), 6.63–6.59 (m, 1H), 6.83 (d,
3
J = 8.7 Hz, 1H), 7.08–7.02 (m, 3H), 7.21 (t, J = 7.4 Hz, 1H), 7.41 (t, J = 8.2 Hz, 2H), 7.59–
13
7.57 (m, 1H), 8.21–8.13 (m, 1H), 8.40 (br s, 1H), 8.61 ppm (d, J = 2.2 Hz, 1H); C NMR
(100 MHz, (CD ) SO): δ = 109.7, 111.8, 118.6, 121.1, 124.0, 126.4, 126.5, 127.5, 130.4,
3 2
+
131.0, 137.7 ppm. HRMS (ESI-positive): calcd for C H ClN O ([M+H] ): 340.0847,
18 14
3 2
found: 340.0851.
5-chloro-2-(2,4-dichlorophenoxy)phenyl pivaloate (3)—To a mixture of 1 (0.26 g,
0.9 mmol) and DMAP (0.02 g, 0.2 mmol) in 8 mL of methylene chloride at 0 °C was added
Et N (0.10 g, 1.0 mmol) and stirred for 10 minutes. To this, pivaloyl chloride (0.12 g, 1.0
3
mmol) was added at 0 °C and the reaction mixture was stirred for 1h. The reaction mixture
was stirred for an additional 2 h at rt. After evaporation of methylene chloride, the reaction
mixture was diluted with ethyl acetate, washed with water followed by brine. The combined
organic layers were extracted by ethyl acetate, dried over Na SO and concentrated under
2
4
vacuum. Purification by flash chromatography (SiO , 3% EtOAc/Hexanes) gave analytically
2
1
product 3 (85%). H NMR (400 MHz, CDCl ): δ = 1.25 (s, 9H), 6.81 (d, J = 8.8 Hz, 1H),
3
13
7.21–7.18 (m, 3H), 7.46 ppm (d, J = 2.4 Hz, 3H); C NMR (100 MHz, CDCl ): δ = 26.5,
3
38.7, 118.7, 120.5, 124.1, 124.8, 126.4, 127.6, 128.5, 129.3, 129.9, 142.2, 145.6, 150.9,
175.6 ppm.
3-(6-Aminopyridin-3-yl)-N-(4-methoxyphenyl)acrylamide (52)—A mixture of 3-(6-
23
aminopyridin-3-yl)-acrylic acid (0.12 g, 0.7 mmol), EDAC·HCl (0.13 g, 0.7 mmol) and
HOBt (0.10 g, 0.7 mmol) in 15 mL DMF was stirred for 10 minutes at rt under Ar. To this, a
mixture of p-anisidine (0.09 g, 0.7 mmol) and triethylamine (0.07 g, 0.7 mmol) in 5 mL of
DMF was added at rt and the reaction mixture was stirred at rt. After 16 h, DMF was
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Tipparaju et al.
Page 15
evaporated, the reaction mixture was diluted with EtOAc, washed with saturated sodium
bicarbonate solution. The organic layer was separated and washed with water followed by
brine. The aqueous layer was extracted twice with ethyl acetate. The combined organic
layers were then dried over Na SO , concentrated under vacuum, and the crude product was
2
4
1
purified by flash chromatography on silica gel (5% MeOH/chloroform) to result in 52. H
NMR (400 MHz, CD OD): δ = 3.79 (s, 3H), 6.56 (d, J = 15.6 Hz, 1H), 6.62 (d, J = 15.6 Hz,
3
1H), 6.90 (d, J = 9.0 Hz, 1H), 7.50 (s, 1H), 7.55 (d, J = 9.1 Hz, 2H), 7.75 (dd, J = 4.4, 2.2
13
Hz, 1H), 8.08 ppm (d, J = 1.8 Hz, 1H); C NMR (100 MHz, CD OD): δ = 54.1, 108.6,
3
113.2, 115.7, 119.7, 121.0, 131.3, 135.1, 137.6, 148.0, 156.1, 159.9, 164.9 ppm. HRMS
+
(ESI-positive): calcd for C H N O ([M+H] ): 270.1237, found: 270.1239.
15 15
3 2
N-(2-Aminobenzyl)-3-(6-aminopyridin-3-yl)acrylamide (53)—A mixture of 3-(6-
23
aminopyridin-3-yl)-acrylic acid (0.13 g, 0.8 mmol), EDAC·HCl (0.15 g, 0.8 mmol), HOBt
(0.10 g, 0.8 mmol), 2-aminobenzylamine (0.09 g, 0.8 mmol) and triethylamine (0.08 g, 0.8
1
mmol) in 15 mL DMF were reacted according to the procedure described above for 52. H
NMR (400 MHz, CD OD): δ = 4.42 (s, 2H), 6.41 (d, J = 15.6 Hz, 1H), 6.62 (d, J = 15.7 Hz,
3
1H), 6.60 (d, J = 8.8 Hz, 1H), 6.68 (t, J = 7.3 Hz, 1H), 6.75 (d, J = 7.9 Hz, 1H), 7.06 (t, J =
7.5 Hz, 1H), 7.11 (d, J = 7.4 Hz, 1H), 7.45 (d, J = 15.7 Hz, 1H), 7.72 (d, J = 8.6 Hz, 1H),
13
8.04 ppm (s, 1H); C NMR (100 MHz, CD OD): δ = 39.4, 108.7, 115.4, 116.1, 117.2,
3
119.7, 122.2, 127.9, 129.3, 135.2, 137.2, 145.1, 147.7, 159.8, 167.1 ppm. HRMS (ESI-
+
positive): calcd for C H N O ([M+H] ): 269.1397, found: 269.1402.
15 16
4
1-(4-Nitrophenyl)-1,3-dihydrobenzimidazol-2-one (46)—1,3-
Dihydrobenzimidazol-2-one (0.50 g, 3.7 mmol), 1-fluoro-4-nitrophenol (0.53 g, 3.7 mmol)
and K CO (1.00 g, 7.5 mmol) in DMSO (8 mL) were heated to 100 °C under nitrogen for
2
3
12 h (Method A above). The reaction mixture was cooled to rt and filtered. The yellow solid
was repeatedly washed with water (3 × 5 mL), EtOAc (3 × 2 mL), ether (3 × 2 mL) and
finally with hexanes (3 × 2 mL). The solid was dried in vacuum to obtain analytically pure
1
46 (0.70 g, 74%). H NMR (400 MHz, (CD ) SO): δ = 6.92 (s, 2H), 7.26 (br s, 1H), 7.34 (br
3 2
s, 1H), 8.00 (d, J = 8.5 Hz, 2H), 8.48 ppm (d, J = 8.7 Hz, 1H).
1-(4-Nitrobenzoyl)-1,3-dihydrobenzimidazol-2-one (48)—To a mixture of 1,3-
dihydrobenzimidazol-2-one (0.51 g, 3.8 mmol) in 9 mL of anhydrous pyridine at 0 °C was
added 4-nitrobenzoyl chloride (1.00 g, 5.6 mmol) portion wise and the reaction mixture was
stirred for 15 minutes. The contents were then refluxed at 80 °C for 4 h. The reaction
mixture solidified when cooled to rt, to which, 10 mL water and 20 mL of EtOAc were
added and stirred for 10 minutes. The organic layer was separated and washed with 1N HCl
followed by water and brine. The aqueous layer was extracted twice with ethyl acetate. The
combined organic layers were then dried over Na SO , concentrated under vacuum to result
2
4
1
in 48 (1.50 g, 98%). H NMR (400 MHz, (CD ) SO): δ = 6.91 (s, 2H), 8.16 (d, J = 8.5 Hz,
3 2
13
2H), 8.31 (d, J = 8.6 Hz, 1H), 10.59 ppm (s 1H); C NMR (100 MHz, CDCl ): δ = 108.9,
3
120.8, 124.1, 130.1, 131.1, 136.9, 150.0, 150.4, 155.7, 166.3 ppm.
1-(4-Nitrobenzyl)-1,3-dihydrobenzimidazol-2-one (45)—1,3-
Dihydrobenzimidazol-2-one (0.20 g, 1.5 mmol), 4-nitrobenzyl bromide(NOTE: strong
lachrymator) (0.32 g, 1.5 mmol) and K CO (0.31 g, 2.2 mmol) in MeOH (6 mL) were
2
3
refluxed at 100 °C under nitrogen for 4 h. The reaction mixture was cooled to rt and MeOH
was evaporated, 10 mL water and 20 mL of EtOAc were added and stirred for 10 minutes.
The organic layer was separated and washed with water followed by brine. The aqueous
layer was extracted twice with ethyl acetate. The combined organic layers were then dried
over Na SO , concentrated under vacuum and the crude product was purified by flash
2
4
1
chromatography on silica gel (15% EtOAc/Hexanes) to result in 45. H NMR (400 MHz,
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Tipparaju et al.
Page 16
(CD ) SO): δ = 4.98 (s, 2H), 6.98 (d, J = 8.5 Hz, 2H), 7.15 (d, J = 8.5 Hz, 2H), 8.28 (d, J =
3 2
13
8.6 Hz, 2H), 11.38 ppm (s, 1H); C NMR (100 MHz, CDCl ): δ = 43.8, 108.4, 108.9,
3
109.5, 120.8, 121.1, 121.7, 122.0, 154.7 ppm. HRMS (ESI-positive): calcd for C H N O
14 11
3 3
+
([M+H] ): 270.0873, found: 270.0875.
1-(4-Nitrobenzenesulfonyl)-1,3-dihydrobenzimidazol-2-one (47)—To a mixture of
1,3-dihydrobenzimidazol-2-one (0.50 g, 3.7 mmol) in 8 mL of methylene chloride and 2 mL
of DMF at 0 °C was added Et N (0.42 g, 4.1 mmol) and stirred for 10 minutes. To this, 4-
3
nitrobenzenesulfonyl chloride (0.83 g, 3.73 mmol) was added at 0 °C and the reaction
mixture was stirred for 15 minutes. The reaction mixture was stirred for an additional 2 h at
rt. The reaction mixture was quenched by adding crushed ice. The precipitated yellow solid
was repeatedly washed with water (3 × 5 mL), EtOAc (3 × 2 mL), ether (3 × 2 mL) and
finally with hexanes (3 × 2 mL). The solid was dried in vacuum to obtain analytically pure
1
46 (0.70 g, 74%). H NMR (400 MHz, (CD ) SO): δ = 6.91 (s, 2H), 7.85 (d, J = 8.4 Hz,
3 2
13
2H), 8.20 (d, J = 8.4 Hz, 2H), 10.56 ppm (s, 1H); C NMR (100 MHz, CDCl ): δ = 108.6,
3
120.5, 123.4, 127.0, 129.7, 155.3 ppm.
Biology
Testing of inhibitors in vitro against T. gondii Tachyzoites—Four-day old
3
confluent cultures of human foreskin fibroblasts (HFF) were infected with 10 tachyzoites of
RH strain of T. gondii and cultured for 1 hour to allow parasite invasion, inhibitors added
3
and cells cultured for 3 days, supplemented with H uracil and incubated for a further day,
whereupon uracil incorporation into cells and thus parasite growth were assessed by liquid
47
scintillation counting. Lack of toxicity for mammalian host cells was demonstrated first by
3
visual inspection of monolayers and in separate H thymidine incorporation assays using
non-confluent cell monolayers.
Cloning, Sequencing, Overexpression, and Purification of TgENR—These were
14,48
performed as described previously.
TgENR inhibitor assay—Activity of TgENR was assayed by monitoring consumption of
−1
−1
NADH (ε340=6220M cm ) with a scanning spectrophotometer. ENR enzymes catalyze
the conversion of trans-2-acyl-ACP to acyl-ACP, however, they are typically assayed with a
18
surrogate substrate, trans-2-butyryl-Coenzyme A (crotonyl-CoA). We used a reaction
mixture which would contain 20nM TgENR, 100mM Na/K phosphate buffer pH7.5,
150mM NaCl, and 100μM NADH (Sigma) when diluted to a final volume of 50μL.
Compounds dissolved in DMSO were added to this mixture (2% final concentration of
DMSO) and reactions were initiated at 25°C by addition of 100μM crotonyl-CoA (Sigma).
This assay had also previously been used to monitor TgENR inhibition by an octaarginine
tagged triclosan compound over a 24-hour period. Hydrolysis of the octaarginine tag
released triclosan, leading to decreasing IC values (11nM at 24 hours) over the time
50
10
course of the experiment . Nonlinear regression analysis was performed using GraphPad
Prism software
Co-crystallization and structure determination of TgENR in complex with
+
NAD and compound 19. Preparation of protein crystals—Crystals of TgENR in
+
complex with NAD and compound 19 were grown using the hanging drop vapor diffusion
technique at 290K and by screening around the conditions which had previously been shown
+
to be appropriate for crystallizing the TgENR/NAD /triclosan complex (0.1M Tris-HCL
14
pH9.0 and 6% PEG 8,000). In order to co-crystallize the TgENR with compound 19 the
poor solubility was overcome by dissolving the inhibitor in DMSO and then diluting with
the appropriate buffer for co-crystallization experiments.
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Tipparaju et al.
Page 17
+
Data collection and image processing—Crystals of both the TgENR/NAD /
compound 19 complex were flash frozen in 20% glycerol and data were collected at 100K to
2.7Å at the Daresbury SRS. The data were subsequently processed using the DENZO/
49
SCALEPACK package (Table 1). The structure of the complex was determined by
+
molecular replacement using the program Amore and the TgENR/NAD /triclosan structure
+
as a search model from which the triclosan and NAD coordinates were omitted. A clear
solution was obtained in Amore and the model was subjected to rigid-body refinement and
50
was subsequently rebuilt and refined in an iterative process using REFMAC5 and
WinCoot. Although omitted in the search model, clear and continuous density could be seen
+
for both the NAD cofactor and compound 19 allowing for their unambiguous fitting. Data
collection and model refinement statistics can be seen in Table 1.
Testing of inhibitors in vivo—Compounds effective in enzyme assays and then in vitro
10
(IC <10μM) were tested in an acute parenteral infection. CD1 mice were infected
50
intraperitoneally with 2000 T. gondii (RH strain, Type 1). This inoculation dose is capable
of killing one hundred percent of mice by day 10 post-infection. Mice were administered
inhibitors intraperitoneally at 10mg/kg doses. Protection was measured by determining
parasite burdens in the peritoneal cavity by microscopy at day 5 post-infection and survival
up to 5 days post infection.
ADMET assays—Cyp450 inhibition and human liver microsomal stability assays were
performed as is standard.
Statistical Analyses—Sample size and number of experiments. There were 6 replicate
samples per group for in vitro experiments and 6 mice for each experimental group. All
experiments were performed with sufficient sample sizes to have an 80% power to detect
differences at the 5% level of significance. Groups included untreated or controls. Results
were compared using students T test, Chi square analysis or Fisher’s exact test.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
Supported by NIAID U01 AI082180-01. We gratefully acknowledge support of this work by gifts from the Finley
John Gubbins Samuel Special Needs Trust, R Blackfoot, R Thewind, A Akfortseven, S Gemma, S Jackson, AK
Bump, S. Pettican, the Rooney Aldens, the Dominique Cornwell and Peter Mann Family Foundation, the Morel,
Rosenstein, Kapnick and Kiewit families, Intervet/Schering Plough, Toxoplasmosis Research Institute, and The
Research to Prevent Blindness Foundation. This work was also supported by the Johns Hopkins Malaria Research
Institute and the Bloomberg Family Foundation (JLZ and STP). We thank J. McCammon and Dr. Marco Pieroni for
their assistance in preparation of this manuscript.
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Figure 1.
Inhibitors of ENR: Structures of potential ENR inhibitors, ID #, MW, and ClogP of
inhibitors (calculated by using ALOGPS 2.1 www.vcclab.org/lap/alogps/), inhibition of T.
gondii in tissue culture and toxicity for HFF at highest concentration tested in simultaneous
experiments; IC enzyme. * = co-crystal. MIC, Inhibition of T. gondii uptake of tritiated
50
uracil; “Tox,” Effect of compound on nonconfluent fibroblast’s uptake of tritiated thymidine
with visual analysis of normal confluent monolayer, (−) represents no toxicity at highest
concentration tested. Dark grey shading indicates most promising inhibitors and light grey
shading indicates next initially most promising inhibitors but with either greater toxicity to
fibroblasts relative to effect on parasite or lower enzyme activity. Shaded compounds were
prescreened in a T. gondii ENR enzyme assay at three concentrations: 0.2, 2, and 20μM.
Compounds with significant inhibition at 2μM were further analyzed to determine IC
50
values with all data collected in triplicate.
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Figure 2.
(a) Synthesis of compound in 3 schemes: Scheme 1. Synthesis of compounds: When X = F
2
3
4
2
3
4
and R = Cl, R = H, R = CN; or when X = F and R = R = H, R = CN, Method A:
2
4
3
K CO or Cs CO , DMSO, 100 °C, 8–12 h. When X = I and R = R = H, R = Cl, Method
2
3
2
3
B: KOtBu, DMF, (CuOTf) ·PhH, 140 °C, 16–20 h. a) Excess BBr , CH Cl , −78 °C to rt,
2
3
2
2
2–6 h; b) n-BuLi, B(OMe) , 2N HCl, −78 °C to rt, 18 h; c) 35% H O , 3N NaOH, EtOH, 30
3
2 2
°C, 18 h; d) 25% NaOH, EtOH, reflux, 20 h; e) NaN , SiCl , CH CN, 90 °C, 6 h; (b)
3
4
3
Scheme 2. Synthesis of compounds: a) Sorbic acid, EDAC?HCl, HOBt, Et N, DMF, rt, 16
3
h; b) Excess BBr , CH Cl , −78 °C to rt, 6 h; c) Pd/C, H , 40 PSI, TEA, EtOH, rt, 16 h; d)
3
2
2
2
Pivaloyl chloride, Et N, THF, rt, 4 h; e) 2-Aminonicotinic acid, EDAC?HCl, HOBt, Et N,
3
3
DMF, rt, 16 h; f) Pivaloyl chloride, Et N, DMAP, CH Cl , 0 °C to rt, 6 h; (c) Scheme 3.
3
2
2
Synthesis of compounds: a) p-Anisidine, EDAC?HCl, HOBt, Et N, DMF, rt, 20 h; b) 2-
3
Aminobenzylamine, EDAC?HCl, HOBt, Et N, DMF, rt, 16 h; c) 4-Fluoronitrobenzene,
3
K CO , DMSO, 100 °C, 16 h; d) 4-Nitrobenzoyl chloride, pyridine, 0−80 °C, 4 h; e) 4-
2
3
Nitrobenzyl bromide, K CO , MeOH, 100 °C, 4 h; f) 4-Nitrobenzenesufonyl chloride, Et N,
2
3
3
DMF, 0 °C–rt, 4 h.
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Figure 3.
(a,b) Representative experiments showing effects of compounds 25 and 19 on parasite
uptake of tritiated uracil and on growth of nonconfluent host cell fibroblasts. (a) Inhibition
of T. gondii uptake of tritiated uracil by compound 19 and lack of inhibition by compound
3
25. (b) Lack of toxicity to fibroblasts (uptake of H Thymidine by nonconfluent HFF) by
compound 19. Toxicity to HFF from compound 25. (c,d,e) Representative experiments
showing inhibition of T. gondii tachyzoites (top panels), lack of inhibition of fibroblast
growth (middle panels) and inhibition of ENR enzyme activity (bottom panels) by
compounds 2, 19, and 39. Error bars in panels A–E show the standard deviation between
triplicate measurements. Compound 2 IC = 45nM and 19 = 20nM, and 39 less active (low
50
micro molar), possibly off target.
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Page 24
Figure 4.
Reduction of parasite burden by 2 and 19 in mice.
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Tipparaju et al.
Page 25
Figure 5.
(a) 2F – 1F
electron density map contoured at 1σ for compound 19 in complex with
calc
obs
+
TgENR/NAD after refinement in REFMAC5. (b) Superposition of TgENR in complex
with triclosan (blue) and compound 19 (green) shown in cartoon format, to show the
conservation of the overall fold between the two complexes. Both the NAD cofactor and
+
compound 19 are displayed in stick format and coloured yellow, red, blue, green and purple
for carbon, oxygen, nitrogen, chlorine and phosphorus, respectively. (c) Stereo diagram of
the TgENR triclosan (green) and compound 19 (blue) complex structures superposed. The
+
NAD cofactor and inhibitors are shown in stick format in addition to Phe243 which display
the greatest change between the different complexes.
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Table 1
Data collection/processing
Space group
P3221
2.7
Resolution (Å)
Unique reflections
Completeness (%)
I/σ(I) >3 (%)
16525
90.85
26
Rmerge (%)
0.378
Refinement statistics
a
b
0.26/0.32
Rcryst (%) /Rfree (%)
R.m.s.d. values
Bond length (Å)
Bond angle (deg.)
0.0054
1.00
c
Ramachandran plot
Most favoured (%)
93.0
7.0
0.0
0.0
Additionally allowed (%)
Generously allowed (%)
Disallowed (%)
Protein/substrate atoms/waters 4486/128/51
Mean B-values (Å2)
Protein
38
Co-factors
Water molecules
38 (compound 19) 36 (NAD)
43
+
Refinement statistics for TgENR/NAD /compound 19 complex.
a
Rcryst = Σ (|F | - |F
hkl obs
|)/Σ |F |.
calc hkl obs
b
c
R
was calculated on 5% of the data omitted randomly.
free
Percentage of residues in regions of the Ramachandran plot, according to PROCHECK.
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Tipparaju et al.
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Table 2
Triclosan
2
a
5.5
4.5
ClogP
b
−4.68
−4.44
ALOGpS
c
29.46
75.28
tPSA
a
calculated with ChemDraw Ultra 7.0, CambridgeSoft;
b
c
Predicted water solubility calculated by using ALOGPS 2.1 (www.vcclab.org/lab/alogps/);
total Polar Surface Area calculated by using http://www.molinspiration.com/cgi-bin/properties.
Reference for vcclab
Tetko, I. V.; Gasteiger, J.; Todeschini, R.; Mauri, A.; Livingstone, D.; Ertl, P.; Palyulin, V. A.; Radchenko, E. V.; Zefirov, N. S.; Makarenko, A. S.;
Tanchuk, V. Y.; Prokopenko, V. V. J. Comput. Aid. Mol. Des., 2005, 19, 453-63.
J Med Chem. Author manuscript; available in PMC 2011 September 9.