Synthesis and biological evaluation of functionalised tetrahydro-β- carboline analogues as inhibitors of Toxoplasma gondii invasion

Article abstract of DOI:10.1039/b902319d

Techniques for the identification of the protein target(s) of small molecules are proving very important following an increase in the use of phenotype-based screening in chemical biology and drug discovery. One approach, known as the yeast-3-hybrid approach, has shown considerable potential. A key factor in the success of this approach is the preparation of a complex molecule referred to as a chemical inducer of dimerisation (CID). The synthesis of two CIDs based on a bioactive tetrahydro-β-carboline core structure is reported and evidence presented that shows the CIDs are of utility in this approach. A series of chemo- and bioinformatic studies coupled with SAR development inspired the choice of CIDs.

Full text of DOI:10.1039/b902319d

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and biological evaluation of functionalised tetrahydro-b-carboline
analogues as inhibitors of Toxoplasma gondii invasion†
Jeffrey G. A. Walton,‡a Stephen Patterson,‡a Gu Liu,‡a Jeralyn D. Haraldsen,‡b Jonathan J. Hollick,a
Alexandra M. Z. Slawin,a Gary E. Wardb and Nicholas J. Westwood*a
Received 4th February 2009, Accepted 22nd May 2009
First published as an Advance Article on the web 17th June 2009
DOI: 10.1039/b902319d
Techniques for the identification of the protein target(s) of small molecules are proving very important
following an increase in the use of phenotype-based screening in chemical biology and drug discovery.
One approach, known as the yeast-3-hybrid approach, has shown considerable potential. A key factor
in the success of this approach is the preparation of a complex molecule referred to as a chemical
inducer of dimerisation (CID). The synthesis of two CIDs based on a bioactive tetrahydro-b-carboline
core structure is reported and evidence presented that shows the CIDs are of utility in this approach. A
series of chemo- and bioinformatic studies coupled with SAR development inspired the choice of CIDs.
unknown. The identification of a compound’s protein target(s) is
Introduction
essential if it is to be used as a small molecule tool.7,8 Here we report
Toxoplasma gondii, a protozoan parasite capable of infecting any
results from our programme to identify the protein target(s) of 1.
warm-blooded animal, is one of the most common parasites of
A chemoinformatic assessment of 1 led to several potential protein
man.1 Infection can be life threatening during pregnancy, or in the
target candidates. However, SAR and bioinformatic investigations
immunocompromised patient.2 T. gondii is also considered a model
for other related, but less experimentally tractable parasites in the
did not support the hypothesis that 1 inhibits T. gondii invasion
by disrupting the function of a T. gondii homologue of a known
phylum Apicomplexa, including Plasmodium, the causative agent
mammalian protein target of 1. As a result we were compelled to
of malaria, and Cryptosporidium, a significant world-wide cause
adopt genome/proteome-wide target identification approaches. In
this context, we report the preparation of novel reagents based on
1 and present data that show these reagents are suitable for this
purpose.
of water-borne disease.3 Apicomplexan parasites are only able to
grow and replicate within cells of their hosts. The process of host
cell invasion by T. gondii and other apicomplexans is therefore
both a key step in the life cycle of the parasite and a potential
point of therapeutic intervention.
Results and discussion
The mechanism of host cell invasion by apicomplexan parasites
has previously been studied in detail,4 in part through the
use of pharmacological inhibitors whose targets have been well
characterised in other systems.3b,5 However, little progress has been
made towards the development of anti-parasitic drugs that target
invasion. In a broader search for compounds that disrupt host
cell invasion by T. gondii, we recently reported the use of a high-
throughput cell-based assay to identify novel inhibitors of this
process.6 The assay delivered 24 non-cytotoxic compounds that
reproducibly inhibit host cell invasion, including the function-
alised tetrahydro-b-carboline 1 (Scheme 1A). However, the mode
of action of 1 and the other inhibitors identified in this screen was
Preliminary SAR
As a number of analogues of 1 are commercially available,
initial studies focused on the biological assessment of 3 racemic
compounds that can be viewed as truncated versions of 1,
compounds 2, 3 and 4 (Scheme 1A). All 3 analogues were
inactive in the invasion assay (no inhibition of invasion at final
concentrations up to 100 mM) and we therefore concluded that
the fused tetracyclic core structure of 1 represents the minimum
bioactive pharmacophore.
Four stereoisomers of 1 exist and the next step focused on
determining the influence that the stereochemistry at C5 and
C11a in 1 had on the observed biological activity. Reacting either
(D)- (5a) or (L)-tryptophan (5b) with m-hydroxybenzaldehyde
under Pictet–Spengler reaction conditions led to formation of
the corresponding cis-acid 2a and its enantiomer 2b respectively
(Scheme 1B).9a In this and the other examples reported here, the
major isomer formed in the Pictet–Spengler reaction had the cis-
C5-C11a relative stereochemistry. Subsequent reaction of cis-2a
aSchool of Chemistry and Biomolecular Sciences Research Complex, Uni-
versity of St Andrews, St Andrews, Fife, KY16 9ST, Scotland, UK. E-mail:
njw3@st-andrews.ac.uk
bDepartment of Microbiology and Molecular Genetics, 316 Stafford Hall,
University of Vermont, 95 Carrigan Drive, Burlington, VT 05405, USA
† Electronic supplementary information (ESI) available: Numbering
scheme used for the b-carboline ring system, NOSEY spectrum of cis-
6b and trans-7a, bioinformatic analysis of T. gondii kinesins, the yeast-
3-hybrid approach, synthesis of C9-substituted analogues 7e–7j, synthesis
and biological activity of analogues 9ii, 9iv, 9x–xvii, NMR characterisation
of CID 23 and CID 15. CCDC reference number 719462. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/b902319d
n
or 2b with butylisocyanate gave cis-6a and 6b, respectively in
good yields. A NOESY experiment carried out on cis-6b showed
a correlation between the C5 and C11a-hydrogens as expected for
a cis-relationship.10 This assignment is consistent with a previous
literature investigation of the hydantoin ring-forming reaction in
‡ These authors contributed equally.
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Scheme 1 A Tetrahydro-b-carboline analogues of 1. B Synthesis of analogues of 1. Reaction conditions: (i) H2SO4, H2O, 100 ◦C, 8 hrs; 70% for◦2a
(R4 = H); 50% 2b (R4 = H); 34% 2c; 52% 2d; (ii) DMSO/acetone, R1NCO, 56 ◦C, 2–4 hrs; 65% 6a; 92% 6b; 74% 6c; 64% 6d; (iii) K2CO3, MeCN, 82 C,
30 min.; 80% 7a; 99% 7b; 53% 7c; 77% 7d; 87% 7k; 82% 7l; 63% 7m; (iv) DMSO/acetone, R1NCS, 56 ◦C, 2 hrs; 56% 8a; 50% 8b. C Known inhibitors of
cGMP-specific 3¢,5¢-cyclic nucleotide phosphodiesterases and kinesins.
this heterocyclic system.9a In order to access the corresponding
trans-isomers 7a and 7b, the C11a protons in cis-6a and cis-
6b were epimerised by treatment with potassium carbonate in
refluxing acetonitrile. X-Ray crystallographic analysis of trans-
7b confirmed the relative stereochemical assignment at C5 and
C11a.10,11 Reaction of cis-2a and 2b with nbutylisothiocyanate led
directly to the corresponding C3-thiocarbonyl analogues that, in
line with literature precedent,9 were assigned as trans-8a and trans-
8b respectively (Scheme 1B).
Interestingly, the trans-(5R, 11aS)-isomer 7a12 proved to be
the most potent inhibitor of T. gondii invasion with a minimum
inhibitory concentration (MIC) of 12.5 mM (Table 1, entry 1).
Its enantiomer trans-7b did not inhibit T. gondii invasion even
at 100 mM, the highest concentration tested (Table 1, entry 2).
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Table 1 Minimum inhibitory concentrations (MIC) in the T. gondii cell
invasion assay for analogues of 1
structurally related to tadalafil (10) (Scheme 1C), a clinically-used
inhibitor of the mammalian cGMP-specific 3¢,5¢-cyclic nucleotide
phosphodiesterase PDE5.13 A bioinformatic analysis of the
T. gondii genome14 revealed that the T. gondii genome encodes
at least 15 kinesins (see also Foth and Soldati15) and 17 cyclic
nucleotide PDEs.
MIC
(mM)
Entry # Cpd # C5 C11a R1
R2
R3
X
1
2
3
4
5
6
7
8
9
7a
7b
6a
6b
7c
7d
6c
6d
8a
8b
9i
R
S
R
S
R
S
R
S
S
R
R
S
S
R
R
S
S
R
S
nButyl OH
nButyl OH
nButyl OH
nButyl OH
H
H
H
H
OH
OH
OH
OH
H
H
H
O
O
O
O
O
O
O
O
S
12.5
>100
50
50
25
Of the 15 kinesins encoded by T. gondii, conserved domain
searches and BLASTP analyses suggested that four belong to
kinesin family-5, of which human Eg5 is a member. Based
on expression data,14b at least three of these four family-5
kinesins (TgME49_097110, TgME49_024880, TgME49_119710)
are expressed in the tachyzoite stage of the parasite used in our
invasion assays. Trans-7a inhibits human Eg5 allosterically and the
amino acids that make up the allosteric inhibitor-binding pocket
have recently been determined.16 The primary sequences of these
four T. gondii kinesins were aligned with several of the known
family-5 kinesins, including human Eg5, and the corresponding
inhibitor-binding regions were compared (see Figure S310). Whilst
comparisons based on inhibitor-binding region 1 (Y125-E145)
were inconclusive, all four of the T. gondii kinesins appear to
have reasonable sequence similarity to human Eg5 in inhibitor-
binding region 2 (I202-L227). The observation that trans-7a is the
most active isomer for both inhibition of Eg512 and inhibition of
T. gondii invasion (Table 1) is also consistent with the hypothesis
that trans-7a acts on a T. gondii Eg5 homologue. However, assay
results obtained with other close structural analogues did not
support this hypothesis. For both trans-7a and monastrol (11)
(Scheme 1C), inhibition of Eg5 involves an important hydrogen
bond between the phenol functional group and the backbone
carbonyl oxygen of either Trp127 or Glu118 in Eg5.17,18 In contrast,
we observed that the phenolic OH in 7a was not essential for
inhibition of T. gondii invasion. The methyl ether analogue 9i
(Scheme 2A) was as potent in the T. gondii invasion assay
as 7a (Table 1, entry 11 vs. 1). In addition, previous studies
on structurally related 4-aryl-tetrahydroisoquinoline inhibitors
(e.g. 12, Scheme 1C), which bind at the same site on human
nButyl
nButyl
nButyl
nButyl
H
H
H
H
25
>100
100
3.1
12.5
12.5
R
S
R
nButyl OH
nButyl OH
nButyl OCH3
10
11
S
O
The other enantiomeric pair in this series, cis-6a and cis-6b, both
exhibited weaker inhibition of T. gondii invasion compared to
trans-7a with MICs of 50 mM (Table 1, entries 3 and 4). A similar
activity trend was observed across a second series of four isomers,
6c, 6d, 7c, 7d, that differed from the 6/7a,b series only in the
position of the phenolic OH (Scheme 1B) with one exception.
The activity associated with trans-7d was significantly increased
compared to that of trans-7b (Table 1, entry 6 vs. entry 2).
Unexpectedly, replacement of the C3 carbonyl in both trans-7a
and trans-7b by a thiocarbonyl moiety9b to give trans-8a and trans-
8b respectively led to a significant increase in potency (Table 1,
entry 9 vs. 1 and 10 vs. 2). Conversion of the phenolic functional
group in trans-7a to the corresponding methyl ether gave trans-
9i (Scheme 2A) that had analogous activity to trans-7a (Table 1,
entry 11 vs. entry 1).
Potential biological targets generated using
chemoinformatics/bioinformatics
Substructure searches using the core structure of 1 revealed that
trans-7a is an inhibitor of the mitotic kinesin Eg512 and 1 is
Scheme 2 A Synthesis of O-substituted analogues of trans-7a. Reaction conditions: (i) PPh3, DEAD, MeOH, THF; 85% for 9i; (ii) RX, K2CO3, CH3CN,
61–84%; for R2 see Table 2. B Synthesis of analogues of trans-7e substituted at R4 (see Table 2). Reaction conditions: (iii) R4B(OH)2, Pd(PPh3)4, Na2CO3,
DME, H2O, 70 ◦C, 24 hrs. For details see ESI.10
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Table 2 Minimum inhibitory concentrations (MIC) in the T. gondii cell invasion assay for analogues 7e–7m and 9i–9xii(see Schemes 1B and 2B for
structures; R3 = H in all cases). For the synthesis and testing of additional O-substituted analogues see supplementary material10
Entry #
Cpd #
C5
C11a
R1
R2
R4
MIC (mM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
7a
R
S
nButyl
OH
OH
OH
OH
OH
OH
OH
OH
H
Br
12.5
25
12.5
12.5
25
25
100
50
7e
S/R
S/R
S/R
S/R
S/R
S/R
R
R/S
R/S
R/S
R/S
R/S
R/S
S
nButyl
7f
nButyl
Ph
4-Cl–C6H4
4-Me–C6H4
4-OMe–C6H4
7g
nButyl
7h
nButyl
7i
7j
nButyl
nButyl
3,4-diCl-C6H4
7k
7l
7m
9i
nOctyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
R
S
CH2CH2CO2Et
p-C6H4-CO2Et
nButyl
OH
OH
OMe
>100
50
R
S
R
S
12.5
6.25
12.5
6.25
12.5
1.6
3.1
3.1
12.5
100
100
25
9ii
9iii
9iv
9v
9vi
9vii
9viii
9ix
9x
9xi
9xii
R
S
nButyl
OEt
R
S
nButyl
OnPr
R
S
nButyl
OnBu
R
S
nButyl
Oallyl
R
S
nButyl
OiPr
R
S
nButyl
OCH2C(CH3)=CH2
Opropargyl
OBn
OCH2p-C6H4-OMe
OCH2m,p-C6H3-Cl2
CH2CONH2
R
S
nButyl
R
S
nButyl
R
S
nButyl
R
S
nButyl
R
S
nButyl
Eg5 as 7a and 11, have demonstrated that analogues containing
a para-phenol are inactive, whereas those containing the phenolic
OH in the meta-position are active.17 This is in contrast to our
observations with 6c, 6d, 7c and 7d which all inhibit T. gondii
invasion (Table 1, entries 5–8). Finally, the replacement of the
C3 carbonyl in both trans-7a and 7b with a thiocarbonyl had a
significant effect on the observed biological activity in our system,
whereas an identical change has previously been reported to have
no impact on Eg5 inhibition.12a In the light of these observations
and the fact that the known Eg5 inhibitors monastrol (11) and
S-trityl-L-cysteine (13) (Scheme 1C) had no effect on parasite
invasion (data not shown), it was concluded that the inhibition of
T. gondii invasion by 1 probably occurs through a different target
class or through a parasite kinesin with unique characteristics.
Of the 17 cyclic nucleotide PDEs encoded by T. gondii, at
least nine are expressed in the tachyzoite stage of the parasite.14
Tadalafil (10) targets a human PDE of the PDE5 family, but
subclassification of the T. gondii cyclic nucleotide PDEs into their
most likely families revealed no PDE5 family members within the
T. gondii genome. PDE6 and PDE11 family members have been
proposed as secondary targets of 1019,20 (and therefore, potentially
of 1), but these PDE families are also absent from the T. gondii
genome. It is therefore unlikely that PDE5, 6 or 11 is the target of
1 in the parasite.
Amongst many challenges in the Y3H approach is the synthesis of
a key compound referred to as a chemical inducer of dimerisation
(CID). This requires linkage of methotrexate (Mtx (14), Scheme 3)
to the molecule under study in a manner that preserves its ability
to bind to its target(s). Confidence that an appropriate position
for Mtx (14) attachment has been chosen comes from SAR
studies.
Extended SAR studies
The effect of incorporating a substituent in the indole aromatic
ring of trans-7a was explored to assess whether this position
may provide a potential site for linker attachment or whether
incorporation of an R4-substituent (Scheme 2B) would lead to an
increase in potency. An analogue of 7a containing a bromine at
R4 (7e) was prepared from commercially available 5-bromo-(DL)-
tryptophan (5c) (Schemes 1B and 2B).10 Racemic trans-7e was
active in the T. gondii invasion assay (Table 2, entry 2) and since
the large bromo-substituent was tolerated, a Topliss22 series of
aryl ring-containing analogues, 7f–j, was prepared from 7e using
Suzuki coupling methodology (Scheme 2B).10 With the exception
of the incorporation of an electron-withdrawing meta-chlorine
atom (c.f. Table 2, entry 4 vs. entry 7), the nature of the substituent
on the phenyl ring had little effect on the observed biological
activity of these analogues. Given the flat SAR for 7f–j and the
relatively low yields associated with substitution at R4, alternative
sites for attachment of the linker were explored and the R4 = H
substitution was retained.
Genome/proteome-wide target identification techniques
As no clear leads to a putative target of 1 in T. gondii could
be reached from the chemo/bioinformatic analyses and initial
SAR studies, we focused on the synthesis of analogues of trans-
7a that could be used for more general target identification
techniques.7 In particular, we decided to prepare reagents that
were applicable to the yeast-3-hybrid (Y3H) approach (see Figure
S4).10,21 This relatively new technique shows considerable promise
for identifying protein binding partners of small molecules.
Variation of the N2-substituent of the hydantoin ring in trans-
7a was considered first as a possible linker attachment site
(Scheme 1A for numbering system). Exchange of the nbutyl
substituent for an noctyl group was achieved by reaction of
n
2a with octylisocyanate followed by epimerisation at C11a to
give trans-7k (Scheme 1B). This analogue retained the desired
biological activity, albeit at a reduced level (Table 2, entry 8). This
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Scheme 3 Synthetic routes to chemical inducers of dimerisation (CIDs) 15 and 23. Reagents and conditions: (i) TsCl, Ag2O, KI, DCM, 84%; (ii) NaN3,
DMF, 110 ◦C, 93%; (iii) PPh3, THF, H2O, 98%; (iv) Boc2O, DCM, 89%; (v) MsCl, DIPEA, DCM, 75%; (vi) NaN3, DMF, 110 ◦C, 89%; (vii) PS-PPh3,
THF, H2O, quant.; (viii) LiOH, THF, H2O, 66%; (ix) 16, TPTU, Et3N, DMF, 45%; (x) TFA, DCM; (xi) 21, TPTU, DIPEA, DMF, 65% over 2 steps;
(xii) TFA, DCM, 55%; (xiii) 18, DEAD, PPh3, THF, 62%; (xiv) TFA, DCM; (xv) 21, EDC, HOBt, DIPEA, DCM, DMF, 63% over 2 steps; (xvi) TFA,
DCM, 66%.
result suggested that the N2-position could be a suitable point of
attachment for a linker unit and led to the synthesis and testing of
trans-7l, an analogue containing a shorter alkyl chain terminating
in an ester functionality (Scheme 1B). It was envisaged that
hydrolysis of the ester would provide a carboxylic acid functional
group that could be used to attach the linker unit using a standard
amide bond-forming reaction. Unfortunately, 7l proved inactive
in the parasite invasion assay. Before abandoning this approach,
however, an alternative ester analogue, trans-7m, was prepared that
contained an aryl spacer between N2 (Scheme 1B) and the required
ester functionality. 7m did inhibit T. gondii invasion (Table 2,
entry 10), thereby providing a suitable structure for synthetic
transformation to the desired CID 15 (Scheme 3). The different
activities of 7l and 7m may be due to differences in the levels to
which the esters are hydrolysed under the assay conditions in live
parasites.
Synthesis of CID 15
Scheme 3 shows the route used to access 15. A key component
of this reaction sequence was synthesis of the PEG linker unit
16, which contained both a primary amine functional group
and a second suitably protected amine allowing for sequential
amide bond-forming reactions. Previous reports23 described the
synthesis of 16 in relatively low yield (17% yield over 5 steps from
hexaethylene glycol (17)). We initially converted 17 to the Boc-
protected amine 18 in a four step sequence. This involved mono-
tosylation of 17 and substitution of the resulting tosylate using
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Table 3 Structure of control CID Dex-Mtx 26. Strain V1019Y was
incubated for 48 hrs at 30 ◦C in medium with and without leucine, in
the presence of the CID and competitors indicated. Entry 3 confirms that
Dex-Mtx can support the growth of V1019Y in selective medium (compare
to entry 2). This growth was competitively inhibited by methotrexate, (14)
(entry 4), 15 and 23 (entries 5–6)
sodium azide, followed by Staudinger reduction of the azide and
N-protection.23a Subsequent conversion of 18 to the corresponding
mesylate, followed by reaction with sodium azide and reduction
of the azide functionality using polymer supported triphenyl
phosphine led to a significant improvement in the overall yield of
16 (46% over 7 steps from 17). The use of the solid phase reagent in
the final step circumvented purification challenges associated with
16. Coupling of the linker unit 16 to trans-7m was then carried
out by hydrolysis of the ethyl ester functionality in 7m to give
the corresponding acid 19. TPTU-mediated coupling of 19 with
16 afforded 20 in acceptable yield (Scheme 3). The preparation
of acid 21,24 a suitably protected version of methotrexate (14),
allowed the synthesis of 15 to be completed. Boc-deprotection of
20 followed by a second TPTU-mediated coupling step, this time
with 21, gave the desired CID 15 after hydrolysis of the tbutyl ester
in 22. Extensive characterisation of 15 using a range of NMR
techniques and mass spectroscopic analysis were consistent with
the structure of 15 as drawn.10
Entry
Medium
CID
Competitor
OD600 (48 hrs)
1
2
3
4
5
6
7
8
9
+ Leu
- Leu
- Leu
- Leu
- Leu
- Leu
+ Leu
+ Leu
- Leu
- Leu
—
—
—
—
—
1.30
0.04
0.93
0.05
0.00
0.00
1.31
1.31
0.03
0.05
10 mM 26
10 mM 26
10 mM 26
10 mM 26
—
—
—
100 mM Mtx (14)
100 mM 15
100 mM 23
100 mM 15
100 mM 23
100 mM 15
100 mM 23
Synthesis of CID 23
Early studies had demonstrated that substitution of the phenolic
oxygen in trans-7a with a methyl group led to an analogue 9i
that retained the desired biological activity (Table 1, entry 11).
Substitution at this position was therefore further investigated
using a parallel synthesis approach (Scheme 2A). Our initial
alkylation protocol used the Mitsunobu reaction giving 9i in
good yield. However, this method was impractical for parallel
synthesis applications. Instead, analogues 9ii–9xii were prepared
by treating trans-7a with one equivalent of the appropriate alkyl
halide in the presence of excess potassium carbonate. This method
allowed the facile isolation of the desired compounds following
filtration through a short plug of silica gel. Variation of the
phenolic substitutent by extending the alkyl chain from methyl
10
—
Chemical inducers of dimerisation (CID) 15 and 23 are suitable for
use as target identification reagents
In order to perform a Y3H screen, it is a prerequisite that the
synthesised CIDs are cell-permeable and can gain access to the
yeast nucleus, the location of the transcription factor fusion
proteins that allow selection of positive small molecule-target
interactions (see Figure S410). The cell- and nuclear-permeability
of 15 and 23, our two CIDs based on 1, were assessed by a
competition assay using a positive control strain of yeast, V1019Y.
This strain has previously been used to validate other CIDs
in a similar manner to the one described here.21b V1019Y is a
leucine (Leu) auxotroph and, in the absence of any CID, is only
able to grow in medium containing leucine. However, V1019Y
is engineered to activate expression of a LEU2 reporter when
there is a positive interaction between a control CID, Dex-Mtx
26 (Mtx (14) linked to dexamethasone, Table 3), and a control
target fusion protein (the rat glucocorticoid receptor fused to
the B42 activation domain) resulting in CID-dependent leucine
prototrophy and restoration of growth in the absence of exogenous
leucine.
n
up to butyl (9i–9iv) had little effect on activity (Table 2 entries
11–14). However, the incorporation of either unsaturated and/or
branched O-alkyl chains led to either retention (9v, Table 1, entry
15) or more frequently an increase in activity. For example, the
O-iso-propyl analogue 9vi consistently inhibited T. gondii host
cell invasion at a final concentration of 1.6 mM (Table 2, entry
16) and 9vii, an analogue containing a 2-methylallyl substituent,
was active at 3.1 mM (Table 2, entry 17). The incorporation
of an O-propargyl group in 9viii also led to an increase in
potency (Table 1, entry 18) but plans to access a series of 1,2,3-
triazole-containing analogues from 9viii were abandoned when
it was determined that incorporation of a structurally related
O-benzyl substituent was detrimental to activity (Table 2, entries
20 and 21).
The ability to substitute the phenolic oxygen in trans-7a with a
range of different alkyl groups whilst retaining activity suggested
to us that this may also be an appropriate place to incorporate
a linker unit. However, as the introduction of an amide group
at this position led to decreased activity (Table 2, entry 22), we
decided to use the previously prepared linker unit 18 (Scheme 3).
Mitsunobu reaction of trans-7a with 18 gave the phenolic ether
24, which was converted to the desired CID 23 via the tbutyl ester
25 in 26% yield over 3 steps. Extensive analysis of 23 using a
range of NMR techniques supported the structural assignment
of 23.10
Strain V1019Y was inoculated into a 96-well plate and growth
was assessed under a variety of conditions by measuring the
OD600 after incubation for 48 hours at 30 ◦C. As expected, in
the absence of any CID, strain V1019Y only grew when leucine
was provided exogenously (Table 3, entry 1; compare to no growth
in the absence of leucine, entry 2). In agreement with previous
reports,21b we showed that incubation of V1019Y with Dex-Mtx
26 (10 mM) was sufficient to induce a transcriptional response
and supported growth in the absence of leucine (Table 3, entry 3).
Also as previously reported,21b the addition of excess Mtx (14) to
the medium was found to abrogate the transcriptional activation
3054 | Org. Biomol. Chem., 2009, 7, 3049–3060
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of the LEU2 reporter through competition with Dex-Mtx 26 for
binding to the dihydrofolate reductase (DHFR)-containing fusion
protein present in V1019Y (see Table 3, entry 4 and Figure S410)
We then proceeded to test whether 15 and 23 could compete with
Dex-Mtx 26 in an analogous manner.
Like Mtx (14), both 15 and 23 (100 mM) are able to inhibit the
CID-dependent growth of V1019Y in the presence of Dex-Mtx
26, indicating that they do indeed compete for binding to DHFR
(Table 3, entries 5–6). Further control experiments demonstrated
that the observed growth inhibition is not due to cytotoxicity
associated with 15 or 23 (Table 3, entries 7 and 8) and that neither
15 or 23 was able to autoactivate the LEU2 reporter in the absence
of the rat glucocorticoid receptor-B42 activation domain fusion
(Table 3, entries 9 and 10). These data strongly suggest that both 15
and 23 are cell- and nuclear-permeable and are therefore suitable
for Y3H screening experiments.
In general, the 1H and 13C NMR spectra of several of the
compounds reported were found to be concentration dependent.
Low and high-resolution mass spectral analysis were recorded
using ES operating in positive or negative ion mode. Details of the
synthesis of additional compounds discussed here are included in
the supplementary material including an improved spectroscopic
characterisation of several literature compounds.
(5R, 11aS)-2-(4-Benzoic acid ethyl ester)-5-(3-hydroxyphenyl)-
6H -1,2,3,5,11,11a-hexahydro-imidazo[1,5-b]-b-carboline-1,3-
dione (7m). 7m was prepared according to the general protocols
described in the ESI from 5a via 2a and the corresponding cis-
isomer.
2a (0.5 g, 1.62 mmol) and 4-(ethoxycarbonyl)phenyl isocyanate
(0.47 g 2.44 mmol) were stirred at reflux for 3 hours in anhydrous
DMSO (0.5 mL) and anhydrous acetone (2 mL). The solution
was diluted with water (5 mL) and extracted into EtOAc (3 ¥
5 mL). The combined organic layers were dried (Na2SO4) and
reduced in vacuo. Purification of the residue by chromatogra-
phy on silica gel (EtOAc:Hex 20:80) afforded (5R,11aR)-2-(4-
benzoic acid ethyl ester)-5-(3-hydroxyphenyl)-6H-1,2,3,5,11,11a-
hexahydro-imidazo[1,5-b]-b-carboline-1,3-dione as a yellow solid
(0.51 g, 1.05 mmol, 65%). m.p. 176.8–177.4 ◦C; nmax (KBr) 3349,
Conclusion
The ability of cell-based high-throughput assays to deliver com-
pounds that elicit the desired response in cells is very powerful.25
For example, our previous studies led to the identification of
24 non-cytotoxic inhibitors of host cell invasion by the parasite
Toxoplasma gondii.6 However, this approach is limited by the
challenges inherent in identifying the mode of action of the
bioactive compounds. Here we describe our follow-up studies on
the T. gondii invasion inhibitor 1. Our initial studies identified
trans-7a as the most active isomer of 1, a result that led us
to propose that parasite kinesins or phosphodiesterases may
be the relevant target(s) following chemoinformatic analysis. A
more detailed analysis of these possibilities using bioinformatic
approaches and a comparison of our SAR data with that existing
in the literature suggested that the mechanism of action of 1 is most
likely through interaction with a different class of protein. A range
of genome/proteome wide approaches to target identification was
therefore considered culminating in a focus on the use of the
recently reported yeast-3-hybrid (Y3H) method.21 A key challenge
in this approach is the synthesis of the required CIDs. Here we
report the synthesis of two such CIDs, 15 and 23, based on 1.
The CIDs differ in the point of attachment of the linker and
methotrexate units. We also demonstrate that 15 and 23 have the
properties necessary to access the nucleus of yeast cells, an absolute
requirement for their use in this approach. Y3H screening using
15 and 23 has recently been initiated and will be the subject of a
future report.
1
2981, 1773, 1729 cm-1; H NMR (400 MHz, DMSO-d4) d 7.99
(2H, d, J 8.8, H3¢), 7.52 (1H, d, J 6.7, H10), 7.47 (2H, d, J 8.8,
H2¢), 7.18–7.06 (4H, m, H7, 5¢¢, 9, 8), 6.82 (1H, d, J 7.8, H6¢¢),
3
6.70 (1H, s, H2¢¢), 6.63 (1H, d, J 8.3, H4¢¢), 5.73 (1H, s, H5),
4.50 (1H, dd, J 11.7, 4.8, H11a), 4.30 (2H, q, J 7.17, H7¢), 3.45
(1H, dd, J 15.6, 4.8, H11syn), 3.12 (1H, dd, J 15.6, 11.7, H11anti),
1.31 (3H, t, J 7.2, H8¢); 13C NMR (100 MHz, DMSO-d6) d 170.0
=
=
=
(C O), 165.9 (C O), 156.2 (C), 153.2 (C O), 140.1 (C), 137.3
(C), 136.8 (C), 135.4 (C), 133.0 (C), 130.3 (CH), 130.2 (CH), 126.1
(C), 125.4 (CH), 122.9 (CH), 120.3 (CH), 119.9 (CH), 118.5 (CH),
115.9 (CH), 114.7 (CH), 111.3 (CH), 106.6 (C), 61.2 (CH2), 57.8
(CH), 56.8 (CH), 22.6 (CH2), 14.3 (CH3); LRMS (ES+) m/z: 482.30
[M + H]+; HRMS (ES+) [M + H]+ m/z expected for C28H24N3O5
482.1716 found 482.1733 [a]D20 = +0.8 (c = 0.007, EtOAc). The cis-
isomer (0.49 g, 1.01 mmol) was then dissolved in anhydrous MeCN
(12 mL) and K2CO3 (0.182 g, 1.32 mmol) was added. After stirring
at reflux for 5 hours, the mixture was filtered and the solvent was
removed in vacuo. Purification of the residue by chromatography
on silica gel (EtOAC:Hex 20:80) gave 7m as a yellow solid (0.31 g,
0.64 mmol, 63%). m.p. 178.2–179.1 ◦C; nmax (KBr) 3349, 2981,
1773, 1729 cm-1; 1H NMR (400 MHz, DMSO-d6) d 10.90 (1H, s,
NH), 9.50 (1H, s, OH), 8.05 (2H, d, J 8.5, H3¢), 7.85 (2H, d, J
8.5, H2¢), 7.57 (1H, d, J 8.0 H10), 7.31 (1H, d, J 8.2, H7), 7.18
(1H, m, H5¢¢), 7.13–7.08 (1H, m, H9), 7.06–7.00 (1H, m, H8), 6.82
(1H, d, J 8.04, H6¢¢), 6.78 (1H, s, H2¢¢), 6.73 (1H, d, J 7.7, H4¢¢),
6.21 (1H, s, H5), 4.60 (1H, dd, J 10.9, 5.6, H11a), 4.33 (2H, q,
J 10.4, H7¢), 3.42 (1H, dd, J 14.8, 5.02, H11syn), 3.08 (1H, dd, J
10.65, 4.53, H11anti), 1.31 (3H, t, J 7.1, H8¢); 13C NMR (100 MHz,
Experimental
Unless otherwise noted, starting materials and reagents were
obtained from commercial suppliers and were used without
further purification. PE 40–60 refers to the fraction of light
PE 40–60 boiling in the range 40–60 ◦C. Melting points were
recorded using an Electrothermal 9100 capillary melting point
apparatus. Values are quoted to the nearest 0.5 ◦C. IR spectra were
measured on a Perkin-Elmer Paragon 1000 FT-IR spectrometer.
1H and 13C NMR spectra were measured on a Bru¨ker Advance
300/500 instrument. J values are quoted in Hz. Chemical shifts
are calibrated with reference to the residual proton and carbon
resonances of the solvent (CDCl3: dH = 7.26, dC = 77.0 ppm).
=
=
=
DMSO-d6) d 171.7 (C O), 165.8 (C O), 165.5 (C), 158.1 (C O),
156.1 (C), 144.4 (C), 136.8 (C), 136.5 (C), 131.4 (C), 130.8 (CH),
130.2 (CH), 129.9 (CH), 126.1 (C), 122.9 (CH), 119.3 (CH), 118.6
(CH), 117.9 (CH), 115.6 (CH), 115.4 (CH), 111.8 (CH), 106.5 (C),
61.3 (CH2), 53.2 (CH), 52.1 (CH), 22.9 (CH2), 14.7 (CH3); LRMS
(ES+) m/z: 482.29 [M + H]+; HRMS (ES+) [M + H]+ m/z expected
for C28H24N3O5 482.1716 found 482.1709 [a]2D0 = -258.0 (c = 0.003,
EtOAc).
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General procedure for the alkylation of 7a
30.3 (CH2), 23.6 (CH2), 22.0 (CH3), 21.9 (CH3), 20.0 (CH2), 13.7
(CH3); LRMS (ES+) m/z: 454.13 (100%) [M + Na]+; HRMS (ES+)
[M + Na]+ m/z expected for C26H29N3O3Na 454.2107 obtained
454.2110; [a]2D0 = -198.2 (c = 0.0011, CHCl3).
To a solution of 7a (200 mg, 0.51 mmol) in anhydrous MeCN
(2 mL) was added K2CO3 (105.7 mg, 0.77 mmol) and the
appropriate alkyl halide (1.0 eq, 0.51 mmol). After stirring at
reflux for 18 hrs, the reaction mixture was diluted with H2O
(2 mL). The mixture was extracted with DCM (2 ¥ 2 mL) and the
combined extracts were dried (Na2SO4) and concentrated in vacuo.
Purification of the residue by filtration through a plug of silica
(EtOAc:Hex 1:9) afforded the desired product.
(5R,11aS)-2-Butyl-5-(3-(2-methyl-allyloxy)phenyl)-6H-1,2,3,
5,11,11a-hexahydro-imidazo[1,5-b]-b-carboline-1,3-dione
(9vii).
Yield 71%, yellow solid. m.p. 94–95 ◦C; nmax (KBr) 3332, 2956,
1765, 1702, 1454, 1237 cm-1; 1H NMR (300 MHz, CDCl3) d 7.78
(1H, s, NH), 7.56 (1H, d, J 7.5, H10), 7.32–7.15 (4H, m, H8, 9, 5¢¢,
7), 6.93–6.88 (3H, m, H6¢¢, 4¢¢, 2¢¢), 6.27 (1H, d, J 1.2, H5), 5.03,
(1H, s, H10¢¢), 4.96 (1H, s, H10¢¢), 4.38 (2H, s, H8¢¢) 4.32 (1H, dd,
J 11.0, 5.5, H11a), 3.56–3.48 (3H, m, H1¢, 11syn), 2.88 (1H, ddd,
J 15.3, 11.0, 1.8, H11anti), 1.79 (3H, s, H11¢¢), 1.65–1.57 (2H, m,
H2¢), 1.40–1.27 (2H, m, H3¢), 0.93 (3H, t, J 7.4, H4¢); 13C NMR
(5R,11aS)-2-Butyl-5-(3-propoxyphenyl)-6H-1,2,3,5,11,11a-
hexahydro-imidazo[1,5-b]-b-carboline-1,3-dione
(9iii). Yield
63%, yellow oil. nmax (KBr) 2960, 1713, 1490, 1222 cm-1; 1H NMR
(400 MHz, CDCl3) d 7.49 (1H, d, J 7.5, H10), 7.25–7.07 (4H,
m, H8, 9, 5¢¢, 7), 6.87–6.78 (3H, m, H4¢¢, 2¢¢, 6¢¢), 6.19 (1H, s,
H5), 4.25 (1H, dd, J 11.0, 5.5, H11a), 3.80 (2H, t, J 6.6, H8¢¢),
3.51–3.39 (3H, m, H1¢, 11syn), 2.81 (1H, dd, J 15.3, 11.0, H11anti,),
1.75–1.64 (2H, m, H9¢¢), 1.59–1.51 (2H, m, H2¢), 1.30–1.22 (2H,
m, H3¢), 0.93 (3H, t, J 7.6, H10¢¢), 0.86 (3H, t, J 7.4, H4¢); 13C
=
=
(75 MHz, CDCl3) d 172.8 (C O), 159.2 (C), 154.9 (C O), 140.6
(C), 140.5 (C), 136.6 (C), 130.4 (CH), 130.1 (C), 126.1 (C), 122.8
(CH), 120.4 (CH), 120.1 (CH), 118.4 (CH), 114.7 (CHx2), 113.1
(CH2), 111.2 (CH), 107.9 (C), 71.7 (CH2), 53.3 (CH), 51.9 (CH),
38.6 (CH2), 30.2 (CH2), 23.5 (CH2), 20.0 (CH2), 19.2 (CH3), 13.6
(CH3); LRMS (ES+) m/z: 466.13 (100%) [M + Na]+; HRMS (ES+)
[M + Na]+ m/z expected for C27H29N3O3Na 466.2107 obtained
466.2087; [a]2D0 = -194.4 (c = 0.00125, CHCl3).
=
=
NMR (100 MHz, CDCl3) d 172.8 (C O), 160.2 (C), 155.0 (C O),
140.7 (C), 136.6 (C), 130.4 (C), 130.2 (CH), 126.2 (C), 122.9
(CH), 120.3 (CH), 120.2 (CH), 118.4 (CH), 114.1 (CH), 114.0
(CH), 111.2 (CH), 108.1 (C), 69.5 (CH2), 53.4 (CH), 51.9 (CH),
38.6 (CH2), 30.2 (CH2), 23.5 (CH2), 22.5 (CH2), 20.0 (CH2), 13.6
(CH3), 10.5 (CH3); LRMS (ES+) m/z: 432.33 (100%) [M + H]+.
[a]2D0 = -204.2 (c = 0.00034, DCM) HRMS (ES+) [M + H]+ m/z
expected for C26H30N3O3 432.2107 obtained 432.2110.
(5R,11aS)-2-Butyl-5-(3-prop-2-ynyloxyphenyl)-6H -1,2,3,
5,11,11a-hexahydro-imidazo[1,5-b]-b-carboline-1,3-dione (9viii).
Yield 72%, yellow solid. m.p. 92–94 ◦C; nmax (KBr) 3329, 2973,
1767, 1700, 1456, 1238 cm-1; 1H NMR (400 MHz, CDCl3) d 7.80
(1H, s, NH), 7.56 (1H, d, J 7.5, H10), 7.32–7.16 (4H, m, H8, 9, 5¢¢,
7), 7.01–6.94 (3H, m, H6¢¢, 4¢¢, 2¢¢), 6.29 (1H, d, J 1.4, H5), 4.65
(2H, d, J 2.4, H8¢¢), 4.32 (1H, dd, J 11.0, 5.5, H11a), 3.56–3.49
(3H, m, H1¢, 11syn), 2.88 (1H, ddd, J 15.3, 11.0, 1.8, H11anti), 2.46
(1H, t, J 2.4, H10¢¢), 1.65–1.58 (2H, m, H2¢), 1.40–1.29 (2H, m,
H3¢), 0.93 (3H, t, J 7.4, H4¢); 13C NMR (75 MHz, CDCl3) d 172.8
(5R,11aS)-2-Butyl-5-(3-allyloxyphenyl)-6H-1,2,3,5,11,11a-
hexahydro-imidazo[1,5-b]-b-carboline-1,3-dione (9v). Yield 74%,
yellow solid. m.p. 85.4–86.0 ◦C; nmax (KBr) 2963, 1710, 1499,
1
1235 cm-1; H NMR (400 MHz, CDCl3) d 7.49 (1H, d, J 7.5,
H10), 7.25–7.07 (4H, m, H8, 9, 5¢¢, 7), 6.87–6.78 (3H, m, H4¢¢,
2¢¢, 6¢¢), 6.19 (1H, s, H5), 5.99–5.87 (1H, m, H9¢¢), 5.30 (1H, d,
J 17.3, H10¢¢trans), 5.12 (1H, d, J 10.5, H10¢¢cis), 4.45–4.39 (2H,
m, H8¢¢), 4.25 (1H, dd, J 11.0, 5.5, H11a), 3.51–3.39 (3H, m,
H1¢, 11syn), 2.81 (1H, dd, J 15.3, 11.0, H11anti), 1.59–1.51 (2H, m,
H2¢), 1.30–1.22 (2H, m, H3¢), 0.86 (3H, t, J 7.4, H4¢); 13C NMR
=
=
(C O), 158.0 (C), 154.9 (C O), 140.7 (C), 136.6 (C), 130.2 (CH),
130.2 (C), 126.1 (C), 122.9 (CH), 121.3 (CH), 120.1 (CH), 118.4
(CH), 114.9 (CH), 114.8 (CH), 111.2 (CH), 108.1 (C), 75.8 (C),
75.6 (CH), 55.8 (CH2), 53.3 (CH), 51.8 (CH), 38.6 (CH2), 30.2
(CH2), 23.5 (CH2), 20.0 (CH2), 13.6 (CH3); LRMS (ES+) m/z:
450.11 (100%) [M + Na]+; HRMS (ES+) [M + Na]+ m/z expected
for C26H25N3O3Na 450.1794 obtained 454.1806; [a]2D0 = -178.3
(c = 0.0012, CHCl3).
=
=
(100 MHz, CDCl3), d 172.8 (C O), 160.2 (C), 155.0 (C O), 140.7
(C), 136.6 (C), 130.2 (CH), 130.4 (C), 130.2 (CH), 126.2 (C), 122.9
(CH), 120.3 (CH), 120.2 (CH), 118.4 (CH), 118.0 (CH2), 114.1
(CH), 114.0 (CH), 111.2 (CH), 108.1 (C), 68.9 (CH2), 53.4 (CH),
51.9 (CH), 38.6 (CH2), 30.2 (CH2), 23.5 (CH2), 20.0 (CH2), 13.6
(CH3); LRMS (ES+) m/z: 430.33 (100%) [M + H]+. [a]2D0 = -206.5
(c = 0.00441, DCM) HRMS (ES+) [M + H]+ m/z expected for
C26H28N3O3 430.1577 obtained 430.1585.
(5R,11aS)-2-Butyl-5-(3-benzyloxyphenyl)-6H -1,2,3,5,11,
11a-hexahydro-imidazo[1,5-b]-b-carboline-1,3-dione (9ix). Yield
84%, yellow solid. m.p. 101–102 ◦C; nmax (KBr) 3328, 2932, 1767,
1701, 1456, 1238 cm-1; 1H NMR (300 MHz, CDCl3) d 8.01 (1H, s,
NH), 7.56 (1H, d, J 7.5, H10), 7.39–7.16 (9H, m, H8, 9, 5¢¢, 7, 10¢¢,
11¢¢, 12¢¢), 6.96–6.60 (3H, m, H6¢¢, 4¢¢, 2¢¢), 6.26 (1H, d, J 1.4, H5),
5.01 (2H, d, J 1.9, H8¢¢), 4.25 (1H, dd, J 11.0, 5.5, H11a), 3.56–
3.45 (3H, m, H1¢, 11syn), 2.87 (1H, ddd, J 15.3, 11.0, 1.8, H11anti),
1.66–1.56 (2H, m, H2¢), 1.37–1.30 (2H, m, H3¢), 0.93 (3H, t, J
(5R,11aS)-2-Butyl-5-(3-isopropoxyphenyl)-6H-1,2,3,5,11,11a-
hexahydro-imidazo[1,5-b]-b-carboline-1,3-dione (9vi). Yield 61%,
yellow solid. m.p. 105–106 ◦C; nmax (KBr) 3320, 2957, 1766, 1701,
1454, 1236 cm-1; 1H NMR (400 MHz, CDCl3) d 7.79 (1H, s, NH),
7.56 (1H, d, J 7.7, H10), 7.31–7.15 (4H, m, H8, 9, 5¢¢, 7), 6.89–6.84
(3H, m, H6¢¢, 4¢¢, 2¢¢), 6.27 (1H, d, J 1.3, H5), 4.52 (1H, sept, J 6.1,
H8¢), 4.33 (1H, dd, J 11.1, 5.6, H11a), 3.57–3.48 (3H, m, H1¢, 11syn),
2.88 (1H, ddd, J 15.3, 11.1, 1.8, H11anti), 1.65–1.57 (2H, m, H2¢),
1.39–1.29 (8H, m, H9, H3¢), 0.92 (3H, t, J 7.4, H4¢); 13C NMR
7.4, H4¢); 13C NMR (75 MHz, CDCl3) d 172.8 (C O), 159.2 (C),
154.9 (C O), 140.6 (C), 136.6 (C), 136.5 (C), 130.3 (C), 130.2
=
=
(CH), 128.6 (CH x2), 128.1 (CH), 127.5 (CH x2), 126.1 (C), 122.8
(CH), 120.6 (CH), 120.1 (CH), 118.4 (CH), 115.0 (CH), 114.7
(CH), 111.2 (CH), 108.0 (C), 70.0 (CH2), 53.2 (CH), 51.9 (CH),
38.6 (CH2), 30.2 (CH2), 23.5 (CH2), 20.0 (CH2), 13.6 (CH3); LRMS
(ES+) m/z: 502.09 (100%) [M + Na]+; HRMS (ES+) [M + Na]+ m/z
=
=
(75 MHz, CDCl3) d 172.9 (C O), 158.5 (C), 155.0 (C O), 140.7
(C), 136.6 (C), 130.5 (C), 130.2 (CH), 126.2 (C), 122.9 (CH), 120.2
(CH), 120.1 (CH), 118.5 (CH), 115.9 (CH), 115.5 (CH), 111.2
(CH), 108.1 (C), 69.9 (CH), 53.3 (CH), 52.0 (CH), 38.6 (CH2),
3056 | Org. Biomol. Chem., 2009, 7, 3049–3060
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expected for C30H29N3O3Na 502.2107 obtained 502.2102; [a]D20
-181.7 (c = 0.0012, CHCl3).
=
H3¢), 7.53–7.45 (3H, m, H7, 2¢), 7.20–7.03 (4H, m, H8, 9, 5¢¢, 7),
6.87–6.55 (3H, m, H4¢¢, 2¢¢, 6¢¢), 6.15 (1H, s, H5), 3.63–3.37 (25H,
m, Hlinker, 11syn), 2.97–2.83 (1H, m, H11anti); MS (ES+) m/z: 716
[M + H]+).
(5R,11aS)-2-(4-Benzoic acid)-5-(3-hydroxyphenyl)-6H-1,2,3,
5,11,11a-hexahydro-imidazo[1,5-b]-b-carboline-1,3-dione (19).
21 (16.4 mg, 0.032 mmol), TPTU (9.31 mg, 0.032 mmol)
and DIPEA (209.2 mL, 1.28 mmol) in anhydrous DMF (1 mL)
was stirred for 18 hrs. A solution of the above amine (23 mg,
0.032 mmol) in anhydrous DMF (1 mL) was added and stirred for
3 days. The solvent was reduced in vacuo and the residue purified by
chromatography on silica gel (0–10% MeOH in DCM) to afford
22 as a bright yellow solid (25.11 mg, 0.021 mmol, 65%). m.p.
74.1–75.5 ◦C; nmax (KBr) 3449, 1715, 1509, 1364, 1224, 1093 cm-1;
1H NMR (400 MHz, DMSO-d6) d 10.97 (1H, s, NH), 9.50 (1H, s,
OH), 8.56 (1H, s, Hmtx), 8.24 (1H, d, J 7.7, Hmtx), 7.85 (2H, d, J
8.4, H3¢), 7.71 (2H, d, J 8.4, H2¢), 7.58–7.51 (3H, m, H7, Hmtx),
7.31 (1H, d, J 7.7, H10), 7.17 (1H, dd, J 7.5, 8.0, H5¢¢), 7.10
(1H, dd, J 7.7, 7.0, H9), 7.03 (1H, dd, J 7.7, 7.0, H8), 6.84–6.72
(5H, m, H6¢¢, H2¢¢, H4¢¢, Hmtx), 6.20 (1H, s, H5), 4.77 (2H, s,
Hmtx), 4.70 (1H, dd, J 10.5, 5.6, H11a), 4.23–4.16 (1H, m, Hmtx),
3.66–3.06 (26H, m, Hlinker, 11anti, 11syn), 3.20 (3H, s, Hmtx), 2.24–
To a solution of 7m (0.200 g, 0.42 mmol) in THF (10 mL)
was added LiOH (0.101 g, 0.42 mmol) in H2O (5 mL), and
left to stir overnight. The solvent was reduced, and the residue
was partitioned between EtOAc (10 mL) and H2O (10 mL).
The aqueous layer was separated, adjusted to pH 4 with 2M
HCl, and extracted with EtOAc (3 ¥ 10 mL). These combined
extracts were washed with brine, dried (Na2SO4) and reduced
in vacuo. Purification of the residue by chromatography on silica
gel (DCM:MeOH 98:2) afforded 19 as a yellow solid (0.132 g,
0.29 mmol, 66%). m.p. 155.7–158.3 ◦C; nmax (KBr) 3352, 1726,
1245, 1123 cm-1; 1H NMR (400 MHz, DMSO-d6) d 10.97 (1H, s,
NH), 9.49 (1H, s, OH), 8.03 (2H, d, J 8.7, H3¢), 7.62–7.55 (3H,
m, H7, 2¢), 7.31 (1H, d, J 8.0, H10), 7.20–7.15 (1H, m, H5¢¢),
7.13–7.08 (1H, m, H9), 7.06–7.00 (1H, m, H8), 6.82 (1H, d, J 7.8,
H6¢¢), 6.77 (1H, s, H2¢¢), 6.74 (1H, d, J 8.0, H4¢¢), 6.22 (1H, s,
H5), 4.60 (1H, dd, J 10.3, 5.8, H11a), 4.05 (1H, s, OH), 3.41 (1H,
dd, J 15.1, 5.8, H11syn), 3.12–3.05 (1H, m, H11anti); 13C NMR
2.11 (2H, m, Hmtx), 2.04–1.82 (2H, m, Hmtx), 1.37 (9H, s, Hmtx);
13
=
=
C NMR (100 MHz, DMSO-d6) d 172.1 (C O), 171.6 (C O),
=
=
(100 MHz, DMSO-d4) d 171.8 (C O), 167.2 (C O), 158.1 (C),
153.3 (C O), 141.5 (C), 136.8 (C), 136.5 (C), 136.3 (C), 131.4 (C),
=
=
=
171.5 (C O), 171.3 (C O), 166.2 (C O), 165.5 (C), 164.1 (C),
162.9 (C), 162.6 (C), 157.6 (C), 155.2 (C), 152.8 (C O), 150.8 (C),
=
=
130.2 (CH), 130.2 (CH), 126.4 (CH), 122.2 (CH), 119.3 (CH),
119.1 (CH), 118.7 (CH), 115.7 (CH), 115.4 (CH), 111.9 (CH),
106.6 (C), 53.3 (CH2), 52.2 (CH2), 23.0 (CH2); LRMS (ES+)
m/z: 454.24 [M + H]+; HRMS (ES+) [M + H]+ m/z expected for
C26H20N3O5 454.1403 found 454.1437 [a]2D0 = -114.5 (c = 0.00031,
MeOH).
149.0 (CH), 141.05 (C), 136.6 (C), 134.2 (C), 133.5 (C), 130.9 (C),
129.7 (CH), 128.8 (CH x2), 127.5 (CH x2), 126.2 (CH x2), 125.7
(C), 121.7 (CH), 121.1 (C), 118.8 (CH), 118.6 (CH), 118.2 (CH),
115.2 (CH), 114.9 (CH), 111.3 (CH), 110.9 (CH x2), 106.0 (C),
80.2 (C), 69.7–68.8 (CH2), 54.8 (CH2), 53.4 (CH), 52.9 (CH), 51.6
(CH), 39.5 (CH3), 31.7 (CH2), 27.6 (CH3), 26.4 (CH2), 22.5 (CH2);
LRMS (ES+) m/z: 1230.76 (100%) [M + Na]+, 1208.87 (80%)
[M + H]+.
Synthesis of 20. TPTU (32.8 mg, 0.11 mmol) was added to a
solution of 19 (50 mg, 0.11 mmol) and Et3N (15.4 uL, 0.11 mmol)
in anhydrous DMF (2 mL) and stirred for 18 hrs. A solution
of 16 (41.91 mg, 0.11 mmol) in anhydrous DMF (2 mL) was
added and stirred for 3 days. The solvent was reduced and the
residue was partitioned between EtOAc (10 mL) and H2O (10 mL).
The organic layer was washed with sat. NaHCO3 (2 ¥ 5 mL),
brine (5 mL), dried (Na2SO4) and reduced in vacuo. Purification of
the residue by chromatography on silica gel (DCM:MeOH 97:3)
afforded 20 as a yellow solid (40.3 mg, 0.049 mmol, 45%). m.p.
Synthesis of CID 15. 22 (150 mg, 0.12 mmol) was dissolved in
DCM (3 mL) and TFA (500 mL, 1:6 TFA:DCM) was added. The
solution was stirred at room temperature for 5 hrs. Toluene (5 mL)
was added and the solvent was removed in vacuo. Purification
of the residue by chromatography on silica gel (0–30% MeOH
in DCM) afforded 15 as a yellow solid (75 mg, 0.065 mmol,
55%). m.p. 76.5–77.0 ◦C; nmax (KBr) 3433, 2925, 1638, 1421, 1204,
1099 cm-1; 1H NMR (400 MHz, (CD3)2SO) d 10.97 (1H, s, NH),
9.50 (1H, s, OH), 8.56 (1H, s, H23¢), 8.24 (1H, d, J 7.4, NH), 7.96
(2H, d, J 8.8, H3¢,), 7.74 (2H, d, J 8.8, H2¢), 7.60–7.53 (3H, m,
H7, Hmtx), 7.31 (1H, d, J 8.1, H10), 7.17 (1H, dd, J 7.7, H5¢¢),
7.13 (1H, dd, J 8.1, 7.7, H9), 7.03 (1H, dd, J 7.6, 7.4, H8), 6.84–
6.72 (5H, m, H6¢¢, H2¢¢, H4¢¢, Hmtx), 6.23 (1H, s, H5), 4.79 (2H, s,
Hmtx), 4.72 (1H, dd, J 10.5, 5.6, H11a), 4.33–4.26 (1H, m, Hmtx),
3.66–3.26 (26H, m, Hlinker, 11syn), 3.22 (3H, s, Hmtx), 3.09 (1H, m,
H11anti), 2.24–2.18 (2H, m, Hmtx), 2.09–1.87 (2H, m, Hmtx); 13C
64.1–64.3 ◦C; nmax (KBr) 3334, 2872, 1720, 1507, 1247 cm-1; H
1
NMR (400 MHz, CDCl3) d 7.86 (2H, d, J 8.7, H3¢), 7.51–7.45 (3H,
m, H7, 2¢), 7.26–7.08 (4H, m, H8, 9, 5¢¢, 7), 6.91–6.80 (3H, m, H4¢¢,
2¢¢, 6¢¢), 6.30 (1H, s, H5), 3.63–3.37 (25H, m, Hlinker, 11syn), 3.05–2.93
(1H, m, H11anti), 1.35 (9H, s, H9¢); 13C NMR (100 MHz, CDCl3)
=
=
=
d 171.6 (C O), 167.0 (C O), 159.6 (C), 153.7 (C O), 140.7 (C),
137.1 (C), 137.0 (C), 136.7 (C), 134.7 (CH), 130.7 (CH), 130.5 (C),
128.4 (CH x2), 126.5 (C), 125.9 (CH x2), 123.4 (CH), 121.1 (CH),
120.6 (CH), 118.9 (CH), 115.4 (CH), 115.3 (CH), 111.8 (CH),
108.2 (C), 70.3–70.9 (CH2 x10), 70.5(C), 53.6 (CH) 52.6 (CH) 40.3
(CH2) 28.8 (CH3), 24.1 (CH2); LRMS (ES+) m/z: 815.20 [M + H]+;
HRMS (ES+) [M + H]+ m/z expected for C43H54N5O11 816.3820
found 816.1753 [a]2D0 = -104.3 (c = 0.00015, DCM).
=
=
NMR (100 MHz, (CD3)2SO) d 173.8 (C O), 171.8 (C O), 171.6
(C O),171.4 (C O), 166.2 (C O), 165.5 (C), 164.1 (C), 162.9
=
=
=
=
(C), 162.8 (C), 157.7 (C), 155.2 (C), 153.0 (C O), 151.0 (C), 149.2
(C), 141.2 (C), 136.8 (C), 134.4 (C), 133.7 (C), 131.1 (C), 129.8
(CH), 128.9 (CHx2), 127.7 (CHx2), 126.3 (CHx2), 125.8 (C), 121.7
(CH), 121.2 (C), 118.9 (CH), 118.6 (CH), 118.3 (CH), 115.2 (CH),
115.0 (CH), 111.5 (CH), 111.1 (CHx2), 106.1 (C), 68.8–69.7 (CH2),
54.9 (CH2), 52.9 (CH), 52.3 (CH), 51.7 (CH), 38.5 (CH3), 32.0
(CH2), 26.6 (CH2), 22.6 (CH2); LRMS (ES-) m/z: 1150.57 (100%)
[M-H]-.
Synthesis of 22. 20 (26 mg, 0.032 mmol) was dissolved in DCM
(3 mL) and TFA (50 mL, 0.7 mmol) was added. The solution
was stirred for 5 minutes then the solvent was removed in vacuo.
The crude reaction product 21 was used in the next step without
purification. (1H NMR (400 MHz, CDCl3) d 7.86 (2H, d, J 8.7,
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Synthesis of 24. Diethyl azidocarboxylate (154.3 mL,
0.98 mmol) was added dropwise to a solution of triphenylphos-
phine (257 mg, 0.98 mmol) in anhydrous THF (12 mL) and stirred
for 30 minutes. A solution of 7a (303.1 mg, 0.78 mmol) and 18
(373.4 mg, 0.98 mmol) in anhydrous THF (15 mL) was added,
and the mixture stirred for 48 hrs. The solvent was reduced,
and the residue partitioned between EtOAc (30 mL) and H2O
(30 mL). The aqueous layer was extracted with EtOAc (2 ¥
10 mL) and the combined organics were dried over Na2SO4 and
reduced. Purification of the residue by chromatography on silica
gel (Hex:EtOAc 1:1 to 1:9 as the eluent) gave 24 as a yellow oil
(366.2 mg, 0.49 mmol, 62.4%). m.p. 153–154 ◦C; nmax (KBr) 3332,
2936, 1760, 1695, 1456, 1231 cm-1; 1H NMR (300 MHz, CDCl3)
d 8.38 (1H, s, NH), 7.56 (1H, d, J 7.0, H10), 7.32–7.13 (4H, m,
H8, 9, 5¢¢, 7), 6.95 (1H, d, J 7.7, H6¢¢), 6.88–6.85 (2H, m, H4¢¢,
2¢¢), 6.27 (1H, d, J 0.8, H5), 5.08–4.98 (1H, br s, NH), 4.23 (1H,
dd, J 11.0, 5.5, H11a), 4.08–4.06 (2H, m, Hlinker), 3.82–3.78 (2H,
m, Hlinker), 3.69–3.46 (21H, m, Hlinker, H11syn, H4¢), 3.25–3.20 (2H,
m, Hlinker), 2.88 (1H, ddd, J 15.3, 11.0, 1.8, H11anti), 1.66–1.56 (2H,
m, H2¢), 1.43 (9H, s, Hlinker), 1.37–1.29 (2H, m, H3¢), 0.92 (3H, t,
m, Hlinker), 3.69–3.39 (21H, m, Hlinker, H11syn, Hmtx), 3.30–3.22 (2H,
m, Hlinker), 3.15 (3H, s, Hmtx), 2.87 (1H, dd, J 14.0, 11.0, H11anti),
2.37–2.32 (2H, m, Hmtx), 2.22–2.12 (2H, m, Hmtx), 1.62–1.56 (2H,
m, H2¢), 1.45 (9H, s, Hmtx), 1.34–1.24 (2H, m, H3¢), 0.91 (3H,
13
=
t, J 7.4, H4¢); C NMR (100 MHz, CDCl3) d 173.0 (C O x2),
=
=
=
172.9 (C O), 171.5 (C O), 162.7 (C), 158.9 (C), 154.8 (C O),
153.1 (C), 152.9 (C), 151.3 (C), 149.5 (CH), 149.3 (C), 141.0 (C),
136.8 (C), 130.7 (C), 129.7 (CH), 128.8 (CH x2), 126.0 (C), 122.3
(CH), 121.8 (C), 121.7 (C), 120.3 (CH), 119.5 (CH), 118.1 (CH),
114.6 (CH), 113.9 (CH), 111.5 (CH x2), 111.4 (CH), 107.0 (C),
81.9 (C), 70.5 (CH2), 70.5 (CH2), 70.4 (CH2), 70.3 (CH2), 70.0
(CH2), 69.6 (CH2), 69.5 (CH2), 67.2 (CH2), 55.7 (CH2), 53.2 (CH),
51.8 (CHx2), 39.3 (CH2), 39.3 (CH3), 38.5 (CH2), 32.4 (CH2),
30.1 (CH2), 28.0 (CH3), 27.8 (CH2), 23.5 (CH2), 19.9 (CH2), 13.6
(CH3); LRMS (ES+) m/z: C59H76N12O12Na [M + Na]+ 1168.21
(100%), [M + H]+ 1146.27 (7%), [M + Na]2+ 584.58 (40%).
Synthesis of CID 23. 25 (179.9 mg, 0.16 mmol) was dissolved
in DCM (2 mL) and TFA (0.6 mL, 7.8 mmol) was slowly added.
The solution was stirred at RT for 5 hours. Toluene (5 mL) was
added and the solvent was removed in vacuo. Purification of the
residue by chromatography on silica gel (DCM:MeOH 99:1 to 4:1
as the eluent) gave 23 as an orange solid (115.8 mg, 0.11 mmol,
66.4%). m.p. 132–134 ◦C; nmax (KBr) 3410, 2942, 1761, 1706, 1456,
J 7.4, H4¢); 13C-NMR (75 MHz, CDCl3) d 172.8 (C O x2), 159.2
=
=
(C), 154.8 (C O), 140.8 (C), 136.7 (C), 130.5 (C), 129.9 (CH),
126.4 (C), 122.5 (CH), 120.5 (CH), 119.7 (CH), 118.2 (CH), 114.7
(CH), 114.2 (CH), 111.3 (CH), 107.4 (C), 77.1 (C), 70.7 (CH2),
70.6 (CH2), 70.6 (CH2), 70.5 (CH2), 70.4 (CH2), 70.1 (CH2), 69.7
(CH2), 69.6 (CH2), 67.3 (CH2), 53.2 (CH), 51.8 (CH), 40.2 (CH2),
38.5 (CH2), 30.1 (CH2), 28.3 (CH3), 23.4 (CH2), 19.9 (CH2), 13.5
(CH3); LRMS (ES+) m/z: 753.5 [M + H]+; HRMS (ES+) [M + H]+
m/z expected for C40H57N4O10 753.4075 obtained 753.4081.
1
1232 cm-1; H NMR (300 MHz, DMSO) d 10.88 (1H, s, NH),
8.57 (1H, s, Hmtx), 8.32 (1H, s, OH), 8.26 (1H, d, J 7.4, NH), 7.89
(1H, t, J 5.4, NH), 7.85–7.79 (1H, br s, NH), 7.73 (2H, d, J 8.9,
Hmtx), 7.66–7.58 (1H, br s, NH), 7.54 (1H, d, J 7.6, H10), 7.31–7.25
(2H, m, H7, 4¢¢), 7.11–7.06 (1H, m, H5¢¢), 7.04–6.99 (1H, m, H9),
6.97–6.87 (3H, m, H8, 6¢¢, 2¢¢), 6.82 (2H, d, J 8.9, Hmtx), 6.80–6.72
(2H, br s, NH), 6.17 (1H, s, H5), 4.79 (2H, s, CHmtx), 4.62 (1H,
dd, J 10.6, 5.4, H11a), 4.31–4.24 (1H, m, Hmtx), 4.12–3.98 (2H,
m, Hlinker), 3.70 (2H, pseudo t, J 4.5, Hlinker), 3.60–3.38 (21H, m,
Hlinker, H11syn), 3.21 (3H, s, Hmtx), 3.20–3.13 (2H, m, H1¢), 2.83–2.71
(1H, m, H11anti), 2.22–2.17 (2H, m, Hmtx), 2.05–1.86 (2H, m, Hmtx),
1.55–1.45 (2H, m, H2¢), 1.28–1.17 (2H, m, H3¢), 0.86 (3H, t, J 7.4,
Synthesis of 25. 24 (339.7 mg, 0.45 mmol) was dissolved in
DCM (8 mL) and TFA (2 mL, mmol) was added. The solution
was stirred for 5 minutes then the solvent was removed in vacuo.
The crude reaction product was used in the next step without
purification. (1H NMR (300 MHz, CDCl3) d 9.35–9.34 (2H, m,
NH), 8.44 (1H, s. NH), 7.55 (1H, d, J 7.2, H10), 7.32–7.14 (4H,
m, H8, 9, 5¢¢, 7), 6.93–6.84 (3H, m, H6¢¢, 4¢¢, 2¢¢), 6.21 (1H, d, J 0.6,
H5), 4.42 (1H, dd, J 11.0, 5.5, H11a), 4.14–4.12 (2H, m, Hlinker),
3.86–3.83 (2H, m, Hlinker) 3.75–3.72 (2H, m, Hlinker), 3.68–3.49 (19H,
m, Hlinker, H11syn, H1¢), 3.18–3.13 (2H, m, Hlinker), 2.92 (1H, ddd, J
15.3, 11.1, 1.7, H11anti), 1.65–1.55 (2H, m, H2¢), 1.40–1.28 (2H, m,
H3¢), 0.92 (3H, t, J 7.4, H4¢)).
The residue was dissolved in DCM (8 mL) and DIPEA
(374.5 mL, 2.15 mmol) was added. To this was added a solution
of 21 (219.5 mg, 0.43 mmol), EDC (92.0 mg, 0.48 mmol) and
HOBt (64.9 mg, 0.48 mmol) in anhydrous DMF (8 mL). After
stirring for 42 hours, the solvent was removed and the residue
partitioned between DCM (20 mL) and H2O (20 mL). The organic
layer was washed with sat NaHCO3 (20 mL), sat NH4Cl (20 mL),
dried (Na2SO4) and reduced in vacuo. Purification of the residue
by chromatography on silica gel (DCM:MeOH 99:1 to 9:1 as
the eluent) gave 25 as an orange solid (309.0 mg, 0.27 mmol,
62.7%). m.p. 146–147 ◦C; nmax (KBr) 3338, 2928, 1765, 1708, 1452,
1244 cm-1; 1H NMR (300 MHz, CDCl3) d 9.76 (1H, s, NH), 8.57
(1H, s, NH), 7.79 (2H, d, J 8.7, Hmtx), 7.52 (1H, d, J 7.9, H10), 7.37
(1H, d, J 7.9, H7), 7.18–7.04 (4H, m, H8, 9, 5¢¢, 6¢¢), 6.85 (1H, s,
Hmtx), 6.76–6.71 (2H, m, H2¢¢, H4¢¢), 6.69 (2H, d, J 8.7, Hmtx), 6.30
(1H, s, H5), 4.70 (2H, s, Hmtx), 4.51–4.47 (1H, m, Hmtx), 4.29 (1H,
dd, J 11.0, 5.6, H11a), 4.07–3.98 (2H, m, Hlinker), 3.77–3.75 (2H,
H4¢); 13C NMR (125 MHz, DMSO) d 173.7 (C O), 172.6 (C O
=
=
=
=
x2), 171.6 (C O), 166.1 (C), 162.6 (C), 162.4 (C O), 158.6 (C),
154.3 (C), 150.8 (C), 149.0 (CH), 146.1 (C), 141.5 (C), 136.6 (C),
131.1 (C), 129.8 (CH), 128.8 (CH x2), 125.6 (C), 121.6 (CH), 121.3
(C), 121.0 (C), 119.8 (CH), 118.7 (CH), 118.1 (CH), 114.0 (CH),
113.6 (CH), 111.3 (CH), 110.9 (CH x2), 105.8 (C), 69.8 (CH2),
69.6 (CH2), 69.6 (CH2), 69.4 (CH2), 68.9 (CH2), 68.8 (CH2), 67.0
(CH2), 54.7 (CH2), 52.9 (CH), 52.2 (CH), 51.4 (CH), 38.4 (CH2),
37.6 (CH2), 31.8 (CH2), 31.5 (CH3), 29.5 (CH2), 26.4 (CH2), 22.6
(CH2), 19.3 (CH2), 13.4 (CH3); LRMS (ES-) m/z: C55H67N12O12
[M - H]- 1087.33.
Biology experimental
Parasite growth and invasion assay. T. gondii tachyzoites (RH
strain) were cultured at 37 ◦C in confluent monolayers of
human foreskin fibroblasts (HFFs) grown in Dulbecco’s Modified
Eagle’s medium (DMEM) (HyClone) containing 1% fetal calf
serum (Invitrogen). Freshly egressed parasites were prepared for
invasion assays by passage through a 26 gauge needle followed
by filtration through a 3.0 mm Nuclepore track etch membrane
filter (Whatman). Invasion assays were performed as previously
described6 except that samples were scored manually rather than
by automated fluorescence microscopy.
3058 | Org. Biomol. Chem., 2009, 7, 3049–3060
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Yeast growth and CID competition assays. Yeast strain
V1019Y26 was grown in synthetic complete (SC) medium (lacking
histidine and tryptophan, and supplemented with 2% galactose
and 2% raffinose) leucine at 30 ◦C with shaking. For CID
competition experiments, CIDs were diluted into the SC medium
prior to inoculation with yeast. 2.0 ¥ 105 yeast cells were inoculated
into a total volume of 150 ml of SC medium ( leucine, CID)
in the wells of a 96-well tissue culture dish (Corning). The 96-well
plate was sealed with parafilm and incubated at 30 ◦C with shaking
for 48 hours. OD600 measurements were taken with a plate reader
(Biotek Instruments). CID 26 was used at 10 mM and competing
CIDs were tested at a 10-fold molar excess (100 mM).
8 For a recent example see: S. Lain, J. J. Hollick, J. Campbell, O. D.
Staples, M. Higgins, M. Aoubala, A. R. McCarthy, V. Appleyard, K. E.
Murray, L. Baker, A. Thompson, J. Mathers, S. J. Holland, M. J. Stark,
G. Pass, J. Woods, D. P. Lane and N. J. Westwood, Cancer Cell, 2008,
13, 377–388.
9 (a) M. L. Lopez-Rodriguez, M. J. Morcillo, M. Garrido, B. Benhamu, V.
Perez and J. G. de la Campa, J.Org.Chem., 1994, 59(6), 1583–5; (b) The
thiocarbonyl-containing analogues 8a and 8b were not progressed
further due to concerns about the lability of this functional group with
respect to conversion to a carbonyl group in cells.
10 See supplementary material for further details.
11 Crystallographic data (excluding structure factors) for 7b has been
deposited with the Cambridge Crystallographic Data Centre as sup-
plementary publication no. CCDC 719462. Copies of the data can
be obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK, (fax: +44 (0)1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk).
Bioinformatics analyses. The Toxoplasma gondii genome
database (ToxoDB release 5.0) was searched by BLAST using the
4 signature motifs of the kinesin heavy chain motor domain26,27
or the catalytic domain of cGMP-specific 3¢, 5¢ cyclic nucleotide
phosphodiesterases26,28 to identify putative targets of 1. The
sequence of each of the identified candidates was further analyzed
by performing a BLAST search and a conserved domain search
against GenBank. Putative classification of T. gondii kinesins was
performed using known family placement of the closest homolog
identified and according to previous reports.29 The identified
cGMP-specific 3¢,5¢ cyclic nucleotide phosphodiesterases were
similarly classified into putative families according to previous
reports.30 Expression data (EST, SAGE, microarray) available in
ToxoDB were also analyzed for each candidate gene.
12 (a) N. Sunder-Plassmann, V. Sarli, M. Gartner, M. Utz, J. Seiler, S.
Huemmer, T. U. Mayer, T. Surrey and A. Giannis, Bioorg.Med.Chem.,
2005, 13(22), 6094–6111; (b) S. Hotha, J. C. Yarrow, J. G. Yang,
S. Garrett, K. V. Renduchintala, T. U. Mayer and T. M. Kapoor,
Angew.Chem. Int.Ed., 2003, 42(21), 2379–2382; (c) A. I. Marcus, U.
Peters, S. L. Thomas, S. Garrett, A. Zelnak, T. M. Kapoor and P.
Giannakakou, J.Biol.Chem., 2005, 280(12), 11569–11577.
13 A. Daugan, P. Grondin, C. Ruault, A.-C. Le Monnier de Gouville, H.
Coste, J. Kirilovsky, F. Hyafil and R. Labaudiniere, J.Med.Chem., 2003,
46(21), 4525–4532.
14 (a) The ToxoDB release 4.1; www.toxodb.org/toxo/home.jsp) was
used in these studies; (b) This analysis was based on ex-
pressed sequence tag [EST], serial analysis of gene expression
[SAGE], and microarray expression data available at (http://roos-
compbio2.bio.upenn.edu/toxo/cgi-bin/gbrowse/gbtoxo_amit/).
15 B. J. Foth, D. Soldati, in Toxoplasma: Molecular and Cellular Biology,
ed. J. W. Ajioka and D. Soldati, Horizon Scientific Press, Norfolk, UK,
1st edn., 2007, ch. 28.
16 S. Brier, D. Lemaire, S. DeBonis, F. Kozielski and E. Forest, Rapid
Commun. Mass Spectrom., 2005, 20(3), 456–462.
17 C. M. Tarby, R. F. Kaltenbach, T. Huynh, A. Pudzianowski, H. Shen,
M. Ortega-Nanos, S. Sheriff, J. A. Newitt, P. A. McDonnell, N. Burford,
C. R. Fairchild, W. Vaccaro, Z. Chen, R. M. Borzilleri, J. Naglich, L. J.
Lombardo, M. Gottardis, G. L. Trainor and D. L. Roussell, Bioorg.
Med. Chem. Letts., 2006, 16(8), 2095–2100.
Acknowledgements
Professor Virginia Cornish for generously providing yeast strain
V1019Y and the control CID, Dex-Mtx 26. We would also like to
thank Christopher Brown and Sarah Wilkinson. This work was
supported by PHS grant AI054961 (GW/NJW) and the Royal
Society (NJW URF Fellowship).
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