Synthesis and Biological Evaluation of CTP Synthetase Inhibitors as Potential Agents for the Treatment of African Trypanosomiasis

Article abstract of DOI:10.1002/cmdc.201200304

Acivicin analogues with an increased affinity for CTP synthetase (CTPS) were designed as potential new trypanocidal agents. The inhibitory activity against CTPS can be improved by increasing molecular complexity, by inserting groups able to establish additional interactions with the binding pocket of the enzyme. This strategy has been pursued with the synthesis of α-amino-substituted analogues of Acivicin and N1-substituted pyrazoline derivatives. In general, there is direct correlation between the enzymatic activity and the invitro anti-trypanosomal efficacy of the derivatives studied here. However, this cannot be taken as a general rule, as other important factors may play a role, notably the ability of uptake/diffusion of the molecules into the trypanosomes.

Full text of DOI:10.1002/cmdc.201200304

MED
DOI: 10.1002/cmdc.201200304
Synthesis and Biological Evaluation of CTP Synthetase
Inhibitors as Potential Agents for the Treatment of African
Trypanosomiasis
Lucia Tamborini,[a] Andrea Pinto,[a] Terry K. Smith,[b] Louise L. Major,[b] Maria C. Iannuzzi,[a]
Sandro Cosconati,[c] Luciana Marinelli,[d] Ettore Novellino,[d] Leonardo Lo Presti,[e]
Pui E. Wong,[f] Michael P. Barrett,[f] Carlo De Micheli,[a] and Paola Conti*[a]
Acivicin analogues with an increased affinity for CTP synthe-
tase (CTPS) were designed as potential new trypanocidal
agents. The inhibitory activity against CTPS can be improved
by increasing molecular complexity, by inserting groups able
to establish additional interactions with the binding pocket of
the enzyme. This strategy has been pursued with the synthesis
of a-amino-substituted analogues of Acivicin and N1-substitut-
ed pyrazoline derivatives. In general, there is direct correlation
between the enzymatic activity and the in vitro anti-trypanoso-
mal efficacy of the derivatives studied here. However, this
cannot be taken as a general rule, as other important factors
may play a role, notably the ability of uptake/diffusion of the
molecules into the trypanosomes.
Introduction
Human African trypanosomiasis (HAT) is a neglected tropical
disease endemic to sub-Saharan Africa. HAT is caused by an
unicellular protozoan belonging to the class of zooflagellates
and transmitted by the bite of tsetse flies to their mammalian
hosts. The disease is characterized by an initial stage in which
parasites proliferate in the hemolymphatic system, and
a second stage in which the parasites have reached the central
nervous system.[1,2] Whereas symptoms are relatively nonspecif-
ic in the first stage, the second stage is marked by progressive
neurological dysfunction, including the breakdown in sleep–
wake patterns that lend the disease its common name of
sleeping sickness. Two subspecies are responsible for causing
the sickness in humans: Trypanosoma brucei (T. b.) gambiense
and T. b. rhodesiense. While T. b. gambiense affects countries in
western and central Africa, causing a chronic form of the dis-
ease, T. b. rhodesiense is confined to eastern and southern
Africa and causes an acute illness within a few weeks of infec-
tion.
(CTP), was suggested to be a potential drug target for the
treatment of HAT.[5] CTPS is the rate-limiting enzyme in the syn-
thesis of cytosine nucleotides, which play an important role in
various metabolic processes and provide the precursors neces-
sary for the synthesis of RNA and DNA. CTPS is expressed both
in humans and parasites, however, T. brucei seems to be more
susceptible to CTPS inhibition due to low rate of the de novo
synthesis and to the lack of the salvage pathways for cytosine
or cytidine.[5]
Acivicin (Figure 1), an antibiotic isolated from the fermenta-
tion broths of Streptomyces sviceus, acts as a covalent inhibitor
of several GATs, including CTPS.[6] Interestingly, its CTPS inhibi-
[a] Dr. L. Tamborini, Dr. A. Pinto, Dr. M. C. Iannuzzi, Prof. C. De Micheli,
Prof. P. Conti
Dipartimento di Scienze Farmaceutiche
Universitꢀ degli Studi di Milano, Via Mangiagalli 25, 20133 Milano (Italy)
E-mail: paola.conti@unimi.it
Chemotherapy is the main way to control this disease, as
there are no effective vaccines, and current treatment depends
on the causative subspecies and the stage of the disease.[3]
The main drawbacks of currently available treatment are poor
efficacy, poor pharmacokinetic properties, cost, and increasing
drug resistance.[1–3] Recent efforts have focused on finding op-
timal therapeutic regimens and on development of combina-
tion therapy with drugs already registered or those used to
treat related diseases. To overcome the difficulties encountered
in the control of HAT, the development of new therapeutic
tools is urgent and efforts have been made to identify new
molecular targets.[4]
[b] Dr. T. K. Smith, Dr. L. L. Major
Biomedical Science Research Complex, St. Andrews, KY16 9ST Scotland (UK)
[c] Dr. S. Cosconati
Dipartimento di Scienze Ambientali
Seconda Universitꢀ di Napoli, 81100 Caserta (Italy)
[d] Dr. L. Marinelli, Prof. E. Novellino
Dipartimento di Chimica Farmaceutica e Tossicologica
Universitꢀ di Napoli “Federico II”, 80131 Napoli (Italy)
[e] Dr. L. Lo Presti
Dipartimento di Chimica, Universitꢀ degli Studi di Milano
Via Golgi 19, 20133 Milano (Italy)
[f] Dr. P. E. Wong, Prof. M. P. Barrett
Wellcome Trust Centre of Molecular Parasitology
Institute of Infection, Immunity and Inflammation
College of Medical, Veterinary and Life Sciences
University of Glasgow, Glasgow, G12 8TA Scotland (UK)
CTP synthetase (CTPS), a glutamine amidotransferase (GAT)
responsible for the de novo synthesis of cytidine triphosphate
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Figure 1. Structure of model and target compounds.
tory activity has been correlated to the observed trypanocidal
activity in bloodstream T. brucei cell cultures.[5]
Figure 2. Binding mode of Acivicin into the CTPS catalytic site represented
as transparent white ribbons. The ligand and the interacting residues are
shown in grey and white sticks, respectively. Hydrogen bonds are represent-
ed with dashed black lines. All hydrogen atoms were removed for clarity.
The target of this project is the study of the structure–activi-
ty relationship of Acivicin and the design and synthesis of new
analogues characterized by an increased affinity for CTPS as
potential new trypanocidal agents.
Acivicin binds to the glutaminase domain of CTPS mimicking
the natural substrate l-Gln. The enzyme is irreversibly inactivat-
ed due to the formation of a covalent adduct produced by nu-
cleophilic attack of the thiol group of a Cys residue to the C3
of the isoxazoline nucleus, with displacement of the chlorine
atom.[7] We previously reported that substituting the 3-chloro
group with bromo to give 3-Br-Acivicin led to a threefold in-
crease of the inhibitory potency against the target enzyme
CTPS. Interestingly this translated into a twelvefold increase in
the in vitro anti-trypanosomal activity, while leaving unaffected
the toxicity against mammalian cells.[8] The observed increased
activity against the enzyme is in accordance with the proposed
mechanism of action.[7]
amino group of Br-Acivicin, which as we said does not seem to
be involved in an ionic interaction, was exploited to design
carbamates 5 and 6. These derivatives, in addition to hydrogen
bonding, may establish further hydrophobic or electronic inter-
actions with the enzyme, thus reinforcing the binding. More-
over, we identified in the glutamine binding site two amino
acid residue, that is, Phe393 and Glu443, that could be the
target of additional interactions. To this aim, the isoxazoline
nucleus of Acivicin was replaced by a pyrazoline ring, which
represents a more versatile scaffold thanks to the ease of func-
tionalization of the N1 position (compounds 7–9) and allows
decoration of the dihydroisoxazole fragment in a fragment-
based drug discovery perspective.
As an extension of our previous work intended at investigat-
ing the role of the C3 substituent of Acivicin, we have now
prepared and tested the 3-methoxy analogue 2, and com- Results and Discussion
pound 3, which, at variance with the other compounds, should
behave as a glutamine mimic without having a good leaving
Chemistry
group at the C3 position, thus possibly inhibiting the enzyme
in a noncovalent manner.
As for the synthesis of Acivicin and Br-Acivicin, previously re-
ported by us,[8,9] the key-step for the synthesis of the isoxazo-
line analogues was a 1,3-dipolar cycloaddition of bromonitrile
oxide, generated in situ by base-promoted dehydrohalogena-
tion of the stable precursor dibromoformaldoxime, to (S)-3-
Furthermore, we have prepared the des-amino analogue of
Br-Acivicin (Æ)-4 to test the importance of the a-amino group
on the biological activity, as our analysis of the crystal structure
of T. brucei CTPS glutaminase domain in complex with Acivicin
(PDB ID: 2W7T, Figure 2) shows that such a group is not direct-
ly involved in an ionic interaction with the binding pocket but
establishes a charge reinforced hydrogen bond with the
Gly392 backbone carbonyl oxygen atom. An additional aim of
this project was to design Acivicin analogues with an increased
affinity for T. brucei CTPS.
(tert-butoxycarbonyl)-2,2-dimethyl-4-vinyloxazolidine
10
(Scheme 1), producing the mixture of diastereomers 11 a and
11 b.[10] Compound (aS,5S)-5 was obtained as previously de-
scribed,[8] being a synthetic intermediate of Br-Acivicin 1, while
derivative (aS,5S)-6 was simply prepared by treating com-
pound (aS,5S)-1 with benzylchloroformate in a mixture of tet-
rahydrofuran and water in the presence of NaHCO3 (Scheme 1).
To obtain derivative 2, we treated the mixture of cycload-
ducts 11 a and 11 b with a MeOH suspension of K2CO3 at 508C.
The acetonide function was removed by treating with a mixture
of acetic acid and water (5:1 v/v) at 408C and the diastereo-
A typical medicinal chemistry approach to enhance the affin-
ity for a target enzyme is to increase the molecular complexity,
by inserting groups able to establish additional interaction
with the binding pocket of the enzyme. In this line, the a-
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followed by ion-exchange chromatography using Amberlite IR
120H afforded derivative (aS,5S)-3 as a zwitterion.
Derivative (Æ)-4 was prepared by 1,3-dipolar cycloaddition
of bromonitrile oxide, generated in situ by base-promoted de-
hydrohalogenation of the stable precursor dibromoformaldox-
ime, to commercially available 3-buten-1-ol. The reaction was
carried out under microwave irradiation, heating for 1 h at
808C. The primary alcohol was oxidized to the corresponding
carboxylic acid (Æ)-4 using ruthenium(IV) oxide and sodium
periodate in a biphasic system, as described above (Scheme 1).
For the synthesis of derivatives 7–9, the pyrazoline nucleus
was generated in a one-pot procedure, through the condensa-
tion of hydrazine to the a,b-unsaturated ester (Æ)-15, followed
by intramolecular cyclization. The a-carboxylate group of
alkene (Æ)-15 had to be appropriately protected to avoid its
participation into the cyclization reaction. In detail, the protec-
tive group chosen for the carboxylic acid was an ortho-ester,
that is, the trioxabicyclo[2.2.2]octane ester (OBO-ester).[12] This
is an attractive protecting group for the carboxylic acid be-
cause it is highly stable in basic reaction media and does not
react toward nucleophiles. Moreover, it can be easily removed
in mild acidic conditions to give the free acids or, through
a transesterification reaction, conveniently converted into the
corresponding methyl ester. On the other hand, the amino
group of (Æ)-15 was protected as a benzylcarbamate, which
guarantees a good stability in the subsequent steps of the
planned reaction sequence, both in basic and in mild acidic
conditions (Scheme 2).
The desired ester (Æ)-15 was obtained as described,[13] and
then reacted with hydrazine hydrate, in ethanol at reflux, to
afford key intermediate (Æ)-16a,b, which was obtained as
1
a 1:1 mixture (based on H NMR spectrum) of two racemic dia-
stereomers, inseparable by column chromatography at this
step. Selective alkylation of the more nucleophilic N1 nitrogen
was accomplished by treating (Æ)-16a,b with benzyl bromide
in the presence of potassium carbonate and a catalytic
amount of sodium iodide, under reflux, affording the mixture
of diastereomers (Æ)-18a,b.[14] At this stage, the OBO ester was
converted into the corresponding methyl ester through a two-
step procedure involving treatment with pyridinium para-tolu-
ene sulfonate (PPTS) in a solution of methanol and water fol-
lowed by transesterification with methanol and potassium car-
bonate.[15] The methyl esters (Æ)-21a and (Æ)-21b could finally
be separated by silica gel column chromatography.
Scheme 1. Synthesis of derivatives 2–6. Reagents and conditions: a) NaHCO3,
EtOAc; b) AcOH/H2O (5:1 v/v), 408C; c) NaIO4, RuO2·H2O, CCl4/H2O/CH3CN;
d) HBr/AcOH; e) Amberlite IR 120H, 1n NH4OH; f) CbzCl, NaHCO3, H2O/THF;
g) K2CO3, MeOH, 508C; h) 30% TFA, CH2Cl2; i) 2n (Me)2NH, THF, 758C; j) 4n
HCl, dioxane; k) NaHCO3, EtOAc, MW, 808C.
meric alcohols 13a and 13b were separated by flash chroma-
tography. The sole erythro cycloadduct (aR,5S)-13a was oxi-
dized to the corresponding carboxylic acid by treating with
ruthenium(IV) oxide in the presence of sodium periodate in
a mixture of water, acetonitrile and carbon tetrachloride.[11] Fi-
nally, the tert-butoxycarbonyl (Boc) protection was cleaved
with a 30% solution of trifluoroacetic acid in dichloromethane,
to give the final amino acid (aS,5S)-2, which was obtained in
its zwitterionic form after ion-exchange chromatography, using
Amberlite IR 120H and eluting the product with a 1n aqueous
solution of ammonia.
Due to low reactivity of the N1 in the nucleophilic substitu-
tion, for the synthesis of derivatives 19 and 20 a different ap-
proach had to be followed. Indeed the selective N1 functional-
ization was achieved through a reductive amination reaction,
using acetaldehyde/NaBH4 or Cbz-glycinal 17/NaBH4, respec-
tively.
As described before for the N1-benzyl derivatives, the next
stages involved the two-step transesterification of the OBO
ester to methyl ester followed by chromatographic separation
of the two diastereomers (Æ)-22a and (Æ)-22b, while in the
case of (Æ)-23a,b the separation was unsuccessful.
Derivative 3 was prepared from (aS,5S)-5 treating with 2n
dimethylamine in tetrahydrofuran in a sealed vial and heating
at 758C for 16 h. When the reaction was completed, the mix-
ture was cooled at 08C to allow the precipitation of dimethyla-
mine hydrobromide, which was easily removed by filtration. Fi-
nally, Boc-deprotection with 4n hydrochloric acid in dioxane
Because 1H NMR spectra did not allow the secure assign-
ment of the relative configuration to the couple of diastereo-
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with phosphoryl chloride in acetonitrile at reflux; the corre-
sponding 3-chloro derivatives were obtained in good yields.
The final amino acids (Æ)-7a, (Æ)-7b, (Æ)-8a and (Æ)-8b
were obtained after deprotection of the amine, by treatment
with BCl3 in dichloromethane, and hydrolysis of the methyl
ester. Because conventional hydrolysis with aqueous sodium
hydroxide led to the partial replacement of the chlorine with
the hydroxy group, this step was performed using a polymer-
supported base Ambersep 900-OH. The carboxylic acid formed
upon hydrolysis was bound by the resin and subsequently re-
leased by washing the resin with 0.05n HCl (Scheme 2).
In the case of derivatives (Æ)-23a,b, due to the presence of
two N-Cbz-protected functions, the synthetic protocol de-
scribed above had to be slightly modified. The two Cbz groups
could simultaneously be removed only by treatment with 33%
HBr in acetic acid. Unfortunately, such a treatment caused the
partial Cl/Br exchange. For such a reason, we removed these
two groups before the chlorination step and then conveniently
replaced them by two Fmoc groups, which could be subse-
quently cleaved in mild basic conditions.
Treatment of (Æ)-26a,b with POCl3 in acetonitrile at reflux,
followed by deprotection of the amino acid function, afforded
final derivative (Æ)-9a,b still as a mixture of diastereomers, as
separation was not possible at any stage of the synthetic pro-
cedure.
Biology
The first step of the biological evaluation concerned the as-
sessment of the inhibitory activity of all new synthesized com-
pounds in comparison with Acivicin and Br-Acivicin 1, against
recombinant CTPS from T. b. brucei. The results are listed in
Table 1.
Subsequently, the compounds were tested as trypanocidal
agents, estimating the cytotoxicity against the trypanosomes
and against human HeLa cells. Where possible, the selectivity
index (SI) was calculated. This parameter expresses the ratio
between EC50 against human cells and EC50 against trypano-
somes. Finally, all derivatives under study were converted into
their corresponding methyl esters and re-tested against blood-
stream form T. b. brucei (strain 427). These experiments were
carried out to evaluate whether the observed differences be-
tween enzymatic and cellular activity could be somehow relat-
ed to an impaired uptake of the derivatives into the target
cells. Due to the hydrophilic nature of Acivicin (calculated
logD=À2.84 at pH 7.4), it is reasonable to hypothesize that its
uptake into the cells takes place via amino acid transporters lo-
cated on the plasma membrane. On the other hand, increasing
the lipophilicity could facilitate the penetration through a pas-
sive diffusion mechanism.
Scheme 2. Synthesis of derivatives 7–9. Reagents and conditions:
a) N2H4·H2O, EtOH, reflux; b) BnBr, K2CO3, NaI, CH3CN, reflux; c) CH3CHO or
CbzNHCH2CHO (17), NaBH4, MeOH; d) i. PPTS, MeOH/H2O; ii. K2CO3, MeOH;
e) POCl3, CH3CN, reflux; f) BCl3, CH2Cl2; g) i. Ambersep 900-OH, H2O/dioxane,
ii. 0.05n HCl; h) 33% HBr, AcOH; i) Fmoc-N-hydroxysuccinimide ester,
Na2CO3, H2O/dioxane; j) piperidine, CH2Cl2.
mers (Æ)-22a and (Æ)-22b, we performed an X-ray crystallo-
graphic analysis on compound 22a. Derivative (Æ)-22a turned
out to possess the (aS*,5R*) configuration; consequently the
(aS*,5S*) configuration was assigned to diastereomer (Æ)-22b.
Subsequently, we assigned the relative stereochemistry to de-
rivatives (Æ)-21a and (Æ)-21b by comparing their 1H NMR
spectra, in particular by comparing the coupling constant of
the a-amino acidic proton.
Discussion
The remaining reaction sequence was accomplished on the
single diastereomers. The first step concerned the halogena-
tion reaction at C3 of the heterocycle, which was performed
by reacting derivatives (Æ)-21a, (Æ)-21b, (Æ)-22a and (Æ)-22b
As previously observed with Br-Acivicin 1, the nature of the
leaving group at the C3 of the isoxazoline ring plays an impor-
tant role in determining the inhibitory efficacy toward CTPS.[8]
In fact, while 1 was threefold more potent than Acivicin as
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not be the ideal prodrug, as intracellular enzymatic hydrolysis
may not occur at a satisfactory rate; thus more suitable pro-
drugs could be designed.
Table 1. Inhibitory activity of Acivicin and derivatives 1–9 (and corre-
sponding methyl esters) against recombinant T. b. brucei CTPS and their
cytotoxicity against T. b. brucei and HeLa cells.
To rationalize the structure–activity relationship data (SARs)
of the newly discovered compounds the X-ray structure of the
covalent Acivicin–CTPS adduct (PDB ID: 2W7T, Figure 2) was
used to run covalent docking calculations on compounds 5
and 6. As represented in Figure 2, the covalent Acivicin–CTPS
adduct is stabilized by several ligand–enzyme interactions and
by the presence of several water molecules. In particular, one
of them (WAT 1 in Figure 2) further reinforces the Coulombic
interaction established by the Acivicin carboxylate group and
R498 side chain by donating and accepting two hydrogen
bonds with the aforementioned groups. In addition, another
ordered water molecule mediates the ligand–enzyme interac-
tions by donating two hydrogen bonds to the oxygen of the
Acivicin isoxazoline ring and to G392 backbone CO. From this
analysis it is clear that covalent and noncovalent ligand–
enzyme interactions and water molecules all have important
roles in CTPS inhibition. Therefore, in our analysis we decided
to include these factors in docking calculations of compound 5
and 6. In particular covalent docking calculations were attained
by employing the AutoDock 4.2 (AD4) software and calculating
a peculiar grid map for the site of attachment of the covalent
ligand (C419 sulfur atom). In particular, a Gaussian function
was constructed with zero energy at the site of attachment
and steep energetic penalties at surrounding areas. This al-
lowed the AD4 simulation to place the ligand atom that forms
the covalent bond within the Gaussian well.[16]
Compound
T. b. brucei
CTPS IC50 [mm]
T. b. brucei
EC50 [mm]
HeLa
EC50 [mm]
SI[a]
Acivicin
Acivicin-
COOMe
1
1-COOMe
2
2-COOMe
3
3-COOMe
4
4-COOMe
5
5-COOMe
6
6-COOMe
7a
7a-COOMe
7b
0.320Æ0.025
0.450Æ0.010
22.4Æ1.3
16.3
41.9Æ3.1
36
2
–
0.098Æ0.010
0.038Æ0.013
2.1Æ0.5
15.1Æ1.2 397
–
12.5Æ1.6
>500
6
>5
–
>5
>17
>5
>3
1.4
3
–
>500
>100
>500
>100
–
–
–
>500
>500
>500
>500
>500
>500
>500
29.8Æ1.7
>100
156.6Æ13.2
0.043Æ0.003
3.4Æ0.4
4.6Æ0.5
–
14.8Æ0.5
43.4Æ2.5
410Æ39
1.740Æ0.400 >100
–
–
–
117.7Æ5.0
39.1Æ3.1
0.3
>10
>24
–
>500
>500
56.2Æ4.3
20.7Æ1.0
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
89.8Æ7.8
10.0Æ0.5
>500
>500
23.7Æ3.9
19.3Æ0.6
7b-COOMe
8a
8a-COOMe
8b
8b-COOMe
9a,b
9a,b-COOMe
–
0.100Æ0.004
>5
>50
–
–
>23
>26
–
>500
–
0.125Æ0.002
–
[a] SI=selectivity index [EC50(HeLa)/EC50(T. b. brucei)]
Interestingly, a further improvement of the AD4 methodolo-
gy has been recently reported in a pioneering work by Forli
and Olson. These authors have developed a new force field
and hydration docking method that allows for the automated
prediction of waters mediating the binding of ligands with
target proteins with no prior knowledge of the apo- or holo-
protein hydration state.[17] Both methodologies, covalent and
hydrated ligand docking, were applied in our inspection. With
the aim of testing the method performances, Acivicin was
docked itself in the enzyme site revealing that the lowest
energy and also most populated conformation calculated by
AD4 was virtually superimposable with the one detected in
the X-ray structure. These encouraging results prompted the
docking of 5 and 6. Analysis of the results achieved for these
compounds revealed that in both cases the ligand isoxazoline
core was placed in the same binding position adopted by Aci-
vicin (Figure 3a and b) so as to allow the Coulombic interac-
tion of the ligand carboxylate group and R498. Interestingly, in
both cases the hydrated covalent docking predicted the pres-
ence of two water molecules that should mediate almost the
same ligand–enzyme interactions detected in the X-ray Acivi-
cin–CTPS structure. The main difference between the binding
poses predicted for 5 and 6 resided in the position of the N-a-
substituents. Indeed while the N-Boc substituent of 5 is pro-
jected toward the outer part of the enzyme active site taking
contacts with M467 and F393, in 6 the N-Cbz substituent is
placed in a rather hydrophilic cleft were unfavorable interac-
tions take place with E443. This should lead to a rather unsta-
a CTPS inhibitor, and 12-fold more efficacious as an anti-trypa-
nosomal agent, derivative 2, in which the halogen was re-
placed by a methoxy was not able to inhibit the enzyme up to
500 mm concentration. Also the insertion of a dimethylamine
residue at the C3 position (compound 3), mimicking the natu-
ral substrate l-Gln, produced a complete loss of the inhibitory
potency against the enzyme, though a modest trypanocidal
activity was detected (EC50 =29.8 mm) when the amino acid
was converted into the corresponding methyl ester.
Concerning the modifications involving the a-amino acid
group, while the absence of the amino group (compound 4)
produced an inactive derivative, the conversion of the amine
into a carbamate gave rise to quite interesting results. While
the N-Cbz protected derivative 6 was fivefold weaker than Aci-
vicin and 17-fold weaker than Br-Acivicin as a CTPS inhibitor,
on the contrary, the N-Boc protected derivative 5 showed an
excellent inhibitory activity against T. b. brucei CTPS (IC50 =
0.043 mm), being twice more potent than Br-Acivicin. The in vi-
tro trypanocidal activity was not as good as expected. In fact,
though compound 5 is provided with low micromolar anti-try-
panosomal activity (EC50 =3.4 mm), this is two order of magni-
tude weaker than that of Br-Acivicin (EC50 =0.038 mm). We hy-
pothesized that such a result could be related to decreased
uptake, due to loss of the amino acidic character, which may
hamper the active transport into the target cell. Conversion of
derivative 5 into its more lipophilic methyl ester did not in-
crease the trypanocidal activity. However, methyl ester may
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Disappointingly, despite their excellent CTPS inhibitory po-
tency, superior to that of Acivicin, both derivatives showed
a trypanocidal activity significantly lower than Acivicin. Upon
conversion into the corresponding methyl esters the trypanoci-
dal activity was improved, more significantly in the case of de-
rivative 8a, the activity of which increased about ninefold. This
may indicate that the pyrazoline analogues have a decreased
affinity for the amino acid transporters responsible for Acivicin
uptake. However, the activity of the methyl esters is still too
weak relative to the very good inhibitory activity against CTPS
of the free amino acids. Possible additional explanations may
involve a not efficient regeneration of the drug from its methyl
ester prodrug, or the instability of the molecule in the intracel-
lular medium.
Concerning the selectivity index, from a drug discovery
point of view, ideally a selectivity index greater than 200
would be required to be encouraged to move lead com-
pounds forward. Rather surprisingly, only Br-Acivicin has a nota-
ble selectivity index (SI 397), which is drastically decreased
upon esterification, whereas pyrazoline derivatives show
a modest increase in trypanocidal activity and selectivity upon
esterification, for example, compound 8a.
Conclusions
The present research project was focused on the study of the
structure–activity relationship of Acivicin. In summary, the re-
sults obtained have shown that while substituting the 3-chloro
with a 3-bromo increased the inhibitory potency of Acivicin
against the target enzyme CTPS threefold, the substitution
with a 3-methoxy group produced a complete loss of the ac-
tivity, confirming the important role played by the leaving
group in the C3 position of the isoxazoline ring. In this respect
it would be highly interesting to test the 3-iodo analogue of
Acivicin; however attempts to synthesize such derivative have
failed so far.
Figure 3. Binding mode of a) 5 and b) 6 into the CTPS catalytic site repre-
sented as transparent white ribbons. The ligands and the interacting resi-
dues are shown in grey and white sticks, respectively. Hydrogen bonds are
represented with dashed black lines. All hydrogen atoms were removed for
clarity.
Another important finding of the present research project is
that the inhibitory activity against CTPS can be increased by
applying a molecular complication approach, that means in-
serting groups able to establish additional interaction with the
binding pocket of the enzyme. This can be realized either func-
tionalizing the nitrogen of the amino acid moiety, or replacing
the isoxazoline ring with a pyrazoline and inserting the select-
ed substituents at the N1 position.
ble covalent adduct thus negatively influencing CTPS inhibitory
potency of 6.
The results obtained with the pyrazoline analogues are more
difficult to interpret. In fact, two of these derivatives, notably
the N-ethyl derivative (Æ)-8a and the N-aminoethyl derivative
(Æ)-9a,b displayed an excellent sub-micromolar inhibition
against CTPS, being about threefold more potent than Acivicin.
It must be noted that the N-aminoethyl derivative (Æ)-9a,b
was preliminarily tested as a diastereomeric mixture, thus test-
ing the single stereoisomer may actually give an even lower
IC50 value. However, no molecular modeling studies were at-
tempted for these compounds. In fact, if for the halogen sub-
stituted isoxazoline ring of 5 and 6 we could assume the same
reactivity and same covalent adduct formation displayed by
Acivicin, no experimental data are available for the pyrazolines
8 and 9 (a and b) to support our modeling studies.
The parallel analysis of the enzymatic activity and the in vitro
anti-trypanosomal activity of the derivatives in this study, leads
to the conclusion that an increased inhibitory activity toward
CTPS may produce a great increase of the anti-trypanosomal
activity, as was the case for Br-Acivicin, but this cannot be
taken as a general rule, as other important factors may play
a role, notably the ability of the molecules to penetrate into
the target cells.
Future efforts will be devoted to understanding the reasons
behind the decreased anti-trypanosomal activity of the potent
CTPS inhibitors identified in the present work and to their opti-
mization, in particular through the design of suitable prodrugs
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washed with H2O (2ꢂ10 mL). The organic layer was dried over an-
hydrous Na2SO4 and concentrated to dryness, obtaining the crude
product (625 mg, 91%) that was directly submitted to the next
step. b) The crude intermediate obtained from the previous step
(625 mg, 2.08 mmol) was treated with a 5:1 mixture of AcOH/H2O
(15 mL) and the solution was stirred at 408C for 48 h. The solvent
was evaporated and the residue was dissolved in EtOAc (10 mL)
and washed with H2O (2ꢂ5 mL). The organic layer was dried over
anhydrous Na2SO4, filtered and evaporated under reduced pres-
sure. The crude material was purified by column chromatography
(cyclohexane/EtOAc 1:1, then EtOAc) to obtain the desired com-
pound (aR,5S)-13a (227 mg) as a yellow oil; Rf: 0.55 (cyclohexane/
able to guarantee a good penetration into the cells followed
by an efficient release of the active drug.
Experimental Section
Materials and methods
All reagents were purchased from Sigma. Enantiomerically pure
Acivicin and Br-Acivicin, used as standard in the biological assays,
were prepared as previously described.[8,9] Dibromoformaldoxime
(DBF) was prepared according to a published procedure.[18] The
diastereomeric mixture of cycloadducts 11 a and 11 b was prepared
as previously described[10] from DBF, and (S)-3-(tert-butoxycarbon-
yl)-2,2-dimethyl-4-vinyloxazolidine (S)-10.[19] Compound (aS,5S)-5
was prepared as previously described.[8] Alkene (Æ)-15 was ob-
tained according to a reported method.[13] N-Cbz-glycinal 17 was
prepared according to a published procedure.[20]
1
EtOAc, 2:8); [a]D20: +9.61 (c=1.0 CHCl3); H NMR (CDCl3): d=1.42
(s, 9H), 2.70–2.80 (m, 1H), 2.90–3.08 (m, 2H), 3.60–3.94 (m, 3H),
3.81 (s, 3H), 4.65 (ddd, J=8.0, 8.0, 16.8, 1H), 5.20 ppm (brd, J=7.7,
1H); 13C NMR (CDCl3): d=28.5, 35.7, 54.18, 57.5, 61.4, 80.1, 80.7,
156.2, 168.5 ppm; MS: 261.2 [M+H]+.
Synthesis of (S)-2-amino-2-((S)-3-methoxy-4,5-dihydroisoxazol-5-
yl)acetic acid [(aS,5S)-2]: a) Compound (aR,5S)-13a (227 mg,
0.87 mmol) was dissolved in a mixture of H2O (2.9 mL), CH3CN
(1.9 mL) and CCl4 (1.9 mL). NaIO4 (744 mg, 3.48 mmol) and a catalyt-
ic amount of Ru2O·H2O (2.3 mg) were added and the suspension
was vigorously stirred at room temperature until disappearance of
the starting material. After 45 min, CH2Cl2 (10 mL) and H2O (10 mL)
were added. The organic layer was separated and evaporated
under reduced pressure. The residue was dissolved in EtOAc
(5 mL), the organic layer was extracted with a 10% aqueous solu-
tion of K2CO3 (3ꢂ5 mL) and the aqueous phase was made acidic
with 2n HCl and newly extracted with EtOAc (3ꢂ5 mL). The organ-
ic extracts were washed with brine, dried over anhydrous Na2SO4,
and the solvent evaporated to yield the desired carboxylic acid
(174 mg, 73% yield) as a white solid, which was used without fur-
ther characterization in the next step. b) The carboxylic acid ob-
tained from the previous step (174 mg, 0.63 mmol) was dissolved
in CH2Cl2 (1.6 mL) and TFA (0.48 mL, 6.30 mmol) was added drop-
wise at 08C. The reaction was stirred for 2 h. The volatiles were re-
moved under reduced pressure and the residue was dissolved in
H2O and submitted to cation exchange chromatography using Am-
berlite IR-120H. The acidic solution was slowly eluted onto the
resin, and then the column was washed with H2O until the pH was
neutral. The compound was eluted off the resin with 1n aq. NH3
and the product-containing fractions (detected with ninhydrin
stain on a TLC plate) were combined. The solvent was freeze-dried
to give compound (aS,5S)-2 (87 mg, 80% yield) as a white solid;
mp: dec >1708C; Rf: 0.28 (BuOH/H2O/AcOH, 4:2:1); [a]D20: +78
(c=0.5 H2O); 1H NMR (D2O): d =3.07 (dd, J=8.0, 17.1, 1H), 3.16
(dd, J=10.5, 17.1, 1H), 3.70 (s, 3H), 3.89 (d, J=3.6, 1H), 5.04 ppm
(ddd, J=3.6, 8.0, 10.5, 1H); 13C NMR (D2O): d=33.8, 56.2, 58.0, 79.2,
169.5, 170.3 ppm; MS: 175.0 [M+H]+; Anal. calcd for C6H10N2O4: C
41.38, H 5.79, N 16.09, found: C 41.08, H 5.62, N 15.81.
1H NMR and 13C NMR spectra were recorded with a Varian Mercury
300 (300 MHz) spectrometer. Chemical shifts (d) are expressed in
ppm, and coupling constants (J) are expressed in Hz. Rotary power
determinations were carried out using a Jasco P-1010 spectropo-
larimeter, coupled with a Haake N3-B thermostat. TLC analyses
were performed on commercial silica gel 60 F254 aluminum
sheets; spots were further evidenced by spraying with a dilute al-
kaline potassium permanganate solution or ninhydrin. Melting
points were determined on a model B 540 Bꢁchi apparatus and
are uncorrected. MS analyses were performed on a Varian 320-MS
triple quadrupole mass spectrometer with ESI source. Microanaly-
ses (C, H, N) of new compounds were within Æ0.4% of theoretical
values.
For biological assays, the final amino acids were converted into
their corresponding methyl esters, by adding an ethereal solution
of diazomethane (3ꢂ20 mL aliquots) to the dried compound, while
on ice. After 30 min the samples were allowed to warm to room
temperature and left to evaporate to dryness in a fume hood. Elec-
trospray mass spectrometry was used to confirm 100% conversion.
Synthesis of (S)-(benzyloxycarbonylamino)-2-((S)-3-bromo-4,5-di-
hydroisoxazol-5-yl)acetic acid [(aS,5S)-6]: To a stirred solution of
(aS,5S)-1 (55 mg, 0.25 mmol) in a 1:1 mixture of H2O and THF
(8 mL) was added NaHCO3 (62 mg, 0.75 mmol). After the gas evolu-
tion finished, benzyl chloroformate (39 mL, 0.28 mmol) was added
dropwise and the reaction was stirred at room temperature for 1 h.
After disappearance of the starting material, the organic solvent
was evaporated under reduced pressure and the aqueous phase
was washed with Et2O (1ꢂ4 mL), made acidic with 2n HCl and ex-
tracted with EtOAc (3ꢂ5 mL). The organic extract was dried over
anhydrous Na2SO4, and the solvent evaporated to give compound
(aS,5S)-6 (83 mg, 93% yield) as a hygroscopic white foam; Rf: 0.35
(CH2Cl2/MeOH, 95:5 + 1% AcOH); [a]D20: +167 (c=0.2H2O);
1H NMR (CDCl3): d=3.18–3.58 (m, 2H), 4.58 (dd, J=3.6, 8.2, 1H),
4.85–4.98 (m, 1H), 5.12 (s, 2H), 5.92 (brd, J=7.7, 1H), 7.25–7.40 (m,
5H), 8.90 ppm (brs, 1H); 13C NMR (CDCl3): d=44.2, 56.6, 67.9, 81.9,
128.4, 128.7, 128.9, 135.9, 138.7, 156.4, 171.7 ppm; MS: 357.0 [M+
H]+; Anal. calcd for C13H13BrN2O5: C 43.72, H 3.67, N 7.84, found: C
43.90; H 3.80, N 7.98.
Synthesis of (S)-2-amino-2-((S)-3-(dimethylamino)-4,5-dihydroi-
soxazol-5-yl)acetic acid [(aS,5S)-3]: a) In a sealed vial, compound
(aS,5S)-5 (300 mg, 0.93 mmol) was treated with a 2n solution of di-
methylamine in THF (10 mL) and the reaction mixture was heated
at 758C until disappearance of the starting material (16 h). The vol-
atiles were removed under reduced pressure and the residue was
dissolved in EtOAc (5 mL) and extracted with 0.1n NaOH (3ꢂ
5 mL). The aqueous phase was made acidic with 1n HCl and ex-
tracted with EtOAc (4ꢂ5 mL). The organic layer was dried over an-
hydrous Na2SO4, filtered and evaporated under vacuum, obtaining
compound (aS,5S)-14 (160 mg, 60% yield), as a white foam; Rf:
Synthesis of tert-butyl (R)-2-hydroxy-1-((S)-3-methoxy-4,5-dihy-
droisoxazol-5-yl)ethylcarbamate [(aR,5S)-13a]: a) The mixture of
diastereomers 11 a,b (800 mg, 2.29 mmol) was dissolved in dry
MeOH (12 mL) and K2CO3 (1.0 g, 7.23 mmol) was added. The reac-
tion was heated at 508C for 2 h. The volatiles were removed under
vacuum and the residue was dissolved in EtOAc (10 mL) and
1
0.16 (CH2Cl2/MeOH, 95:5+1% AcOH). H NMR (CDCl3): d=1.45 (s,
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9H), 2.82 (s, 6H), 3.19 (dd, J=10.5, 15.8, 1H), 3.38 (dd, J=5.3, 15.8,
1H), 4.25–4.35 (m, 1H), 4.70–4.82 (m, 1H); 5.65 (brd, J=7.4, 1H),
7.95 ppm (brs, 1H); 13C NMR (CDCl3): d=28.5, 37.2, 39.7, 56.3, 80.5,
80.8, 155.8, 162.5, 171.5 ppm; MS: 288.3 [M+H]+. b) Compound
(aS,5S)-14 obtained from the previous step (120 mg, 0.42 mmol)
was treated with a 4n solution of HCl in dioxane (10 mL) at 08C.
The reaction was stirred at room temperature for 2 h. The volatiles
were removed under vacuum and the residue was dissolved in
H2O and submitted to cation exchange chromatography using Am-
berlite IR-120H. The acidic solution was slowly eluted onto the
resin, and then the column was washed with H2O until the pH was
neutral. The compound was eluted off the resin with 1n aq. am-
monia, and the product-containing fractions (detected with ninhy-
drin stain on a TLC plate) were combined. The solvent was freeze-
was purified by column chromatography on silica gel (CH2Cl2/
MeOH, 95:5 to 90:10 gradient) to give an inseparable mixture of
the two diastereomers (Æ)-16a,b (4.41 g, 85% yield, 1:1 mixture of
diastereomers), as a white foam; Rf: 0.55 (CH2Cl2/MeOH 9:1);
1H NMR (CDCl3): d=1.79 (s, 3H), 2.32 (dd, J=7.4, 16.8, 0.5H), 2.48–
2.60 (m, 1.5H), 3.88 and 3.90 (s, 6H), 4.00–4.18 (m, 2H) 5.09 (s, 2H),
5.20 (brd, J=8.5, 0.5H), 5.54 (brd, J=9.6, 0.5H), 7.30–7.42 ppm (m,
5H); MS: 378.2 [M+H]+.
Synthesis of benzyl (1-benzyl-3-hydroxy-4,5-dihydro-1H-pyrazol-
5-yl)(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)methylcarba-
mate [(Æ)-18a,b]: Compound (Æ)-16a,b (1.02 g, 2.7 mmol) was
dissolved in CH3CN (50 mL). Benzyl bromide (0.32 mL, 2.7 mmol),
K2CO3 (373 mg, 2.7 mmol) and NaI (41 mg, 0.27 mmol) were added
to the solution. The reaction mixture was held at reflux for 90 min.
After disappearance of the starting material, the solvent was
evaporated under vacuum and the residue was diluted with EtOAc
(50 mL). The organic layer was washed with 3% aq. NH4Cl (50 mL),
dried over anhydrous Na2SO4, filtered and evaporated to dryness.
The product (Æ)-18a,b (870 mg, 69% yield) was isolated by
column chromatography (silica gel, cyclohexane/EtOAc, 1:1, 100%
EtOAc gradient) and was used in the next step without further
characterization.
dried to give compound (aS,5S)-3 (60 mg, 76% yield) as a white
20
solid; mp: dec >1938C; Rf: 0.19 (BuOH/H2O/AcOH, 4:2:1); [a]D
:
1
+105 (c=0.5 H2O); H NMR (D2O): d=2.69 (s, 6H), 3.13 (dd, J=7.7,
16.5, 1H), 3.13 (dd, J=11.5, 16.5, 1H), 3.81 (d, J=3.9, 1H),
4.85 ppm (ddd, J=3.9, 7.7, 11.5, 1H); 13C NMR (D2O): d=35.6, 38.8,
56.0, 77.9, 164.1, 170.7 ppm; MS: 188.1 [M+H]+; Anal. calcd for
C7H13N3O3. C 44.91, H 7.00, N 22.45, found: C 45.10, H 7.22, N 22.70.
Synthesis of 2-(3-bromo-4,5-dihydroisoxazol-5-yl)acetic acid
[(Æ)-4]: a) Solid NaHCO3 (0.84 g, 10.0 mmol) and DBF (0.40 g,
2.0 mmol) were added to a 5% solution of 3-buten-1-ol (0.14 g,
2.00 mmol) in EtOAc (2.8 mL). The reaction mixture was heated in
the microwave at 808C for 1 h, and the progress of the reaction
was monitored by TLC (cyclohexane/EtOAc 8:2). Few drops of H2O
were added and the solvent was decanted. The organic phase was
washed with H2O (2ꢂ10 mL), dried over anhydrous Na2SO4 and
evaporated under reduced pressure. Purification of the crude ma-
terial by column chromatography (cyclohexane/EtOAc 1:1) afforded
the desired cycloadduct (237 mg, 61% yield), as a yellow oil. Rf:
0.26 (cyclohexane/EtOAc 1:1); 1H NMR (CDCl3): d=1.80–2.03 (m,
2H), 2.59 (brs, 1H), 2.94 (dd, J=8.5, 17.3, 1H), 3.32 (dd, J=10.2,
17.3, 1H), 3.72–3.79 (m, 2H), 4.84 ppm (dddd, J=5.2, 8.5, 8.5, 10.2,
1H); 13C NMR (CDCl3): d=37.5, 47.0, 59.2, 80.25, 138.1 ppm. b) The
product obtained in the previous step (237 mg, 1.22 mmol) was
dissolved in a mixture of H2O (4.0 mL), CH3CN (2.7 mL) and CCl4
(2.7 mL). NaIO4 (1.04 g, 4.88 mmol) and a catalytic amount of
Ru2O·H2O (2.5 mg) were added and the suspension was vigorously
stirred at room temperature. After disappearance of the starting
material (45 min), CH2Cl2 (10 mL) and H2O (10 mL) were added. The
organic layer was separated and evaporated under reduced pres-
sure. The residue was dissolved in EtOAc (5 mL), the organic layer
was extracted with a 10% aqueous solution of K2CO3 (3ꢂ5 mL)
and the aqueous phase was made acidic with 2n HCl and newly
extracted with EtOAc (3ꢂ5 mL). The organic extracts were washed
with brine, dried over anhydrous Na2SO4, and the solvent evaporat-
ed to yield the desired product (Æ)-4 (185 mg, 73% yield) as
a white solid; crystallized from hexane/EtOAc as white prisms; mp:
Synthesis of benzyl (S*)-(methoxycarbonyl)((R*)-1-benzyl-4,5-di-
hydro-3-hydroxy-1H-pyrazol-5-yl)methylcarbamate [(Æ)-21a] and
benzyl
(S*)-(methoxycarbonyl)((S*)-1-benzyl-4,5-dihydro-3-hy-
droxy-1H-pyrazol-5-yl)methylcarbamate [(Æ)-21b]: a) To a solution
of (Æ)-18a,b (870 mg, 1.9 mmol) in MeOH (12 mL) and bi-distilled
H2O (1.7 mL), a catalytic amount of PPTS (47 mg, 0.19 mmol) was
added. The reaction mixture was stirred for 1 h at room tempera-
ture until completion. After evaporation of MeOH under reduced
pressure, the residue was dissolved in EtOAc (5 mL) and washed
with bi-distilled H2O (2ꢂ5 mL). The organic phase was dried over
Na2SO4, filtered and concentrated in vacuo. The residue was dis-
solved in MeOH (18 mL) and K2CO3 (54 mg, 0.4 mmol) was added
to the solution. The reaction mixture was stirred for 3 h at room
temperature. The solvent was evaporated in vacuo and the residue
was dissolved in EtOAc (5 mL) and washed with 3% aq. NH4Cl
(10 mL). The organic phase was dried over anhydrous Na2SO4, fil-
tered and concentrated to dryness. Purification via column chro-
matography (silica gel, eluent: EtOAc) allowed the separation of
the two diastereomers. (Æ)-21a: 322 mg (43% yield); crystallized
from EtOAc/hexane as a foamy white solid; mp: 123–1258C; Rf:
0.66 (EtOAc); 1H NMR (CDCl3): d=2.32 (d, J=17.3, 1H), 2.80 (dd,
J=9.4, 17.3, 1H), 3.58 (s, 3H), 3.72 (d, J=12.7, 1H), 3.80 (d, J=12.7,
1H), 3.87–3.98 (m, 1H), 4.46 (dd, J=2.5, 9.4, 1H), 5.05 (d, J=12.1,
1H), 5.13 (d, J=12.1, 1H), 5.70 (d, J=9.4, 1H), 7.18–7.40 (m, 10H),
7.78 ppm (s, 1H); 13C NMR (CDCl3): d=31.8, 52.8, 58.4, 61.9, 64.8,
67.6, 128.3, 128.4, 128.7, 128.8, 128.9, 129.9, 135.4, 136.3, 157.2,
170.5, 173.8 ppm; MS: 398.2 [M+H]+; Anal. calcd for C21H23N3O5: C
63.46, H 5.83, N 10.57, found: C 63.66, H 5.95, N 10.74; (Æ)-21b:
325 mg (43% yield); crystallized from EtOAc/hexane as a foamy
1
82–838C; Rf: 0.40 (CH2Cl2/MeOH, 95:5+1% AcOH); H NMR (CDCl3):
d=2.54 (dd, J=7.3, 16.8, 1H), 2.90 (dd, J=6.0, 16.8, 1H), 3.00 (dd,
J=7.3, 17.4, 1H), 3.45 (dd, J=10.4, 17.4, 1H), 5.05 ppm (dddd, J=
6.0, 7.3, 7.3, 10.4, 1H); 13C NMR (CDCl3): d=39.2, 46.8, 77.5, 137.6,
175.3 ppm; MS: 207.8, 205.8 [MÀH]À; Anal. calcd for C5H6BrNO3: C
28.87, H 2.91, N, 6.73, found: C 29.10, H 3.05, N 6.90.
1
white solid; mp: 108–1108C; Rf: 0.51 (EtOAc); H NMR (CDCl3): d=
2.47 (d, J=17.1, 1H), 2.80 (dd, J=9.1, 17.1, 1H), 3.68 (s, 3H), 3.63–
3.75 (m, 1H), 3.79 (d, J=12.5, 1H), 3.84 (d, J=12.5, 1H), 4.27 (dd,
J=4.1, 8.1, 1H), 4.97 (d, J=12.3, 1H), 5.04 (d, J=12.3, 1H), 5.67 (d,
J=8.1, 1H), 7.20–7.40 ppm (m, 11H); 13C NMR (CDCl3): d=31.6,
52.8, 56.6, 63.5, 64.5, 67.3, 128.3, 128.5, 128.7, 128.8, 129.0, 129.8,
135.5, 136.3, 155.7, 170.4, 173.8 ppm; MS: 398.2 [M+H]+; Anal.
calcd for C21H23N3O5: C 63.46, H 5.83, N 10.57, found: C 63.63, H
5.94, N 10.71.
Synthesis of benzyl (3-hydroxy-4,5-dihydro-1H-pyrazol-5-yl)(4-
methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)methylcarbamate
[(Æ)-16a,b]: To
a
solution of compound (Æ)-15 (5.18 g,
13.75 mmol) in EtOH (110 mL) was added hydrazine monohydrate
(3.33 mL, 68.73 mmol) and the reaction mixture was held at reflux
for 3 h. The solvent was removed under vacuum and the crude
Synthesis of benzyl (S*)-(methoxycarbonyl)((R*)-1-benzyl-3-
chloro-4,5-dihydro-1H-pyrazol-5-yl)methylcarbamate [(Æ)-24a]:
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CTP Synthetase Inhibitors
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POCl3 (0.85 mL, 9.3 mmol) was added to a stirred solution of com-
pound (Æ)-21a (370 mg, 0.93 mmol) in CH3CN (40 mL). The reac-
tion mixture was held at reflux for 3 h, until complete conversion
was observed by TLC analysis. After removal of the solvent under
vacuum, the residue was dissolved in EtOAc (50 mL) and the solu-
tion was added dropwise to a crushed ice solution (30 mL). The or-
ganic layer was separated and washed with H2O (50 mL) and brine
(50 mL). The pooled organic layers were dried over Na2SO4 filtered
and evaporated. The obtained reaction crude was purified by
column chromatography on silica gel (eluent: cyclohexane/EtOAc
8:2, then 7:3) to obtain the desired product (Æ)-24a (342 mg, 88%
yield); crystallized from diisopropyl ether as white prisms; mp:
(Æ)-7b was synthesized following the procedure reported for (Æ)-
7a starting from intermediate (Æ)-24b. (Æ)-7b: white solid, 60%
1
yield mp: dec> 1568C; Rf: 0.58 (butanol/H2O/AcOH, 4:2:1); H NMR
(D2O): d=2.82 (dd, J=11.4, 18.2, 1H), 3.01 (dd, J=11.4, 18.2, 1H),
3.83 (d, J=4.1, 1H), 4.05 (ddd, J=4.1, 11.4, 11.4, 1H), 4.17 (q, J=
13.8, 2H), 7.21–7.39 ppm (m, 5H); 13C NMR (D2O): d=38.3, 53.3,
58.4, 63.2, 128.5, 129.1, 129.8, 135.4, 145.5, 169.8 ppm; MS: 268.1
[M+H]+; Anal. calcd for C12H15Cl2N3O2: C 47.38, H 4.97, N 13.81,
found: C 47.01, H 5.02, N 13.70.
Synthesis of benzyl (1-ethyl-3-hydroxy-4,5-dihydro-1H-pyrazol-
5-yl)(4-methyl-2,6,7-trioxabicyclo[2.2.2]octan-1-yl)methylcarba-
mate [(Æ)-19a,b]: To a solution of compound (Æ)-16a,b (1.67 g,
4.42 mmol) in MeOH (100 mL) was added acetaldehyde (0.41 mL,
6.84 mmol) at room temperature. After 30 min, NaBH4 (245 mg,
6.61 mmol) was added and the reaction mixture was allowed to
stir for an additional hour. After disappearance of the starting ma-
terial, the solvent was evaporated under reduced pressure, the resi-
due was dissolved in EtOAc (50 mL) and washed with a 10% solu-
tion of NH4Cl (3ꢂ50 mL). The organic phase was dried over Na2SO4
and evaporated under reduced pressure to give the product (Æ)-
19a,b (1.52 g, 84% yield), as a white foam; Rf: 0.28 (CH2Cl2/MeOH
95:5); MS: 406.2 [M+H]+. The product was used in the next step
without further characterization.
1
124–1268C; Rf: 0.30 (cyclohexane/EtOAc, 8:2); H NMR (CDCl3): d=
2.86 (dd, J=10.6, 17.6, 1H), 2.98 (dd, J=11.7, 17.6, 1H), 3.74 (d, J=
14.4, 1H), 3.79 (s, 3H), 4.10 (dd, J=10.6, 11.7, 1H), 4.26 (d, J=14.4,
1H), 4.46 (d, J=9.4, 1H), 5.16 (s, 1H), 5.63 (d, J=9.4, 1H), 7.20–
7.41 ppm (m, 10H); 13C NMR (CDCl3): d=41.8, 53.0, 55.5, 59.1, 63.9,
67.6, 128.1, 128.3, 128.5, 128.7, 128.8, 129.8, 135.6, 136.2, 143.1,
157.0, 171.1 ppm; MS: 416.1 [M+H]+; Anal. calcd for C21H22ClN3O4:
C 60.65, H 5.33, N 10.10, found: C 60.42, H 5.26, N 9.97.
Synthesis of (S*)-2-amino-2-((R*)-1-benzyl-3-chloro-4,5-dihydro-
1H-pyrazol-5-yl)acetic acid hydrochloride [(Æ)-7a]: a) A 1m solu-
tion of BCl3 in CH2Cl2 (1.64 mL, 1.64 mmol) was added dropwise to
a solution of (Æ)-24a (342 mg, 0.82 mmol) in CH2Cl2 (30 mL)
cooled at À108C. The reaction mixture was allowed to warm up to
room temperature and stirred for 2 h. After complete conversion,
the reaction was quenched by the addition of 0.2n HCl (17 mL).
The aqueous layer was separated, made basic with aq. K2CO3
(pH 8) and extracted with EtOAc (3ꢂ30 mL). The combined organic
layers were dried over Na2SO4, filtered and evaporated under
vacuum. Purification via column chromatography (silica gel, EtOAc)
afforded the free amine, which was immediately dissolved in a mix-
ture of dioxane (9.8 mL) and bi-distilled H2O (4.5 mL). Ambersep
900-OH resin (300 mg) was added. The reaction mixture was stirred
for 4 h, until disappearance of the starting material. The resin was
filtered and washed with dioxane, bi-distilled H2O and Et2O to
remove impurities. The product was then eluted off the resin with
0.05n HCl and the product-containing fractions (detected with nin-
hydrin on a TLC plate) were combined and concentrated in vacuo
to obtain the desired hydrochloride amino acid (Æ)-7a (142 mg,
57% yield), as a white solid; mp: dec >1418C; Rf: 0.62 (butanol/
H2O/AcOH, 4:2:1); 1H NMR (D2O): d=2.85 (dd, J=10.0, 18.5, 1H),
3.17 (dd, J=12.3, 18.5, 1H), 3.75 (d, J=14.2, 1H), 3.91 (d, J=2.2,
1H), 4.13 (ddd, J=2.2, 10.0, 12.3, 1H), 4.20 (d, J=14.2, 1H), 7.15–
7.35 ppm (m, 5H); 13C NMR (D2O): d=41.2, 55.0, 58.8, 62.0, 128.3,
128.8, 130.0, 135.4, 146.0, 171.2 ppm; MS: 268.1 [M+H]+; Anal.
calcd for C12H15Cl2N3O2: C 47.38, H 4.97, N 13.81, found: C 47.03, H
5.00, N 13.71.
Synthesis of benzyl (S*)-(methoxycarbonyl)((R*)-1-ethyl-4,5-dihy-
dro-3-hydroxy-1H-pyrazol-5-yl)methylcarbamate [(Æ)-22a] and
benzyl
(S*)-(methoxycarbonyl)((S*)-1-ethyl-4,5-dihydro-3-hy-
droxy-1H-pyrazol-5-yl)methylcarbamate
[(Æ)-22b]:
a) PPTS
(93 mg, 0.37 mmol) was added to a solution of (Æ)-19a,b (1.52 g,
3.70 mmol) in a mixture of MeOH (32 mL) and bi-distilled H2O
(3.9 mL). The reaction mixture was stirred for 90 min at room tem-
perature, until disappearance of the starting material. After evapo-
ration of the organic solvent under reduced pressure, the residue
was dissolved in EtOAc (20 mL) and washed with H2O (3ꢂ20 mL).
The organic layer was dried over Na2SO4 and concentrated under
vacuum. The residue was dissolved in MeOH (34 mL) and K2CO3
(94 mg, 0.68 mmol) was added to the solution. The reaction mix-
ture was stirred for 1 h at room temperature. After disappearance
of the starting material, the solvent was evaporated under vacuum
and the residue was dissolved in EtOAc (20 mL). The organic layer
was washed with 3% aq. NH4Cl (20 mL), brine (20 mL) and dried
over Na2SO4, filtered and concentrated to dryness. The crude was
purified by column chromatography (CH2Cl2/iPrOH, 97:3), to obtain
the two diastereomers. (Æ)-22a: 513 mg (41% yield); crystallized
from EtOAc/hexane as a white foam; mp: 94–968C; Rf: 0.49
(CH2Cl2/iPrOH 97:3); 1H NMR (CDCl3): d=1.00 (t, J=7.1, 3H), 2.35
(dd, J=2.5, 17.3, 1H), 2.68 (q, J=7.1, 2H), 2.92 (dd, J=9.6, 17.3,
1H), 3.72 (s, 3H), 3.75–3.85 (m, 1H), 4.41 (dd, J=2.5, 9.6, 1H), 5.09
(d, J=12.4, 1H), 5.12 (d, J=12.4, 1H), 5.65 (d, J=9.6, 1H), 7.25–
7.40 (m, 5H), 8.57 ppm (brs, 1H); 13C NMR (CDCl3): d=12.2, 31.9,
52.8, 55.0, 58.4, 62.4, 67.6, 128.2, 128.4, 128.7, 136.3, 157.2, 170.7,
174.0 ppm; MS: 336.1 [M+H]+; Anal. calcd for C16H21N3O5: C 57.30,
H 6.31, N 12.53, found: C 57.05, H 6.22, N 12.40; (Æ)-22b: 515 mg
(41% yield); colorless oil; Rf: 0.35 (CH2Cl2/iPrOH 97:3); 1H NMR
(CDCl3): d=1.00 (t, J=7.1, 3H), 2.49 (d, J=17.3, 1H), 2.72 (q, J=
7.1, 2H), 2.92 (dd, J=9.1, 17.3, 1H), 3.53–3.62 (m, 1H), 3.72 (s, 3H),
4.29 (dd, J=4.7, 8.0, 1H), 5.10 (s, 2H), 5.90 (d, J=8.0, 1H), 7.20–
7.40 (m, 5H), 8.40 ppm (brs, 1H); 13C NMR (CDCl3): d=12.4, 31.7,
52.8, 54.7, 56.6, 63.9, 67.4, 128.4, 128.5, 128.8, 136.3, 156.0, 170.6,
174.2; MS: 336.1 [M+H]+; Anal. calcd for C16H21N3O5: C 57.30, H
6.31, N 12.53, found: C 57.09, H 6.26, N 12.41.
Synthesis of benzyl (S*)-(methoxycarbonyl)((S*)-1-benzyl-3-
chloro-4,5-dihydro-1H-pyrazol-5-yl)methylcarbamate [(Æ)-24b]:
Compound (Æ)-24b was synthesized following the procedure re-
ported for (Æ)-24a starting from intermediate (Æ)-21b. (Æ)-24b:
1
colorless oil, 77% yield; Rf: 0.26 (cyclohexane/EtOaAc, 8:2); H NMR
(CDCl3): d=2.82 (dd, J=11.5, 16.9, 1H), 3.10 (dd, J=11.5, 16.9, 1H),
3.74 (s, 3H), 3.76–3.84 (m, 1H), 4.07 (d, J=14.0, 1H), 4.31 (d, J=
14.0, 1H), 4.44–4.52 (m, 1H), 5.11 (s, 2H), 5.51 (d, J=6.3, 1H), 7.22–
7.40 ppm (m, 10H); 13C NMR (CDCl3): d=40.5, 53.1, 55.0, 58.9, 65.9,
67.5, 128.0, 128.1, 128.2, 128.3, 128.7, 129.8, 135.8, 136.2, 142.1,
156.0, 170.3 ppm; MS: 416.2 [M+H]+; Anal. calcd for C21H22ClN3O4:
C 60.65, H 5.33, N 10.10, found: C 60.75, H 5.40, N 10.23.
Synthesis of (S*)-2-amino-2-((S*)-1-benzyl-3-chloro-4,5-dihydro-
1H-pyrazol-5-yl)acetic acid hydrochloride [(Æ)-7b]: Compound
Synthesis of (S*)-2-amino-2-((R*)-3-chloro-1-ethyl-4,5-dihydro-
1H-pyrazol-5-yl)acetic acid hydrochloride [(Æ)-8a]: a) POCl3
ChemMedChem 0000, 00, 1 – 13
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P. Conti et al.
MED
(0.98 mL, 10.74 mmol) was added to a stirred solution of com-
pound (Æ)-22a (360 mg, 1.07 mmol) in CH3CN (52 mL). The reac-
tion mixture was held at reflux for 3 h, until complete conversion
was observed by TLC analysis. After removal of the solvent in va-
cuo, the residue dissolved in EtOAc (50 mL) and the solution was
added dropwise to a crushed ice solution (30 mL). The organic
layer was separated and washed with H2O (50 mL) and brine
(50 mL). The pooled organic layers were dried over Na2SO4 filtered
and evaporated. The obtained reaction crude was purified by
column chromatography on silica gel (eluent: cyclohexane/EtOAc
8:2, then 7:3) to obtain (Æ)-25a (332 mg, 88% yield) as a colorless
oil; Rf: 0,54 (cyclohexane/EtOAc 7:3); 1H NMR (CDCl3): d=1.12 (t,
J=7.0, 3H), 2.68 (dq, J=7.0, 13.5, 1H), 2.85 (dq, J=7.0, 13.5, 1H),
2.93 (dd, J=11.3, 17.3, 1H), 3.10 (dd, J=11.3, 17.3, 1H), 3.75 (s,
3H), 4.05 (ddd, J=1.7, 11.3, 11.3, 1H), 4.45 (dd, J=1.7, 9.6, 1H),
5.14 (s, 2H), 5.56 (brd, J=9.6, 1H), 7.38 (s, 5H) ppm; 13C NMR
(CDCl3): d=12.0, 41.7, 50.1, 52.9, 55.0, 65.5, 67.6, 128.3, 128.5,
128.8, 136.2, 142.7, 156.9, 171.1 ppm; MS: 354.1 [M+H]+; Anal.
calcd for C16H20ClN3O4: C 54.32, H 5.70, N 11.88, found: C 54.50, H
5.82, N 12.05. b) A 1m solution of BCl3 in CH2Cl2 (1.88 mL,
1.88 mmol) was added dropwise to a solution of compound (Æ)-
25a (332 mg, 0.94 mmol) in CH2Cl2 (35 mL) cooled at À108C. The
reaction mixture was allowed to warm up to room temperature
and stirred for 2 h. After complete conversion, the reaction was
quenched by the addition of 0.2n HCl (20 mL). The aqueous layer
was separated, made basic with aq. K2CO3 (pH 8) and extracted
with EtOAc (3ꢂ30 mL). The combined organic layers were dried
over Na2SO4, filtered and evaporated under vacuum. Purification
via column chromatography (silica gel, cyclohexane/EtOAc 1:1 then
EtOAc) afforded the desired amine which was directly dissolved in
a mixture of dioxane (3.4 mL) and bi-distilled H2O (3.4 mL). Amber-
sep 900-OH resin (500 mg) was added. The reaction mixture stirred
for 40 min, until disappearance of the starting material. The resin
was filtered off and washed with dioxane, bi-distilled H2O and Et2O
to remove impurities. The product was then eluted off the resin
with 0.05n HCl and the product-containing fractions (detected
with ninhydrin on a TLC plate) were combined and concentrated in
vacuo to obtain the desired hydrochloride amino acid (Æ)-8a
(118 mg, 52% yield), as a white solid; mp: >1628C dec; Rf: 0.25
(butanol/H2O/AcOH 4:2:1); 1H NMR (D2O): d=0.98 (t, J=7.0, 3H),
2.70 (dq, J=7.0, 13.5, 1H), 2.90 (dd, J=10.8, 18.5, 1H), 2.98 (dq, J=
7.0, 13.5, 1H), 3.35 (dd, J=10.8, 18.5, 1H), 3.80 (d, J=2.1, 1H),
4.12 ppm (ddd, J=2.1, 10.8, 10.8, 1H); 13C NMR (D2O): d=10.5,
41.2, 49.7, 54.6, 62.4, 145.8, 170.9 ppm; MS: 206.0 [M+H]+; Anal.
calcd for C7H13Cl2N3O2: C 34.73, H 5.41, N 17.36, found: C 34.47, H
5.46, N 17.20.
for C7H13Cl2N3O2: C 34.73, H 5.41, N 17.36, found: C 34.38, H 5.48, N
17.15.
Synthesis of benzyl (1-(2-benzyloxycarbonylaminoethyl)-3-hy-
droxy-4,5-dihydro-1H-pyrazol-5-yl)(4-methyl-2,6,7-trioxabicyclo-
[2.2.2]octan-1-yl)methylcarbamate [(Æ)-20a,b]: A solution of N-
Cbz-glycinal 17 (2.3 g, 11.91 mmol) in MeOH (18 mL) was added to
a solution of (Æ)-16a,b (1.5 g, 3.97 mmol) in MeOH (30 mL) at
room temperature. After 1 h, NaBH4 (0.4 g, 11.91 mmol) was added
and the reaction was stirred for an additional hour. After disap-
pearance of the starting material, the solvent was evaporated
under reduced pressure, the residue was dissolved in EtOAc
(50 mL) and washed with a 10% solution of NH4Cl (3ꢂ50 mL). The
organic phase was dried over Na2SO4 and evaporated under re-
duced pressure. Purification via column chromatography (silica gel,
cyclohexane/EtOAc 1:1, 100% EtOAc) afforded the desired product
(Æ)-20a,b (1.6 g, 73% yield), as a white foam; Rf: 0.46 (EtOAc); MS:
555.2 [M+H]+. The product was used in the next step without fur-
ther characterization.
Synthesis of benzyl (methoxycarbonyl)(1-(2-benzyloxycarbonyla-
minoethyl)-4,5-dihydro-3-hydroxy-1H-pyrazol-5 yl)methylcarba-
mate hydrochloride [(Æ)-23a,b]: a) PPTS (73 mg, 0.29 mmol) was
added to a solution of (Æ)-20a,b (1.6 g, 2.89 mmol) in a mixture of
MeOH (25 mL) and bi-distilled H2O (3.1 mL). The reaction mixture
was stirred for 2 h at room temperature, until disappearance of the
starting material. After evaporation of the organic solvent under re-
duced pressure, the residue was dissolved in EtOAc (20 mL) and
washed with H2O (3ꢂ20 mL). The organic layer was dried over
Na2SO4 and concentrated in vacuo. The residue was dissolved in
MeOH (30 mL) and K2CO3 (100 mg, 0.72 mmol) was added to the
solution. The reaction mixture was stirred for 2 h at room tempera-
ture. After disappearance of the starting material, the solvent was
evaporated under vacuum and the residue was dissolved in EtOAc
(20 mL). The organic layer was washed with 3% aq. NH4Cl (20 mL),
brine (20 mL) and dried over Na2SO4, filtered and concentrated to
dryness. The product (Æ)-23a,b (945 mg, 67% yield, 1:1 mixture of
diastereomers) was isolated after column chromatography (silica
gel, eluent: EtOAc), as white foam; Rf: 0.43 (CH2Cl2/MeOH 9:1);
1H NMR (CDCl3): d=2.30–2.50 (m, 1H), 2.65–3.00 (m, 3H), 3.07–3.20
(m, 1H), 3.25–3.45 (m, 1H), 3.50–3.62 (m, 0.5H), 3.65 and 3.68 (s,
3H), 3.75–3.85 (m, 0.5H), 4.30–4.38 (m, 0.5H), 4.42–4.50 (m, 0.5H),
5.02–5.20 (m, 4H), 5.08–5.12 (m, 0.5H), 5.25–5.32 (m, 0.5H); 5.60
(brd, J=8.2, 0.5H), 5.88 (brd, J=8.2, 0.5H), 7.25–7.40 (m, 10H),
8.05 (bs, 0.5H), 8.20 ppm (bs, 0.5H). MS 485.2 [M+H]+.
Synthesis of methyl 2-(((9H-fluoren-9-yl)methoxy)carbonylami-
no)-2-(1-(2-(((9H-fluoren-9-yl)methoxy)carbonylamino)ethyl)-3-
hydroxy-4,5-dihydro-1H-pyrazol-5-yl)acetate [(Æ)-26a,b]: a) A so-
lution of compound (Æ)-23a,b (660 mg, 1.36 mmol) in a 33% solu-
tion of hydrobromic acid in AcOH (5 mL) was stirred for 30 min at
room temperature. After the disappearance of the starting material
monitored by TLC, Et2O was added and a white solid precipitate
from the solution. The solid was filtered, washed with MeOH and
dried under vacuum to afford the desired amine which was directly
dissolved in a 1:1 mixture of H2O and dioxane (50 mL). After cool-
ing with an ice bath, solid Na2CO3 (664 mg, 6.26 mmol) and Fmoc
N-hydroxysuccinimide ester (918 mg, 2.72 mmol) were added. The
reaction mixture was stirred overnight at room temperature and
then the organic solvent was evaporated. The residue was diluted
with H2O (50 mL) and EtOAc (50 mL). The aqueous phase was ex-
tracted with EtOAc (3ꢂ50 mL) and the combined organic layers
were dried over Na2SO4 and evaporated in vacuo. The crude was
purified by column chromatography (silica gel, eluent: EtOAc) to
give the product (Æ)-26a,b (476 mg, 53% yield, 1:1 mixture of dia-
Synthesis of (S*)-2-amino-2-((S*)-3-chloro-1-ethyl-4,5-dihydro-
1H-pyrazol-5-yl)acetic acid hydrochloride [(Æ)-8b]: Compound
(Æ)-8b was synthesized following the procedure reported for (Æ)-
8a starting from intermediate (Æ)-22b. (Æ)-25b: colorless oil; 80%
1
yield; Rf: 0.41 (cyclohexane/EtOAc 7:3); H NMR (CDCl3): d=1.20 (t,
J=6.9, 3H), 2.82 (dq, J=6.9, 13.2, 1H), 2.85 (dd, J=11.6, 17.1, 1H),
3.05 (dd, J=12.1, 17.1, 1H), 3.09 (dq, J=6.9, 13.2, 1H), 3.79 (s, 3H),
3.75–3.85 (m, 1H), 4.50–4.60 (m, 1H), 5.10 (s, 2H), 5.60–5.70 (m,
1H), 7.35–7.60 ppm (m, 5H); 13C NMR (CDCl3): d=12.4, 40.1, 49.5,
53.1, 54.7, 67.1, 67.6, 128.4, 128.6, 128.8, 136.2, 141.7, 156.0,
170.4 ppm; MS: 354.1 [M+H]+; Anal. calcd for C16H20ClN3O4: C
54.32, H 5.70, N 11.88, found: C 54.02, H 5.59, N 11.65. (Æ)-8b:
white solid; 51% yield mp: >1798C dec.; Rf: 0.48 (butanol/H2O/
1
AcOH 4:2:1); H NMR (D2O): d=1.00 (t, J=7.1, 3H), 2.77–2.88 (m,
1H), 2.84 (dd, J=7.0, 13.4, 1H), 2.98–3.10 (m, 1H), 3.04 (dd, J=7.0,
13.4, 1H), 3.98–4.10 ppm (m, 2H); 13C NMR (D2O): d=11.1, 38.0,
48.5, 53.4, 63.5, 145.1, 170.7 ppm; MS: 206.0 [M+H]+; Anal. calcd
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CTP Synthetase Inhibitors
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1
stereomers) as a white foam; Rf: 0.55 (CH2Cl2/MeOH 9:1); H NMR
(DMSO): d=1.90–2.02 (m, 1H), 2.60–3.15 (m, 5H), 3.45–3.70 (m,
4H), 3.98–4.05 (m, 1H), 4.10–4.40 (m, 6H), 7.22–7.42 (m, 8H), 7.55–
7.70 (m, 4H), 7.82–7.90 ppm (m, 4H); MS: 661.4 [M+H]+.
ring is present. CCDC 885136 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
Synthesis of 2-amino-2-(1-(2-aminoethyl)-3-chloro-4,5-dihydro-
1H-pyrazol-5-yl)acetic acid monohydrochloride [(Æ)-9a,b]:
a) POCl3 (0.66 mL, 7.26 mmol) was added to a stirred solution of
compound (Æ)-26a,b (476 mg, 0.72 mmol) in CH3CN (18 mL). The
reaction mixture was held at reflux for 2 h, until complete conver-
sion was observed by TLC analysis. After removal of the solvent
under vacuum, the residue dissolved in EtOAc (20 mL) and the so-
lution was added dropwise to a crushed ice solution (30 mL). The
organic layer was separated and washed with H2O (50 mL) and
brine (50 mL). The pooled organic layers were dried over Na2SO4
filtered and evaporated. The obtained reaction crude was purified
by column chromatography on silica gel (eluent: cyclohexane/
EtOAc 7:3, then 1:1). The product obtained was dissolved in CH2Cl2
(20 mL) and treated with piperidine (1.64 mL, 28 mmol) and stirred
for 15 min at room temperature. The solvent was evaporated
under reduced pressure and the residue was dissolved in bi-dis-
tilled H2O (30 mL) and washed with Et2O (3ꢂ15 mL). The aqueous
layer was evaporated in vacuo; EtOH was added and the precipitat-
ed was filtered off under vacuum to yield the free amine, directly
submitted to next step. The intermediate obtained from the previ-
ous step was dissolved in a mixture of dioxane (5 mL) and bi-dis-
tilled H2O (5 mL). Ambersep 900-OH resin (500 mg) was added. The
reaction mixture stirred for 40 min, until disappearance of the start-
ing material. The resin was filtered off and washed with dioxane,
bi-distilled H2O and Et2O to remove impurities. The product was
then eluted off the resin with 0.05n HCl and the product-contain-
ing fractions (detected with ninhydrin on a TLC plate) were com-
bined and concentrated in vacuo to obtain the desired amino acid
(Æ)-9a,b monohydrochloride (60 mg, 32% yield, 1:1 mixture of dia-
Covalent hydrated docking
The new version of the docking program AutoDock (version4.2,
AD4),[23] as implemented through the graphical user interface
called AutoDockTools (ADT), was used to dock Acivicin, 5 and 6.
Ligand structures were built using the builder in the Maestro pack-
age of Schrçdinger Suite 2012 and optimized using a version of
MacroModel also included. The constructed compounds were con-
verted into AD4 format files using ADT, hydrated using the wet.py
suite and edited to include the correct atom type for the ligand
atom that forms the covalent bond with the enzyme C419 sulfur
atom. ADT was also used to convert the receptor in the AD4
format file. The docking area was centered around the putative
binding site. A set of grids of 60ꢂ60ꢂ60 ꢃ with 0.375 ꢃ spacing
was calculated around the docking area for the ligand atom types
using AutoGrid4. An additional grid map was calculated for the
water molecules using the mapwater.py suite. For each ligand, 100
separate docking calculations were performed. Each docking calcu-
lation consisted of 10 million energy evaluations using the La-
marckian genetic algorithm local search (GALS) method. The GALS
method evaluates a population of possible docking solutions and
propagates the most successful individuals from each generation
into the subsequent generation of possible solutions. A low-fre-
quency local search according to the method of Solis and Wets is
applied to docking trials to ensure that the final solution repre-
sents a local minimum. All dockings described in this paper were
performed with a population size of 250, and 300 rounds of Solis
and Wets local search were applied with a probability of 0.06. A
mutation rate of 0.02 and a crossover rate of 0.8 were used to gen-
erate new docking trials for subsequent generations, and the best
individual from each generation was propagated over the next
generation. The docking results from each of the 100 calculations
were clustered on the basis of root-mean square deviation (RMSD)
(solutions differing by <2.0 ꢃ) between the Cartesian coordinates
of the atoms and were ranked on the basis of free energy of bind-
ing (DGAD4). The top-ranked compounds were visually inspected for
good chemical geometry. Because AD4 does not perform any
structural optimization and energy minimization of the complexes
found, a molecular mechanics/energy minimization (MM/EM) ap-
proach was applied to refine the AD4 output. The computational
protocol applied consisted of the application of 100000 steps of
the Polak–Ribiꢄre conjugate gradients (PRCG) or until the deriva-
tive convergence was 0.05 kJmolÀ1. All complexes pictures were
rendered employing the UCSF Chimera software.[24]
1
stereomers), as a white solid; H NMR (D2O): d=2.80–3.40 (m, 6H),
3.75–4.10 ppm (m, 2H); MS: 221.0 [M+H]+; Anal. calcd for
C7H14Cl2N4O2: C 32.70, H 5.49, N 21.79, found: C 32.33, H 5.55, N
21.54.
Single crystal X-ray diffraction analysis of benzyl (S*)-(methoxy-
carbonyl)((R*)-1-ethyl-4,5-dihydro-3-hydroxy-1H-pyrazol-5-yl)me-
thylcarbamate (Æ)-22a: A single crystal suitable for the X-ray dif-
fraction analysis has been obtained by slow crystallization from
a 1:1 mixture of EtOAc and hexane at T=48C. A 100% complete
sphere of data has been collected at room temperature up to a 2#
Bragg angle of 52.78 using graphite-monochromated MoKa radia-
tion on a three-circle Bruker Smart Apex diffractometer equipped
with a CCD area detector. The measured structure factor ampli-
tudes have been corrected for beam anisotropy effects by using
the program SADABS.[21] The substance crystallizes in the non-cen-
trosymmetric orthorhombic Pca21 space group, with eight formu-
lae in cell and the following cell parameters (ꢃ, ꢃ3): a=14.6960(6),
b=9.3401(9), c=25.4311(15), V=3490.7(4). The structure has been
solved by direct methods and refined within the spherical atom
approximation implemented in the software SHELX,[22] giving final
agreement factors R1(F)=0.0405 for 4154 unique data with I>
2s(I), goodness-of-fit as high as 1.032 and greatest Fourier residues
as low as Æ0.20 eꢃÀ3. The asymmetric unit is made up by a pair of
symmetry-independent enantiomers, with the corresponding ste-
reocenters showing aS*, 5R* (and aR*, 5S*) relative configurations.
Some structural disorder affects the methyl ester carbonyl of one
of the two molecules, with occupation coefficients of the Oxygen
atom as high as 0.49(2) and 0.51(2). Interestingly, in the solid state
only the pyrazolidinone NÀNHÀC=O form of the dihydropyrazole
CTPS enzyme assays
T. b. brucei CTPS was expressed recombinantly and purified as de-
scribed elsewhere.[5c] A coupled spectrophotometric assay was
used consisting of a final volume of 200 mL containing: 50 mm
MOPS pH 7.6, 150 mm KCl, 12 mm MgCl2, 1 mm ATP, 1 mm DTT,
1 mm phosphoenolpyruvate, 0.5 mm NADH, 1.5 units of pre-mixed
pyruvate kinase and lactate dehydrogenase, 300 mm GTP, 1 mm
UTP, 750 mm l-glutamine and 25 mg of purified T. b. brucei CTPS.[5c]
Prior to assaying various concentrations of test compounds were
pre-incubated with T. b. brucei CTPS at room temperature, assays
were started with addition of pre-incubated CTPS to reaction mix
ChemMedChem 0000, 00, 1 – 13
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P. Conti et al.
MED
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152, 1155–1171.
(substrates, buffer, coupled enzymes and test compounds) and de-
creasing absorbance at l 341 nm was monitored.
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unpublished results.
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De Micheli, M. P. Barrett, T. K. Smith, ChemMedChem 2011, 6, 329–333.
[9] A. Pinto, P. Conti, L. Tamborini, C. De Micheli, Tetrahedron: Asymmetry
2009, 20, 508–511.
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[16] G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S. Good-
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In vitro toxicity assays for T. b. brucei and HeLa mammalian
cells
T. b brucei (strain 427) was cultured to the optimum density of 1–
2ꢂ106 cellsmLÀ1 in HMI-9 supplemented with 10% fetal calf serum
(FCS) under environmental conditions of 378C and 5% CO2. Solu-
tions of test compounds were prepared in culture media at stock
concentration of 200 mm and diluted serially (1:2) across the 96-
well, flat-bottom solid white plates to give a total of 11 decreasing
concentrations (100 mL wellÀ1). The last well of each series was left
blank, that is, “drug free” (negative control). Cells were prepared at
the concentration of 4ꢂ104 cellsmLÀ1 and was added to each well
of the respective compound series (100 mL wellÀ1). Plates were in-
cubated at 378C/5% CO2 for 48 h prior to addition of Alamar Blue
solution (20 mLwellÀ1, 0.49 mm in 1ꢂ PBS, pH 7.4) followed by a fur-
ther 24 hour. Assay end points were measured fluorimetrically with
the fluorescence spectrometer (FluoStar, BMG LabTech, Germany)
and Optima program set at l excitation 544 nm and l emission
590 nm. Data were analyzed using Prism 5.0 software to obtain
EC50 values. Experiment was performed in duplicate and repeated
three times. Similar Alamar Blue assay was carried out with HeLa
cells, cultured in DMEM supplemented with 10% FCS and 2 mm l-
glutamine. HeLa cells were plated at initial cell concentration of 3ꢂ
105 cellsmLÀ1 (100 mLwellÀ1) and incubated with test compounds
for 16 h prior to addition of Alamar Blue solution.
Acknowledgements
This work was funded by MIUR-PRIN 2009. T.K.S. was supported
by Wellcome Trust project grant (086658 and 093228).
[24] E. F. Pettersen, T. D. Goddard, C. G. Huang, G. S. Couch, D. M. Greenblatt,
E. C. Meng, T. E. Ferrin, J. Comput. Chem. 2004, 25, 1605–1612.
Keywords: amino acids · CTP synthetase · isoxazolines ·
pyrazolines · trypanosoma
Received: June 19, 2012
[1] R. Brun, J. Blum, F. Chappuis, C. Burri, Lancet 2010, 375, 148–159.
Published online on && &&, 0000
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FULL PAPERS
Increasing molecular complexity is
a tool to improve the activity of CTPS
inhibitors: New potent inhibitors of
CTPS were designed based on the Acivi-
cin scaffold as potential anti-trypanoso-
mal agents. Compounds that can estab-
lish additional interactions with CTPS
have greater inhibitory efficacy.
L. Tamborini, A. Pinto, T. K. Smith,
L. L. Major, M. C. Iannuzzi, S. Cosconati,
L. Marinelli, E. Novellino, L. Lo Presti,
P. E. Wong, M. P. Barrett, C. De Micheli,
P. Conti*
&& – &&
Synthesis and Biological Evaluation of
CTP Synthetase Inhibitors as Potential
Agents for the Treatment of African
Trypanosomiasis
ChemMedChem 0000, 00, 1 – 13
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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These are not the final page numbers!