Clotrimazole scaffold as an innovative pharmacophore towards potent antimalarial agents: Design, synthesis, and biological and structure-activity relationship studies

1278

J. Med. Chem. 2008, 51, 1278–1294

Clotrimazole Scaffold as an Innovative Pharmacophore Towards Potent Antimalarial Agents:

Design, Synthesis, and Biological and Structure–Activity Relationship Studies

Sandra Gemma,^†,‡Giuseppe Campiani,*^,†,‡Stefania Butini,^†,‡Gagan Kukreja,^†,‡Salvatore Sanna Coccone,^†,‡

Bhupendra P. Joshi,^†,‡Marco Persico,^†,§Vito Nacci,^†,‡Isabella Fiorini,^†,‡Ettore Novellino,^†,|Ernesto Fattorusso,^†,§

Orazio Taglialatela-Scafati,^†,§Luisa Savini,^†,‡Donatella Taramelli,^†,Nicoletta Basilico,^†,#Silvia Parapini,^†,Giulia Morace,

Vanessa Yardley,^†,3Simon Croft,^†,3Massimiliano Coletta, Stefano Marini,^#and Caterina Fattorusso^†,§

European Research Centre for Drug DiscoVery and DeVelopment and Dipartimento Farmaco Chimico Tecnologico, UniVersità di Siena, Via

Aldo Moro, 53100 Siena, Italy, Dipartimento di Chimica delle Sostanze Naturali and Dipartimento di Chimica Farmaceutica e Tossicologica,

UniVersità di Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy, Dipartimento di Sanità Pubblica-Microbiologia-Virologia,

UniVersita′ di Milano, Via Pascal 36, 20133 Milano, Italy, Department of Infectious, Tropical Diseases, London School of Hygiene and

Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom, and Dipartimento di Medicina Sperimentale e Scienze Biochimiche,

UniVersità degli Studi di Roma Tor Vergata, Rome, Italy

ReceiVed October 2, 2007

We describe herein the design, synthesis, biological evaluation, and structure–activity relationship (SAR)

studies of an innovative class of antimalarial agents based on a polyaromatic pharmacophore structurally

related to clotrimazole and easy to synthesize by low-cost synthetic procedures. SAR studies delineated a

number of structural features able to modulate the in vitro and in vivo antimalarial activity. A selected set

of antimalarials was further biologically investigated and displayed low in vitro toxicity on a panel of human

and murine cell lines. In vitro, the novel compounds proved to be selective for free heme, as demonstrated

in the ꢀ-hematin inhibitory activity assay, and did not show inhibitory activity against 14-R-lanosterol

demethylase (a fungal P450 cytochrome). Compounds 2, 4e, and 4n exhibited in vivo activity against P.

chabaudi after oral administration and thus represent promising antimalarial agents for further preclinical

development.

Introduction

interaction with free heme and exacerbation of oxidative stress

are supposed to be the mode of action of two of the most popular

and effective classes of antimalarial agents, namely, 4-amino-

quinolines and endoperoxides. Before the appearance of resis-

tance, chloroquine (CQ) was one of the most successful

antimicrobial agents ever developed, while artemisinins repre-

sent at the moment the most effective therapeutic option for

the treatment of resistant malaria, although there is concern of

overgrowing evidence for the in vitro appearance of artemisinin-

resistant plasmodia.⁴The high efﬁcacy of these antimalarials

resides in their ability to speciﬁcally target free heme (in its

Fe(III) or Fe(II) forms, respectively) and to interfere with the

fragile redox balance necessary for plasmodium survival.

Moreover, their low propensity to induce resistance (resistance

to CQ only occurred after many years of heavy drug pressure)

is due to the lack of interaction with a speciﬁc protein target.

Consequently, speciﬁc interaction with free heme aimed at

inhibiting heme detoxiﬁcation and increasing oxidative stress

still represents a valuable strategy for the discovery of potential

antimalarial drugs selectively toxic for the plasmodium.

Despite the considerable efforts of academic and industrial

research, malaria exacts a devastating social and economic cost

across the globe. The disease causes 2–3 million deaths per

annum as well as incalculable suffering. It strikes hardest at

some of the poorest nations and is itself a signiﬁcant cause and

consequence of poverty. Its effects are not only limited to the

developing nations, but malaria is of serious concern to travelers,

especially in the absence of a vaccine and in the face of

widespread resistance to several antimalarial drugs.¹

The etiological agent of malaria is Plasmodium falciparum

(Pf), a highly evolved unicellular parasite whose life cycle,

shared between an Anopheline mosquito vector and the human

host, exhibits striking biological features that have been

exploited to seek intervention strategies for malaria therapy. The

detoxiﬁcation of free heme released in the food vacuole (FV)

of Pf during host hemoglobin (Hb) catabolism represents a

unique biochemical process. It imposes a signiﬁcant oxidative

burden²that has to be managed by the plasmodium through

speciﬁc mechanisms of heme detoxiﬁcation such as crystalliza-

tion of heme to hemozoin and degradation in the presence of

reduced glutathione.³Interference with such processes through

Within a program aimed at developing new antimalarial

drugs,⁵we recently reported the identiﬁcation of a new

polyaromatic antimalarial pharmacophore based on clotrimazole

(CLT, 1, Chart 1) scaffold.⁶

CLT is a well-known antimycotic drug which exhibits a weak

in vitro antimalarial activity against different Pf strains and

importantly, irrespective of their CQ sensitivity.^7,8Biological

activities of CLT are mediated by its ability to interact with

ferri-protoporphyrin IX (Fe(III)-FP), which is present as the

prosthetic group in several enzymes, such as (i) 14R-lanosterol-

demethylase (14-LD), the fungal cytochrome inhibited by CLT,

(ii) human P450 cytochromes, which are inhibited by CLT

causing altered metabolism of both xenobiotics and endogenous

* To whom correspondence should be addressed. Phone: 0039-0577-

234172. Fax: 0039-0577-234333. E-mail: campiani@unisi.it.

†

European Research Centre for Drug Discovery, Università di Siena.

Dipartimento Farmaco Chimico Tecnologico, Università di Siena.

Dipartimento di Chimica delle Sostanze Naturali, Università di Napoli.

Dipartimento di Chimica Farmaceutica e Tossicologica, Università di

‡

§

|

Napoli.

3

London School of Hygiene and Tropical Medicine.

Dipartimento di Sanità Pubblica-Microbiologia-Virologia, Universita′

di Milano.

#

Università degli Studi di Roma Tor Vergata.

10.1021/jm701247k CCC: $40.75

2008 American Chemical Society

Published on Web 02/16/2008

Clotrimazole Scaffold as a Pharmacophore

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1279

Chart 1. Reference and Title Compounds

rings, and at the heme complexing moiety. Among the com-

pounds synthesized, the most active were selected for further

biological investigation to establish (i) the antiplasmodial activity

in vivo against murine plasmodia such as P. berghei and P.

chabaudi, (ii) the antifungal activity predictive for their capabil-

ity to interact with P450 cytochromes (protein-bound heme),

and (iii) their ability to interact with Fe(III)-FP in vitro.

Compounds 4e and 4n were identiﬁed as promising antimalarial

agents suitable for further development.

Chemistry

Compounds 3a-g reported in Table 1 were synthesized

according to Schemes 1 and 2. Intermediate 7a (Scheme 1) was

synthesized starting from 6,¹⁵transformed into the corresponding

Grignard reagent, and coupled with 5, while 7b was obtained

starting from the bromo derivative 8,⁶after its transformation

into the Boc-piperazine intermediate 9. Subsequently, carbinols

7a,b were transformed into the corresponding chlorides by

exposure to thionyl chloride, followed by reaction with imida-

zole or 1H-1,2,4-triazole, in the presence of triethylamine, to

afford the ﬁnal compounds 3a-c. Finally, treatment of N-Boc

derivative 3c with triﬂuoroacetic acid in dichloromethane (DCM)

gave the free piperazine-derivative 3d.

Compounds 3e-g were synthesized as depicted in Scheme

2. Commercially available aldehyde 11a was reacted with

3-chlorophenylmagnesium bromide to afford alcohol 13, while

carbinols 14a,b were synthesized coupling the Grignard reagent

obtained from 12 with the appropriate aldehydes 11a,b. Alde-

hyde 11b was in turn prepared starting from the 4,7-dichloro-

quinoline 10 by a two-step procedure involving the introduction

of a methyl group at the 4-position¹⁶of the quinoline system,

followed by selenium dioxide oxidation. Conversion of carbinols

13 and 14a,b into the corresponding chlorides, followed by

treatment with the sodium salt of imidazole in N,N-dimethyl-

formamide (DMF) afforded the ﬁnal compounds 3e-g.

Compounds 4a-e,o,p were obtained as described in Scheme

3. The alkylation of bromobenzyl intermediate 15 with dieth-

ylamine, morpholine, or N-Boc-piperazine furnished the cor-

responding ketones 16a-c. The latter were in turn reacted with

phenylmagnesium bromide to afford carbinols 17a-c. On the

other hand, carbinols 18a-c were prepared starting from 16d,⁶

by reaction with p-ﬂuorophenylmagnesium bromide (18a) or

with the Grignard reagent derived from 12 (18b) or with

phenylmagnesium bromide (18c). Following a described pro-

cedure,¹⁷triarylcarbinols 17a-c and 18a,b were converted into

the corresponding unstable chlorides which on reaction with

imidazole in the presence of triethylamine afforded the target

compounds 4a-e. The chloride derivative of 18c⁶was used as

a substrate for the synthesis of 4o,p by reaction with benzimi-

dazole or piperazine, respectively.

chemicals, and (iii) hemoperoxidase,⁹a Pf-derived enzyme,

which is inhibited by CLT, in the presence of H₂O₂, by a

mechanism based on CLT-one electron oxidation product.

Moreover, CLT is also able to form in vitro complexes with

free Fe(III)-FP in which the imidazole ring behaves as a Fe(III)

axial ligand and is able to inhibit in vitro the crystallization of

free Fe(III)-FP into ꢀ-hematin.^10,11

Based on the aforementioned properties of CLT and consid-

ering its peculiar chemical structure, characterized by (i) an

imidazole ring, known to mediate electron transfer reactions in

biological systems,^12,13and (ii) a triphenylmethyl system, known

to form and stabilize a radical intermediate (the triphenylmethyl

radical was a landmark discovery as it marked the beginning

of organic free radical chemistry¹⁴), we hypothesized that CLT,

in the unique FV chemical environment, could interact with

hemoglobin-derived ferro-protoporphyrin IX (Fe(II)-FP) and to

get activated to form toxic trityl radicals able to kill the parasite

by oxidative damage.

Consequently, our design strategy mainly focused on the

synthesis of analogues with speciﬁc electronic and chemical

features to better penetrate and accumulate into the FV and to

generate active radical intermediates therein (pH 5.5 and

presence of heme). Furthermore, we envisioned to improve the

selectivity for free heme over heme as a prosthetic group to

minimize side effects. These studies led to the identiﬁcation of

the CLT analogue 2,⁶a novel hit endowed with potent

antimalarial activity in vitro whose structure is characterized

by the presence of a protonatable pyrrolidinylmethyl group,

which was rationally introduced to improve the pharmacokinetic

properties, to increase tropism for the acidic FV and to attain

selectivity for free heme over heme into cytochromes (due to

the presence of conserved protonated amino acid residues at

the lip of the cytochrome active site).

Here we report the synthesis and the biological evaluation

of two series of compounds (3a-h and 4a-r) based on the

innovative polyaromatic pharmacophore previously disclosed.⁶

Structure–activity relationships for the novel series of antima-

larials were investigated through structural modiﬁcations per-

formed at the protonatable lateral chain of 2, at the polyaromatic

scaffold, introducing various heterocycles and substituted phenyl

The heterocyclic analogues 4f-l were prepared according to

Scheme 4. 3-Chlorophenylmagnesium bromide was reacted with

two equivalents of the corresponding commercially available

carboxaldehydes 19a-e to afford ketones 20a-e, while the

corresponding p-chloro-substituted derivatives 21a,b were ob-

tained starting from 4-chlorophenylmagnesium bromide and

aldehydes 19a,b. The ketones thus obtained were subjected to

a second Grignard reaction to afford carbinols 22a-e and 23a,b,

which were in turn transformed to ﬁnal compounds 4f-l, as

described above.

For the synthesis of the quinoline-derivatives 4m,n (Scheme 5),

the intermediate ketones 26a,b were prepared starting from

7-chloro-4-methoxyquinoline 24.¹⁸Accordingly, compound 24 was

reacted with phenylacetonitrile or 4-ﬂuorophenylacetonitrile, in the

1280 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5

Table 1. Antiplasmodial Activity of Compounds 3a-h

Gemma et al.

^aPercentage of ionic form in brackets; TP ) triprotonated form; DP ) diprotonated form; P ) protonated form; and N ) neutral form (ACD/pKa DB

version 10.00 software – Advanced Chemistry Development, Inc., Toronto, Canada-). ^bIC₅₀s are the mean of at least three determinations. Standard errors

were all within 10% of the mean; n.a. ) Not Active (IC₅₀> 10 µM). ^cCQ-S clone. ^dCQ-R clone. ^eRef. 6.

presence of sodium hydride, to afford nitriles 25a and 25b,

sponding ketone derivatives 26a,b.¹⁹Following the synthetic

respectively, which were subsequently converted into the corre-

procedure already described, Grignard reaction, followed by

Clotrimazole Scaffold as a Pharmacophore

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1281

Scheme 3^a

Scheme 1^a

^aReagents and conditions: (a) N-Boc-piperazine, Et₃N, MeCN, 0 °C, 2 h;

(b) NaBH₄, THF/H₂O 2:1, 80 °C, 1.5 h; (c) Mg turnings, THF, 80 °C; (d) (i)

SOCl₂, DCM, 0 °C to rt, 4 h; (ii) imidazole (for 3a,c) or 1H-1,2,4-triazole (for

3b), Et₃N, MeCN, 0 to 80 °C, 4 h; (e) TFA, DCM, 0–5 °C, 3 h.

Scheme 2^a

^aReagents and conditions: (a) Et₂NH (for 16a), morpholine (for 16b),

N-Boc-piperazine (for 16c), Et₃N, MeCN, 0 °C, 1 h; (b) phenylmagnesium

bromide, THF, 80 °C, 6 h; (c) p-ﬂuorobromobenzene (for 18a), 12 (for

18b), Mg, THF, 80 °C, 6 h; (d) (i) SOCl₂, DCM, 0 to 45 °C, 4 h, (ii)

imidazole, Et₃N, MeCN, 0 to 80 °C, 4 h; (e) (i) SOCl₂, DCM, 0 °C to rt,

4 h, (ii) benzimidazole (for 4o), piperazine (for 4p), Et₃N, MeCN, 0 to 80

°C, 4 h.

Scheme 4^a

^aReagents and conditions: (a) MeMgBr, [1,2-bis(diphenylphosphino)-

ethane]dichloronickel(II), Et₂O, 0 °C to rt, 24 h; (b) SeO₂, bromobenzene,

170-175 °C, 18 h; (c) 3-chlorophenylmagnesium bromide, THF, 80 °C,

6 h; (d) n-BuLi, THF, -78 °C to rt, 24 h; (e) SOCl₂, DCM, 0 °C to rt, 5 h;

(f) imidazole sodium salt, DMF, 80 °C, 3 h.

^aReagents and conditions: (a) m-chlorophenylmagnesium bromide or

p-chlorophenylmagnesium bromide, THF, 80 °C, 3 h; (b) 12, Mg, THF, 80

°C, 6 h; (c) (i) SOCl₂, DCM, 0 °C to rt, 4 h; (ii) imidazole, Et₃N, MeCN,

0 to 80 °C, 4 h.

(CQ-S) D10 and 3D7 and the CQ-resistant (CQ-R) W2 and K1

strains. The antimalarial activity (IC₅₀, nM) was quantiﬁed as

inhibition of parasite growth, measured with the production of

parasite lactate dehydrogenase (D10 and W2 strains, asynchro-

nous culture) or the incorporation of [³H]-hypoxanthine (3D7

and K1 strains, synchronous culture). Results are reported in

Tables 1,2.

chlorination of the resulting carbinols 27a,b and subsequent

treatment with imidazole and triethylamine, afforded the desired

compounds 4m,n.

Results and Discussion

1. In Vitro Antimalarial Activity and Structure–Activ-

ity Relationship. All synthesized compounds were tested in

vitro against a series of Pf strains, namely, the CQ-sensitive

Based on the above-mentioned CLT properties, we used its

scaffold to develop novel antimalarial agents characterized by

1282 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5

Gemma et al.

Scheme 5^a

for antimalarial activity. Indeed, the potency strongly increased

when an additional phenyl ring or an appropriate heterocycle

was introduced (3h vs 4q and 4g, 3g vs 4m, Tables 1 and 2),

indicating that a triarylmethyl moiety represents the minimal

feature for high antimalarial activity.

(b). Key Role of the Heme Complexing Group. Following

our design hypothesis, the imidazole ring, protonated at the FV

pH and having the dual role of interacting with free heme and

of being the substrate for the redox reaction generating a trityl

radical, was the heme interacting group of choice.^9,12,13,20In

the diarylmethyl series, introduction of a triazole ring resulted

in the loss of antimalarial activity with respect to the imidazole

analogue (3a vs 3b), and in the triarylmethyl series, a similar

effect was observed when a piperazine ring was introduced (4p

vs 2). This conﬁrms that the introduction of groups capable of

chelating iron but unable to promote the formation of the radical

intermediate are detrimental for the activity. Indeed, the triazole

nitrogen-centered radical is destabilized through the unfavorable

dipole–dipole interaction of the radical electron with lone pair

electrons of the adjacent nitrogen atom.²⁰Moreover, the triazole

moiety is not protonated at the FV pH, causing a decreased

accumulation of the compound. On the other hand, the pipera-

zine nitrogen is protonated at the FV pH but is unable to

generate a radical through a conjugation-mediated electron

transfer. Accordingly, 4o was found to be more potent than 4p

but slightly less potent than 2 because its benzimidazole ring is

able to chelate iron and to generate free radicals²¹but is less

protonated at FV pH than imidazole. This ﬁnding further

supports our design hypothesis.⁶

^aReagents and conditions: (a) phenylacetonitrile (for 25a), 4-ﬂuorophe-

nylacetonitrile (for 25b), NaH (60% in mineral oil), THF, 70 °C, 30 min;

(b) NaH (60% in mineral oil), O₂, THF, rt, 2 h; (c) 12, Mg, THF, 80 °C,

6 h; (d) (i) SOCl₂, DCM, 0 to 45 °C, 24 h, (ii) imidazole, Et₃N, MeCN, 0

to 80 °C, 4 h.

(c). Protonatable Lateral Chain. Introduction of an extra

protonatable chain was a key ﬁnding to highly improve potency

and FV accumulation of the novel antimalarials (2 vs CLT).⁶

The antimalarial activity could also be modulated by varying

the nature of the protonatable lateral chain. Substitution of the

pyrrolidine group by a piperazine led to a moderate increase of

antimalarial activity against asynchronous D10 and W2 strains

(3d vs 3h and 4c vs 2). This data could be explained taking

into consideration that also piperazine is able to form complexes

with iron,^5athus suggesting that an additional metal interaction

point, as in 3d and 4c, could be responsible of the increased in

vitro antimalarial activity with respect to 2 (a mechanism already

exploited by other antimalarial agents used for iron chelation

therapy²²). In the triarylmethyl series, the introduction of a

diethylamino group (4a) resulted in a decreased activity against

both CQ-S strains, while a 10-fold potentiation was observed

against the K1 CQ-R strain (4a vs 2). On the other hand, the

presence of the less protonatable morpholine ring (4b) reduced

the antimalarial activity with respect to 2 against both D10 and

W2 strains. Such a dependence of antimalarial activity from

the nature of the protonatable lateral chain has been extensively

described for CQ derivatives, in which introduction of bulkier

groups at the distal nitrogen generally resulted in an increased

activity against CQ-R strains²³and is probably suggestive of a

possible altered interaction with a protein transporter, which

has been hypothesized as a mechanism leading to CQ-resistance.

the selective binding to free heme but lacking interaction with

a deﬁnite Pf or host target protein (to minimize resistance), with

improved antimalarial potency and pharmacological proﬁle over

CLT and reduced side effects. To achieve activity against both

CQ-S and CQ-R Pf strains, we reasoned that an effective

antimalarial should rapidly accumulate and be activated inside

the microenvironment of the parasite FV (i.e., pH 5.5; redox

milieu -250 mV; presence of free heme), where the spontaneous

oxidation of hemoglobin-derived Fe(II) heme to Fe(III) heme

promotes the formation of superoxide ions generating H₂O₂and

hydroxyl radicals. On these bases, our design strategy focused

on novel antimalarials characterized by improved penetration

into the FV and with appropriate electronic features that would

generate radical intermediates in the Pf FV environment. Starting

from CLT structure, to increase FV accumulation, we introduced

an extra protonatable function, while to modulate the interaction

with free heme and the generation and stabilization of a radical

intermediate, we investigated the effect of different heme

complexing groups and of different aromatic substituents.

(a). Diarylmethyl versus Triarylmethyl Derivatives. We

dismantled the structure of CLT and consequently synthesized

the diphenylmethyl derivatives 3a-g (Table 1) to identify the

minimal structural requirement for antimalarial activity, which

could subsequently be optimized to meet the pharmacodynamic

and pharmacokinetic prerequisites to enter into further develop-

ment. Compounds 3a-g were found to be signiﬁcantly less

potent than CLT against both CQ-S and CQ-R Pf strains (Table

1). According to our hypothesized mechanism of action, the

lower activity of the diaryl analogues with respect to CLT

(triaryl system) can be explained in terms of interference with

the formation and stabilization of a radical intermediate, critical

(d). Halogen vs Methoxy Substituents at the Trityl

System. Electron-donating and electron-withdrawing substitu-

ents at the trityl system differently affect the capability of the

system to generate and stabilize radical intermediates and, by

consequence, the antimalarial potency of the corresponding

compounds. In general, electron-donating or -withdrawing

groups may signiﬁcantly modulate the reactivity of triaryl-

methyls and, consequently, the pharmacological properties. In

fact, while the p-Cl substituent was responsible for a ﬁne-tuning

Clotrimazole Scaffold as a Pharmacophore

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1283

Table 2. Antiplasmodial Activity of Compounds 4a-r

^aPercentage of ionic form in brackets; TP ) triprotonated form; DP ) diprotonated form; P ) protonated form; and N ) neutral form (ACD/pKa DB

version 10.00 software – Advanced Chemistry Development, Inc., Toronto, Canada-). ^bIC₅₀s are the mean of at least three determinations. Standard errors

were all within 10% of the mean; n.t. ) Not Tested. ^cCQ-S clone. ^dCQ-R clone. ^eRef. 6.

1284 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5

Gemma et al.

Table 3. AM1 Partial Charges of the CR (ipso Phenyl Carbons) and the

Central Methine Carbon of 2, 4d-4h, 4q, and 4r in their Prevalent

Ionic Forms at Cytoplasmatic (7.2) and FV (5.5) pH

to the trityl central methine carbon (Chart 1), which is

detrimental for the formation of the intermediate trityl radical.

We also explored the effect produced by the introduction of

ﬂuorine atoms at the trityl system to increase the metabolic

stability. When a p-ﬂuorine substituent was introduced, the

resulting compound 4d showed antimalarial properties similar

to 2 against asynchronous strains D10 and W2 and the

synchronous CQ-S strain 3D7. However, it proved to be less-

potent against K1. This could be explained considering that each

Pf strain, sensitive or not to CQ, may present a different

chemical environment at the FV level (i.e., pH value associated

to CQ resistance²⁴). Thus, in the case of 4d, the efﬁcacy on the

different strains can be explained by taking into consideration

the electron distribution of the monoprotonated and diprotonated

forms whose relative amount is determined by the different FV

environment of each strain (Table 2). Accordingly, the dipro-

tonated form of 4d has a partial charge distribution disfavoring

radical formation with respect to the corresponding monopro-

tonated form (Figure 1 and Table 3).

central

CR1

DP^a

CR2

CR3

methine carbon

cmpd

P^a

P

DP

P

DP

P

DP

2

-0.11 -0.15 -0.12 -0.16 -0.03 -0.06 +0.23 +0.23

-0.11 -0.15 -0.14 -0.18 -0.03 -0.06 +0.23 +0.24

4d

4e^b-0.12 -0.18 -0.07 -0.09 -0.05 -0.08 +0.22 +0.24

4f

-0.11 -0.14 -0.45 -0.54 -0.04 -0.07 +0.27 +0.28

-0.10 -0.15 -0.36 -0.45 -0.04 -0.07 +0.26 +0.28

-0.10 -0.14 -0.19 -0.26 -0.05 -0.07 +0.26 +0.28

-0.14 -0.18 -0.06 -0.11 -0.04 -0.06 +0.23 +0.24

-0.10 -0.13 -0.11 -0.16 -0.04 -0.07 +0.23 +0.24

-0.15 -0.20 -0.11 -0.15 -0.04 -0.06 +0.24 +0.24

4g

4h

4n

4q

4r

^aP ) protonated form; pH 7.2; DP ) diprotonated form; pH 5.5.

^bCompound 4e can only exist in the diprotonated (pH 7.2) and triprotonated

(pH 5.5) form.

(e). Modulation of the Electronic Features of the Triaryl-

methyl System: Effect of Five-Membered Heterocycles. To

further explore the role played by speciﬁc electronic features

in modulating the formation/stabilization of a radical intermedi-

ate and the antimalarial activity of these polycyclic antimalarials,

we replaced one aromatic group by ﬁve-membered heterocycles

and their partial charge distribution was calculated by AM1

quantomechanical methods. Even though it has been demon-

strated that replacement of a phenyl by a thiophene does not

change dramatically the reactivity of the trityl system, we

observed in our series of compounds (ﬁve-membered hetero-

cycles combined to the presence of an imidazole ring) signiﬁ-

cantly different electron distributions in the polyaromatic

systems taken into consideration. Indeed, as shown in Figure 2

and Table 3, by comparison of the electron distribution of 4q,

to the one of the thiophene (4f), thiazole (4g), and furan (4h)

derivatives, an increased electron density at the CR (ipso aryl

carbons, Chart 1) was observed, thus reducing the generation

of a radical intermediate and lowering the antimalarial potency

with respect to 4q. In general, the increased polarization and

electron density of the heterocycles introduced was inversely

related to their antimalarial potency (Figure 2). In fact, the

thiophene derivative 4f, which displayed the higher electron

density at CR was the least potent compound of the subseries,

while activity increased introducing a furan or a thiazole ring,

following the rank order of potency 4h > 4g > 4f. The same

SAR was observed for the corresponding p-Cl analogues 4k

and 4l. The higher activity of 4l compared to 4g conﬁrmed the

ﬁne-tuning of the antimalarial activity operated by the p-Cl

substituent even in this heterocyclic series of analogues.

Figure 1. Resulting MOPAC (AM1) conformers of 2 and 4d in their

prevalent ionic forms at the cytoplasmatic pH (A and C, respectively)

and at the FV pH (B and D, respectively). Left: the compounds are

colored by atom type (green for C, white for H, blue for N, light-green

for Cl, and dark-green for F). AM1 partial charges of CR (ipso phenyl

carbons), central methine carbon, heteroatoms, and protonatable groups

are showed. All hydrogens, except those of protonable nitrogens, have

been omitted for clarity. Right: the compounds are colored by AM1

charge distribution (the atoms are colored from red (-0.5) to blue

(+0.5) depending upon their partial charge value).

(f). Pyridine versus Quinoline: Increasing the Antima-

larial Potency. When a 4-pyridyl (4j) or a 2-pyridyl (4i)

heterocycles were taken into consideration, a lower antimalarial

activity with respect to 4q was observed. Although the replace-

ment of a phenyl ring by a piridyl ring in a triphenylmethyl

radical does not change dramatically its stability, however, the

pyridyl group delocalizes radicals less than the phenyl ring.¹⁴

On these bases, to increase the ability to stabilize a radical

intermediate and, consequently, the antimalarial activity, we

planned to replace the 4-pyridyl by a 7-chloroquinolin-4-yl

system (a pyridine with a fused 4-Cl benzo group at C2-C3),

which stabilizes the radical by a higher π delocalization.

Accordingly, the 7-Cl-quinoline derivative 4m, although lacking

the favorable electron-withdrawing substituent on the phenyl

ring (see paragraph (d), Halogen vs Methoxy Substituents at

of activity against CQ-S and CQ-R strains (2 vs 4q), a dramatic

decrease of activity was observed when the electron-withdrawing

chlorine atom was replaced by the electron-donating methoxy

group (4r vs 2). AM1 quantomechanical calculation showed

that the partial charge distribution at the trityl system in the

monoprotonated and diprotonated forms of both 2 and 4r was

highly affected by the p-substituent (Table 3). In 4r, the presence

of the p-methoxy-substituted phenyl ring determined an in-

creased electron density at the CR1 (ipso phenyl carbon 1) linked

Clotrimazole Scaffold as a Pharmacophore

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1285

Figure 3. Hematin inhibitory activity assay of compounds CQ, CLT,

2, and 4e.

Table 4. ꢀ-Hematin Inhibitory Activity Assay of Compounds 4e, 2,

CLT (1), and CQ

a

cmpd

4e

2

CLT, 1

CQ

IC₅₀

2.85

2.53

2.50

1.69

^aThe IC₅₀represents the molar equivalents of compound, relative to

hemin, that inhibit ꢀ-hematin formation by 50%. Data are the mean of three

different experiments in triplicate. Standard errors were all within 10% of

the mean.

at CR (Chart 1; AM1 calculations, Table 3), increasing the

stabilization of a putative trityl radical intermediate responsible

for antimalarial activity. These data together with the high

potency of 4e further conﬁrm the hypothesis of the generation

of a radical intermediate for this class of antimalarials.

Compound 4e was thus selected for further biological investiga-

tion (see paragraph 5).

Figure 2. Resulting MOPAC (AM1) conformers of 4q (A), 4h (B),

4g (C), and 4f (D) in their prevalent ionic forms at the FV pH. Left:

the compounds are colored by atom type (green for C, white for H,

blue for N, light-green for Cl, red for O, and yellow for S). AM1 partial

charges of CR (ipso phenyl carbons), central methine carbon, heteroa-

toms, and protonatable groups are showed. All hydrogens, except those

of the protonable nitrogens, have been omitted for clarity. Right: the

compounds are colored by AM1 charge distribution (the atoms are

colored from red (-0.5) to blue (+0.5) depending upon their partial

charge value).

2. ꢀ-Hematin Inhibitory Activity Assay. Two of the most

active compounds (2 and 4e) were screened for inhibition of

ꢀ-hematin formation by using the ꢀ-hematin inhibitory activity

(BHIA) assay.²⁵Both compounds showed a dose-dependent

inhibition of ꢀ-hematin formation (Figure 3 and Table 4).

Although compounds 2 and 4e were found much more potent

than CLT on different Pf strains, their in vitro BHIA was found

to be very similar to that of CLT, suggesting a similar interaction

with free heme. On the other hand, although with a similar

protonation state, CQ and 2 (Table 2) differ for their in vitro

antimalarial potency (2 >> CQ) and their inhibitory potency

in the BHIA assay (CQ >>2). This suggests that other

physicochemical characteristics of the molecules and/or a

different mechanism of action, besides inhibition of hemozoin

formation, may be responsible for killing the parasite. Com-

pound 4e, triprotonated at the FV pH (Table 2), proved to be

even less potent than CQ and 2 in the BHIA assay, while

maintaining a high potency against CQ-S and CQ-R strains.

3. Antifungal Activity. Selected compounds were also tested

to measure their antifungal activity (Table 1, Supporting

Information (SI)), as the lack of antifungal activity could be

predictive for a low propensity to interfere with cytochrome

P450 activity. CLT and the other antifungal azoles are known

to exert their activity by interfering with fungal cytochrome

P450s (such as 14-LD), and some of their side effects are linked

to interference with human cytochromes. On the basis of our

docking studies and bioinformatic analysis, the basic side chain

of 2 and analogues, besides to increase the pharmacokinetic

properties with respect to CLT, were also designed to improve

selectivity for free heme over heme as cytochromes P450

prosthetic group. To hit this mark, CLT was subjected to ﬂexible

the Trityl System), was endowed with a higher antimalarial

activity against both CQ-R and CQ-S strains than 4j, in perfect

agreement with its electronic properties. The higher potency of

4m might also be explained taking into account that the

7-chloroquinoline system may interact with heme, competing

with the imidazole. According to the SARs discussed in

paragraph (d), a further increase of activity was then obtained

by introducing a p-F substituent (4n) at the phenyl ring of 4m

(4n vs 4m). Nevertheless, contrary to what was observed for

compound 4d, the activity on the K1 strain of 4n is preserved

(Table 2), according to its partial charge distribution (Table 3).

(g). Achiral Antimalarials. Most of the compounds designed

and tested incorporate a chiral center that, when resolved into

the pure enantiomers, may cause an increase of synthetic costs,

thus compromising the potentiality of industrial development.

Consequently, to improve the drugability of these antimalarials,

we speciﬁcally removed the chiral center by introducing an extra

protonatable pyrrolidinylmethyl system (4e), maintaining sig-

niﬁcant FV penetration and accumulation and ameliorating the

solubility (2 logD: pH 7.4 ) 3.33, pH 7.2 ) 3.13, pH 5.5 (FV)

) 1.72; 4e logD: pH 7.4 ) 1.56, pH 7.2 ) 1.40, pH 5.5 (FV)

) 0.67). Similar to 2, compound 4e was found to be highly

potent against all tested strains and was more potent against

CQ-R than CQ-S strains. In addition, the presence of two

pyrrolidinylmethyl substituents decreased the electron density

1286 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5

Gemma et al.

different compounds.²⁸The data processing included the

Student’s t-test with p < 0.05 taken as signiﬁcance level.

Cytotoxicity of compounds 4d,i,l-n,p was assessed by using

KB cells, a cell line derived from a human carcinoma of the

nasopharynx. All compounds tested showed low cytotoxicity

in the MTT tests, thus demonstrating a good therapeutic index

(>300).

5. In Vivo Antimalarial Activity. Compound 2 and CLT

were tested in female CD1, 20–25 g mice infected with P.

berghei ANKA, while compounds 4e-g,n, together with 2 and

CLT, were tested in mice infected with P. chabaudi AS. For

both murine strains of Plasmodium, in vivo antimalarial activity

evaluation was performed according to the four-day suppressive

test of Peters et al.²⁹Compounds were orally or intraperitoneally

administered once daily for 4 days, with the ﬁrst drug

administered 2 h after i.v. parasite inoculation. Parasitaemia in

untreated control mice generally reached 40% for P. chabaudi

and 10% for P. berghei.

Table 5. Cytotoxicity Assays for Selected Compounds

TC₅₀(µM)^a

NSO

Daudi

cells^c

Normal human

lymphocytes^d

KB

cmpd

cells^b

cells^e

2

4b

4c

4d

4e

4g

4i

51.5

16.0

16.2

n.t.

47.0

52.9

n.t.

58.5

15.6

15.8

n.t.

58.7

57.5

n.t.

70.3

13.3

13.7

n.t.

68.5

80.5

n.t.

n.t.^f

n.t.

8.2

n.t.

27.2

64.2

51.8

33.0

20.9

88.1

207.0

4l

n.t.

4m

4n

4p

CLT

CQ

^aStandarderrorsneverexceeded5%ofthereportedvalues.^bPlasmocytoma

murine cell line. ^cHuman lymphoblastoid cell line. ^dNormal human

lymphocytes PHA-stimulated. ^eHuman carcinoma of the nasopharynx cell

line. ^fn.t. ) not tested.

As displayed in Table 6, at an oral dose of 150 mg/kg, CLT

only caused a 28% suppression of parasitaemia against P.

berghei, and, to our surprise, one of the most active compounds

in vitro against CQ-S strains, namely, 2, was inactive at an oral

dose of 30 mg/kg (data not shown) and only poorly active (37%)

after i.p. administration, while the control drug CQ was able to

suppress parasitaemia by 89% at a dose of 10 mg/kg. However,

we found that 2, but not CLT, was active in vivo against P.

chabaudi with parasitaemia suppression of 94.8% after oral

administration at a dose of 50 mg/kg. These rodent species

notably differ in their ability to invade mature or immature

erythrocytes and in their degrees of synchronism. At the

moment, we have no explanation for the different activity of 2

against these strains, and further studies are in progress to

address this question. However, it is of note that, in vitro,

compound 2 and its analogues generally display higher IC₅₀

values against synchronous 3D7 and K1 strains than asynchro-

nous D10 and W2 strains. Consequently, P. chabaudi was used

for the further in vivo evaluation of 2 analogues, and both 4e

and 4n demonstrated high antimalarial activity in vivo. Doses

of 50 mg/kg achieved 94.0 and 99.4% of parasitaemia suppres-

sion, respectively. On the other hand, at the same dose,

compound 4g showed 73.1% parasitaemia suppression, comple-

menting its lower in vitro antimalarial activity with respect to

4e and 4n.

Table 6. In Vivo Antimalarial Activity of Selected Compounds against

P. berghei and P. chabaudi after Oral Administration

% suppression on day 4^a(mg/Kg)

cmpd

P. chabaudi AS

P. berghei ANKA

2

4e

4g

4n

CLT (1)

CQ

94.8 (50)^b

94.0 (50)^b

73.1 (50)^b

99.4 (50)^b

6.0 (50)^b

37.6 (30)^c

n.t.^d

n.t.

28.3 (150)^b

89.1 (10)^b

100 (10)^b

^aPercent suppression ) [(C - T)/C] × 100; where C ) parasitaemia in

control group and T ) parasitaemia in treated group after oral/i.p.

administration. Five mice per group were used ^bOral administration.

^cIntraperitoneal administration. ^dn.t. ) not tested.

docking studies into 14-LD homology model²⁶of C. albicans.

At this purpose, it has to be underlined that the unfavorable

electronic and steric interactions with the residues present in

the active site of 14-LD may not be the only determinant for

substrate selectivity, as the external surface and the access

channel properties may play a key role.²⁷Indeed we considered

the presence of positively charged residues on the external

surface of the active site and in the putative substrate access

channel of 14-LD. Most of the positively charged residues

identiﬁed in C. albicans 14-LD, including the K108 at the active

site, are conserved in the other fungi P450 orthologs as well as

in the active site of CYP3A4, the most important enzyme for

drug metabolism in humans, where two arginine residues are

placed in the same region occupied by K108. Our studies led

to the hypothesis that the introduction of a protonatable group

could decrease interaction with heme as P450s prosthetic group.

These results were conﬁrmed by determination of the

antifungal activity of compounds 2, 4e, and 4n against a panel

of fungal species (Aspergillus spp., Candida spp., and one isolate

of Cryptococcus neoformans). As shown in the SI, Table 1,

none of the new compounds had any signiﬁcant activity against

these fungi, conﬁrming the selectivity for free heme as the key

factor for antimalarial activity of these innovative antimalarial

agents.

Conclusions

This work led to the discovery of potent antimalarial agents

structurally unrelated to known antiplasmodial drugs currently

used in therapy or under development. The design strategy was

focused on the development of polyaromatic compounds with

high antimalarial activity especially against CQ-R strains and

with high selectivity for free heme over P450 cytochrome

prosthetic groups. The in vitro and in vivo antimalarial activity

of this innovative class of antimalarials is dependent upon

several key structural features: (i) the protonatable lateral chain,

responsible for improved pharmacokinetic properties with

respect to CLT, for selectivity for free heme over P450

cytochrome heme and for higher FV accumulation; (ii) the

imidazole ring, with its dual role of heme-complexing group

and substrate for the redox reaction generating the trityl radical;

and (iii) the aryl/heteroaryl systems whose structural and

electronic properties are key factors to inﬂuence the stabilization

of a trityl radical intermediate.

4. Cytotoxicity: MTT Assays. Cytotoxicity for compounds

2 and 4b,c,e,g was evaluated using plasmocytoma murine cell

line (NSO cells), normal human lymphocytes PHA-stimulated,

and human lymphoblastoid cell line (Daudi cells). Data are

shown in Table 5 and were obtained by the 3-(4,5-dimethylthi-

azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test aimed

to analyze cell proliferation in cells cultured in the presence of

Exploiting this novel pharmacophore potent antimalarials

were identiﬁed, characterized by low in vitro toxicity against

Clotrimazole Scaffold as a Pharmacophore

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1287

was treated with water and extracted with CHCl₃. The combined

organic layers were washed with brine, dried over Na₂SO₄, and

evaporated. The residue was chromatographed (1% MeOH in DCM)

to afford 9 as a yellow oil (1.2 g, 74%): ¹H NMR (200 MHz,

CDCl₃) δ 7.75–7.71 (m, 3H), 7.65–7.61 (m, 1H), 7.55–7.51 (m,

1H), 7.46–7.39 (m, 3H), 3.56 (s, 2H), 3.43 (t, 4H, J ) 5.0 Hz),

2.39 (t, 4H, J ) 4.9 Hz), 1.43 (s, 9H); ESI MS m/z (M + H)⁺415.

different human and murine cell lines and with no activity

against fungal pathogens.

The most promising antimalarial compounds 2, 4e, and 4n

were further evaluated to assess their efﬁcacy in vivo and

resulted in activity against P. chabaudi in CD1 mice after oral

administration in the four-day suppressive test. The development

of these compounds is characterized by a limited number of

synthetic steps and by low cost of goods. The straightforward

synthetic pathway was further simpliﬁed with compound 4e,

where the chiral center was removed thus avoiding racemate

resolution and improving drugability.

In summary, both antimalarials 2 and 4e, which combine low

propensity to generate rapid resistance, potent in vitro and in

vivo activity against various plasmodia, and endowed with oral

bioavailability, prove to be promising candidates for early

preclinical development. These novel compounds meet the

requirements necessary for their potential development as

antimalarials in poverty-stricken regions where oral bioavail-

ability, stability of the formulation, and affordability are strict

selection criteria beside activity against drug-resistant strains

and rapid mode of action.

(()-(3-Chlorophenyl){4-[(N-tert-butoxycarbonylpiperazin-4-

yl)methyl]phenyl}methanol (7b). To a solution of 9 (0.746 g, 1.8

mmol) in ethanol (5 mL), sodium borohydride (140 mg, 3.7 mmol)

was added portionwise at 0 °C. The resulting mixture was stirred

at rt for 30 min and then treated with H₂O and concentrated under

reduced pressure. The residue was extracted with EtOAc, and the

combined organic layers were washed with brine, dried over

Na₂SO₄, and evaporated. The residue was chromatographed (2%

1

MeOH in DCM) to afford 7b as a yellow oil (540 mg, 72%): H

NMR (200 MHz, CDCl₃) δ 7.38–7.36 (m, 2H), 7.27–7.22 (m, 6H),

5.76 (s, 1H), 3.46 (s, 2H), 3.39 (t, 4H, J ) 4.9 Hz), 2.34 (t, 4H, J

) 5.0 Hz), 1.44 (bs, 1H), 1.42 (s, 9H); ESI MS m/z (M + H)⁺

417.

7-Chloroquinoline-4-carbaldehyde (11b). A solid mixture of

4,7-dichloroquinoline 10 (2.97 g, 15.1 mmol) and [1,2-bis(diphe-

nylphosphino)ethane]-dichloronikel(II) (60.0 mg, 0.114 mmol) was

evacuated and ﬂushed with argon several times. Dry diethylether

(40 mL) was then added and the solution was stirred at 0 °C.

Thereafter, a 3.0 M solution of methylmagnesium bromide in

diethylether (5.0 mL, 15.1 mmol) was added dropwise. The reaction

mixture was allowed to warm at rt and stirred for 24 h; thereafter,

it was poured into saturated NH₄Cl solution, and the ether layer

was separated, dried over Na₂SO₄, and evaporated under reduced

pressure to afford a crude solid that was puriﬁed by ﬂash

chromatography (15% EtOAc in Hex) to afford 4-methyl-7-

chloroquinoline as a white solid (1.5 g, 56%): mp 59–61 °C (lit.

mp 57–58 °C).¹⁶A stirred mixture of the above compound (1.17

g, 6.6 mmol) and selenium dioxide (1.17 g, 10.6 mmol) in

bromobenzene (16 mL) was heated to 170 °C for 18 h. The selenium

was ﬁltered off and washed with DCM and the ﬁltrate was

evaporated to dryness. The residue was taken up in DCM, ﬁltered

through celite, and evaporated to dryness, affording 11b as a

colorless crystalline solid (740 mg, 57%): mp (EtOH) 108–110 °C

(lit. mp 107–108 °C).³¹

Experimental Section

Chemistry. Reagents were purchased from Aldrich and were

used as received. Reaction progress was monitored by TLC using

Merck silica gel 60 F₂₅₄(0.040–0.063 mm) with detection by UV.

Merck silica gel 60 (0.040–0.063 mm) was used for column

chromatography. Melting points were determined in Pyrex capillary

tubes using an Electrothermal 8103 apparatus and are uncorrected.

¹H NMR and ¹³C NMR spectra were recorded on Bruker 200 MHz,

Varian 300 MHz, or Bruker 400 MHz spectrometer with TMS as

internal standard. Splitting patterns are described as singlet (s),

doublet (d), triplet (t), quartet (q), and broad (br); the value of

chemical shifts (δ) are given in ppm and coupling constants (J) in

Hertz (Hz). ESI-MS spectra were performed by an Agilent 1100

Series LC/MSD spectrometer. Elemental analyses were performed

in a Perkin-Elmer 240C elemental analyzer and the results were

within (0.4% of the theoretical values, unless otherwise noted.

Yields refer to puriﬁed products and are not optimized. All moisture-

sensitive reactions were performed under argon atmosphere using

oven-dried glassware and anhydrous solvents. All the organic layers

were dried using anhydrous sodium sulfate.

(()-(3-Chlorophenyl)[4-(N,N-diethylaminomethyl)phenyl]-

methanol (7a). To a stirred suspension of magnesium turnings (122

mg, 5.0 mmol) in anhydrous tetrahydrofurane (THF; 8.0 mL), a

solution containing N-(4-bromobenzyl)-N-ethylethanamine 6³⁰(1.32

g, 5.0 mmol) in dry THF (8.0 mL) was slowly added. The reaction

mixture was stirred and heated under reﬂux until all magnesium

was consumed (1–2 h). Thereafter, the reaction was cooled to 0

°C and a solution of 3-chlorobenzaldehyde 5 (506 mg, 3.6 mmol)

in 10 mL of anhydrous THF was added during 2 h, maintaining

the temperature between -5 and 0 °C. The reaction mixture was

quenched by the dropwise addition of 20% ammonium chloride

solution (30 mL). The layers were separated and the aqueous layer

was extracted with EtOAc. The organic layer was washed with 10%

Na₂CO₃solution and dried over Drierite. Evaporation of the solvent

afforded a residue, which was puriﬁed by ﬂash chromatography

(2% MeOH in DCM) to give 7a as a light yellow oil (1.0 g, 92%):

¹H NMR (200 MHz, CDCl₃) δ 7.38–7.21 (m, 8H), 5.67 (s, 1H),

3.79 (bs, 1H), 3.52 (s, 2H), 2.47 (q, 4H, J ) 7.1 Hz), 1.01 (t, 6H,

J ) 7.1 Hz); ESI MS m/z (M + H)⁺304.

(()-(3-Chlorophenylphenyl)(quinolin-4-yl)methanol (13). A

solution of 11a (0.487 g, 3.1 mmol) in dry THF (25 mL) was added

dropwise to a solution of 3-chlorophenylmagnesium bromide (0.5

M in THF, 3.1 mmol, 6.2 mL), and the resulting solution was heated

to 75 °C for 6 h. The reaction mixture was quenched with 20%

ammonium chloride solution. The aqueous layer was extracted with

EtOAc, and the combined organic layers were washed with brine,

dried over Na₂SO₄, and evaporated under reduced pressure. The

crude residue was puriﬁed by ﬂash chromatography (2% MeOH

1

in CHCl₃) to give 13 as a colorless oil (0.40 g, 48%): H NMR

(200 MHz, CDCl₃) δ 8.77 (d, 1H, J ) 8.0 Hz), 8.10 (d, 1H, J )

6.0 Hz), 7.92 (d, 1H, J ) 8.1 Hz), 7.68–7.45 (m, 3H), 7.40–7.12

(m, 4H), 6.39 (s, 1H), 3.55 (bs, 1H); ESI MS m/z (M + H)⁺270.

(()-[4-(Pyrrolidin-1-ylmethyl)phenyl](quinolin-4-yl)meth-

anol (14a). To a stirred solution of 12 (0.624 g, 2.6 mmol) in dry

THF (15 mL) was added dropwise n-butyllithium (1.6 M in Hex,

1.8 mL, 2.9 mmol) at -78 °C and was further stirred at -60 °C

for 3 h. Thereafter, a solution of 4-quinolinecarbaldehyde (0.456

g, 2.9 mmol) in dry THF was added dropwise, and the temperature

was maintained at -60 °C for 2 h. Subsequently, the reaction

mixture was allowed to cool to rt, stirred for 24 h, and ﬁnally

quenched with a saturated solution of NH₄Cl. The aqueous layer

was extracted with EtOAc and the combined organic layers were

washed with brine, dried over Na₂SO₄, and evaporated under

reduced pressure. The crude residue was puriﬁed by ﬂash chro-

matography (6% MeOH in DCM) to afford 14a as a brown oil

(0.27 g, 32%): ¹H NMR (200 MHz, CDCl₃) δ 8.80 (d, 1H, J ) 4.2

Hz), 8.06 (d, 1H, J ) 8.2 Hz), 7.90 (d, 1H, J ) 8.2 Hz), 7.68–7.60

(m, 2H), 7.56–7.29 (m, 1H), 7.24–7.20 (m, 4H), 6.41 (s, 1H), 3.71

(()-(3-Chlorophenyl){4-[(N-tert-butoxycarbonylpiperazin-4-

yl)methyl]phenyl}methanone (9). To a stirred solution of 8⁶(1.21

g, 3.9 mmol) in dry MeCN (15 mL) at 0 °C was added a solution

of N-Boc-piperazine (0.860 g, 4.6 mmol) and Et₃N (0.64 mL, 4.6

mmol) in MeCN (5 mL), and the reaction mixture was allowed to

stir for 2 h. Thereafter, the reaction was quenched with H₂O and

the solvent was evaporated under reduced pressure. The residue

1288 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5

Gemma et al.

(bs, 1H), 3.52 (s, 2H), 2.42 (m, 4H), 1.71 (m, 4H); ESI MS m/z

(M + H)⁺319.

stirred at 0 °C for 20 min and thereafter at rt for 5 h. After

evaporation of the solvent, the residue was dissolved in EtOAc and

the organic layer was washed with saturated NaHCO₃, H₂O, and

brine and dried over Na₂SO₄. The crude product was puriﬁed by

ﬂash chromatography (2% MeOH in DCM) to afford 4-{chloro[4-

(pyrrolidin-1-ylmethyl) phenyl]methyl} quinoline as a yellow oil

(0.33 g, 78%): ¹H NMR (200 MHz, CDCl₃) δ 8.92 (d, 1H, J ) 4.3

Hz), 8.13 (d, 1H, J ) 7.8 Hz), 7.96 (d, 1H, J ) 8.0 Hz), 7.70–7.58

(m, 2H), 7.58–7.45 (m, 1H), 7.36–7.24 (m, 4H), 6.77 (s, 1H), 3.56

(s, 2H), 2.46 (m, 4H), 1.73 (m, 4H); ESI MS m/z (M + H)⁺337.

To a stirred solution of the above compound (250 mg, 0.74 mmol)

in dry DMF (1 mL) was added imidazole sodium salt (136 mg, 1.5

mmol), and the reaction mixture was heated to 80 °C for 3 h. The

solvent was evaporated under reduced pressure and the residue was

treated with H₂O and extracted with EtOAc. The combined organic

layers were washed with brine, dried over Na₂SO₄, and evaporated

under reduced pressure. The crude residue was puriﬁed by ﬂash

chromatography (2% MeOH in DCM) to give 3f as a brown oil

(()-[4-(Pyrrolidin-1-ylmethyl)phenyl](7-chloroquinolin-4-yl)-

methanol (14b). Starting from 11b (170 mg, 0.89 mmol), the title

compound was prepared following the above-described procedure

1

and was obtained as a brown oil (62.0 mg, 20%): H NMR (200

MHz, CDCl₃) δ 8.87 (d, 1H, J ) 4.5 Hz), 8.06 (d, 1H, J ) 1.9

Hz), 7.83 (d, 1H, J ) 9.0 Hz), 7.67 (d, 1H, J ) 4.3 Hz), 7.38–7.25

(m, 5H), 6.36 (s, 1H), 3.68 (bs, 1H), 3.54 (s, 2H), 2.45 (m, 4H),

1.73 (m, 4H); ESI MS m/z (M + H)⁺353.

(()-1-{(3-Chlorophenyl)[4-(N,N-diethylaminomethyl)phenyl]-

methyl}-1H-imidazole (3a). To a solution of 7a (75.9 mg, 0.25

mmol) and a drop of DMF in dry DCM (10 mL) was added SOCl₂

(63.5 µL, 0.87 mmol) in dry DCM (2 mL) at 0 °C, and the mixture

was stirred at the same temperature for 20 min and thereafter at

room temperature for 3.5 h. The volatiles were removed, and the

residue was washed with dry MeCN and concentrated under reduced

pressure to remove residual SOCl₂. The resulting hydrochloride salt

(89.7 mg, 0.25 mmol) was suspended in dry MeCN (5 mL) and to

this suspension was slowly added a solution containing Et₃N (70.0

µL, 0.50 mmol) and imidazole (34.1 mg, 0.50 mmol) in MeCN (5

mL) at 0 °C. Thereafter, the reaction mixture was heated to 80 °C

for 4 h. The solvent was evaporated under reduced pressure, and

the residue was treated with H₂O and extracted with EtOAc. The

combined organic layers were dried over Na₂SO₄and concentrated

in vacuo. The crude product was puriﬁed by ﬂash column

chromatography (1% MeOH in DCM) to afford 3a as a brown oil

(60.2 mg, 68%): ¹H NMR (200 MHz, CDCl₃) δ 7.39–7.27 (m, 5H),

7.09–6.94 (m, 3H), 6.82 (s, 3H), 6.45 (s, 1H), 3.56 (s, 2H), 2.52

(q, 4H, J ) 7.0 Hz), 1.03 (t, 6H, J ) 7.0 Hz); MS m/z (M + H)⁺

354. Anal. (C₂₁H₂₄ClN₃) C, H, N.

1

(140 mg, 52%): H NMR (200 MHz, CDCl₃) δ 8.84 (d, 1H, J )

4.0 Hz), 8.14 (d, 1H, J ) 8.2 Hz), 7.72–7.64 (m, 2H), 7.48–7.30

(m, 4H), 7.25–7.05 (m, 4H), 6.81 (s, 1H), 6.71 (d, 1H, J ) 4.5

Hz), 3.58 (s, 2H), 2.47 (m, 4H), 1.75 (m, 4H); ESI MS m/z (M +

H)⁺369. Anal. (C₂₄H₂₄N₄) C, H, N.

(()-7-Chloro-4-{(1H-Imidazol-1-yl)[4-(pyrrolidin-1-ylmeth-

yl)phenyl]methyl} quinoline (3g). Starting from 14b (35.0 mg,

0.099 mmol), the title compound was prepared following the above-

described procedure and was obtained as a brown oil (22.0 mg,

1

55%): H NMR (200 MHz, CDCl₃) δ 8.85 (d, 1H, J ) 4.0 Hz),

8.14 (s, 1H), 7.64 (d, 1H, J ) 9.0 Hz), 7.42–7.32 (m, 4H), 7.25–7.04

(m, 4H), 6.81 (s, 1H), 6.69 (d, 1H, J ) 4.3 Hz), 3.60 (s, 2H), 2.49

(m, 4H), 1.76 (m, 4H); ESI MS m/z (M + H)⁺403. Anal.

(C₂₄H₂₃ClN₄) C, H, N.

(()-1-{(3-Chlorophenyl)[4-(N,N-diethylaminomethyl)phenyl]-

methyl}-1H-1,2,4-triazole (3b). Starting from 7a (66.8 mg, 0.22

mmol), the title compound was prepared following the above-

described procedure and was obtained as brown oil (52.0 mg, 68%):

¹H NMR (200 MHz, CDCl₃) δ 8.01 (s, 1H), 7.86 (s, 1H), 7.39–7.28

(m, 5H), 7.08–7.01 (m, 3H), 6.93–6.89 (m, 1H), 3.56 (s, 2H), 2.51

(q, 4H, J ) 7 Hz), 1.03 (t, 6H, J ) 6.9 Hz); ESI MS m/z (M +

H)⁺355. Anal. (C₂₀H₂₃ClN₄) C, H, N.

(4-Chlorophenyl)[4-(diethylaminomethyl)phenyl]methanone-

(16a). To a stirred solution of 15 (3.5 g, 11.3 mmol) in dry MeCN

(75 mL), cooled to 0 °C, diethylamine (1.41 mL, 16.9 mmol) and

triethylamine (3.15 mL, 22.6 mmol) were added, and the resulting

mixture was allowed to stir for 1 h at 0 °C. Thereafter, the reaction

was quenched with H₂O and the solvent was evaporated under

reduced pressure. The residue was treated with H₂O and extracted

with CHCl₃. The combined organic layers were washed with brine,

dried over Na₂SO₄, and evaporated. The residue was chromato-

graphed (2% MeOH in DCM) to afford 16a as a yellow oil (2.8 g,

82%): ¹H NMR (300 MHz, CDCl₃) δ 7.74–7.70 (m, 4H), 7.47–7.41

(m, 4H), 3.62 (s, 2H), 2.53 (q, 4H, J ) 7.0 Hz), 1.04 (t, 6H, J )

7.0 Hz); ESI MS m/z (M + H)⁺302.

(4-Chlorophenyl){4-[(morpholin-4-yl)methyl]phenyl}meth-

anone (16b). Starting from 15 (3.00 g, 9.7 mmol) and morpholine

(1.02 mL, 11.6 mmol), the title compound was prepared following

the above-described procedure and was obtained as a white low

melting amorphous solid (2.5 g, 81%): ¹H NMR (300 MHz, CDCl₃)

δ 7.76–7.72 (m, 4H), 7.47–7.44 (m, 4H), 3.74–3.71 (m, 4H), 3.57

(s, 2H), 2.48–2.45 (m, 4H); ESI MS m/z (M + 1)⁺316.

(4-Chlorophenyl){4-[1-(tert-butoxycarbonyl)piperazin-4-

ylmethyl]phenyl}methanone (16c). Starting from 15 (2.88 g, 9.3

mmol) and N-Boc-piperazine (2.08 g, 11.2 mmol), the title

compound was prepared following the above-described procedure

and was obtained as a white low melting amorphous solid (2.0 g,

51%): ¹H NMR (300 MHz, CDCl₃) δ 7.73–7.69 (m, 4H), 7.43–7.40

(m, 4H), 3.55 (s, 2H), 3.43–3.40 (m, 4H), 2.40–2.37 (m, 4H), 1.42

(s, 9H); ESI MS m/z (M + H)⁺415.

(()-(4-Chlorophenyl)[4-(diethylaminomethyl)phenyl]phe-

nylmethanol (17a). The Grignard reagent was prepared in the usual

manner as described for 7a from bromobenzene (0.60 mL, 5.7

mmol), magnesium turnings (138 mg, 5.7 mmol), and a catalytic

amount of iodine in dry THF (35 mL). A solution of 16a (1.15 g,

3.8 mmol) in dry THF (25 mL) was added dropwise to the Grignard

reagent, and the resulting solution was heated to 75 °C for 6 h.

The reaction mixture was quenched with 20% ammonium chloride

solution. The aqueous layer was extracted with EtOAc, and the

combined organic layers were washed with brine, dried over

Na₂SO₄, and evaporated under reduced pressure. The crude residue

(()-1-{(3-Chlorophenyl)[4-[(N-tert-butoxycarbonylpiperazin-

4-yl)methyl]phenyl]methyl}-1H-imidazole (3c). Starting from 7b

(0.54 g, 1.3 mmol), the title compound was prepared following the

above-described procedure and was obtained as brown oil (0.37 g,

61%): ¹H NMR (200 MHz, CDCl₃) δ 7.42 (s, 1H), 7.37–7.21 (m,

4H), 7.04–6.93 (m, 5H), 6.80 (s, 1H), 6.43 (s, 1H), 3.47 (s, 2H),

3.39 (t, 4H, J ) 4.7 Hz), 2.35 (t, 4H, J ) 4.7 Hz), 1.42 (s, 9H);

ESI MS m/z (M + H)⁺467. Anal. (C₂₆H₃₁ClN₄O₂) C, H, N.

(()-1-{(3-Chlorophenyl)[4-[(piperazin-1-yl)methyl]phenyl]-

methyl}-1H-imidazole (3d). A solution of 3c (98.1 mg, 0.21 mmol)

in a 1:1 TFA/DCM mixture (3.5 mL) was stirred at 0–5 °C for 3 h.

Then the solvent was evaporated and the residue was treated with

2.5 M NaOH. The aqueous phase was extracted with DCM, and

the organic layer was dried over Na₂SO₄and concentrated in vacuo.

The crude residue was puriﬁed by ﬂash column chromatography

(8% MeOH in CHCl₃) to afford 3d as a yellow oil (67.0 mg, 87%):

¹H NMR (200 MHz, CDCl₃) δ 7.37 (s, 1H), 7.32–7.22 (m, 4H),

7.08–6.91 (m, 5H), 6.81 (s, 1H), 6.43 (s, 1H), 3.47 (s, 2H), 2.87 (t,

4H, J ) 4.7 Hz), 2.39 (t, 4H, J ) 4.3 Hz); 2.22 (bs, 1H); ESI MS

m/z (M + H)⁺367. Anal. (C₂₁H₂₃ClN₄) C, H, N.

(()-4-[(1H-Imidazol-1-yl)(3-chlorophenyl)methyl]quinoline

(3e). Starting from 13 (0.38 g, 1.4 mmol), the title compound was

prepared following the above-described procedure and was obtained

1

as pale yellow oil (0.29 g, 65%): H NMR (200 MHz, CDCl₃) δ

8.88 (d, 1H, J ) 8.0 Hz), 8.14 (d, 1H, J ) 6.0 Hz), 7.94 (d, 1H,

J ) 8.1 Hz), 7.68–7.45 (m, 3H), 7.43–7.10 (m, 6H), 6.89 (s, 1H),

6.74 (d, 1H, J ) 4.4 Hz); ESI MS m/z (M + H)⁺320. Anal.

(C₁₉H₁₄ClN₃) C, H, N.

(()-4-{(1H-Imidazol-1-yl)[4-(pyrrolidin-1-ylmethyl)phenyl]-

methyl}quinoline (3f). To a solution of 14a (0.382 g, 1.2 mmol)

and a drop of DMF in dry DCM (5 mL) was added SOCl₂(263

µL, 3.6 mmol) in dry DCM (5 mL) at 0 °C, and the mixture was

Clotrimazole Scaffold as a Pharmacophore

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1289

was puriﬁed by ﬂash chromatography (2% MeOH in CHCl₃) to

give 16a as a yellow oil (220 mg, 45%): ¹H NMR (300 MHz,

CDCl₃) δ 7.32–7.16 (m, 13H), 3.59 (m, 2H), 3.0 (bs, 1H), 2.55 (q,

4H, J ) 7.0 Hz), 1.06 (t, 6H, J ) 7.0 Hz); ESI MS m/z (M + H)⁺

379.

(()-(4-Chlorophenyl){4-[(morpholino-4-yl)methyl]phenyl}-

phenylmethanol (17b). Starting from 16b (0.347 g, 1.1 mmol),

the title compound was prepared following the above-described

procedure and was obtained as a yellow oil (0.35 g, 81%): ¹H NMR

(300 MHz, CDCl₃) δ 7.29–7.18 (m, 13H), 3.64–3.61 (m, 4H), 3.46

(s, 2H), 2.41–2.38 (m, 4H); ESI MS m/z (M+H)⁺394.

(()-(4-Chlorophenyl){4-[1-(tert-butoxycarbonyl)piperazin-4-

ylmethyl]phenyl}phenylmethanol (17c). Starting from 16c (0.661

g, 1.6 mmol), the title compound was prepared following the above-

described procedure and was obtained as a yellow oil (0.40 g, 51%):

¹H NMR (300 MHz, CDCl₃) δ 7.28–7.17 (m, 13H), 3.47 (s, 2H),

3.40–3.37 (m, 4H), 2.37–2.34 (m, 4H), 1.44 (s, 9H); ESI MS m/z

(M + H)⁺493.

(()-(4-Chlorophenyl)(4-ﬂuorophenyl)[4-(pyrrolidin-1-ylmeth-

yl)phenyl]methanol (18a). Starting from 16d⁶(1.11 g, 3.7 mmol)

and p-ﬂuorobromobenzene, the title compound was prepared as

described for 17a and was obtained as a brown oil (0.94 g, 65%):

¹H NMR (300 MHz, CDCl₃) δ 7.33–7.16 (m, 10H), 7.01–6.99 (m,

2H), 3.66 (s, 2H), 2.78 (bs, 1H), 2.58 (m, 4H), 1.83–1.82 (m, 4H);

ESI MS m/z (M + H)⁺396.

(4-Chlorophenyl)bis[4-(pyrrolidin-1-ylmethyl)phenyl]meth-

anol (18b). Starting from 16d⁶(222 mg, 0.74 mmol) and 12,⁶the

title compound was prepared following the above-described pro-

cedure and was obtained as a brown oil (240 mg, 70%): ¹H NMR

(300 MHz, CDCl₃) δ 7.27–7.15 (m, 12H), 3.59 (s, 4H), 3.59 (bs,

1H), 2.50–2.47 (m, 8H), 1.78–1.76 (m, 8H); ESI MS m/z (M +

H)⁺461.

(3-Chlorophenyl)(thien-2-yl)methanone (20a). To a stirred

solution of 2-thiophenecarbaldehyde 19a (2.38 g, 10.7 mmol) in

dry THF (10 mL) was added dropwise 3-clorophenylmagnesium-

bromide (0.5 N solution in THF, 10.6 mL, 5.3 mmol), and the

mixture was reﬂuxed for 3 h. After cooling to rt, the reaction

mixture was quenched by dropwise addition of a saturated solution

of NH₄Cl. The aqueous phase was extracted with ethyl acetate and

washed with brine. The combined organic layers were dried over

Na₂SO₄and evaporated. The crude residue was puriﬁed by ﬂash

chromatography (20% EtOAc in Hex) to afford the title compound

as colorless prisms (0.99 g, 84%): mp (n-Hex) 95–96 °C; ¹H NMR

(300 MHz, CDCl₃) δ 7.82–7.80 (m, 1H), 7.75–7.70 (m, 2H),

7.63–7.61 (m, 1H), 7.56–7.52 (m, 1H), 7.45–7.42 (m, 1H),

7.18–7.15 (m, 1H); ESI MS m/z (M + H)⁺223.

19e (1.0 g, 9.3 mmol), the title compound was prepared following

the above-described procedure and was obtained as colorless prisms

(0.63 g, 64%): mp (n-Hex) 99–100 °C; ¹H NMR (300 MHz, CDCl₃)

δ 8.82 (d, 1H, J ) 1.7 Hz), 8.80 (d, 1H, J ) 1.5 Hz), 7.79–7.77

(m, 1H), 7.67–7.64 (m, 1H), 7.62–7.58 (m, 1H), 7.56–7.54 (m, 2H),

7.47–7.41 (m, 1H); MS m/z (M + H)⁺218.

(4-Chlorophenyl)(thien-2-yl)methanone (21a). Starting from

4-chlorophenylmagnesium bromide and 2-thiophenecarbaldehyde

19a (1.98 g, 17.8 mmol), the title compound was prepared following

the above-described procedure and was obtained as colorless prisms

(1.6 g, 80%): mp (n-Hex) 91–92 °C; ¹H NMR (CDCl₃) δ 7.84–7.79

(m, 2H), 7.74 (dd, 1H, J ) 1.1 Hz, 4.7 Hz), 7.62 (dd, 1H, J ) 1.1

Hz, 3.8 Hz), 7.49–7.45 (m, 2H), 7.16 (dd, 1H, J ) 3.8 Hz, 4.7

Hz); ESI MS m/z (M + 1)⁺223.

(4-Chlorophenyl)(thiazol-2-yl)methanone (21b). Starting from

4-chlorophenylmagnesium bromide and 2-thiazolecarbaldehyde 19b

(2.50 g, 22.1 mmol), the title compound was prepared following

the above-described procedure and was obtained as colorless prisms

(2.3 g, 91%): mp (n-Hex) 79–80 °C; ¹H NMR (300 MHz, CDCl₃)

δ 8.44–8.41 (m, 2H), 7.98 (d, 1H, J ) 2.7 Hz), 7.52 (d, 1H, J )

3.0 Hz), 7.45–7.42 (m, 2H); ESI MS m/z (M + H)⁺224.

(()-(3-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](thien-

2-yl)methanol (22a). Starting from 12, magnesium turnings, and

20a (289 mg, 1.30 mmol), the title compound was prepared as

described for 17a and was obtained as colorless prisms (340 mg,

66%): mp (EtOAc/n-Hex) 70–71 °C; ¹H NMR (300 MHz, CDCl₃)

δ 7.43–7.42 (m, 1H), 7.38–7.22 (m, 8H), 6.96–6.93 (m, 1H),

6.72–6.71 (m, 1H), 3.73 (s, 2H), 3.10 (bs, 1H), 2.57–2.53 (m, 4H),

1.79–1.75 (m, 4H); ESI MS m/z (M + H)⁺384.

(()-(3-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](thiazol-

2-yl)methanol (22b). Starting from 12, magnesium turnings, and

20b (1.21 g, 5.4 mmol), the title compound was prepared following

the above-described procedure and was obtained as colorless prisms

1

(0.9 g, 46%): mp (EtOAc/n-Hex) 73–74 °C; H NMR (200 MHz,

CDCl₃) δ 7.81 (d, 1H, J ) 3.2 Hz), 7.47 (s, 1H), 7.33 (d, 1H, J )

3.2 Hz), 7.31–7.25 (m, 7H), 4.24 (br, 1H), 3.62 (s, 2H), 2.54–2.49

(m, 4H), 1.80–1.76 (m, 4H); ESI-MS m/z (M + H)⁺385.

(()-(3-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](furan-

3-yl)methanol (22c). Starting from 12, magnesium turnings, and

20c (268 mg, 1.3 mmol), the title compound was prepared following

the above-described procedure and was obtained as yellow prisms

(300 mg, 26%): mp (EtOAc/n-Hex) 80–81 °C; ¹H NMR (300 MHz,

CDCl₃) δ 7.44–7.43 (m, 1H), 7.41–7.40 (m, 1H) 7.26 (m, 5H),

7.23- 7.22 (m, 3H), 7.05 (dd, 1H, J ) 1.4, 0.9 Hz), 6.29 (dd, 1H,

J ) 1.6, 0.9 Hz), 3.57 (s, 2H), 3.22 (br, 1H), 2.50–2.46 (m, 4H),

1.76–1.72 (m, 4H); ESI MS m/z (M + H)⁺368.

(3-Chlorophenyl)(thiazol-2-yl)methanone (20b). Starting from

3-chlorophenylmagnesium bromide and 2-thiazolecarbaldehyde 19b

(2.45 g, 22.1 mmol), the title compound was prepared following

the above-described procedure and was obtained as colorless prisms

(2.3 g, 93%): mp (n-Hex) 87–88 °C; ¹H NMR (300 MHz, CDCl₃)

δ 8.41–8.39 (m, 2H), 8.03 (d, 1H, J ) 2.9 Hz), 7.66 (d, 1H, J )

2.9 Hz), 7.47–7.44 (m, 2H); ESI MS m/z (M + H)⁺224.

(3-Chlorophenyl)(furan-3-yl)methanone (20c). Starting from

3-chlorophenylmagnesium bromide and 3-furancarbaldehyde 19c

(1.47 g, 15.3 mmol), the title compound was prepared following

the above-described procedure and was obtained as colorless prisms

(0.70 g, 45%): mp (n-Hex) 94–95 °C; ¹H NMR (300 MHz, CDCl₃)

δ 7.93 (dd, 1H, J ) 1.4, 1.0 Hz), 7.83–7.81 (m, 1H), 7.74–7.71

(m, 1H), 7.58–7.54 (m, 1H), 7.53–7.51 (m, 1H), 7.46–7.40 (m, 1H),

6.89 (dd, 1H, J ) 1.7, 1.0 Hz); ESI MS m/z (M + H)⁺207.

(3-Chlorophenyl)(pyridin-2-yl)methanone (20d). Starting from

3-chlorophenylmagnesium bromide and 2-pyridinecarboxaldehyde

19d (1.00 g, 9.3 mmol), the title compound was prepared following

the above-described procedure and was obtained as colorless prisms

(0.40 g, 40%): mp (n-Hex) 82–83 °C; ¹H NMR (300 MHz, CDCl₃)

δ 8.81–8.78 (m, 2H), 7.74–7.72 (m, 1H), 7.60–6.98 (m, 1H),

7.59–7.52 (m, 1H), 7.56–7.52 (m, 2H), 7.45–7.38 (m, 1H); MS

m/z (M + 1)⁺218; ESI MS m/z (M + H)⁺.

(()-(3-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](pyri-

din-2-yl)methanol (22d). Starting from 12, magnesium turnings,

and 20d (413 mg, 1.9 mmol), the title compound was prepared

following the above-described procedure and was obtained as

colorless prisms (340 mg, 55%): mp (EtOAc/n-Hex) 68–69 °C;

¹H NMR (300 MHz, CDCl₃) δ ¹H NMR (300 MHz, CDCl₃) δ

8.56 (m, 1H), 7.65–7.59 (m, 1H), 7.37–7.01 (m, 10H), 6.36 (br,

1H), 3.60 (s, 2H), 2.52–2.48 (m, 4H), 1.78–1.74 (m, 4H); ESI-MS

m/z (M + H)⁺379.

(()-(3-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](pyri-

din-4-yl)methanol (22e). Starting from 12, magnesium turnings,

and 20e (413 mg, 1.9 mmol), the title compound was prepared

following the above-described procedure and was obtained as

colorless prisms (375 mg, 52%): mp (EtOAc/n-Hex) 75–76 °C;¹H

NMR (300 MHz, CDCl₃) δ 8.44–8.42 (m, 2H), 7.33–7.20 (m, 7H),

7.16–7.09 (m, 3H), 3.62 (s, 2H), 2.56–2.52 (m, 4H), 1.80–1.76 (m,

4H); ESI MS m/z (M + H)⁺379.

(()-(4-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](thien-

2-yl)methanol (23a). Starting from 12, magnesium turnings, and

21a (623 mg, 2.8 mmol), the title compound was prepared following

the above-described procedure and was obtained as colorless prisms

(860 mg, 80%): mp (EtOAc/n-Hex) 69–70 °C; ¹H NMR (300 MHz,

CDCl₃) δ 7.35–7.34 (m, 1H), 7.32–7.31 (m, 1H), 7.28–7.24 (m,

7H), 6.92 (dd, 1H, J ) 3.5, 4.9 Hz), 6.70 (dd, 1H, J ) 1.1, 3.5

(3-Chlorophenyl)(pyridyl-4-yl)methanone (20e). Starting from

3-chlorophenylmagnesium bromide and 4-pyridinecarboxaldehyde

1290 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5

Gemma et al.

Hz), 4.39 (br s, 1H), 3.56 (s, 2H), 2.46–2.44 (m, 4H), 1.73–1.70

(m, 4H); ESI-MS m/z (M + H)⁺385.

(()-1-{(4-Chlorophenyl)[4-(N,N-diethylaminomethyl)phe-

nyl]phenylmethyl}-1H-imidazole (4a). To a solution of 17a (323

mg, 0.85 mmol) and a drop of DMF in dry DCM (15 mL), cooled

to 0 °C, was added SOCl₂(180 µL, 2.5 mmol) in dry DCM (10

mL), and the mixture was stirred at 0 °C for 20 min and thereafter

was heated to 45 °C for 4 h. The volatiles were removed and the

residue was treated with dry MeCN and concentrated under reduced

pressure to remove residual SOCl₂. The resulting hydrochloride salt

(360 mg, 0.85 mmol) was suspended in dry MeCN (20 mL) and to

this solution was slowly added a solution containing Et₃N (580

µL, 4.2 mmol) and imidazole (280 mg, 4.2 mmol) in MeCN (10

mL) at 0 °C. Thereafter, the reaction mixture was heated to 80 °C

for 4 h. The solvent was evaporated under reduced pressure and

the residue was treated with water and extracted with EtOAc. The

combined organic extracts were dried over Na₂SO₄and concen-

trated. The crude residue was puriﬁed by ﬂash column chroma-

tography (2% MeOH in CHCl₃) to afford 4a as a yellow oil (30.0

mg, 26% yield): ¹H NMR (200 MHz, CDCl₃) δ 7.43 (s, 1H),

7.32–7.27 (m, 7H), 7.13–7.04 (m, 7H), 6.79 (s, 1H), 3.58 (s, 2H),

2.55 (q, 4H, J ) 7.0 Hz), 1.05 (t, 6H, J ) 7.0 Hz); ESI-MS m/z

(M - imidazole)⁺364. Anal. (C₂₇H₂₈ClN₃) C, H, N.

(()-(4-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](thiazol-

2-yl)methanol (23b). Starting from 12, magnesium turnings, and

21b (1.21 g, 5.4 mmol), the title compound was prepared following

the above-described procedure and was obtained as colorless prisms

1

(1.1 g, 55%): mp (EtOAc/n-Hex) 78–79 °C; H NMR (300 MHz,

CDCl₃) δ 7.70 (d, 1H, J ) 3.2 Hz), 7.34–7.38 (m, 2H), 7.26–7.21

(m, 7H), 5.61 (br, 1H), 3.53 (s, 2H), 2.42 (m, 4H), 1.70 (m, 4H).

ESI-MS m/z (M + 1)⁺385.

(()-2-(7-Chloroquinolin-4-yl)-2-phenylacetonitrile (25a). A

60% oil dispersion of NaH (70.0 mg, 1.74 mmol) was washed with

Hex and suspended in THF; thereafter, benzylcyanide (0.17 mL,

1.5 mmol) in dry THF (10 mL) was added, and the mixture was

heated under reﬂux for 30 min. Thereafter, compound 24 (0.15 g,

0.77 mmol) was added and the mixture was reﬂuxed until

completion. The solvent was evaporated under reduced pressure,

the residue was treated with H₂O, neutralized with 5% acetic acid,

and extracted with CHCl₃. The organic extracts were dried over

Na₂SO₄, the solvent was removed, and the residue was puriﬁed by

ﬂash chromatography, eluting (15% EtOAC in Hex) to afford 25a

(0.18 g, 85%) as a viscous yellow oil. ¹H NMR (300 MHz, CDCl₃)

δ 8.93 (d, 1H, J ) 4.5 Hz), 8.12 (d, 1H, J ) 2.1 Hz), 7.81 (d, 1H,

J ) 8.7 Hz), 7.52–7.43 (m, 2H), 7.37–7.26 (m, 5H), 5.77 (s, 1H);

ESI-MS m/z (M + H)⁺279.

(()-1-{(4-Chlorophenyl)[4-(morpholin-4-ylmethyl)phenyl]phe-

nylmethyl}-1H-imidazole (4b). Starting from 17b (351 mg, 0.89

mmol), the title compound was prepared following the above-

described procedure and was obtained as a light yellow viscous oil

(260 mg, 66%): ¹H NMR (300 MHz, CDCl₃) δ 7.41 (s, 1H),

7.31–7.25 (m, 7H), 7.10–7.03 (m, 7H), 6.77 (m, 1H), 3.68 (m, 4H),

3.47 (s, 2H), 2.42 (m, 4H); ESI MS m/z (M - imidazole)⁺376.

Anal. (C₂₇H₂₆ClN₃O) C, H, N.

(()-2-(7-Chloroquinolin-4-yl)-2-(4-ﬂuorophenyl)acetonitrile

(25b). Starting from 24 (0.45 g, 2.3 mmol) and 4-ﬂuorophenylac-

etonitrile (0.55 mL, 4.6 mmol), the title compound was prepared

following the above-described procedure and was obtained as a

1

viscous yellow oil (0.58 g, 85%): H NMR (300 MHz, CDCl₃) δ

(()-1-{(4-Chlorophenyl)[4-(piperazin-4-ylmethyl)phenyl]phe-

nylmethyl}-1H-imidazole (4c). Starting from 17c (286 mg, 0.58

mmol), the title compound was prepared following the above-

described procedure and was obtained as a brown amorphous solid

(138 mg, 53%): ¹H NMR (300 MHz, CDCl₃) δ 7.40 (s, 1H),

7.30–7.24 (m, 8H), 7.09–7.01 (m, 6H), 6.76 (m, 1H), 3.46 (s, 2H),

2.84 (m, 4H), 2.38 (m, 4H), 1.73 (br, 1H); ESI MS m/z (M -

imidazole)⁺375. Anal. (C₂₇H₂₇ClN₄) C, H, N.

8.97 (d, 1H, J ) 4.8 Hz), 8.17 (d, 1H, J ) 2.4 Hz), 7.78 (d, 1H,

J ) 9.0 Hz), 7.53–7.49 (m, 2H), 7.33–7.26 (m, 2H), 7.11–7.04 (m,

2H), 5.75 (s, 1H); ESI-MS m/z (M + H)⁺297.

(7-Chloroquinolin-4-yl)phenylmethanone (26a). A 60% oil

dispersion of NaH (0.31 g, 0.79 mmol) was washed with Hex and

suspended in THF; thereafter, 25a (0.220 g, 0.79 mmol) in dry

THF (10 mL) was added at rt and stirred for 5 min, until evolution

of H₂ceased. A current of O₂was passed into the yellow solution

until the solution turn to colorless. The solvent was evaporated under

reduced pressure and residue was treated with H₂O and extracted

with CHCl₃. The organic extracts were dried over Na₂SO₄, the

solvent was removed under reduced pressure and the residue was

puriﬁed by ﬂash chromatography (15% EtOAc in Hex) to afford

26a as a viscous oil (0.20 g, 93%): ¹H NMR (300 MHz, CDCl₃) δ

8.98 (d, 1H, J ) 6.3 Hz), 8.15 (d, 1H, J ) 2.4 Hz), 7.81–7.78 (m,

3H), 7.64–7.58 (m, 1H), 7.48–7.42 (m, 3H), 7.37 (d, 1H, J ) 4.2

Hz).

(7-Chloroquinolin-4-yl)(4-ﬂuorophenyl)methanone (26b). Start-

ing from 25b (0.46 g, 1.56 mmol), the title compound was prepared

following the above-described procedure and was obtained as a

viscous oil (0.36 g, 90%): ¹H NMR (300 MHz, CDCl₃) δ 9.03 (d,

1H, J ) 4.2 Hz), 8.20 (d, 1H, J ) 2.4 Hz), 7.89–7.79 (m, 3H),

7.52–7.48 (m, 1H), 7.39 (d, 1H, J ) 4.5 Hz), 7.19–7.13 (m, 2H).

(()-[4-(Pyrrolidin-1-ylmethyl)phenyl](7-chloroquinolin-4-

yl)phenylmethanol (27a). Starting from 12, magnesium turnings,

and 26a (0.209 g, 0.78 mmol), the title compound was prepared as

described for 17a and was obtained as colorless prisms (0.11 g,

32%): mp (EtOAc/n-Hex) 124–125 °C; ¹H NMR (300 MHz,

CDCl₃) δ 8.64 (d, 1H, J ) 4.8 Hz), 8.10 (d, 1H, J ) 9.0 Hz), 8.02

(d, 1H, J ) 2.4 Hz), 7.38–7.16 (m, 10H), 6.79 (d, 1H, J ) 4.8

Hz), 3.98 (br, 1H), 3.61 (s, 2H), 2.52 (m, 4H), 1.80–1.75 (m, 4H);

ESI MS m/z (M + H)⁺429.

(()-1-{(4-Chlorophenyl)(4-ﬂuorophenyl)[4-(pyrrolidin-1-yl-

methyl)phenyl]methyl}-1H-imidazole (4d). Starting from 18a

(0.348 g, 0.88 mmol), the title compound was prepared as described

for 4a and was obtained as brown oil (0.35 g, 88%): ¹H NMR

(300 MHz, CDCl₃) δ 7.39 (s, 1H), 7.31–7.26 (m, 4H), 7.08–6.99

(m, 9H), 6.75 (s, 1H), 3.60 (s, 2H), 2.53–2.49 (m, 4H), 1.79–1.75

(m, 4H); ESI MS m/z (M - imidazole)⁺378. Anal. (C₂₇H₂₅ClFN₃)

C, H, N.

1-{(4-Chlorophenyl)bis[4-(pyrrolidin-1-ylmethyl)phenyl]me-

thyl}-1H-imidazole (4e). Starting from 18b (143 mg, 0.31 mmol),

the title compound was prepared following the above-described

procedure and was obtained as brown oil (100 mg, 65%): ¹H NMR

(200 MHz, CDCl₃) δ 7.40 (s, 1H), 7.31–7.25 (m, 6H), 7.06–7.01

(m, 8H), 3.62 (s, 4H), 2.53 (m, 8H), 1.78 (m, 8H); ESI MS m/z (M

- imidazole)⁺444. Anal. (C₃₂H₃₅ClN₄) C, H, N.

(()-1-{(3-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](th-

ien-2-yl)methyl}-1H-imidazole (4f). Starting from 22a (0.345 g,

0.90mmol), the title compound was prepared following the above-

described procedure and was obtained as a brown oil (77.0 mg,

20%): ¹H NMR (300 MHz, CDCl₃) δ 7.40 (m, 1H), 7.37–7.30 (m,

4H), 7.27–7.25 (m, 1H), 7.13–7.12 (m, 1H), 7.09–7.07 (m, 2H),

7.06–7.05 (m, 1H), 7.03–6.99 (m, 2H), 6.88–6.85 (m, 2H), 3.63

(s, 2H), 2.54–2.50 (m, 4H), 1.81–1.77 (m, 4H); ESI MS m/z, (M

- imidazole)⁺366. Anal. (C₂₅H₂₄ClN₃S) C, H, N.

(()-(4-Fluorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](7-chlo-

roquinolin-4-yl)methanol (27b). Starting from 12, magnesium

turnings, and 26b (0.485 g, 1.7 mmol), the title compound was

prepared following the above-described procedure and was obtained

as colorless prisms (0.30 g, 40%): mp (EtOAc/n-Hex) 110–111 °C;

¹H NMR (300 MHz, CDCl₃) δ 8.65 (d, 1H, J ) 4.5 Hz), 8.06 (d,

1H, J ) 9.3 Hz), 8.03 (d, 1H, J ) 2.1 Hz), 7.32–6.98 (m, 9H),

6.78 (d, 1H, J ) 4.8 Hz), 3.93 (bs, 1H), 3.61 (s, 2H), 2.52 (m,

4H), 1.79–1.77 (m, 4H); ESI MS m/z (M + H)⁺447.

(()-1-{(3-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](thi-

azol-2-yl)methyl}-1H-imidazole (4g). Starting from 22b (0.60 g,

1.56 mmol), the title compound was prepared following the above-

described procedure and was obtained as a colorless oil (0.50 g,

1

74%): H NMR (400 MHz, CDCl₃) δ 7.92 (d, 1H, J ) 3.2 Hz),

7.55 (s, 1H), 7.37–7.23 (m, 5H), 7.11–6.94 (m, 6H), 3.63 (s, 2H),

2.58–2.45 (m, 4H), 1.87–1.76 (m, 4H); ESI-MS m/z (M -

imidazole)⁺367. Anal. (C₂₄H₂₃ClN₄S) C, H, N.

Clotrimazole Scaffold as a Pharmacophore

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1291

(()-1-{(3-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](fu-

ran-3-yl)methyl}-1H-imidazole (4h). Starting from 22c (0.184 g,

0.50 mmol), the title compound was prepared following the above-

described procedure and was obtained as a colorless oil (42.0 mg,

20%): ¹H NMR (300 MHz, CDCl₃) δ 7.48–7.47 (m, 1H), 7.33–7.29

(m, 4H), 7.26 (m, 2H), 7.13 (m, 2H), 7.08 (m, 1H), 7.03 (m, 1H),

7.01 (m, 1H), 6.96–6.94 (m, 1H), 6.26 (s, 1H), 3.62 (s, 2H), 2.52

(m, 4H), 1.79 (m, 4H); ESI MS m/z (M - imidazole)⁺350. Anal.

(C₂₅H₂₄ClN₃O) C, H, N.

(()-1-{(3-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](py-

ridin-2-yl)methyl}-1H-imidazole (4i). Starting from 22d (0.417

g, 1.1 mmol), the title compound was prepared following the above-

described procedure and was obtained as a colorless oil (200 mg,

42%): ¹H NMR (300 MHz, CDCl₃) δ 8.69–8.66 (m, 1H), 7.69–7.63

(m, 1H), 7.58–7.57 (m, 1H), 7.31–7.23 (m, 5H), 7.10–7.04 (m, 3H),

6.99–6.91 (m, 4H), 3.60 (s, 2H), 2.52–2.48 (m, 4H), 1.79 (m, 4H);

ESI-MS m/z (M - imidazole)⁺361. Anal. (C₂₆H₂₅ClN₄) C, H, N.

(()-1-{(3-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](py-

ridin-4-yl)methyl}-1H-imidazole (4j). Starting from 22e (0.379

g, 1.0 mmol), the title compound was prepared as described for 4a

and was obtained as a colorless oil (230 mg, 53%): ¹H NMR (300

MHz, CDCl₃) δ 8.63 (d, 2H, J ) 6.2 Hz), 7.42–7.26 (m, 6H),

7.10–7.04 (m, 6H), 6.78 (s, 1H), 3.68 (s, 2H), 2.59 (m, 4H), 1.83

(m, 4H); ESI MS m/z (M - imidazole)⁺361. Anal. (C₂₆H₂₅ClN₄)

C, H, N.

the above-described procedure and was obtained as brown oil (51.0

mg, 51%): ¹H NMR (200 MHz, CDCl₃) 7.42–7.15 (m, 13H), 3.55

(s, 2H), 2.99 (m, 6H), 2.52 (m, 4H), 2.22 (m, 3H), 1.79–1.76 (m,

4H); ESI MS m/z (M - piperazine)⁺360. Anal. (C₂₈H₃₂ClN₃) C,

H, N.

1. Molecular Modeling. Molecular modeling calculations were

performed on SGI Origin 200 8XR12000, while molecular modeling

graphics were carried out on SGI Octane 2 and Octane workstations.

Apparent pK_avalues of CQ, CLT, 2, 3g, 4q,r, and the newly

designed compounds (3a-f and 4a-p) were calculated by using

the ACD/pK_aDB version 10.00 software (Advanced Chemistry

Development Inc., Toronto, Canada), accordingly, percentage of

neutral/ionized forms were computed at pH 7.4 (blood), 7.2

(cytoplasm),and5.5(Pffoodvacuole)usingtheHanderson-Hasselbach

equation.

All the compounds were built taking into account the prevalent

ionic forms at the considered different pH values using the Insight

2005 Builder module.

Partial charges of the compounds, considered protonated at the

imidazole and benzimidazole moieties as consequence of the

estimation of apparent pK_avalues, were assigned by comparing

partial charges assigned by CFF91 force ﬁeld³²with those calculated

by MNDO³³semiempirical 1 SCF calculations performed on the

neutral and the ionized compounds. In particular, CFF91 force ﬁeld

partial charges were added to the algebraic difference between

MNDO partial charges of the protonated form and MNDO partial

charge of the neutral form.

The conformational space of all compounds was sampled through

200 cycles of simulated annealing (CFF91 force ﬁeld³²) by

following our standard protocol. The system was heated up to 1000

K over 2000 fs (time step ) 3.0); the temperature of 1000 K was

applied to the system for 2000 fs (time step ) 3.0), with the aim

of surmounting torsional barriers; successively, temperature was

linearly reduced to 300 K in 1000 fs (time step ) 1.0). Resulting

structures were subjected to energy minimization within Insight

2005 Discover 3 module (CFF91 force ﬁeld,³²conjugate gradient

algorithm;³⁴ꢀ ) 80*r) until the maximum rms derivative was less

than 0.001 kcal/Å and subsequently ranked in different families,

taking into account their (i) conformational energy and (ii) torsional

angles values, with the exception of those of lateral chains.

To properly analyze the electronic properties, the most stable

conformer of each family was subjected to a full geometry

optimization by semiempirical calculations, using the quantum

mechanical method AM1 in the Mopac 6.0 package³⁵in Ampac/

Mopac module of Insight 2000.1. GNORM value was set to 0.5.

To reach a full geometry optimization, the criteria for terminating

all optimizations was increased by a factor of 100 using the keyword

PRECISE.

(()-1-{(4-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](th-

ien-2-yl)methyl}-1H-imidazole (4k). Starting from 23a (0.883 g,

2.3 mmol), the title compound was prepared following the above-

described procedure and was obtained as a colorless oil (150 mg,

15%): ¹H NMR (200 MHz, CDCl₃) δ 7.40 (s, 1H), 7.34–7.27 (m,

5H), 7.10–7.04 (m, 5H), 7.00 (dd, 1H, J ) 3.8, 5.2 Hz), 6.86 (dd,

1H, J ) 1.1, 3.8 Hz), 6.85 (s, 1H), 3.63 (s, 2H), 2.55–2.50 (m,

4H), 1.81–1.77 (m, 4H); ESI-MS m/z (M - imidazole)⁺366. Anal.

(C₂₅H₂₄ClN₃S) C, H, N.

(()-1-{(4-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](thi-

azol-2-yl)methyl}-1H-imidazole (4l). Starting from 23b (0.355 g,

1.0 mmol), the title compound was prepared following the above-

described procedure and was obtained as a colorless oil (0.32 g,

1

72%): H NMR (300 MHz, CDCl₃) δ 7.89 (d, 1H, J ) 3.4 Hz),

7.53 (m, 1H), 7.36–7.26 (m, 5H), 7.08–6.98 (m, 6H), 3.58 (s, 2H),

2.48–2.44 (m, 4H), 1.75–1.71 (m, 4H); ESI-MS m/z (M -

imidazole)⁺367. Anal. (C₂₄H₂₃ClN₄S) C, H, N.

(()-1-{[4-(Pyrrolidin-1-ylmethyl)phenyl](7-chloroquinolin-4-

yl)phenylmethyl}-1H-imidazole (4m). Starting from 27a (90.1 mg,

0.21 mmol), the title compound was prepared as described for 4a

1

and was obtained as a brown oil (57.0 mg, 52%): H NMR (300

MHz, CDCl₃) δ 8.87 (d, 1H, J ) 4.5 Hz), 8.12 (d, 1H, J ) 2.4

Hz), 7.39–7.07 (m, 13H), 6.87 (d, 1H, J ) 4.8 Hz), 6.73 (s, 1H),

3.61 (s, 2H), 2.52 (m, 4H), 1.81–1.77 (m, 4H); ESI MS m/z (M -

imidazole)⁺411. Anal. (C₃₀H₂₇ClN₄) C, H, N.

The dipole moments were calculated using partial charges

obtained by the quantum mechanical method AM1 (Ampac/Mopac,

Accelrys, San Diego) and visualized as vector (Decipher, Accelrys,

San Diego). Connolly surfaces were computed and colored by AM1

charge distribution. LogD values of 2 and 4e at pH 7.4 (blood),

7.2 (cytoplasm), and 5.5 (Pf FV) were calculated by using the ACD/

pK_aDB version 10.00 software (Advanced Chemistry Development

Inc., Toronto, Canada).

(()-1-{(4-Fluorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl](7-

chloroquinolin-4-yl)methyl}-1H-imidazole (4n). Starting from 27b

(0.380 g, 0.85 mmol), the title compound was prepared following

the above-described procedure and was obtained as a brown

1

amorphous solid (0.25 g, 60%): H NMR (300 MHz, CDCl₃) δ

8.86 (d, 1H, J ) 4.8 Hz), 8.10 (d, 1H, J ) 1.8 Hz), 7.36–7.38 (m,

3H), 7.10–6.99 (m, 9H), 6.83 (d, 1H, J ) 4.8 Hz), 6.70 (s, 1H),

3.60 (s, 2H), 2.50 (m, 4H), 1.77 (m, 4H); ESI MS m/z (M -

imidazole)⁺429. Anal. (C₃₀H₂₆ClFN₄) C, H, N.

Docking studies were carried out on CLT (global minimum

conformer) into the homology model²⁶of lanosterol 14R-demeth-

ylase of C. Albicans using a docking methodology (Afﬁnity,

SA_Docking; Insight2005, Accelrys, San Diego) that considers all

(()-1-{(4-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl]phe-

nylmethyl}-1H-benzo[d]imidazole (4o). Starting from 17c⁶(94.0

mg, 0.22 mmol) and 1H-benzo[d]imidazole, the title compound was

prepared following the above-described procedure and was obtained

as brown oil (51.0 mg, 51%): ¹H NMR (200 MHz, CDCl₃) δ 7.83

(s, 1H), 7.77 (d, 1H, J ) 8.1 Hz), 7.32–7.25 (m, 7H), 7.19–7.06

(m, 7H), 6.93–6.86 (m, 1H), 6.44 (d, 1H, J ) 8.3 Hz), 3.59 (s,

2H), 2.50 (m, 4H), 1.81–1.76 (m, 4H); ESI MS m/z (M -

benzimidazole)⁺360. Anal. (C₃₁H₂₈ClN₃) C, H, N.

the systems (i.e., ligand and protein) ﬂexible. Heme29.frc,²⁶

a

forceﬁeld including heme parameters, was used during all molecular

simulations. Although in the subsequent ﬂexible docking protocol

all the systems were perturbed by means of Monte Carlo and

simulated annealing procedures, nevertheless, the ﬂexible docking

procedure formally requires a reasonable starting structure. Ac-

cordingly, CLT was manually positioned into the active site, taking

into account the binding mode of ﬂuconazole (PDB code: 1EA1)

and ketoconazole (PDB code:1JIN) in cytochromes P450. The

obtained complex was subjected to preliminary energy minimization

(steepest descendent algorithm; ꢀ ) 80*r) until the maximum rms

(()-1-{(4-Chlorophenyl)[4-(pyrrolidin-1-ylmethyl)phenyl]phe-

nylmethyl}piperazine (4p). Starting from 17c6 (94.0 mg, 0.22

mmol) and piperazine, the title compound was prepared following

1292 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5

Gemma et al.

derivative was less than 5 kcal/Å to generate roughly docked starting

structure, as required by the Afﬁnity docking procedure. Succes-

sively, ﬂexible docking was achieved through the Afﬁnity module

in the Insight2005 suite, using the SA_Docking procedure³⁶and

using the Cell_Multipole³⁷method for nonbond interactions.

Lanosterol 14R-demethylase binding domain area was deﬁned as

a ﬂexible subset around the ligand containing all residues having

at least one atom within a 6 Å radius from any given ligand atom.

All atoms included in the binding domain area and the ligand were

left free to move during the entire docking calculations, with the

exception of the coordinating nitrogen, heme pyrroles, and the iron,

which were tethered with a force constant of 30 kcal/Å². A Monte

Carlo/minimization approach for random generation of a maximum

of 20 structures was used, with an energy tolerance of 10⁶kcal/

mol to ensure a wide variance of the input structures to be

minimized (2500 iterations; ꢀ ) 80*r). During this step, the ligand

is moved by a random combination of translation, rotation, and

torsional changes (Flexible_Ligand option, considering all rotatable

bonds), to sample both the conformational space of the ligand and

its orientation with respect to the enzyme. Van der Waals (vdW)

and coulombic terms were scaled to a factor of 0.1 to avoid very

severe divergences in the coulombic and vdW energies. The

Metropolis test, at a temperature of 310 K, and a structure similarity

check (rms tolerance ) 0.3 kcal/Å) were applied to select acceptable

structures. A total of 50 stages of simulated annealing (100 fs each)

were applied on the resulting complexes. Over the course of the

simulated annealing, system temperature was linearly decreased

from 500 to 300 K; concurrently, the van der Waals and coulombic

scale factors were similarly decreased from their initial values

(deﬁned above as 0.1) to their ﬁnal value (1.0). A ﬁnal round of

10⁵minimization steps was applied at the end of the molecular

dynamics. Resulting docked complexes were ranked by their

conformational energy, and the geometry of imidazole-iron coor-

dination bond. The coordination bond distances and angles were

compared with those found by analyzing the iron-imidazole

complexes found in CSDS (Cambridge Structural Database System,

CSDS codes: CELWIZ, FEWXAG, KIWNIN, SIBZIM, SIBZOS,

YILZIC, YILZOI, YIMYUO). The lowest energy complex showing

the best coordination geometry was selected as representing

structure for the most probable binding mode. Connolly surface

was computed and colored on the basis of amino acids formal

charge.

(OD650) by measuring the activity of the parasite lactate dehy-

drogenase (pLDH), according to a modiﬁed version of the method

of Makler in control and drug-treated cultures.³⁹The antimalarial

activity is expressed as 50% inhibitory concentrations (IC₅₀); each

IC₅₀value is the mean and standard deviation of at least three

separate experiments performed in duplicate.

3D7 and K1 Strains. All samples were tested in triplicate against

the 3D7 and K1 strains. The cultures were maintained in continuous

log phase growth in a RPMI1640 medium supplemented with 5%

wash human A+ erythrocytes, 25 mM HEPES, 32 nM NaHCO₃,

and Albu-MAXII (lipid-rich bovine serum albumin). All cultures

and assays were conducted at 37 °C under an atmosphere of 5%

CO₂, 5% O₂, and 90% N₂. Stock compound solutions were prepared

in 100% DMSO at 5 mg/mL. The compounds were further diluted

using a complete RPMI1640 medium supplemented with cold

hypoxanthine and AlbuMAXII. Assays were preformed on sterile

96-well microtiter plates, with each plate containing 100 µL of the

parasite culture (1% parasitaemia, 2.5% hemacrit). After 24 h of

incubation at 37 °C, 3.7 Bq of [³H]hypoxanthine was added to each

well. Cultures were incubated for a further 24 h before they were

harvested onto glass-ﬁber ﬁlter mats, and the radioactivity was

counted using a Wallac Microbeta 1450 scintillation counter.⁴⁰

3. BHIA Assay. A total of 50 µL of an 8 mM solution of hemin

dissolved in DMSO was distributed in 96-well U-bottom micro-

plates (0.4 µmol/well; Costar 3799); 50 µL of different compounds

in water, in doses ranging from 1 to 8 mol equiv to hemin, was

added to triplicate test wells. In control wells, 50 µL of water was

added. Water-insoluble compounds were solubilized in 25 µL of

DMSO and then added to hemin prepared at 16 mM and distributed

into the wells in 25 µL aliquots. The ﬁnal concentration of DMSO/

well was kept constant at 25%. ꢀ-Hematin formation was initiated

by the addition of 100 µL of 8 M acetate buffer (pH 5). Plates

were incubated at 37 °C for 18 h to allow for complete reaction

and then centrifuged at 3300g for 15 min. The soluble fraction of

unprecipitated material was collected (fraction I). The remaining

pellet was resuspended with 200 µL of DMSO to remove unreacted

hematin. Plates were then centrifuged again at 3300g for 15 min.

The DMSO-soluble fraction (fraction II) was collected and the

pellet, consisting of a pure precipitate of ꢀ-hematin, was dissolved

in 0.1 M NaOH (fraction III) for spectroscopic quantitation. A 150

µL aliquot of each fraction (I, II, or III) was transferred onto a

new plate and serial 4-fold dilutions in 0.1 M NaOH were made.

The amount of hematin was determined by measuring the absor-

bance at 405 nm using a microtiter plate reader (Molecular Devices).

A standard curve of hematin dissolved in 0.1 M NaOH was used

to calculate the amount of porphyrin present in each fraction. The

drug concentration required to inhibit ꢀ-hematin formation by 50%

(IC₅₀) was determined for each compound.²⁵

The experimentally determined structures of cytochromes P450

(PDB IDs: 1EA1, 1JIN, 1E9X, 1OXA, 1W0E, 1W0F, 1W0G) were

downloaded from the Protein Data Bank (PDB; http://www.rcsb.org/

pdb/). Hydrogens were added to all the PDB structures, considering

a pH value of 7.2. P450 X-ray structures were superimposed by

ﬁtting heme atoms.

The sequences of cytochromes P450 were downloaded from the

Sequence Retrieval System (http://srs.embl-heidelberg.de:8000/

srs5). Sequence alignments were performed using ClustalW (WWW

service at the European Bioinformatics Institute, http://www.ebi.

ac.uk/clustalw) and Multiple_Alignment (Accelrys, San Diego).

Results were analyzed using the Insight2005 Homology Module

(Accelrys, San Diego).

2. In Vitro Antiplasmodial Activity Studies. D10 and W2

Strains. P. falciparum cultures were carried out according to Trager

and Jensen’s, with slight modiﬁcations.³⁸The CQ-S, strain D10,

and the CQ-R, strain W2, were maintained at 5% hematocrit (human

type A-positive red blood cells) in RPMI 1640 (EuroClone, Celbio;

NaHCO₃24 mM) medium with the addition of 10% heat inactivated

A-positive human plasma, 20 mM Hepes, 2 mM glutamine. All

the cultures were maintained at 37 °C in a standard gas mixture

consisting of 1% O₂, 5% CO₂, 94% N₂. Compounds were dissolved

in either water or DMSO and then diluted with medium to achieve

the required concentrations (ﬁnal DMSO concentration < 1%,

which is nontoxic to the parasite). Drugs were placed in 96-well,

ﬂat-bottom microplates (COSTAR) and serial dilutions were made.

Asynchronous cultures with parasitaemia of 1–1.5% and 1% ﬁnal

hematocrit were aliquoted into the plates and incubated for 72 h at

37 °C. Parasite growth was determined spectrophotometrically

4. Cytotoxicity. Cell Lines. All cell lines were obtained from

ATCC. The cells were cultured in RPMI 1640 supplemented with

5% FCS, 0.1 mM glutamine, 1% penicillin, and streptomycin. Cells

were grown in Nunc clone plastic bottles (TedNunc, Roskilde,

Denmark) and split twice weekly at different cell densities according

to standard procedure. Perypheral blood mononuclear cells (MNC)

were separated from heparinized whole blood obtained from a

healthy donor on a Fycoll-Hypaque gradient. MNC thus obtained

were washed twice with RPMI 1640 supplemented with 10% FCS,

glutamine, and antibiotics, suspended at 200.000 viable cells/mL

in medium containing, as mitogen, 5 µg/mL PHA (Sigma) and used

in toxicity tests.

Chemicals. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-

razolium bromide) was purchased from Aldrich. It was dissolved

at a concentration of 5 mg/mL in sterile PBS at room temperature,

and the solution was further sterilized by ﬁltration and stored at 4

°C in a dark bottle. SDS was obtained from Sigma. Lysis buffer

was prepared as follows: 20% w/v of SDS was dissolved at 37 °C

in a solution of 50% of each DMF and demineralised water; pH

was adjusted to 4.7 by adding 2.5% of an 80% acetic acid and

2.5% 1 N HCl solution.

Toxicity Experiments. Cells were plated at different concentra-

tions on ﬂat bottom 96-well microplates (0.1 mL/well). Lympho-

Clotrimazole Scaffold as a Pharmacophore

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1293

cytes were plated out at 20000 cells/well. NSO cells (plasmocytoma

murine cell line) were plated out at 3000 cells/well, and Daudi cells

(human lymphoblastoid cell line) were plated at 300 cells/well. A

total of 12 h after plating, different concentrations of each compound

dissolved in DMSO were added to each well. After 48 h, MTT

assay was performed to analyze cytotoxicity of the different

compounds. All experiments were performed at least two times in

triplicate.

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JM701247K

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Clotrimazole scaffold as an innovative pharmacophore towards potent antimalarial agents: Design, synthesis, and biological and structure-activity relationship studies

DOI: 10.1021/jm701247k

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