Falcipain inhibitors: Optimization studies of the 2-pyrimidinecarbonitrile lead series

J. Med. Chem. 2010, 53, 6129–6152 6129

DOI: 10.1021/jm100556b

Falcipain Inhibitors: Optimization Studies of the 2-Pyrimidinecarbonitrile Lead Series^†

‡

^

‡

ꢀ

Jose M. Coteron, David Catterick, Julia Castro, Marı

a J. Chaparro,^‡Beatriz Dı

az, Esther Fernandez,

ꢀ

´ ´

Santiago Ferrer,^§Francisco J. Gamo,^§Mariola Gordo,^‡Jiri Gut, Laura de las Heras,^‡Jennifer Legac, Maria Marco,^‡

Juan Miguel, Vicente Munoz, Esther Porras, Juan C. de la Rosa, Jose R. Ruiz, Elena Sandoval, Pilar Ventosa,

Philip J. Rosenthal,*^,and Jose M. Fiandor*^,‡

‡

~

^‡Department of Drug Discovery Chemistry, and ^§Department of Drug Discovery Biology, Diseases of the Developing World (DDW),

GlaxoSmithKline, Severo Ochoa, 2 (PTM), Tres Cantos, Madrid 28760, Spain, Department of Medicine, San Francisco General Hospital,

University of California, San Francisco, Box 0811, San Francisco, California 94143, and ^{^}GlaxoSmithKline, Synthetic Chemistry,

Old Powder Mills, Tonbridge, Kent, TN11 9AN, U.K.

Received May 11, 2010

Falcipain-2 and falcipain-3 are papain-family cysteine proteases of the malaria parasite Plasmodium

falciparum that are responsible for host hemoglobin hydrolysis to provide amino acids for parasite

protein synthesis. Different heteroarylnitrile derivatives were studied as potential falcipain inhibitors

and therefore potential antiparasitic lead compounds, with the 5-substituted-2-cyanopyrimidine

chemical class emerging as the most potent and promising lead series. Through a sequential lead opti-

mization process considering the different positions present in the initial scaffold, nanomolar and

subnanomolar inhibitors at falcipains 2 and 3 were identified, with activity against cultured parasites in

the micromolar range. Introduction of protonable amines within lead molecules led to marked improve-

ments of up to 1000 times in activity against cultured parasites without noteworthy alterations in other

SAR tendencies. Optimized compounds presented enzymatic activities in the picomolar to low nano-

molar range and antiparasitic activities in the low nanomolar range.

Introduction

falcipain-1, falcipain-2, falcipain-2⁰, and falcipain-3.⁹Falci-

pain-2 and falcipain-3 (65% amino acid identity) are predo-

minantly expressed in the acidic food vacuole of trophozoites

and schizonts. Falcipain-3 is expressed later in the life cycle

and appears to be a more efficient hemoglobinase than

falcipain-2.^10-12Importantly, cysteine protease inhibitors

that inhibit falcipain-2 and falcipain-3 consistently block

hemoglobin hydrolysis and parasite development, suggesting

that these two proteases are key target enzymes. Knockout of

falcipain-2 led to a block in hemoglobin hydrolysis in tropho-

zoites, although parasites recovered from this defect, presum-

ably because of expression of falcipain-3 later in the life cycle.

Knockout of falcipain-3 was not successful, although replace-

ment of the falcipain-3 gene witha functional copy was readily

achieved, strongly suggesting that falcipain-3 is an essential

enzyme in erythrocytic parasites.¹³Falcipain-2⁰, a nearly

identical copy of falcipain-2, also has unknown function, as

disruption of the falcipain 2⁰gene did not lead to noticeable

alterations in erythrocytic parasites.¹⁴Finally, the role of

falcipain-1 is unknown; knockout of this protease led to no

apparent changes in erythrocytic parasites.¹⁵In any event,

cysteine protease inhibition, in particular the inhibition of

falcipain-2 and falcipain-3, blocks parasite development.

Falcipain-2 and related plasmodial cysteine proteases are thus

logical targets for antiparasitic chemotherapy, and therefore,

we were interested in the development of inhibitors of these

enzymes as antiparasitic drugs.

Malaria is one of the major disease problems of the devel-

oping world. The most virulent malaria parasite is Plasmo-

dium falciparum, which is the cause of hundreds of millions of

cases of malaria and about 1 million deaths each year, mostly

in African children.^1,2One problem encountered in the treat-

ment of malaria is the development of resistance to most

available drugs. Thus, new antimalarials are urgently needed.

Plasmodium falciparum relies on hemoglobin hydrolysis to

supply amino acids for protein synthesis and to maintain

osmotic stability. Hemoglobin is transported to an acidic

(pH 4-6) food vacuole, where it is hydrolyzed by multiple

enzymes, including food vacuole cysteine, aspartic, and metal-

loproteases, before further action against hemoglobin frag-

ments by a cytosolic aminopeptidase.^3,4Hemoglobin degra-

dation is blocked by cysteine protease inhibitors, causing a

characteristic morphological abnormality in which the food

vacuole fills with undegraded hemoglobin and parasite devel-

opment is blocked.^5,6Hence, plasmodial hemoglobinases con-

stitute potential therapeutic targets. Efforts to identify the

enzymes responsible for hemoglobin degradation led to the

characterization of “falcipain” as a trophozoite food vacuole

cysteine protease.^7,8More recent advances identified and char-

acterized a family of papain-like cysteine proteases comprising

†

ꢀ

Dedicated to Prof. Federico Gomez de las Heras, DDW-GlaxoS-

mithKline, on the occasion of his retirement.

*To whom correspondence should be addressed. For P.J.R.: phone,

(415) 206-8845; fax, (415) 648-8425; e-mail, prosenthal@medsfgh.ucsf.edu.

For J.M.F.: phone, þ34 918070554; fax, þ34918070550; e-mail, jose.m.

fiandor@gsk.com.

Alkyl- and arylnitriles are well established inhibitors of

cysteine proteases.^16-24The active site of papain and related

proteases is large and can be divided into pockets at each side

r

2010 American Chemical Society

Published on Web 07/30/2010

pubs.acs.org/jmc

6130 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16

Scheme 1. Synthesis of Pyridine and Pyrimidine Derivatives^a

Coteroꢀn et al.

^aReagents and conditions: (a) i-PrOH, 0 ꢀC f rt, then H₂, PtO₂, rt; (b) Pd₂(dba)₃, BINAP, NaO^tBu, toluene, rt; (c) Zn(CN)₂, Pd₂(dba)₃, dppf, Zn,

DMA, 120 ꢀC.

Scheme 2. Synthesis of 5-Cyanopyrimidine Derivatives^a

^aReagents and conditions: (a) POCl₃, PCl₅, 115 ꢀC; (b) DIPEA, toluene or i-PrOH, rt.

of the catalytic site, S1-S4 and S1⁰-S3⁰, with each pocket

accommodating one amino acid residue of the peptide

substrate.²⁵The general structure of a cysteine protease

inhibitor (Figure 1) should comprise an electrophilic moiety

for interaction with the cysteine residue of the enzyme and one

or two series of substituents, P1-P4 and/or P1⁰-P3⁰, respon-

sible for interactions with the differentpockets of the protease.

Ideally, the X-ray structure of the complex formed between

the enzyme and an inhibitor should be a valuable source of

information about interactions in the complex and potential

interactions to guide the design of new inhibitors. Despite the

lack of X-ray structures at the time of initiation of this project,

falcipains were perceived as novel and extremely promising

targets, and we began lead optimization studies with the

arylnitrile series following an empirical approach in which

optimization of the initial hits took place considering interac-

tions with both the active site cysteine and P1-P3 positions.

ability of the appropriately substituted heteroaryl precursors,

different approaches were followed.

Palladium catalyzed amination and cyanation reactions

were necessary in the synthesis of pyridine and pyrazine deri-

vatives starting out from the corresponding dihalogen deri-

vatives (Scheme 1). Aromatic substitution of 2,6-dibromo-

pyridine with the hydrazido derivative 1 failed when DIPEA^a

and isopropanol were used at 130 ꢀC, while palladium-

catalyzed aminations (both using palladium(II) acetate/BI-

NAP/potassium carbonate and Pd₂(dba)₃/BINAP/sodium

tert-butoxide) yielded the desired product when 2,6-dichlor-

opyridine was used as starting material, diamination being

^aAbbreviations: ACN, acetonitrile; AcO/OAc, acetate; AcOH, acetic

acid; anh, anhydrous; aq, aqueous; BINAP, 2,2⁰-bis(diphenyl-

phosphino)-1,1⁰-binaphthyl; DABCO, 4-diazabicyclo[2.2.2]octane;

dba, dibenzylideneacetone; DCM, dichloromethane; DIPEA, diisopro-

pylethylamine; DMA, dimethylacetamide; DME, dimethoxyethane;

DMF, dimethylformamide; DMSO, dimethyl sulfoxide; dppf, 1,1⁰-bis-

(diphenylphosphino)ferrocene; DTT, dithiothreitol; EtOAc, ethyl acet-

ate; Et₃N, triethylamine; EtOH, ethanol; FP-2, falcipain-2; FP-3,

falcipain-3; g, gram; h, hour; H-D-VLR-AFC, HD-valyl-leucyl-argi-

nyl-7-amido-4-trifluoromethylcoumarin; HetAr, heteroaryl; HMPA,

hexamethylphosphoramide; HPLC, high performance liquid chroma-

tography; IC₅₀, inhibitory concentration 50; i-PrOH, isopropanol;

LCMS, liquid chromatography-mass spectrometry; M, molar; min,

minute; MeOH, methanol; MHz, megahertz; mL, milliliter; mM, milli-

molar; mmol, millimole; N, normal; NBS, N-bromosuccinimide; ND,

not determined; NaOEt/EtONa, sodium ethoxide; NaO^tBu, sodium

tert-butoxide; nM, nanomolar; NMR, nuclear magnetic resonance;

ꢀC, degree centigrade; PBS, phosphate buffer solution; ppm, parts per

million; RPMI, Roswell Park Memorial Institute; rt, room temperature;

sat., saturated; TFA, trifluoroacetic acid; THF, tetrahydrofuran; μM,

micromolar; vs, versus.

Chemistry

Synthesis of Heteroaryl Nitriles for Optimization of the

Electrophilic Moiety and P1 Position. The first step of the

optimization process was the identification of the most

promising heteroarylnitrile designed to contain both the

electrophilic moiety (nitrile group) and the P1 substituent

(heteroaryl ring). In order to carry out a fair comparison

among different compounds, substitutions at positions P2

(2-methylpropyl-) and P3 (tert-butyloxycarbonyl-) were fixed

for all derivatives. The synthesis of tert-butyloxycarbonyl

derivatives of heteroarylnitrile falcipain inhibitors is out-

lined in Schemes 1-13. Depending on the commercial avail-

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6131

observed when 2,6-dibromopyridine was subjected to these

reaction conditions. Cyanation of the intermediate 1,1-di-

methylethyl-2-(6-bromo-2-pyridinyl)-2-(2-methylpropyl)-

hydrazinecarboxylate 2a failed when nickel(II) bromide and

sodium cyanide were used under the conditions described by

Leadbeater and Arvela.²⁶Negative results followed the

exploration of different solvents as well as the use of other

cyanating agents, such as copper(I) cyanide, sodium cyanide/

copper(I) iodide/N,N⁰-dimethylethylenediamine/potassium

iodide,²⁷zinc(II) cyanide/tetrakis(triphenylphosphine)palla-

dium(0) or sodium cyanide/tetrakis(triphenylphosphine)palla-

dium(0)/copper(I) iodide. Finally, the use of zinc(II) cyanide/

tris(dibenzylideneacetone)dipalladium(0)/dppf/Zn²⁸yielded

the desired product 4. With these reaction conditions in hand,

the corresponding pyrazine derivative 5 was synthesized start-

ing out from 2,6-dichloropyrazine through intermediate 3.

The 4-, 5- and 6-nitrile pyrimidine derivatives (Schemes

2 and 3) were prepared from the corresponding dichloro-

pyrimidines by chlorine displacement with the hydra-

zide derivative following the literature procedure given

by Bagley et al.²⁹5-Carbonitrilepyrimidines 6 and 7

were obtained by alkylation of 5-cyano-2,4-dichloro-

pyrimidine with intermediate 1 (Scheme 2), while 4-

and 6-nitrile substituted pyrimidines 8 and 9 were ob-

tained following an alkylation-cyanation sequence start-

ing from 4,6- and 2,4-dichloropyrimidine, respectively

(Scheme 3).

Triazine derivative 10 was synthesized using milder reac-

tion conditions because of the reactivity of the starting 2,4-

dichloro-6-methoxy-1,3,5-triazine (Scheme 4). Chlorine dis-

placement with the hydrazido derivative 1 in the absence of

base followed by cyanation using sodium cyanide afforded

the desired product.

The synthesis of 2-pyrimidinenitrile 12 was accomplished

using the synthetic route described in Scheme 5 through an

alkylation and subsequent cyanation process starting from

2,6-dichloropyrimidine and compound 1.

The tert-butyloxycarbonyl protected 2-pyrimidinenitriles

presenting different substitutions in positions 5 and 6 of the

ring are outlined in Schemes 6-13. Selection of the appro-

priate synthetic approach was made individually for each

compound on the basis of commercial availability of the sub-

stituted pyrimidine precursors.

6-Substituted 2-cyanopyrimidines were prepared accord-

ing to Schemes 6 and 7. 6-Substituted 2,4-dichloropyrimi-

dines were used as starting materials, and chlorine displace-

ment by the hydrazide derivative 1 was achieved following

the literature procedure given by Bagley et al.²⁹Intermediates

13a,b were reacted with KCN in DMSO/H₂O to yield

14a,b, respectively. 6-Methoxy substituted pyrimidine 17 was

Figure 1. General structure of 2-pyrimidinecarbonitrile derivatives

showing positions P1, P2, and P3.

Scheme 3. Synthetic Route for 4- and 6-Nitrile Substituted Pyrimidines^a

^aReagents and conditions: (a) DIPEA, toluene or isopropanol; (b) potassium cyanide, 1,4-diazabicyclo[2.2.2]octane, 85:15 DMSO-H₂O, 65-70 ꢀC.

Scheme 4. Synthesis of Triazine Derivative 10^a

^aReagents and conditions: (a) THF, 0 ꢀC to rt; (b) NaCN, DMSO.

Scheme 5. Synthetic Route for 2-Pyrimidinenitrile Derivative 12^a

^aReagents and conditions: (a) triethylamine or DIPEA, EtOH or isopropanol; (b) potassium cyanide, 1,4-diazabicyclo[2.2.2]octane, 9:1

DMSO-H₂O.

6132 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16

Scheme 6. General Synthesis of 6-Substituted Pyrimidines^a

Coteroꢀn et al.

^aReagents and conditions: (a) i-PrOH, 0 ꢀC f rt, then H₂, PtO₂, rt; (b) triethylamine, EtOH; (c) potassium cyanide, 1,4-diazabicyclo[2.2.2]octane,

85:15 DMSO-H₂O, 80-100 ꢀC or tetraethylammonium cyanide, trimethylamine, THF, rt.

Scheme 7. Synthesis of 6-Methoxypyrimidine Derivative^a

^aReagents and conditions: (a) EtOH, rt; (b) sodium methoxide, MeOH, rt; (c) tetraethylammonium cyanide, trimethylamine, ACN, rt.

Scheme 8. General Synthetic Route for 5-Substituted Pyrimidines^a

^aReagents and conditions: (a) triethylamine or DIPEA, EtOH or isopropanol; (b) potassium cyanide, 1,4-diazabicyclo[2.2.2]octane, 9:1 DMSO-

H₂O.

synthesized (Scheme 7) by reaction of 2,4,6-trichloropyrimi-

dine with the hydrazide intermediate 1 followed by chlorine

displacement in intermediate 15 with sodium methoxide.

Substitution at the 2-position was favored over the 6-posi-

tion in a 9:2 ratio; however, no attempt was made to optimize

these reaction conditions. Final cyanation of intermediate 16

took place using tetramethylammonium cyanide to yield

compound 17.

5-Substituted-2-cyanopyrimidine derivatives were pre-

pared following the procedures described in Schemes 8-13.

Whenever availability of starting materials made it pos-

sible, the chlorine atom at the 4-position was substituted

by the hydrazide derivative 1 in a basic alcoholic medium. Re-

markably, little or no substitution at the 2-position of the pyrim-

idine ring was observed with some of these derivatives, in

particular halogens or nitro groups. Cyanation of intermediates

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6133

Scheme 9. Synthesis of 5-Trifluoromethyl Derivative^a

^aReagents and conditions: (a) copper(I) iodide, hexamethylphosphoramide, DMF, 80 ꢀC.

Scheme 10. Synthesis of 5-Benzylamine Derivative^a

^aReagents and conditions: (a) Pd(OAc)₂, (()-BINAP, K₂CO₃, DMF, microwave at 60 ꢀC.

Scheme 11. Synthesis of 5-Alkyne Substituted Derivative^a

^aReagents and conditions: (a) Pd(PPh₃)₂Cl₂, copper(I) iodide, Et₃N, DMF, 50 ꢀC.

Scheme 12. Synthesis of 5-Methoxy Derivative^a

^aReagents and conditions: (a) conc HCl, H₂O, reflux; (b) POCl₃, dimethylaniline, reflux; (c) DIPEA, isopropanol, reflux; (d) potassium cyanide, 1,4-

diazabicyclo[2.2.2]octane, 9:1 DMSO-H₂O, microwave at 60 ꢀC.

18a-e using KCN afforded derivatives 19a-e. A significant

influence of the substituent present at the 5-position on the

reaction temperature was observed.

By use of the corresponding 5-bromopyrimidine deriva-

tive 19b as precursor, compounds 20-22 were prepared

(Schemes 9-11). Reaction of 19b with methyl 2,2-difluoro-

2-(fluorosulfonyl)acetate in the presence of CuI and HMPA

afforded the corresponding 5-trifluoromethyl derivative 20,

whereas coupling between 19b and benzylamine using Pd-

(OAc)₂and (()-BINAP as catalyst and ligand, respectively,

afforded compound 21 (Scheme 10). The preparation of

22 was performed by Sonogashira coupling of 19b and

commercially available 4-ethynyl-R,R,R-trifluorotoluene in

the presence of Pd(PPh₃)₂Cl₂and CuI catalysts. Cyclization

6134 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16

Scheme 13. Synthesis of 5-Methoxymethylene Derivative^a

Coteroꢀn et al.

^aReagents and conditions: (a) POCl₃, PCl₅, reflux; (b) Cs₂CO₃, THF, rt; (c) MeONa, MeOH, rt; (d) potassium cyanide, 1,4-diazabicyclo[2.2.2]octane,

9:1 DMSO-H₂O, rt.

resulting in an opening of the ring, thus yielding the corre-

sponding hydrazine carboxylate which ultimately gave 37.

Likewise, compound 38 was synthesized from 2,2-dimethy-

loxirane, the corresponding hydrazinecarboxylate being for-

med via epoxide opening.

Figure 2. General structure of the P2 series of 2-cyanopyrimidine

The corresponding hydrazine carboxylate derivative 31

derivatives.

was reacted with 5-bromo-2,4-dichloropyrimidine according

to the literature procedure given in Bagley et al.²⁹The cor-

side products were observed when high palladium loadings

responding intermediates 32 were reacted with KCN in

were used and/or triphenylphosphine was used as ligand.

DMSO-H₂O to yield compounds 33-78, respectively.

The corresponding 5-methoxy derivative 26 was synthe-

Derivatives 79 were obtained by hydrazine deprotection

sized starting out from 5-methoxy-2-sulfanyl-4-pyrimidinol

using p-toluenesulfonic acid, and subsequent coupling with

(Scheme 12). Reaction with chloroacetic acid afforded the

4-bromomethylbenzoyl bromide yielded the corresponding

uracil derivative 23 which was then chlorinated using POCl₃

derivatives 80. Nucleophilic substitution using N-methylpi-

to yield the corresponding 2,4-dichloro-5-methoxypyrimi-

perazine yielded the amine containing products 81-119.

Synthesis of Isobutyl-2-pyrimidinenitrile Derivatives for

Optimization of the P3 Position. For the optimization of the

P3 position, the previously optimized substituents at positions

P1 (2-nitrile-5-chloro(or bromo)-6-pyrimidyl) and P2 (2-met-

hylpropyl) wereselected(Figure3). Derivatives121-269 were

synthesized following the procedure outlined in Scheme 15,

analogous to that used for compounds 33-78. Hence, start-

ing from the commercially available isobutyraldehyde, the

corresponding imine was formed by reaction with tert-butyl

carbazate, followed by reduction using NaBH₃CN/AcOH.

Then the corresponding hydrazinecarboxylate derivative 1

was reacted either with 5-bromo-2,4-dichloropyrimidine or

2,4,5-trichloropyrimidine²⁹to yield the corresponding inter-

mediates 18b-c which were reacted with KCN in DMSO-

H₂O to give compounds 19b and 19c, respectively. Inter-

mediate 120 was obtained by hydrazine deprotection with

p-toluenesulfonic acid, and subsequent coupling with the

corresponding acid chloride or carboxylic acid yielded the

final products 121-269.

dine 24.³⁰Subsequent chlorine displacement with hydrazide

1 and consecutive cyanation of intermediate 25 finally

yielded compound 26. The chlorine atom at the 2-position

proved to be quite unreactive, requiring harsh but at the same

time balanced reaction conditions to stop product decomposi-

tion. In an analogous manner, compound 30 was prepared

from 5-(hydroxylmethyl)uracil hydrate (Scheme 13).³¹Prior

to the final cyanation reaction, substitution at the chloro-

methylene end with MeONa to obtain intermediate 29 was

accomplished.

Synthesis of tert-Butyloxy-2-pyrimidinenitrile Derivatives

for Optimization of the P2 Position. The second step of the

optimization process was the study of the P2 position (Figure 2).

Following a similar approach to that used in the first step of

the optimization process, substitutions at the P1 and P3

positions were fixed for comparative purposes as 2-nitrile-5-

chloro(or bromo)-6-pyrimidyl and tert-butyloxycarbonyl,

respectively. Synthesis of all derivatives included in this

study, compounds 33-78, was carried out following the

procedure outlined in Scheme 14. Starting from the com-

mercially available aldehydes or ketones, the corresponding

imine was formed by reacting with tert-butyl carbazate,

followed by reduction using H₂/PtO₂or NaBH₃CN/AcOH.

In those cases where the desired aldehyde was not available,

it was prepared via Weinreb amide from the corresponding

carboxylic acid or ester.

Results and Discussion

The known structures of cysteine proteases, in which a

central nucleophilic moiety (SH group of the cysteine) is

flanked by two sets of pockets where interactions with the

side chains of peptidic substrates take place, make the rational

design of potent enzymatic inhibitors a challenging task

because a compound may interact with either or both sets of

Derivative 37 was prepared from tetrahydro-2-furancarbal-

dehyde, reduction of the corresponding imine via hydrogenation

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6135

Scheme 14. General Synthetic Procedure for 2-Cyanopyrimidines^a

^aReagents and conditions: (a) aldehyde or ketone, NaBH₃CN, AcOH, rt; (b) 5-bromo-2,4-dichloropyrimidine, DIPEA, i-PrOH; (c) potassium

cyanide, 1,4-diazabicyclo[2.2.2]octane, 9:1 DMSO-H₂O; (d) p-toluenesulfonic acid, ACN, rt; (e) 4-bromomethylbenzoyl bromide, DIPEA; (f) N-methyl-

piperazine, DIPEA, THF, rt.

capacity was observed when the hydrazine derivative was

placed at position 2 and one of the pyrimidine nitrogen atoms

was between the substituents. A similar effect, although milder,

was observed for 9, with the nitrile group at position 4 and

hydrazine group at position 2, and for 12, with the nitrile group

Figure 3. General structure of the P3 series of 2-cyanopyrimidine

at position 2 and hydrazine group at position 4. The latter

derivatives.

derivative presented enzymatic inhibition values 3-5 times

pockets. X-ray structures of complexes between proteases and

small inhibitors are a useful tool for the design of improved

inhibitors.

greater than the former.

Replacement of the heteroaryl group by a triazine

(compound 10) yielded the most active compound of this

set (Table 1), with activity against falcipain-2 in the tens of

nanomolar range and against falcipain-3 in the hundreds of

nanomolar range. These data correlate well with the different

calculated electrophilicities of the cyano groups anticipated

by Oballa et al.;³⁵a nitrile substituent in a triazine moiety was

an excellent electrophile, whereas the electrophilicity of a

pyridine derivative was calculated as modest. Among pyr-

imidine derivatives, a nitrile group at the 2-position had the

highest calculated electrophilicity.

Cocrystallization of falcipains with small molecules to

obtain diffraction quality crystals has proven to be difficult,

hampering the rational design of potent inhibitors of these

enzymes. However, a few X-ray structures of falcipain-

inhibitor complexes have been published.^32-34With limited

available structures, we used an empirical optimization pro-

cess starting from the electrophilic nitrile group and then con-

sidering the P1, P2 and P3 positions to obtain potent inhibi-

tors of the falcipains.

Optimization Studies on the P1 Position. In order to study

the influence of the heteroaromatic ring on the nitrile elec-

trophilic nature, several derivatives were prepared and tested

(Table 1). 2-Cyanopyridine derivative 4 presented submicro-

molar inhibition of falcipain-2 but proved to be inactive

versus falcipain-3. When pyrazine was used instead, com-

pound 5 demonstrated tens of nanomolar activity against

falcipain-2 and micromolar activity against falcipain-3.

If the pyrazine was replaced by a pyrimidine ring, greater

variability was found depending on the position of the nitrile

group. No activity was observed when the hydrazine deriva-

tive and the cyano groups occupied positions 4 and 6, re-

spectively (compound 8), whereas falcipain-2 was moder-

ately inhibited by derivative 6, which bears the nitrile group

at position 5, the hydrazine moiety at position 4, and a chlo-

rine atom at the 2-position. On the other hand, better results

were obtained when the cyano group was at the 5-position,

the chlorine atom at position 4, and the hydrazine derivative

at position 2 (compound 7), with nanomolar inhibition

against falcipain-2. The 2-cyano-4-hydrazinopyrimidine de-

rivative 12 was the most potent inhibitor of this series, with

submicromolar inhibition of both falcipain enzymes.

The importance of the relative positioning between the

cyano and hydrazine groups can be illustrated by comparing

compounds 6 and 7. Both derivatives present the cyano

group at the 5-position, although enhancement of the inhibitory

Results in Table 1 indicate a clear advantage in terms of

enzymatic activity for the triazine and 2-pyrimidine nitrile

motifs, with values in the submicromolar range against both

falcipain enzymes. The limited variability of results for sub-

stitution around the triazine moiety prompted us to focus

our efforts on substitution around positions 5 and 6 of the

pyrimidine ring in order to identify an improved scaffold for

the inhibition of falcipains.

The effect of substitution at the 6-position of the pyrimi-

dine ring on the enzymatic activity of these compounds is

reflected in Table S1 (Supporting Information). Insertion of

methyl or methoxy groups (compounds 14a and 17, respec-

tively) had little effect on the inhibitory capacity of these

compounds compared to the reference compound 12; slightly

decreased inhibition of falcipain-3 was observed in the case

of the 6-methyl derivative. A moderate improvement in

activity, 2 and 3 times against falcipain-2 and falcipain-3,

respectively, was observed with the ester group in 14b. Thus,

substitution in position 6 of the pyrimidine ring did not exert

enough influence on the enzymatic potency of 2-cyano-

pyrimidine derivatives to consider further studies on this

position.

A greater influence on the enzymatic activity of these

derivatives was observed when the substituent was placed

at the 5-position (Table 2). An overall loss of falcipain inhi-

bition was observed with a methoxymethylene group, 30,

6136 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16

Coteroꢀn et al.

Scheme 15. General Synthetic Procedure for 2-Cyanopyrimidines^a

^aReagents and conditions: (a) NaBH₃CN, AcOH, rt; (b) 5-bromo-2,4-dichloropyrimidine or 2,4,5-trichloropyrimidine, N,N-diisopropylethylamine,

i-PrOH; (c) potassium cyanide, 1,4-diazabicyclo[2.2.2]octane, 9:1 DMSO-H₂O; (d) p-toluenesulfonic acid, ACN, rt; (e) P₃derivative as acid or acid

chloride.

Table 1. Effect on Falcipain Activity of Different Heterocyclic Rings

Table 2. Cyanopyrimidines: Influence of the Substituent at Position-5

on in Vitro Enzymatic Activity

^aThis value is in fact for a dimethylamino group, but it has been used

for comparative purposes.

methyl substituent as in 19e had a minimal impact on

activity, correlating well with the electron-donating capacity

of these functional groups.

^aAll compounds were characterized and purity was assessed using ¹H

NMR and LCMS.

An enhancement of the inhibitory capacity of 2-cyanopyri-

midine derivatives was observed for compounds bearing

electron-withdrawing groups, 19a-d and 20. Within the

5-halogen derivatives, a 2- and 6-fold improvement against

both enzymes was observed with fluorine and chlorine

and to a lesser extent with an alkyne group, 22, with

falcipain-3 remarkably affected by these changes. Other

electron-donating groups, such as benzylamine, 21, or meth-

oxy, 26, also resulted in less active compounds, whereas a

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6137

Table 3. Comparison between 2-Chloro- and 2-Cyanopyrimidine Deri-

vatives

Table 4. Effect on in Vitro Falcipain Activity of Different P2 Alicyclic

Substituents

compound

R

FP2 IC₅₀(μM)

FP3 IC₅₀(μM)

18d

19d

Cl

CN

>25

0.002

>25

0.026

substituents, whereas the bromine atom proved to have a

greater effect, with an inhibitory capacity 10- and 20-fold

better against falcipain-3 and falcipain-2, respectively. Re-

placement of Br by CF₃, 20, yielded only a marginal increase

in activity. A greater boost in activity was observed when a

nitro group was introduced, 19d, leading to a compound that

was 30 times more potent.

Our inhibitor results correlated reasonably well with the

Hammett parameter, σ_p, described for 4-substituted benzene

rings,³⁶suggesting that these substituents could be modulat-

ing the electrophilic nature of the nitrile group. Hence, aug-

mentation of the electrophilic character of the nitrile group

resulted in a more potent interaction with the nucleophilic

thiol group of the falcipain enzymes, which in turn translated

into better inhibitory activity. However, these observations

raised concerns regarding inhibition due to the liberation of

cyanide caused by the displacement of the cyano by the thiol

group of the falcipains, an alternative to the expected mec-

hanism of action in which the nucleophilic attack takes place

on the carbon atom of the nitrile unit. In order to discard this

hypothesis, the antiparasitic activity of intermediate 18d,

which would liberate chloride and thus be active if this

hypothesis was confirmed, was tested against both falcipain

enzymes (Table 3). The compound was inactive, thus proving

that the observed activity was not triggered by cyanide release.

In summary, the most promising and developable nitrile-

containing moieties in the P1 position were 2-cyano-5-chloro-

6-pyrimidyl and 2-cyano-5-bromo-6-pyrimidyl. These two

chemical units were used in subsequent optimization steps as

the preferred P1 substituents.

Optimization Studies on the P2 Position. For P2, in an

attempt to optimize interaction with the S2 pocket, a wide

range of different structural motifs were tested, while the

tert-butyloxycarbonyl group was maintained as the substi-

tuent in position P3.

^aAll compounds were characterized and purity was assessed using ¹H

NMR and LCMS.

In order to study the depth of the S2 pocket in falcipain-2

and falcipain-3, compounds with different linear aliphatic

substituents were synthesized and evaluated (Table S2, Sup-

porting Information). Our results indicated that the S2

pocket of both enzymes accommodates well linear groups

with up to five carbon atoms, with the optimal interaction

observed when the chain was four carbon atoms long (34)

and diminished inhibition with the six carbon atom substi-

tuent (36). Interestingly, substitution of the last carbon atom

in compound 36 by a polar group (OH) in compound 37 led

to the recovery of enzymatic activity up to the levels observed

for compound 34, suggesting the possibility of a polar

interaction at the end of the S2 pocket.

leucine-like isobutyl moiety (compound 19b) was the most

potent derivative against both enzymes in the branched

aliphatic chain class. Further changes in polarity (com-

pounds 19b and 38), branching distance (compounds 19b,

39 and 46) or length of branches in either asymmetrical

(compounds 19b, 44, and 45) or symmetrical (compounds

19b, 41, 42, and 43) compounds, led to either similar inhibi-

tion profiles or loss of enzymatic activity but not to an

improvement in activity. Hence, it seemed that additions to

the chains should not be larger than a methylene, as larger

moieties did not seem to fit in the P2 pocket. On the other

hand, when the branched chain was directly attached to the

nitrogen atom (46, 47), a loss of activity was observed against

both enzymes, even if the substituents were as small as a

methyl group, as in 46.

In order to study the width of the S2 pocket in falcipain-2

and falcipain-3, compounds with different branched aliphatic

chains were synthesized and evaluated (Table S3, Supporting

Information). Not surprisingly, based on prior data, the

Replacement of the acyclic moiety in compound 19b

by alicyclic substituents (Table 4) showed that enzymatic

6138 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16

Coteroꢀn et al.

Table 5. Effect on in Vitro Falcipain Activity of Different P2 Cycloalkyl

Substituents Directly Attached to the Nitrogen Atom

Table 6. Effect on in Vitro Falcipain Activity of Different P2 Hetero-

cyclic Substituents

^aAll compounds were characterized and purity was assessed using ¹H

NMR and LCMS.

activity against falcipain-2 and falcipain-3 increased with

ring size up to the five-atom cycle (compounds 48-50) and

then decreased for the six-membered ring (compound 51).

Hence, the optimal interaction with the enzyme was found

for cyclopentyl derivative 50, with inhibition in the low nano-

molar range. Further lengthening of the aliphatic moiety

with an extra methylene (compounds 53 and 54) led to a loss

of activity against both enzymes. In addition, the use of

aromatic moieties had a detrimental effect (55 and 56), and

the introduction of unsaturation yielded mixed results with

respect to the saturated compounds, with a similar activity

profile for the five-membered rings (compare compounds 50

and 57) and improvement in the case of the six-membered

rings (compare compounds 51 and 58).

Suppression of the methylene spacer yielding alicyclic

moieties directly attached to the nitrogen atom maximized

interaction with the lipophilic S2 pocket of the falcipains

(compounds 60-67, Table 5). Results obtained for these

alicyclic compounds were somewhat surprising when

compared with those for the corresponding acyclic deri-

vatives (compounds 46 and 47, Table S3 in Supporting

Information), and an explanation for these differences

remains elusive. The most favorable result was achieved

with the cyclohexyl group (62), showing similar activity to

that displayed by 50. Introduction of methyl substituents in the

cyclopentyl ring (63-66) only showed a clear positive effect

when present at the 2-position trans to the nitrogen atom

(compound 66), and this effect was most pronounced against

falcipain-3. The presence of unsaturation in the cyclopentyl ring

(compound 67) did not affect activity strongly.

^aAll compounds were characterized and purity was assessed using ¹H

NMR and LCMS.

The study of heterocyclic moieties in the P2 position

included oxygen-containing (compounds 68-73) and nitro-

gen-containing (compounds 74-78) derivatives (Table 6).

The presence of oxygen yielded similar inhibitory capacities

regardless of the nature of the cyclic moiety (aliphatic,

aromatic, tetrahydrofuran or tetrahydropyran derivatives),

with activities in the low nanomolar range against falcipain-2

and in the high tens to low hundreds of nanomolar range

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6139

against falcipain-3. The nitrogen-containing group of inhi-

bitors showed very different results depending on the nature

of the nitrogen atom involved in the interaction. Good to ex-

cellent activity profiles were observed for compounds con-

taining low polarity nitrogen atoms, as 74, 77, or 78, and very

modest activity profiles were seen for compounds where the

nitrogen atom presented a more polar character, as in com-

pound 76. The latter result is not surprising given the known

preference for leucine, reflecting the lipophilic character of the

P2 pocket in related cysteine proteases.³⁷Overall, the most

potent inhibitors against both enzymes with heterocyclic moi-

eties were the tetrahydropyran derivative directly attached to the

nitrogen atom, 73, and the corresponding N-acetylpiperidine

derivative 77, which was a picomolar falcipain-2 inhibitor.

In summary, some very active compounds against both

falcipain enzymes (low nanomolar or picomolar inhibitors of

falcipain-2 and units of nanomolar or low tens of nanomolar

inhibitors of falcipain-3) were identified with modifications

of position P2 of 2-cyano-5-bromo-6-pyrimidyl derivatives

having a tert-butyloxycarbonyl group in the P3 position and

using compound 19b (P2: isobutyl) as reference. Preferred

substitution at position P2 included the branched alkyl group

cyclopentylmethyl (compound50; FP2IC₅₀= 0.001 μM, FP3

IC₅₀= 0.01 μM), the cycloalkyl group directly attached to the

nitrogen atom cyclohexyl (compound 62;FP2IC₅₀=0.001μM,

FP3 IC₅0 = 0.008 μM), the cycloalkyl group directly attached to

the nitrogen atom trans-2-methylcyclopentyl (compound 66;

FP2 IC₅₀= 0.003 μM, FP3 IC₅₀= 0.006 μM), the heterocyclic

group directly attached to the nitrogen atom tetrahydropyran

(compound 73; FP2 IC₅₀= 0.002 μM, FP3 IC₅₀= 0.028 μM),

and the heterocyclic group directly attached to the nitrogen atom

N-acetyl-4-piperidine (compound 77; FP2 IC₅₀< 0.0005 μM,

FP3 IC₅₀= 0.033 μM).

Table 7. Effect on in Vitro Antiparasitic Activity of Different P2

Branched Alkyl Substituents

All of the compounds described above, despite their promising

enzymatic inhibitory capacities, showed only modest anti-

parasitic activity in the micromolar range (data not shown).

However, we had previously noticed that antiparasitic inhibitory

capacity within the nitrile family improved when amino groups

protonable at the food vacuole pH (4.0-6.0) were present.

Lysosomatropism,³⁸defined as the ability of compounds to

accumulate in acidic compartments, is a property of weakly

basic, lipophilic molecules. These neutral species are able to freely

diffuse across the cell membrane but become membrane im-

permeable once protonated within the acidic compartment. The

degree of compound accumulation is dependent on its pK_aand

the pH difference between the cytosol and the lysosome (or food

vacuole). In addition, compound lipophilicity can affect accu-

mulation because of aggregation or interaction with lipid mem-

branes. In order to test this “amine hypothesis” with the

2-cyanopyrimidine class, a benzylic N-methylpiperazinyl moiety

was introduced in position P3 of the general structure and a

similar SAR exploration of the P2 position was carried out for

this chemical class. Activities against falcipain-2 and falcipain-3

and against cultured P. falciparum were tested (Tables 7-10,

Table S4 Supporting Information).

These in vitro results confirmed the previously observed

tendencies in terms of the best possible substituents for the

P2 position. In addition, introduction of a protonable amine

consistently led to a marked improvement in activity against

cultured parasites, confirming the “amine hypothesis”. When

linear alkyl substituents were introduced (Table S4, Supporting

Information), the best results in terms of enzymatic and anti-

parasitic activities were observed with the n-butyl chain

(compound 82). An improvement in enzymatic inhibition was

^aAll compounds were characterized and purity was assessed using ¹H

NMR and LCMS.

observed when compared to tert-butyloxycarbonyl derivatives.

The improvement was generally moderate but more noticeable

for the n-hexyl derivative 84 (compare with compound 36).

In the case of branched alkyl substituents (Table 7), again the

isobutyl group represented an advantageous interaction with

the S2 pocket (compound 85). Further augmentation of steric

hindrance, as in the tert-butyl group (compound 86), resulted

in a loss of inhibitory capacity, especially against falcipain-3

and cultured parasites. Elongation of the methylene spacer as

in 87 and 88 did not affect enzymatic activities as much as

antiparasitic activities, the same effect being observed when the

branch lengths were prolonged (89-91). Interaction with the

lipophilic S2 pocket was not affected when only one of the

branches was extended, with activities in the tens of nanomolar

range (92). Further elongation or introduction of hindrance

around the nitrogen atom (93-95) led to a diminution in

potency. Overall, the introduction of the N-methylpiperazine

moiety in these derivatives yielded more potent enzymatic

inhibitors than N-tert-butyloxycarbonyl derivatives, which in

turn rendered better inhibitors of parasite growth.

When cycloalkyl substituents were introduced (Table 8),

improved enzymatic potencies over the N-tert-butyloxycarbo-

nyl derivatives were again observed; the most noticeable effects

6140 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16

Coteroꢀn et al.

Table 8. Effect on in Vitro Antiparasitic Activity of Different P2

Cycloalkyl Substituents

Table 9. Effect on in Vitro Antiparasitic Activity of Different P2

Cycloalkyl Substituents Directly Attached to the Nitrogen Atom

^aAll compounds were characterized and purity was assessed using ¹H

NMR and LCMS.

responding N-tert-butyloxy derivatives but led to markedly

improved antiparasitic activity. The most potent derivatives

of this group were the tetrahydropyran and N-acetylpiper-

idine (117 and 118), with activities in the picomolar range

against falcipain-2 and in the tens of nanomolar range

against both falcipain-3 and cultured parasites.

^aAll compounds were characterized and purity was assessed using ¹H

NMR and LCMS.

were with compounds 99-101. The cyclopentylmethyl deriva-

tive 98 and its unsaturated analogue 104 confirmed the

tendency previously observed when no protonable amine

was present (compounds 50 and 57, respectively). These

compounds were very potent enzyme inhibitors and showed

good antiparasitic activities.

In summary, the S2 subsite showed preference for a

number of substituents in the P2 position, including 2-methyl-

propyl (compound 19b), cyclopentylmethyl (compounds 50

and 57), or cyclopentyl (compounds 61, 64, 66, and 67), all

presenting enzymatic activities in the low nanomolar range

against falcipain-2 and in the tens of nanomolar range

against falcipain-3, with antiparasitic activities in the micro-

molar range. The introduction of a protonable amine mark-

edly boosted the antiparasitic activity of these derivatives,

with all being active against cultured parasites in the tens of

nanomolar range (compounds 85, 98, 106, 109, and 111).

Furthermore, the protonable amine series confirmed the pre-

viously observed S2 subsite SAR for enzyme inhibition.

Optimization Studies on the P3 Position. The last step of

the optimization process was the study of position P3.

Substitution in the P1 position was kept as 2-cyano-5-chloro-

(or bromo)-6-pyrimidyl on the basis of the results obtained in

the optimization studies described above. Compounds bear-

ing a phenyl substituent in position P3 demonstrated acti-

vity at units of nanomolar against falcipain-2 and tens of

nanomolar against falcipain-3, regardless of the halogen

substituent at the 5-position of the pyrimidine ring (com-

pounds 121 and 122, Table S5 Supporting Information).

Selection of the most appropriate substitution at position P2

Our results also confirmed the advantage of having the

cycloalkyl substituent directly attached to the nitrogen atom

(Table 9) over the corresponding open chain substituent

(compare, for example, compounds 105 and 94 or com-

pounds 106 and 95), as previously observed for the N-tert-

butyloxy derivatives. Improvements in activity against both

falcipains and cultured parasites were observed. Among the

most active compounds identified in this group of com-

pounds, equivalent potencies were observed for cyclohexyl

and cyclopentyl derivatives (106 and 107), while the most

potent derivative resulted from the introduction of a trans-

methyl substituent at the 2-position of the cycloalkyl ring (111),

yielding picomolar activity against falcipain-2 and single-digit

nanomolar inhibition against falcipain-3 and against cultured

parasites.

The introduction of a protonable amine on derivatives

bearing heterocyclic substituents at P2 (Table 10) again had

little effect on enzymatic activities when compared to the cor-

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6141

Table 10. Effect on in Vitro Antiparasitic Activity of Different P2

Heteroatom Containing Cycloalkyl Substituents

profile of the 4-pyridyl derivative (compound 126) was clearly

worse than that for the 3-pyridyl derivative (compound 128),

and the 2-pyridyl derivative 129 was the best inhibitor of

both enzymes in this series. Replacement of a second carbon

atom by nitrogen did not affect activity against falcipain-2

but led to some differences in activity versus falcipain-3, as

shown for compounds 130, 131, and 132.

In the case of five-membered heteroaromatic rings (Table S6

Supporting Information, compounds 133-150), most of the

compounds studied showed activities against falcipain-2 in

the tens of nanomolar range and against falcipain-3 in the

tens to low hundreds of nanomolar range regardless of the

heteroatom present (oxygen, sulfur, or nitrogen, e.g., comp-

ounds 133, 136, and 139, respectively), the number of hetero-

atoms (one or two, e.g., compounds 139 and 146), or their

disposition within the ring (2- or 3-, e.g., compounds 133 and

134 or compounds 136 and 137). Exceptions to this rule were

compounds 147 and 150. Derivatives bearing two heteroa-

toms in a relative 1,3-position (compounds 141-146) ex-

hibited similar activities. On the other hand, when the hetero-

atoms had a relative 1,2-disposition (compounds 147-150),

an enhancement of their enzymatic inhibitory capacity was

observed when the oxygen atom was adjacent to the carbonyl

group (compounds 148 and 149).

The use of fused (hetero)-aromatic rings yielded variable

results (Table 11, compounds 151-179). When both rings

were six-membered (compounds 151-157), the introduction

of nitrogen atoms distant relative to the carbonyl moiety ring

(compound 152) had a detrimental effect against falcipain-3.

Similar activities were observed when the nitrogen atom was

placed in the ring nearer the carbonyl attachment, regardless

of its positioning (compounds 153-157) and always repre-

senting a clear improvement in activity against falcipain-2

and a more modest enhancement against falcipain-3 over the

reference compound 151. No difference was observed when a

2-benzofuranyl moiety replaced the naphthyl group and

substitution within the condensed rings was explored

(compounds 159-161). The introduction of a methyl group

adjacent to the carbonyl attachment yielded a loss of in-

hibitory capacity (159 vs 158). A similar effect was observed

when a 5-methoxy substituent was present, whereas moving

this group to position 7 of the benzofuran substituent yielded

improved inhibitory capacity (compounds 160 and 161). An

improvement in activity was observed when attachment to

the carbonyl group was at position 5 (compound 162), con-

trasting with the quinoxaline ring (compound 152). If a

benzothiophene group was used instead (compounds 163-

165), analogous results were observed, whereas an indole

moiety yielded mixed results depending upon the substitu-

tion position in the bicyclic structure (compounds 166-170).

Introduction of two heteroatoms, as in 1,2-benzoisoxazole

(compound 171), resulted in the loss of inhibitory capacity

against falcipain-3, while a 2-benzothiazolyl group (com-

pound 172) yielded a quite potent inhibitor. The results

previously observed, with benzofuranyl and benzothiophe-

nyl groups affording more potent inhibitors when the het-

eroatom was placed in the distant ring, were confirmed with

the 1H-benzimidazolyl and 1H-1,2,3-benzotriazolyl deriva-

tives (compounds 173-175), yielding the most potent deri-

vatives of these series, with subnanomolar activities against

falcipain-2. Comparing compounds 159 and 176, the influ-

ence of the heteroatom on the second ring was once more

seen, yielding a compound 2 orders of magnitude more

potent against falcipain-2 and 30 times more potent against

^aAll compounds were characterized and purity was assessed using ¹H

NMR and LCMS.

was more problematic because of the number of available

substituents presenting similarly good enzymatic and whole-

cell activity profiles. We decided that the 2-methylpropyl

moiety was the best balanced substituent among the poten-

tial candidates, considering lack of chirality, low lipophili-

city, low molecular weight, and low predicted cost of goods.

It is worth noting that whole-cell activity was as important as

the enzymatic activity profile in our considerations because it

could reflect the contribution of a substituent to membrane

permeability or transport to the target enzyme, and this point

was crucial for selection of 2-methylpropyl as the substituent

in position P2. Thus, position P3 was studied using deriva-

tives with the 2-cyano-5-chloro (or bromo)-6-pyrimidyl unit

at position P1 and the 2-methylpropyl unit at position P2.

When six-membered aromatic rings were used (Table S5

Supporting Information, compounds 121-132), no improve-

ment of enzymatic activity with regard to reference compounds

121 and 122 was obtained, although some interesting trends

could be observed. Substitution in the 4-position of the phenyl

ring (compound 123) did not affect the activity profile, while

substitution in the 3-position of the ring (compounds 124 and

125) led to a significant loss of activity versus both falcipain

enzymes. Theoppositeeffectwasobserved withreplacement of

one carbon atom of the phenyl ring by nitrogen; the activity

6142 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16

Coteroꢀn et al.

Table 11. Effect on in Vitro Falcipain Inhibition of Different Fused (Hetero)Aromatic Ring Substituents as P₃

^aAll compounds were characterized and purity was assessed using ¹H NMR and LCMS.

falcipain-3. Using tricyclic derivatives such as fluorene or

fluorenone (compounds 177-179), the influence of the

position of the carbonyl group in the tricyclic structure was

apparent: the derivative with carbonyl groups further apart,

178, was 10 times more potent against falcipain-2, but it was

still modestly potent against falcipain-3.

When the two (hetero)aromatic cycles were not fused

(Table 12, compounds 180-223), greater variability in the

results was observed. With two phenyl rings (compounds

180-186), quite a significant improvement was detected

when the two rings were angled toward each other (180 vs 181).

This effect was also seen when an oxygen linker between the

rings was present (185 vs 186), while the influence of the diff-

erent substitution positions in the phenyl ring was only notice-

able in the degree of inhibition of falcipain-3 (compounds

184, 185, and 186). The introduction of one or two nitrogen

atoms in any of the rings (compounds 187-193) proved to be

highly successful, with several derivatives presenting subna-

nomolar potencies against falcipain-2 and low tens of nano-

molar activities against falcipain-3 (188-191). When phenyl

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6143

Table 12. Effect on in Vitro Falcipain Activity of Different Not Fused Bicyclic (Hetero)Aromatic Ring Substituents as P₃

^aAll compounds were characterized and purity was assessed using ¹H NMR and LCMS.

6144 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16

Coteroꢀn et al.

Table 13. Effect on in Vitro Falcipain Inhibition of Different Benzylic

Aromatic Ring Substituents as P₃

falcipain-3 (199 vs 203). For oxazolyl, thiazolyl, and imida-

zolyl derivatives (205-212), no improvements in activity

over the reference compound were observed, while a negative

influence on the enzymatic activity of a methyl group in the

position vicinal to the carbonyl substituent, especially

against falcipain-3, was confirmed (compounds 202, 208,

209, and 211). The use of isoxazolyl derivatives with varying

positioning and substitutions (compounds 213-218) and of

pyrazolyl (compounds 219-220) and 1,2,3-triazolyl deriva-

tives (compound 221) showed no benefits. Also, substitution

of the most distant phenyl ring by thiophene (compounds

222 and 223) did not affect the inhibition of either falcipain-2

or falcipain-3.

Finally, benzylic derivatives were explored (Table 13,

compounds 224-239). Very potent inhibitors against both

enzymes were obtained with very simple substitution of the

phenyl ring (compounds 224-229). Attempts to stabilize the

benzylic position both with methyl groups or different rings

(230, 233, and 236) resulted in a loss of activity against both

enzymes. When para-chloro substituted phenyl derivatives

were compared (231, 234, 235, 237, and 238), there seemed to

be a small pocket around the benzylic position, with the

optimal substituent being dimethyl, a cyclopropyl ring

allowed, but with other aliphatic rings resulting in a loss of

potency, in particular against falcipain-3.

All the P3-substituted compounds described above were

micromolar inhibitors of cultured parasites, a situation similar

to that observed in the optimization process for position P2. In

order to confirm the “amine hypothesis” described above,

different amines were introduced using the most potent scaf-

folds at P3 identified so far, and the activities of the resulting

derivatives were tested against cultured malaria parasites

(Table 14, compounds 240-269). When the phenyl ring was

attached to the carbonyl group (compounds 240-243), very

potent falcipain inhibitors were obtained, although their anti-

parasitic activity was variable. Directly attached N-alkylpiper-

azinyl derivatives such as compounds 240 and 241 showed

superior antiparasitic activity compared to compounds with

an oxygen-ethylene linker between the amine and the phenyl

ring (compounds 242-243). The amine hypothesis was once

again confirmed with compound 244; this compound had an

enzymatic activity profile similar to that observed for com-

pound 240 but was a much weaker inhibitor of cultured

parasites (over 60 times less active), reflecting the lack of a

basic amine in its structure. The use of five-membered hetero-

aromatic rings (compounds 245-247) yielded very good anti-

parasitic activity, with minimal differences in their respective

enzymatic activities observed. In the case of benzofuran

derivatives (compounds 248-250), no significant differences

in enzymatic or whole-cell activities were observed for com-

pounds with substitution at either position 5 or position 7.

Pyridinyl-phenyl combinations (compounds 251-254) were

markedly active, yielding nanomolar antiparasitic inhibitors

when the phenyl rings were angled toward each other, con-

firming the results previously observed for compounds lacking

a basic amine (180 vs 181). This result suggests the importance

of the relative disposition of the rings rather than the nature

of the heteroatoms present in them, as the furanyl-

phenyl derivative 255 was equipotent. Benzylic derivatives

(compounds 256-265) confirmed the tendency previously

observed with directly attached phenyl rings; N-alkylpiperazi-

nyl derivatives were the most potent. Attempts to stabilize the

benzylic position with dimethyl groups once more resulted in a

loss of activity in all cases; also, the overall lipophilicity of these

^aAll compounds were characterized and purity was assessed using ¹H

NMR and LCMS.

five-membered heterocyclic ring combinations were evalu-

ated (compounds 194-198), potent inhibitors of falcipain-2

and moderate inhibitors of falcipain-3 were obtained. When

the order was reversed, i.e., five-membered heterocycle-

phenyl combination (compounds 199-221), a greater influ-

ence of the heteroatoms and/or substituents was detected.

When the heterocycle was a furan or a thiophene ring,

compounds with inhibition of falcipain-2 in the units to tens

of nanomolar range and inhibition of falcipain-3 in the tens

to hundreds of nanomolar range were obtained (199-204),

although the furan seems to offer an advantage in terms of

enzyme inhibition over the thiophene, particularly against

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6145

Table 14. Effect on in Vitro Antiparasitic Activity of Different Substituents as P₃Bearing an Amine

^aAll compounds were characterized and purity was assessed using ¹H NMR and LCMS.

molecules was increased. Introduction of a protonable amine as

a substituent of the second nitrogen atom of the hydrazide

group (compounds 266-269) yielded inhibitors in the low

hundreds of nanomolar range against cultured parasites.

Optimizationstudies on the electrophile and P1position led to

identification of 2-cyano-5-chloropyrimidine and 2-cyano-5-

bromopyrimidine scaffolds as the most promising, showing a

clear advantage in terms of enzymatic activity over other

nitriles. On the basis of these results and studies on the P2

position, compounds with substituents including 2-methyl-

propyl, cyclopentylmethyl, or cyclopentyl at the hydrazide

nitrogen position, with improved enzymatic activity profiles,

were obtained. Further studies on the P3 position using the

Conclusions

A potent new class of falcipain inhibitors based on a

pyrimidine nitrile scaffold has been discovered and optimized.

6146 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16

Coteroꢀn et al.

parasites, parasites were fixed with 1% formaldehyde in PBS,

pH 7.4, for 48 h at room temperature and then labeled with

YOYO-1 (1 nM; Molecular Probes) in 0.1% Triton X-100 in

PBS. Parasitemias were determined from dot plots (forward

scatter vs fluorescence) acquired on a FACSort flow cytometer

using CELLQUEST software (Becton Dickinson). IC₅₀values

for growth inhibition were determined with GraphPad Prism

software from plots of percentage parasitemia compared to

controls (untreated parasites) over inhibitor concentration. In

each case, the goodness of curve fit was documented by R²

values of g0.95.

Chemistry. Starting materials were obtained from commer-

cial suppliers and used without further purification unless

otherwise stated. Flash chromatography was carried out using

prepacked Isolute Flash or Biotage silica gel columns as the

stationary phase and analytical grade solvents as the eluent un-

less otherwise stated.

NMR spectra were determined on a Varian Unity spectro-

meter (300 MHz). Chemical shifts are reported as δ values (ppm)

downfield from tetramethylsilane, used as an internal standard

in the solvent indicated. All coupling constants are reported in

hertz (Hz), and multiplicities are labeled br (broad), s (singlet),

br s (broad singlet), d (doublet), t (triplet), q (quartet), dd

(doublet of doublets), dt (doublet of triplets), td (triplet of

doublets), ddd (double-doubledoublet), and m (multiplet).

Total ion current traces were obtained for electrospray

positive and negative ionization (ESþ/ES-) on a Waters

ZMD 2000.

Figure 4. Most potent 2-cyanopyrimidine derivatives identified.

2-methylpropyl moiety as the reference P2 substitution led to

identification of a group of promising falcipain inhibitors,

with subnanomolar inhibition of falcipain-2 and nanomolar

inhibition of falcipain-3, although inhibition of cultured

parasites was in the micromolar range. Introduction of a

protonable amine in the structure of these compounds con-

firmed the “amine hypothesis”, with the presence of a pro-

tonable amine markedly improving antiparasitic activity.

Compounds presenting excellent in vitro enzymatic and anti-

parasitic activities included85, 98, 106, 107, 240, 241, 245, 246,

253, and 254 (summarized in Figure 4). These results have

encouraged further investigation of these compounds, includ-

ing evaluation of in vitro and in vivo metabolic stability,

selectivity against human cysteine proteases, toxicity, and

in vivo efficacy evaluation. These results will be published in

due course. On the whole, 2-cyanopyrimidines have afforded

a broad range of potent in vitro antimalarials, with the variety

of moieties allowed, especially at the P3 position, facilitating the

modulation of the properties of these promising derivatives.

Analytical chromatographic conditions used for the LC/MS

analysis were as follows. The column was ACE, 4.6 mm ꢀ 30

mm, and the stationary phase particle size was 3 μm. Solvent A

was an aqueous solvent consisting of water þ 0.1% formic acid.

Solvent B was an organic solvent consisting of acetonitrile þ

0.1% formic acid. The composition of the solvent over 5 min is

shown in Table 15. Additional chromatographic parameters

were as follows: flow rate, 1 mL/min; injection volume, 5 μL;

column temperature, 30 ꢀC; UV wavelength range, 220-330 nm.

The purity of all tested compounds was g95% using the

analytical method described above unless stated otherwise.

1,1-Dimethylethyl 2-(2-Methylpropyl)hydrazinecarboxylate (1).

A solution of 1,1-dimethylethyl hydrazinecarboxylate (9.2 g,

70 mmol) in isopropanol (50 mL) was treated at 0 ꢀC with iso-

butyraldehyde (6.4 mL, 70 mmol) over 15 min, and the mixture

was stirred at 0 ꢀC for 2 h. Then the mixture was stirred for 5 h at

room temperature. To this solution containing the intermediate

hydrazone was added PtO₂, and the suspension was hydrogenated

at room temperature and 2.6 bar for 48 h. The suspension was

filtered and the solvent was removed under reduced pressure to

give the title compound. ¹H NMR(300 MHz, CDCl₃) δ ppm: 6.02

(br s, 1H), 3.92 (br s, 1H), 2.66 (d, J = 6.7 Hz, 2H), 1.73 (m, 1H),

1.46 (s, 9H), 0.93 (d, J = 6.9 Hz, 6H). ESIMS m/z: 189 (MH^þ).

1,1-Dimethylethyl 2-(6-Cyano-2-pyridinyl)-2-(2-methylpropyl)-

hydrazinecarboxylate (4). Under a nitrogen atmosphere, Pd₂(dba)₃

(0.077 g, 0.08 mmol), (()-2,2⁰-bis(diphenylphosphino)-1,1⁰-bi-

naphthalene (BINAP, 0.01 g, 0.02 mmol), 1,1-dimethylethyl

2-(2-methylpropyl)hydrazinecarboxylate (1, 0.715 g, 3.8 mmol),

2,6-dibromopyridine (1 g, 4.22 mmol), and sodium tert-butox-

ide (0.568 g, 5.9 mmol) were dissolved in toluene (20 mL). The

resulting reaction mixture was stirred at room temperature for

3 h. The mixture was then diluted with DCM (100 mL), filtered

over a Celite pad, and concentrated in vacuo. The crude mixture

was purified by flash chromatography on silica gel (eluting with

Hex/EtOAc mixtures from 100:0 to 0:100%) and then by pre-

parative HPLC (ACN/H₂O, 0.1%TFA, gradient 30-100%) to

give 1,1-dimethylethyl 2-(6-bromo-2-pyridinyl)-2-(2-methyl-

propyl)hydrazinecarboxylate 2a (0.34 g, 26%). ¹H NMR

(300 MHz, DMSO-d₆) δ ppm: 9.41-8.95 (br, 1H), 7.45 (t,

J = 7.9 Hz, 1H), 6.83 (d, J = 7.5 Hz, 1H), 6.57 (d, J = 8.5

Hz, 1H), 3.77-3.15 (br, 2H), 1.98-1.7 (m, 1H), 1.42-1.28 (br,

9H), 0.87 (d, J = 6.7 Hz, 6H).

Experimental Section

Biological Assays. Enzyme Inhibition. Falcipain-2 and falci-

pain-3 were recombinantly expressed in E. coli and refolded to

the active enzyme as previously described.³⁹IC₅₀values against

recombinant falcipain-2 and falcipain-3 were determined as

follows. The substrate (H-^D-Val-Leu-Arg-AFC), added to a

final concentration of 30 μM for FP-2 and 150 μM for FP-3,

was mixed at room temperature with different concentrations of

tested inhibitors in the reaction buffer (100 mM sodium acetate,

pH 5.5, 500 μM CHAPS, and 10 mM DTT). Inhibitor solutions

were prepared from stocks in DMSO (maximum concentration

of DMSO in the assay was 1%).

The reaction was started with the addition of the recombinant

enzymes, 0.25 nM FP-2 and 2 nM FP-3. Fluorescence was moni-

tored for 1 h at room temperature in a SpectraMax Plus 384

spectrofluorometer. IC₅₀values were determined from plots of

percent activity over concentration using GraFit software.

Inhibition of Cultured Malaria Parasites. The effects of inhi-

bitors were studied against W2-strain P. falciparum, obtained

from the Malaria Research and Reference Reagent Resource

Center and cultured with human erythrocytes at 2% hematocrit

in RPMI medium with either 10% human serum or 0.5%

AlbuMAX II serum substitute. Synchrony was maintained with

sorbitol. Cultures were incubated with serial dilutions of test

compounds from 1000ꢀ stocks in DMSO for 48 h beginning

at the ring stage. The medium was changed after 24 h, with

maintenance of the appropriate inhibitor concentration. After

48 h, when control cultures contained nearly all new ring-stage

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6147

Table 15. Analytical Chromatography: Composition of Solvent over

5 min

(br, 9H), 1.11-1.81 (br, 6H). ¹³C NMR (300 MHz, CDCl₃) δ

ppm: 164.6, 163.9, 162.3, 115.1, 98.6, 89.9, 83.6, 58.2, 28.1, 26.2,

20.0. ESIMS m/z: 326 (MH^þ).

time (min)

% solvent B

1,1-Dimethylethyl 2-(6-Cyano-4-pyrimidinyl)-2-(2-methylpro-

pyl)hydrazinecarboxylate (8). 4,6-Dichloropyrimidine (0.15 g,

1 mmol) was dissolved in isopropanol (5 mL), and 1,1-dimethy-

lethyl 2-(2-methylpropyl)hydrazinecarboxylate (1, 0.17 g, 0.9 mmol)

and DIPEA (0.35 mL, 2.0 mmol) were added to the solution.

The resulting mixture was heated under microwave irradiation

at 130 ꢀC for 90 min. Solvent was evaporated in vacuo and the

residue was taken up in DCM and washed with 1 N NH₄Cl and

brine. The organic layer was dried over anhydrous MgSO₄,

and solvent was removed under reduced pressure to yield 1,1-

dimethylethyl 2-(6-chloro-4-pyrimidinyl)-2-(2-methylpropyl)-

hydrazinecarboxylate (0.23 g, 76%). ¹H NMR (300 MHz,

CDCl₃) δ ppm: 8.44 (s, 1H), 6.77-6.31 (br, 2H), 3.97-3.25

(br, 2H), 2.18-1.94 (m, 1H), 1.68-1.23 (m, 9H), 0.95 (d, J =

6.7 Hz, 6H).

1,1-Dimethylethyl 2-(6-chloro-4-pyrimidinyl)-2-(2-methylpro-

pyl)hydrazinecarboxylate (0.225 g, 0.75 mmol) was dissolved

in a 85:15 DMSO-H₂O mixture (3 mL), and DABCO (0.084 g,

0.75 mmol) and KCN (0.098 g, 1.5 mmol) were added to the

solution. The resulting mixture was heated to 65 ꢀC for 30 h, and

then it was poured into ice-water. The reddish solid that pre-

cipitated was filtered off and purified by flash chromatography

on silica gel (eluting with a 50:1 DCM/MeOH mixture) first and

then by preparative HPLC (ACN/H₂O, gradient 30-100%) to

yield the title compound 8. ¹H NMR (300 MHz, CDCl₃) δ ppm:

8.68 (s, 1H), 7.02 (br s, 1H), 6.71-6.40 (m, 1H), 4.31-2.99 (m,

2H), 2.16-1.96 (m, 1H), 1.81-1.32 (br, 9H), 0.97 (d, J = 6.3 Hz,

6H). ESIMS m/z: 290 (MH^-).

0

10

90

10

0.2

3.5

4

4.01

5

In a round-bottomed flask and under a nitrogen atmosphere,

1,1-dimethylethyl 2-(6-bromo-2-pyridinyl)-2-(2-methylpropyl)-

hydrazinecarboxylate 2a (0.1 g, 0.29 mmol), Pd₂(dba)₃(0.053 g,

0.006 mmol), dppf (0.064 g, 0.01 mmol), zinc(II) cyanide (0.02 g,

0.17 mmol), and zinc dust (0.0023 g, 0.035 mmol) were dissolved

in DMA (10 mL). The mixture was heated to 120 ꢀC for 2 h and

then diluted with DCM, filtered over a Celite pad, and concen-

trated to dryness. The residue was purified by flash chromatog-

raphy on silica gel (eluting with Hex/EtOAc mixtures from 100:0

to 0:100%) to yield the title compound 4 (0.07 g, 83% yield). ¹H

NMR (300 MHz, DMSO-d₆) δ ppm: 9.51-9.06 (br, 1H),

7.77-7.71 (m, 1H), 7.27 (d, J = 7.2 Hz, 1H), 6.91 (d, J = 8.8

Hz, 1H), 3.79-3.33 (br, 2H), 2.01-1.85 (m, 1H), 1.43-1.27 (br,

9H), 0.89 (d, J = 6.6 Hz, 6H). ESIMS m/z: 235 (MH^þ- 56).

1,1-Dimethylethyl 2-(6-Cyano-2-pyrazinyl)-2-(2-methylpro-

pyl)hydrazinecarboxylate (5). Following analogous procedures,

compound 5 was synthesized. ¹H NMR (300 MHz, DMSO-d₆)

δ ppm: 9.81-9.24 (br, 1H), 8.46-8.29 (br, 2H), 4.22-3.13 (br,

2H), 2.03-1.86 (m, 1H), 1.53-1.19 (br m, 9H), 0.9 (d, J = 6.6

Hz, 6H). ESIMS m/z: 292 (MH^þ).

1,1-Dimethylethyl 2-(2-Chloro-5-cyano-4-pyrimidinyl)-2-(2-

methylpropyl)hydrazinecarboxylate (6). 5-Cyanouracil (2 g,

14.6 mmol) and PCl₅(11.68 g, 56.1 mmol) were added to

POCl₃(27 mL, 290.6 mmol), and the resulting mixture was

heated to 115 ꢀC for 4 h. Excess POCl₃was distilled, and the

mixture was added dropwise to a cooled solution of saturated

aqueous NaHCO₃. Product was extracted with DCM several

times, and the combined organic layers were washed with

water and brine and dried over anhydrous Na₂SO₄. Solvent

was evaporated under reduced pressure and the crude mixture

was purified by flash chromatography on silica gel (eluting

with Hex/EtOAc mixtures from 100:0 to 70:30%) to yield 2,4-

dichloro-5-pyrimidinecarbonitrile as a white solid (1.63 g,

64%). ¹H NMR (300 MHz, CDCl₃) δ ppm: 8.63 (s, 1H).

¹³C NMR (300 MHz, CDCl₃) δ ppm: 192.7, 163.8, 163.3,

111.75, 109.2.

1,1-Dimethylethyl 2-(4-Cyano-2-pyrimidinyl)-2-(2-methylpro-

pyl)hydrazinecarboxylate (9). A mixture of 1,1-dimethylethyl

2-(2-methylpropyl)hydrazinecarboxylate (1, 2.38 g, 12.6 mmol),

2,4-dichloropyrimidine (2.07 g, 13.9 mmol), and DIPEA (4.4

mL, 25.3 mmol) in toluene (10 mL) was refluxed for 14 h.

Solvent was removed under reduced pressure, and the residue

was taken up in DCM and washed with 1 N NH₄Cl and brine

and dried over anhydrous MgSO₄. After evaporation of the

solvent in vacuo, the crude mixture was purified by flash chro-

matography on silica gel (eluting with Hex/EtOAc mixtures

from 10:1 to 1:1) to yield intermediate 1,1-dimethylethyl 2-(4-

chloro-2-pyrimidinyl)-2-(2-methylpropyl)hydrazinecarboxylate

(0.787 g, 21%). ¹H NMR (300 MHz, DMSO-d₆) δ ppm: 9.31 (s,

1H), 8.37 (d, J = 4.97 Hz, 1H), 6.87 (d, J = 4.97 Hz, 1H),

3.83-3.37 (br, 2H), 2.04-1.85 (m, 1H), 1.43-1.24 (m, 9H),

0.92-0.85 (m, 6H). ESIMS m/z: 299 (MH^-).

DABCO (0.589 g, 5.25 mmol) and KCN (0.342 g, 5.25 mmol)

were added to a solution of intermediate 1,1-dimethylethyl 2-(4-

chloro-2-pyrimidinyl)-2-(2-methylpropyl)hydrazinecarboxylate

(0.787 g, 2.6 mmol) in a 85:15 DMSO-H₂O mixture (12 mL) at

room temperature. The resulting reaction mixture was then

heated to 70 ꢀC. After 5 h, the reaction mixture was cooled

down to room temperature and then poured into ice-water and

kept at low temperature in order to favor product precipitation.

A gum was filtered off and redissolved in a DCM-MeOH

mixture. Solvent was removed under reduced pressure to yield

a brown oil which was purified by flash chromatography on

silica gel (eluting with a 4:1 Hex/EtOAc mixture) to yield the title

compound 9 (0.28 g, 37%). ¹H NMR (300 MHz, DMSO-d₆) δ

ppm: 9.37-8.98 (m, 1H), 8.77-8.68 (m, 1H), 7.36-7.27 (m, 1H),

3.83-3.16 (br, 2H), 2.03-1.87 (m, 1H), 1.43-1.26 (m, 9H),

0.98-0.78 (m, 6H). ESIMS m/z: 290 (MH^-).

Intermediate 2,4-dichloro-5-pyrimidinecarbonitrile (0.4 g,

2.3 mmol) and 1,1-dimethylethyl 2-(2-methylpropyl)hydrazine-

carboxylate (1, 0.315 g, 1.7 mmol) were dissolved in isopropanol

(5 mL), and DIPEA (0.52 mL, 3.0 mmol) was added to the

mixture. After stirring at room temperature for 22 h, solvent was

evaporated in vacuo and the residue was taken up in DCM and

washed with 1 N NH₄Cl, water, and brine. The organic layer was

dried over anhydrous Na₂SO₄, and solvent was removed under

reduced pressure. The crude mixture was purified by flash

chromatography on silica gel (eluting with Hex/EtOAc mixtures

from 100:0 to 70:30%) followed by preparative HPLC (ACN/

H₂O, 0.1% TFA, gradient 20-100%) to yield the title com-

1

pound 6 (0.121 g, 12%). H NMR (300 MHz, CDCl₃) δ ppm:

8.50 (s, 1H), 6.69-6.56 (br, 1H), 3.81-3.66 (m, 2H), 2.26-1.96

(m, 1H), 1.66-1.23 (m, 9H), 0.95 (d, J = 6.7 Hz, 6H). ¹³C NMR

(300 MHz, CDCl₃) δ ppm: 162.8, 162.6, 162.3, 155.1, 114.3,

98.3, 82.4, 58.1, 28.1, 26.6, 19.9. ESIMS m/z: 324 (MH^-).

1,1-Dimethylethyl 2-(4-Chloro-5-cyano-2-pyrimidinyl)-2-(2-

methylpropyl)hydrazinecarboxylate (7). Replacement of isopro-

panol by toluene in the reaction between 2,4-dichloro-5-pyrimi-

dinecarbonitrile and 1,1-dimethylethyl 2-(2-methylpropyl)-

hydrazinecarboxylate yielded a mixture of 6 and 7, the latter

being the minor one. ¹H NMR (300 MHz, CDCl₃) δ ppm: 8.47

(s, 1H), 6.85 (s, 1H), 3.41-2.83 (br, 2H), 2.20-1.97 (m, 1H), 1.54

1,1-Dimethylethyl 2-[4-Cyano-6-(methyloxy)-1,3,5-triazin-2-

yl]-2-(2-methylpropyl)hydrazinecarboxylate (10). To a solution

of 2,4-dichloro-6-(methyloxy)-1,3,5-triazine (1 g, 5.53 mmol) in

anhydrous THF (40 mL) at 0 ꢀC, 1,1-dimethylethyl 2-(2-methyl-

propyl)hydrazinecarboxylate (1, 1.05 g, 3.8 mmol) was added.

The mixture was left to reach room temperature overnight. It

was then evaporated to dryness and the crude was purified by

6148 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16

Coteroꢀn et al.

flash chromatography on silica gel (eluting with Hex/EtOAc

mixtures from 0:100 to 50:50%) to yield 1,1-dimethylethyl 2-[4-

chloro-6-(methyloxy)-1,3,5-triazin-2-yl]-2-(2-methylpropyl)hyd-

3.93-3.57 (br, 1H), 3.45-3.11 (br, 1H), 2.35 (s, 3H), 2.04-1.86

(m, 1H), 1.56-1.22 (br, 9H), 1.02-0.72 (br, 6H). ¹H NMR

(300 MHz, DMSO-d₆þ D₂O) δ ppm: 6.80-6.52 (br, 1H),

3.96-3.64 (br, 1H), 3.40-3.09 (br, 1H), 2.34 (s, 3H), 2.02-

1.84 (m, 1H), 1.52-1.18 (br, 9H), 0.99-0.70 (br, 6H). ESIMS

m/z: 306 (MH^þ).

Methyl 2-Cyano-6-[2-{[(1,1-dimethylethyl)oxy]carbonyl}-1-(2-

methylpropyl)hydrazino]-4-pyrimidinecarboxylate (14b). A solu-

tion of 1 (1 g, 5.3 mmol) in EtOH was added to methyl 2,6-

dichloro-4-pyrimidinecarboxylate (1 g, 4.83 mmol) previously

dissolved in EtOH (20 mL). The resulting reaction mixture was

stirred at room temperature overnight. The mixture was then

concentrated under vacuum and the crude mixture purified by

flash chromatography on silica gel (eluting with 5:1 Hex/EtOAc)

to yield intermediate 13b (0.46 g, 26%).

A solution of 13b (0.02 g, 0.06 mmol) and tetraethylammo-

nium cyanide (0.04 g, 0.3 mmol) in THF (2 mL) was cooled at

-78 ꢀC. Then excess liquid trimethylamine was added and the

mixture was allowed to warm to room temperature. After 24 h,

the mixture was concentrated in vacuo and the residue taken up

with DCM and washed with water. The organic layer was dried

over anhydrous MgSO₄, filtered, and solvent was then removed

under reduced pressure. The brown oil obtained was purified by

flash chromatography on silica gel (eluting with 50:1 DCM/

MeOH) to yield the title compound 14b (0.02 g, quantitative

yield). ¹H NMR (300 MHz, DMSO-d₆) δ ppm: 10.17-10.04 (br,

1H), 7.31-7.22 (br, 1H), 4.03-3.75 (br, 4H), 3.51-3.3 (br, 1H),

2.06-1.91 (m, 1H), 1.53-1.21 (br m, 9H), 1.01-0.79 (br, 6H).

ESIMS m/z: 350 (MH^þ).

1,1-Dimethylethyl 2-[2-Cyano-6-(methyloxy)-4-pyrimidinyl]-

2-(2-methylpropyl)hydrazinecarboxylate (17). To a solution of

2,4,6-trichloropyrimidine (2.9 g, 16 mmol) in EtOH (30 mL), 1

(3 g, 16 mmol) was added. The mixture was stirred at room

temperature. After 16 h, the mixture was concentrated under

vacuum and the yellow solid obtained was purified by flash

chromatography on silica gel (eluting with Hex/EtOAc mixtures

from 6:1 to 2:1). The solid obtained was washed with EtOH to

yield intermediate 15 (2.5 g, 47%) as a white solid.

1

razinecarboxylate (0.975 g, 53% yield). H NMR (300 MHz,

CDCl₃) δ ppm: 6.83-6.47 (br, 1H), 3.98-3.95 (m, 3H), 3.69-

3.59 (br, 2H), 2.17-2.02 (m, 1H), 1.58-1.33 (br, 9H), 0.97 (d,

J = 6.7 Hz, 6H).

1,1-Dimethylethyl 2-[4-chloro-6-(methyloxy)-1,3,5-triazin-2-

yl]-2-(2-methylpropyl)hydrazinecarboxylate(0.109g, 0.33 mmol)

was dissolved in DMSO (3 mL). To the resulting solution, NaCN

(0.033 g, 0.66 mmol) was added. The reaction mixture was stirred

atroomtemperaturefor 18 h, and thenitwas diluted withEtOAc.

The organic phase was washed withwater several times andbrine.

Solvent was evaporated under reduced pressure and the crude

mixture was purified by flash chromatography on silica gel

(eluting with Hex/EtOAc mixtures from 95:5 to 40:60%) to yield

the title compound 10 (0.06 g, 56% yield). ¹H NMR (300 MHz,

DMSO-d₆) δ ppm: 9.83-9.25 (br, 1H), 3.95-3.92 (m, 3H), 3.62-

3.41 (br, 2H), 2.04-1.9 (m, 1H), 1.48-1.26 (m, 9H), 0.89 (d, J =

6.7 Hz, 6H). ESIMS m/z: 350 (MH^þ).

1,1-Dimethylethyl 2-(2-Cyano-4-pyrimidinyl)-2-(2-methylpro-

pyl)hydrazinecarboxylate (12). A mixture of compound 1 (12.07 g,

64 mmol), 2,4-dichloropyrimidine (19.07 g, 128 mmol), triethyla-

mine (10.7 mL, 145 mmol), and dry EtOH (100 mL) was stirred at

room temperaturefor 3 days. The mixturewas concentrated under

reduced pressure and the residue partitioned between DCM and

1MNH₄Cl. The organic layer was treated with brine and dried over

MgSO₄. The residue was purified by flash chromatography (eluent,

Hex/AcOEt mixtures 87:13 to 50:50%) to give intermediate 11. ¹H

NMR (300 MHz, DMSO-d₆) δ ppm: 9.91-9.21 (br, 1H), 8.15 (d,

J = 6.0 Hz, 1H), 6.72-6.46 (br, 1H), 4.02-3.56 (br, 1H), 3.42-

3.00 (br, 1H), 2.05-1.8 (m, 1H), 1.53-1.17 (br, 9H), 1.02-0.73 (br,

6H). ESIMS m/z 299 (MH^-).

Potassium cyanide (3.5 g, 54.6 mmol) was added to a suspen-

sion of intermediate 11 (13.69 g, 45.51 mmol) and DABCO (5.10 g,

45.51 mmol) in a mixture of 85:15 DMSO-H₂O (195 mL) at

room temperature. The reaction mixture was stirred at 100 ꢀC

for 1.7 h and poured into ice-water (250 mL). The white solid

was filtered off and dried. The compound was purified by flash

chromatography (eluent, Hex/AcOEt mixtures 4:1 to 3:1) to

To a solution of 15 (0.4 g, 1.2 mmol) in MeOH (10 mL),

sodium methoxide (0.13 g, 2.4 mmol previously prepared from

sodium and MeOH) was added. The resulting mixture was

stirred at room temperature. After 16 h, the mixture was

concentrated under vacuum and the residue partitioned between

DCM and water. The organic layer was washed with brine and

the crude product was purified by flash chromatography on

silica gel (eluting with 10:1 Hex/EtOAc) to yield intermediate 16

(0.06 g, 15%).

1

give compound 12. H NMR (300 MHz, DMSO-d₆) δ ppm:

10.06-9.23 (br s, 1H), 8.37 (br, 1H), 7.42-6.67 (br, 1H), 3.92-

3.56 (br, 1H), 3.46-3.02 (br, 1H), 2.1-1.82 (m, 1H), 1.58-1.08

(br, 9H), 1.02-0.67 (br, 6H). ESIMS m/z 290 (MH^-).

1,1-Dimethylethyl 2-(2-Cyano-6-methyl-4-pyrimidinyl)-2-(2-

methylpropyl)hydrazinecarboxylate (14a). To a solution of 2,4-

dichloro-6-methylpyrimidine (1.43 g, 8.8 mmol) in EtOH (4 mL),

a solution of 1 (0.82 g, 4.4 mmol) in EtOH (4 mL) and Et₃N

(0.73 mL, 5.3 mmol) were added. The resulting reaction mixture

was stirred at room temperature for 2 days and then refluxed

overnight. The mixture was washed with saturated NH₄Cl and

brine. The organic layer was dried over anhydrous MgSO₄. The

residue was purified by chromatography on silica gel (eluting

with Hex/EtOAc mixtures from 30:1 to1:1) followed by pre-

parative HPLC purification (ACN/H₂O, 0.1% TFA, gradient

30-100%) to give 1,1-dimethylethyl 2-(2-chloro-6-methyl-4-

pyrimidinyl)-2-(2-methylpropyl)hydrazinecarboxylate 13a (0.5 g,

36%). ¹H NMR (300 MHz, CDCl₃) δ ppm: 6.66-6.46 (br, 1H),

6.46-6.29 (br, 1H), 3.82-3.32 (br, 2H), 2.37 (s, 3H), 2.12-1.95

(m, 1H), 1.73-1.36 (br, 9H), 0.96 (d, J = 6.6 Hz, 6H).

To a solution of 13a in a 9:1 DMSO-H₂O mixture (7 mL),

DABCO (0.18 g, 1.6 mmol) and KCN (0. g, 3.1 mmol) were

added, and the mixture was heated to 80 ꢀC overnight. More

KCN (0.1 g, 1.6 mmol) and DABCO (0.09 g, 0.8 mmol) were

added, and stirring was kept overnight. The mixture was poured

into ice-water, and the white precipitate was filtered off. The

crude product was purified by flash chromatography on silica

gel (eluting with Hex/EtOAc mixtures from 100:0 to 50:50%) to

yield the above product 14a (0.28 g, 58%). ¹H NMR (300 MHz,

DMSO-d₆) δ ppm: 9.93-9.70 (br, 1H), 6.78-6.58 (br, 1H),

A solution of 16 (0.05 g, 0.15 mmol) and tetraethylammonium

cyanide (0.1 g, 0.6 mmol) in THF (3 mL) was cooled at -78 ꢀC.

Then excess liquid trimethylamine was added and the mixture

was allowed to warm to room temperature. After 48 h, the

mixture was concentrated in vacuo and the residue taken up

with DCM and washed with water. The organic layer was

washed with brine and solvent was removed under reduced

pressure to yield the title compound 17 (0.04 g, 91%) as a yellow

1

solid. H NMR (300 MHz, DMSO-d₆) δ ppm: 9.94-9.58 (br,

1H), 6.22-5.85 (br, 1H), 4.04-3.62 (br, 4H), 3.33-3.09 (br,

1H), 2.02-1.82 (m, 1H), 1.56-1.25 (br, 9H), 1.04-0.75 (br, 6H).

ESIMS m/z: 322 (MH^þ) .

1,1-Dimethylethyl 2-(2-Cyano-5-fluoro-4-pyrimidinyl)-2-(2-

methylpropyl)hydrazinecarboxylate (19a). To a solution of

5-fluoro-2,4-dichloropyrimidine (1.5 g, 9.1 mmol) and 1 (1.9 g,

9.8 mmol) in i-PrOH (40 mL), DIPEA (2 mL, 11.3 mmol) was

added. The resulting reaction mixture was refluxed overnight.

The mixture was concentrated under reduced pressure and the

residue partitioned between DCM and 1 M NH₄Cl. The organic

layer was washed with water and brine and dried over anhy-

drous Na₂SO₄. The residue was purified by flash chromatogra-

phy on silica gel (elute, Hex/EtOAc 9:1) to give the intermediate

18a as a colorless oil (2.88 g, 99%). ¹H NMR (300 MHz, CDCl₃)

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6149

δ ppm: 7.98 (d, J = 5.4 Hz, 1H), 6.77-6.65 (br, 1H), 3.68-3.45

(br, 2H), 2.18-1.96 (m, 1H), 1.65-1.31 (br, 9H), 0.95 (d, J = 6.6

Hz, 6H). ESIMS m/z: 319 (MH^þ).

(b s, 1H), 8.56-8.51 (br, 1H), 3.98-3.71 (br, 1H), 3.30-3.07 (br,

1H), 2.08-1.90 (m, 1H), 1.52-1.2 (br, 9H), 0.99-0.79 (br, 6H).

¹H NMR (300 MHz, DMSO-d₆, 80 ꢀC) δ ppm: 9.95-9.5 (br,

1H), 8.47 (s, 1H), 3.80-3.34 (br, 2H), 2.11-1.95 (m, 1H), 1.41

(br s, 9H), 0.93 (d, J = 6.7 Hz, 6H). ESIMS m/z: 326 (MH^þ).

1,1-Dimethylethyl 2-(2-Cyano-5-nitro-4-pyrimidinyl)-2-(2-met-

hylpropyl)hydrazinecarboxylate (19d). To a solution of 2,4-

dichloro-5-nitropyrimidine (0.2 g, 1 mmol) and 1 (0.2 g, 1 mmol)

in i-PrOH (7 mL), DIPEA (0.2 mL, 1.1 mmol) was added. The

resulting reaction mixture was stirred at room temperature for

1.5 h. The mixture was concentrated under reduced pressure and

the residue partitioned between DCM and 1 M NH₄Cl. The

organic layer was washed with water and brine and dried over

anhydrous MgSO₄. The residue was purified by preparative

HPLC (ACN/H₂O, 0.1%TFA, gradient 30-100%) to give inter-

mediate 1,1-dimethylethyl 2-(2-chloro-5-nitro-4-pyrimidinyl)-

2-(2-methylpropyl)hydrazinecarboxylate 18d (0.22 g, 62%).

¹H NMR (300 MHz, CDCl₃) δ ppm: 8.55 (s, 1H), 6.66-

6.44 (br, 1H), 4.53-4.01 (br, 1H), 3.45-2.99 (br, 1H), 2.18-

1.98 (m, 1H), 1.41 (br s, 9H), 1.00 (d, J = 6.4 Hz, 6H). ESIMS

m/z: 346 (MH^þ).

Potassium cyanide (0.5 g, 7.7 mmol) was added to a suspen-

sion of 18a (2.2 g, 7 mmol) and DABCO (0.8 g, 7 mmol) in a 9:1

DMSO-H₂O mixture (65 mL) at room temperature. The reac-

tion mixture was stirred at 65 ꢀC for 5 h and then poured into

ice-water. The cream color solid that precipitated was filtered

off, washed abundantly with water, and dried under air. The

compound was purified by flash chromatography (eluting with

Hex/EtOAC mixtures from 19:1 to 4:1) to give the title com-

1

pound 19a as a white solid (0.26 g, 9%). H NMR (300 MHz,

CDCl₃) δ ppm: 8.16 (d, J = 5.6 Hz, 1H), 6.86-6.56 (br, 1H),

3.83-3.45 (br, 2H), 2.20-1.98 (m, 1H), 1.72-1.31 (br, 9H), 0.97

(d, J = 6.7 Hz, 6H). ESIMS m/z: 310 (MH^þ).

1,1-Dimethylethyl 2-(5-Bromo-2-cyano-4-pyrimidinyl)-2-(2-

methylpropyl)hydrazinecarboxylate (19b). To a solution of 5-

bromo-2,4-dichloropyrimidine (3.0 g, 13 mmol) and 1 (2.5 g,

13 mmol) in i-PrOH (80 mL), DIPEA (3 mL, 17 mmol) was

added. The resulting reaction mixture was refluxed for 5 h. The

mixture was concentrated under reduced pressure and the

residue partitioned between DCM and 1 M NH₄Cl. The organic

layer was washed with water and brine and dried over anhydrous

Na₂SO₄. The residue was purified by flash chromatography

on silica gel (elute, Hex/EtOAc 9:1) to give intermediate 1,1-

dimethylethyl 2-(5-bromo-2-chloro-4-pyrimidinyl)-2-(2-methy-

lpropyl)hydrazinecarboxylate 18b as a white solid (4.12 g, 82%).

¹H NMR (300 MHz, CDCl₃) δ ppm: 8.27 (br s, 1H), 6.85-6.40

(br, 1H), 4.15-3.00 (br, 2H), 2.16-1.96 (m, 1H), 1.55-1.30 (br,

9H), 0.97 (d, J = 6.6 Hz, 6H).

Potassium cyanide (0.9 g, 13 mmol) was added to a suspen-

sion of 18b (4.1 g, 11 mmol) and DABCO (1.2 g, 11 mmol) in a

9:1 DMSO-H₂O mixture at room temperature. The reaction

mixture was stirred at room temperature for 3 h and then poured

into ice-water. The cream color solid that precipitated was

filtered off, washed abundantly with water, and dried under air.

The compound was purified by flash chromatography on silica

gel (eluent, Hex/EtOAC 19:1) to give the title compound 19b as a

white foam (3.68 g, 92%). ¹H NMR (300 MHz, CDCl₃) δ ppm:

8.46 (br s, 1H), 6.85-6.45 (br, 1H), 4.15-3.0 (br, 2H), 2.17-1.95

(m, 1H), 1.52-1.31 (br, 9H), 0.97 (d, J = 6.6 Hz, 6H). ¹H NMR

(300 MHz, DMSO-d₆, 80 ꢀC) δ ppm: 9.94-9.43 (br, 1H), 8.59 (s,

1H), 3.94-3.23 (br, 2H), 2.11-1.97 (dt, J = 6.7, 13.5 Hz, 1H),

1.52-1.29 (br, 9H), 0.92 (d, J = 6.7 Hz, 6H). ¹³C NMR (90

MHz, DMSO-d₆) δ ppm: 160.7, 159.6, 153.6, 140.6, 115.7, 104.7,

80.4, 58.6, 27.7, 25.5, 20.0. ESIMS m/z: 370 (MH^þ).

1,1-Dimethylethyl 2-(5-Chloro-2-cyano-4-pyrimidinyl)-2-(2-

methylpropyl)hydrazinecarboxylate (19c). To a solution of 2,4,5-

trichloropyrimidine (0.5 g, 2.7 mmol) and 1 (0.6 g, 3.1 mmol) in

i-PrOH (15 mL), DIPEA (0.7 mL, 3.7 mmol) was added. The

resulting reaction mixture was refluxed for 3 h. The mixture was

concentrated under reduced pressure and the residue parti-

tioned between DCM and 1 M NH₄Cl. The organic layer was

washed with water and brine and dried over anhydrous Na₂SO₄.

The residue was purified by flash chromatography (elute,

Hex/EtOAc 19:1) to give intermediate 18c as a white solid

(0.76 g, 83%). ¹H NMR (300 MHz, CDCl₃) δ ppm: 8.11

(br s, 1H), 6.85-6.70 (br, 1H), 3.80-3.50 (br, 2H), 2.17-1.97

(m, 1H), 1.63-1.29 (br, 9H), 0.96 (d, J = 6.7 Hz, 6H). ESIMS

m/z: 337 (MH^þ).

Potassium cyanide (0.05 g, 0.7 mmol) was added to a suspen-

sion of 18d (0.2 g, 0.6 mmol) and DABCO (0.07 g, 0.6 mmol) in a

9:1 DMSO-H₂O mixture (7 mL) at room temperature. The

reaction mixture was stirred at room temperature for 1.5 h and

then poured into ice-water. The cream color solid that pre-

cipitated was filtered off, washed abundantly with water, and

dried under air. Compound was further extracted from the

aqueous filtrate with n-butanol. The resulting crude mixture of

compounds was purified by preparative HPLC (ACN/H₂O,

gradient 50-100%) to give the title compound 19d as a yellow

1

solid (0.07 g, 22%). H NMR (300 MHz, DMSO-d₆) δ ppm:

10.19-10.02 (br, 1H), 8.93 (br s, 1H), 4.05-3.85 (br, 1H),

3.22-3.06 (br, 1H), 2.10-1.92 (m, 1H), 1.49-1.20 (br, 9H),

1.03-0.75 (br, 6H). ESIMS m/z: 337 (MH^þ).

1,1-Dimethylethyl 2-(2-Cyano-5-methyl-4-pyrimidinyl)-2-(2-

methylpropyl)hydrazinecarboxylate (19e). To a solution of 2,4-

dichloro-5-methylpyrimidine (1.5g, 9 mmol) and1 (0.8g, 4 mmol)

in EtOH (8 mL), triethylamine (0.7 mL, 5 mmol) was added. The

resulting reaction mixture was stirred at room temperature for

72 h and then refluxed for 40 h to reach completion. The mixture

was concentrated under reduced pressure and the residue parti-

tioned between DCM and 1 M NH₄Cl. The organic layer was

washed with water and brine and dried over anhydrous MgSO₄.

The residue was purified by flash chromatography on silica gel

(eluting with Hex/EtOAc mixtures from 100:0 to 1:1) to give

intermediate 18e (0.87 g, 63%). ¹H NMR (300 MHz, DMSO-d₆)

δ ppm: 9.83-9.41 (br, 1H), 8.01-7.91 (br, 1H), 4.02-3.71 (br,

1H), 3.12-2.92 (br, 1H), 2.14 (s, 3H), 2.01-1.85 (m, 1H), 1.49-

1.23 (br, 9H), 1.00-0.75 (br, 6H).

Potassium cyanide (0.4 g, 6 mmol) was added to a suspension

of 18e (0.9 g, 3 mmol) and DABCO (0.3 g, 3 mmol) in a 85:15

DMSO-H₂O mixture (12 mL) at room temperature. The reac-

tion mixture was stirred at 80 ꢀC for 24 h, then poured into

ice-water. The solid that precipitated was filtered off, washed

abundantly with water, and dried under air. The compound

was purified by flash chromatography on silica gel (eluting

with Hex/EtOAC mixtures from 100:0 to 1:1) to give the

title compound 19e (0.16 g, 20%). ¹H NMR (300 MHz,

DMSO-d₆) δ ppm: 9.93-9.51 (br, 1H), 8.24-8.19 (br, 1H),

4.02-3.73 (br, 1H), 3.14-2.92 (br, 1H), 2.23 (s, 3H), 2.06-1.89

(m, 1H), 1.41-1.23 (br, 9H), 1.00-0.74 (br, 6H). ESIMS m/z:

306 (MH^þ).

Potassium cyanide (0.2 g, 2.7 mmol) was added to a suspen-

sion of 18c (0.7 g, 2.2 mmol) and DABCO (1.2 g, 11 mmol) in a

9:1 DMSO-H₂O mixture (20 mL) at room temperature. The

reaction mixture was stirred at room temperature for 9 h and

then poured into ice-water. The light-yellow solid that pre-

cipitated was filtered off, washed abundantly with water, and

dried under air. The compound was purified by flash chroma-

tography on silica gel (eluting with Hex/EtOAC mixtures from

19:1 to 9:1) to give the title compound 19c as a white solid

1,1-Dimethylethyl 2-[2-Cyano-5-(trifluoromethyl)-4-pyrimidi-

nyl]-2-(2-methylpropyl)hydrazinecarboxylate (20). Under an in-

ert atmosphere, 19b (0.1 g, 0.27 mmol) and copper(I) iodide

(0.07 g, 0.35 mmol) were dissolved in dry DMF (5 mL). The

resulting solution was preheated to 80 ꢀC, prior to the addition

of methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (0.26 g, 1.34 mmol)

and hexamethylphosphoramide (0.25 g, 1.38 mmol). The resulting

1

(0.62 g, 85%). H NMR (300 MHz, DMSO-d₆) δ ppm: 10.09

6150 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16

Coteroꢀn et al.

reaction mixture was heated at 80 ꢀC for a further 3 h, before the

reaction reached completion. Solvent was evaporated in vacuo

and the residue partitioned between EtOAc and 1 M NH₄Cl.

The organic layer was treated water, brine and dried over

anhydrous Na₂SO₄. The residue was purified by flash chro-

matography on silica gel (eluting with Hex/EtOAc mixtures

from 100:0 to 3:2) to give the title compound 20 as a colorless

To a solution of intermediate 24 (0.97 g, 5.4 mmol) in i-PrOH

(25 mL), intermediate 1 (1.2 g, 6.3 mmol) and DIPEA (1.2 mL,

7.1 mmol) were added. The mixture was refluxed overnight.

Solvent was removed under reduced pressure, and the residue

was partitioned between DCM and 1 N NH₄Cl. The organic

layer was washed with water and brine and dried over anhy-

drous Na₂SO₄. The crude material was purified by flash chro-

matography on silica gel (eluting with Hex/EtOAc mixtures

from 100:0 to 60:40%) to yield intermediate 1,1-dimethylethyl

2-[2-chloro-5-(methyloxy)-4-pyrimidinyl]-2-(2-methylpropyl)-

hydrazinecarboxylate 25 (1.7 g, 94%) as a colorless oil. ¹H

NMR (300 MHz, CDCl₃) δ ppm: 7.76 (s, 1H), 6.82-6.61 (br,

1H), 3.84 (s, 3H), 3.77-3.52 (br, 2H), 2.15-2.01 (m, 1H), 1.58-

1.32 (br, 9H), 0.94 (d, J = 6.6 Hz, 6H).

1

oil (0.02 g, 24%). H NMR (300 MHz, CDCl₃) δ ppm: 8.65

(s, 1H), 6.70 (s, 1H), 4.53-4.25 (br, 1H), 3.16-2.89 (br, 1H), 2.21-

1.95 (m, 1H), 1.5-1.28 (br, 9H), 1.08-0.83 (br, 6H). ESIMS

m/z: 360 (MH^þ).

1,1-Dimethylethyl 2-{2-Cyano-5-[(phenylmethyl)amino]-4-

pyrimidinyl}-2-(2-methylpropyl)hydrazinecarboxylate (21). In a

microwave vial, 19b (0.2 g, 0.5 mmol), benzylamine (0.24 mL,

2.2 mmol), palladium(II) acetate (0.06 g, 0.03 mmol), (()-

BINAP (0.02 g, 0.03 mmol), and potassium carbonate (0.15 g,

1.1 mmol) were dissolved in DMF (5 mL). The resulting mixture

was heated under microwave irradiation at 60 ꢀC for 30 min.

Solvent was evaporated to dryness, and the residue was taken up

with DCM and washed with 1 N NH₄Cl, saturated NaHCO₃,

and brine. The organic layer was dried over anhydrous MgSO₄,

filtered, and solvent was removed in vacuo. The crude material

was purified by preparative HPLC (ACN/H₂O, 0.1% TFA,

gradient 30-100%) to give the title compound 21 (0.006 g, 3%).

¹H NMR (300 MHz, DMSO-d₆) δ ppm: 9.53-9.21 (br, 1H),

7.72 (s, 1H), 7.47-7.17 (m, 5H), 6.44-6.24 (br, 1H), 4.46 (d, J =

6 Hz, 2H), 3.60-3.25 (br, 2H), 1.98-1.79 (m, 1H), 1.49-1.06

(br, 9H), 0.92 (d, J = 6.6 Hz, 6H). ESIMS m/z: 397 (MH^þ).

1,1-Dimethylethyl 2-(2-Cyano-5-{[4-(trifluoromethyl)phenyl]-

ethynyl}-4-pyrimidinyl)-2-(2-methylpropyl)hydrazinecarboxylate

(22). Under inert atmosphere, 19b (0.103 g, 0.3 mmol), copper(I)

iodide (0.006 g, 0.03 mmol), and bis(triphenylphosphine)-

palladium(II) chloride (0.01 g, 0.01 mmol) were dissolved in

dry DMF (5 mL). The resulting reaction mixture was stirred at

room temperature for 5 min before 4-ethynyl-R,R,R-trifluoro-

toluene (0.04 mL, 0.2 mmol) and triethylamine (0.06 mL,

0.4 mmol) were added. The resulting reaction mixture was then

heated to 50 ꢀC for 22 h. The mixture was concentrated under

reduced pressure and the residue partitioned between EtOAc

and 1 M NH₄Cl. The organic layer was washed with water and

brine and dried over anhydrous Na₂SO₄. The residue was puri-

fied by preparative HPLC (ACN/H₂O, 0.1%TFA, gradient 50-

100%) to give the title compound 22 as a yellow solid (0.04 g,

In a microwave vial, intermediate 25 (0.05 g, 0.2 mmol),

potassium cyanide (0.03 g, 0.5 mmol), and DABCO (0.02 g,

0.2 mmol) were dissolvedin a 9:1 DMSO-H₂O mixture (0.5 mL).

The resulting reaction mixture was heated under microwave

irradiation to 60 ꢀC for 90 min. Solvent was removed under vac-

uum and the crude material was purified by preparative HPLC

(ACN/H₂O, 0.1%TFA, gradient 20-100%) to give the title

1

compound 26 as a colorless oil (0.02 g). H NMR (300 MHz,

CDCl₃) δ ppm: 7.92 (s, 1H), 6.91-6.77 (br, 1H), 3.92 (s, 3H),

3.80-3.59 (br, 2H), 2.18-2.01 (m, 1H), 1.65-1.31 (br, 9H), 0.96

(d, J = 6.6 Hz, 6H). ESIMS m/z: 322 (MH^þ).

1,1-Dimethylethyl 2-{2-Cyano-5-[(methyloxy)methyl]-4-pyri-

midinyl}-2-(2-methylpropyl)hydrazinecarboxylate (30). 5-(Hydro-

xymethyl)uracil hydrate (2.5 g, 15.6 mmol) and phosphorus

pentoxide (13.7 g, 65.7 mmol) were added to phosphorus oxy-

chloride (31.5 mL, 339 mmol), and the resulting mixture was

heated to 115 ꢀC overnight. Excess POCl₃was distilled, and the

residue was carefully added dropwise into a saturated NaHCO₃

solution previously cooled at 0 ꢀC. Product was extracted with

DCM, and the combined organic layers were washed with water

and brine and dried over anhydrous Na₂SO₄. The crude material

was purified by flash chromatography on silica gel (elute, Hex/

EtOAc 19:1) to yield intermediate 2,4-dichloro-5-(chloromethyl)-

pyrimidine 27 (2.1 g, 69%) as a pearl colored solid. ¹H NMR (300

MHz, CDCl₃) δ ppm: 8.66 (s, 1H), 4.64 (s, 2H). ¹³C NMR (90

MHz, CDCl₃) δ ppm: 161.73, 160.16, 160.11, 128.53, 38.80.

Cesium carbonate (1.66 g, 5.1 mmol) was added to a solution

of 27 (0.99 g, 5 mmol) and 1 (0.98 g, 5.2 mmol) in THF (30 mL),

and the resulting reaction mixture was stirred at room tempera-

ture for 48 h before more cesium carbonate (0.83 g, 2.55 mmol)

was added. Stirring at room temperature continued for further

24 h, and more cesium carbonate (0.83 g, 2.55 mmol) was added

to drive the reaction to completion. After further 6 h, solvent

was removed under reduced pressure. The residue obtained was

partitioned between DCM and 1 M NH₄Cl, and the aqueous

phase was further extracted with DCM. The combined organic

layers were washed with water and brine and dried over anhy-

drous Na₂SO₄. The residue was purified by flash chromatogra-

phy on silica gel (elute, Hex/EtOAc 9:1) to give intermediate

1,1-dimethylethyl 2-[2-chloro-5-(chloromethyl)-4-pyrimidinyl]-

2-(2-methylpropyl)hydrazinecarboxylate 28 (1 g, 57%) as a

white solid. ¹H NMR (300 MHz, DMSO-d₆, 25 ꢀC) δ ppm:

8.80-8.63 (br, 1H), 8.07-7.37 (br, 1H), 4.06-3.65 (br, 2H),

2.66-2.49 (br, 2H), 1.72-1.56 (m, 1H), 1.49-1.08 (br, 9H),

1

24%). H NMR (300 MHz, DMSO-d₆, 80 ꢀC) δ ppm: 10.07-

9.88 (br, 1H), 8.70-8.56 (br s, 1H), 7.94-7.72 (m, 4H), 4.01-

3.46 (br, 2H), 2.17-1.98 (m, 1H), 1.27 (br s, 9H), 0.96 (d,

J = 6.7 Hz, 6H). ESIMS m/z: 460 (MH^þ).

1,1-Dimethylethyl 2-[2-Cyano-5-(methyloxy)-4-pyrimidinyl]-

2-(2-methylpropyl)hydrazinecarboxylate (26). To a solution of

5-methoxy-2-sulfanyl-4-pyrimidinol (2 g, 12.7 mmol) in water

(50 mL), chloroacetic acid (2 g, 21.3 mmol) was added. The

resulting reaction mixture was refluxed for 2 h. After the mixture

was cooled to room temperature, concentrated HCl (8 mL) was

added and the mixture was heated overnight. A precipitate

appeared once the mixture cooled to room temperature. It was

filtered off, washed abundantly with water, and dried under air

toyieldintermediate 5-(methyloxy)-2,4(1H,3H)-pyrimidinedione

23 as a brown solid (1.65 g, 92%). ¹H NMR (300 MHz, DMSO-d₆)

δ ppm: 11.32-11.01 (br, 1H), 10.60-10.23 (br, 1H), 7.00 (s, 1H),

3.57 (s, 3H).

To a suspension of intermediate 23 (1.64 g, 11.5 mmol) in

phosphorus oxychloride (8.3 mL), N,N-dimethylaniline (1.64 mL)

was added. The resulting reaction mixture was heated to reflux

for 20 h. A precipitate appeared upon cooling to room tempera-

ture. It was filtered off, and the mother liquors were extracted

with Et₂O. The combined organic layers were washed with brine

and dried over anhydrous Na₂SO₄. Intermediate 2,4-dichloro-

5-(methyloxy)pyrimidine 24 (1.3 g, 64%) was obtained as a pale-

yellow solid. ¹H NMR (300 MHz, CDCl₃) δ ppm: 8.20 (s, 1H),

4.03 (s, 3H).

1

0.97-0.81 (m, 6H). H NMR (300 MHz, DMSO-d₆, 80 ꢀC) δ

ppm: 8.73 (s, 1H), 7.70-7.49 (br, 1H), 3.92 (br, 2H), 2.6 (d, J =

7.0 Hz, 2H), 1.76-1.63 (m, 1H), 1.27 (br s, 9H), 0.92 (d, J = 6.6

Hz, 6H). ¹³C NMR (90 MHz, DMSO-d₆) δ ppm: 161.78, 157.44,

154.70, 129.57, 78.13, 65.14, 55.14, 28.10, 27.92, 25.94, 20.45.

To a solution of 28 (0.1 g, 0.3 mmol) in dry MeOH (5 mL),

sodium methoxide (0.02 g, 0.3 mmol) was added. The mixture

was stirred at room temperature for 16 h. More sodium meth-

oxide (0.02 g, 0.4 mmol) was added, and stirring was continued

for further 4 h. Solvent was removed under reduced pressure.

The residue was taken up with DCM and washed with 1 N NH₄Cl,

water, and brine, and it was dried over anhydrous Na₂SO₄. The

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6151

crude material was purified by flash chromatography on silica

gel (eluting with Hex/EtOAc mixtures from 100:0 to 0:100%) to

yield intermediate 1,1-dimethylethyl 2-{2-chloro-5-[(methyloxy)-

methyl]-4-pyrimidinyl}-2-(2-methylpropyl)hydrazinecarboxylate

29 (0.04 g, 44%) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ

ppm: 8.51-8.27 (br, 1H), 5.72-4.80 (br, 1H), 4.03 (s, 3H), 3.98-

3.55 (br, 2H), 2.77-2.40 (br, 2H), 1.86-1.65 (m, 1H), 1.40 (s,

9H), 0.94 (d, J = 6.6 Hz, 6H).

To a solution of 29 (0.04 g, 0.1 mmol) in a 9:1 DMSO-H₂O

mixture (2 mL), potassium cyanide (0.02 g, 0.2 mmol) and

DABCO (0.02 g, 0.15 mmol) were added. The resulting reaction

mixture was stirred at room temperature for 22 h. Product was

extracted with n-butanol, and the combined organic layers were

washed with brine and dried over anhydrous Na₂SO₄. The crude

material was purified by preparative HPLC (ACN/H₂O, 0.1%

TFA, gradient 10-100%) to give the title compound 30 (0.02 g,

40%). ¹H NMR (300 MHz, CDCl₃) δ ppm: 8.72-8.53 (br, 1H),

5.73-4.96 (br, 1H), 4.05 (s, 3H), 4.04-3.69 (br, 2H), 2.75-2.44

(br, 2H), 1.84-1.66 (m, 1H), 1.40 (s, 9H), 0.96 (d, J = 6.6 Hz,

6H). ESIMS m/z: 336 (MH^þ), 334 (MH^-).

General Procedure for the Synthesis of N-tert-Butoxycarbo-

nyl-2-cyanopyrimidine Derivatives 33-78. To a solution of tert-

butyl hydrazinecarboxylate (1 mmol) in dry MeOH, the appro-

priate aldehyde or ketone (1 mmol), NaBH₃CN (2 mmol), and

glacial AcOH (0.2 mL/mmol) were added. The mixture was

stirred at room temperature until disappearance of starting

materials (16-18 h, TLC analysis). After the reaction mixture

was cooled in an ice bath, it was neutralized with 2 N NaOH.

Solvent was evaporated, and the residue was partitioned be-

tween DCM and water. The organic phase was washed with

brine, dried and solvent evaporated. The residue was purified on

silica gel column chromatography to yield the corresponding

intermediate hydrazine carboxylate 31.

To a solution of 5-bromo-2,4-dichloropyrimidine (1.2 mmol)

and the corresponding intermediate hydrazine carboxylate 31

(1 mmol) in EtOH or i-PrOH, DIPEA (2 mmol) was added. The

resulting reaction mixture was refluxed until disappearance of

starting materials (3-18 h, HPLC analysis). The mixture was

concentrated under reduced pressure and the residue parti-

tioned between DCM and 1 M NH₄Cl. The organic layer was

washed with water and brine and dried over anhydrous Na₂SO₄.

The residue was purified by flash chromatography on silica gel

to give the corresponding intermediate N-tert-butoxycarbonyl-

2-chloropyrimidine 32.

and DIPEA (2 mmol) were added. The reaction mixture was

stirred at room temperature overnight. Then solvent was re-

moved in vacuo and the residue partitioned between DCM and

water. The organic layer was washed with 1 N aqueous NaOH

and brine, dried over anhydrous MgSO₄, and concentrated to

dryness. The residue was dissolved in a minimum amount of

DCM and the solution stirred with hexane, intermediate

bromobenzoyl derivative 80 precipitating off and used without

any further purification.

To a solution of bromobenzoyl derivative 80 (1 mmol) in

THF, DIPEA (1.5 mmol) and N-methylpiperazine (1.21 mmol)

were added. The resulting reaction mixture was stirred at room

temperature until it reached completion. Solvent was removed

under reduced pressure and the resulting crude product was

purified by either HPLC or flash chromatography on silica gel

to yield the final products 81-119 (Supporting Information

Table S8).

General Procedure for the Synthesis of 2-Cyanopyrimidine

Derivatives 121-269. To a solution of the corresponding inter-

mediate 19b or 19c (1 mmol) in dry ACN, p-toluenesulfonic acid

monohydrate (3 mmol) was added. The mixture was stirred at

room temperature overnight. The reaction mixture was concen-

trated, and the residue was dissolved in DCM and washed with

saturated NaHCO₃. The organic phase was dried over MgSO₄,

filtered and solvent evaporated in vacuo. The residue was

purified by flash chromatography on silica gel to yield inter-

mediate hydrazine derivative 120.

To a solution of the corresponding intermediate derivative

120 (1 mmol) in dry THF or DCM, the corresponding acid

chloride (1 mmol) and DIPEA (2 mmol) were added. The re-

action mixture was stirred at room temperature until reaction

was complete. Then solvent was removed in vacuo and the

residue partitioned between DCM and water. The organic layer

was washed with 1 N aqueous NaOH and brine, dried over

anhydrous MgSO₄, and concentrated to dryness. The resulting

crude product was purified by either HPLC or flash chro-

matography on silica gel to yield the final products 121-269

(Table S9 in Supporting Information).

In cases where the corresponding carboxylic esters were

commercially available, they were hydrolyzed to the corre-

sponding acid using standard procedures unless stated other-

wise. Whenever the corresponding esters, acids, or acyl chlorides

were not commercially available, specific procedures were fol-

lowed in order to synthesize compounds 239, 245-248, 250-

255, 266-269 (see Supporting Information).

1,4-Diazabicyclo[2.2.2]octane (1 mmol) was added to a sus-

pension of the corresponding intermediate N-tert-butoxycarbo-

nyl-2-chloropyrimidine 32 (1 mmol) in a 9:1 DMSO-H₂O mix-

ture at room temperature. Potassium cyanide (1.2 mmol) was

then added to the mixture which was then stirred at the appro-

priate temperature (room temperature to 100 ꢀC) until reaction

was complete. The reaction mixture was cooled to room tem-

perature and then poured into ice-water and kept at low tem-

perature in order to favor product precipitation. The solid was

filtered off, washed abundantly with water, and dried to yield

the title compound. Whenever further purification of the final

product was necessary, HPLC or flash chromatography on

silica gel was used and compounds 33-78 were obtained

(Supporting Information Table S7).

Acknowledgment. We thank people from other GSK Dis-

ease of the Developing World departments (C.A.S.C. Chem-

istry, Compound Management, D.D. Biology) for their assi-

stance. This workwas supportedby the MedicinesforMalaria

Venture. P.J.R. is a Doris Duke Charitable Foundation

Distinguished Clinical Scientist.

Supporting Information Available: Spectroscopic data and

LCMS data for all listed compounds and specific synthetic

procedures. This material is available free of charge via the

Internet at http://pubs.acs.org.

General Procedure for the Synthesis 4-[(4-Methyl-1-piperazi-

nyl)methyl]benzoic Acid 2-Cyanopyrimidine Derivatives 81-119.

To a solution of the corresponding compounds 31-76 (1 mmol)

in dry ACN, p-toluenesulfonic acid monohydrate (3 mmol) was

added. The mixture was stirred at room temperature overnight.

The reaction mixture was concentrated, and the residue was

dissolved in DCM and washed with saturated NaHCO₃. The

organic phase was dried over anhydrous MgSO₄, filtered and

solvent evaporated. The residue was purified by flash chromato-

graphy on silica gel to yield intermediate hydrazine derivative 79.

To a solution of the corresponding intermediate derivative 79

(1 mmol) in dry THF, 4-bromomethylbenzoyl bromide (1 mmol)

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Article Doi

Falcipain inhibitors: Optimization studies of the 2-pyrimidinecarbonitrile lead series

DOI: 10.1021/jm100556b

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Article abstract of DOI:10.1021/jm100556b

Full text of DOI:10.1021/jm100556b

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