Structure-activity relationship of pyrrolyl diketo acid derivatives as dual inhibitors of HIV-1 integrase and reverse transcriptase ribonuclease H domain

Article abstract of DOI:10.1021/jm501799k

The development of HIV-1 dual inhibitors is a highly innovative approach aimed at reducing drug toxic side effects as well as therapeutic costs. HIV-1 integrase (IN) and reverse transcriptase-associated ribonuclease H (RNase H) are both selective targets

Full text of DOI:10.1021/jm501799k

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Article
Structure-Activity Relationship of Pyrrolyl Diketo
Acid Derivatives as Dual Inhibitors of HIV-1 Integrase
and Reverse Transcriptase Ribonuclease H Domain
Giuliana Cuzzucoli Crucitti, Mathieu Métifiot, Luca Pescatori, Antonella Messore,
Valentina Noemi Madia, Giovanni Pupo, Francesco Saccoliti, Luigi Scipione,
Silvano Tortorella, Francesca Esposito, Angela Corona, Marta Cadeddu, Christophe
Marchand, Yves Pommier, Enzo Tramontano, Roberta Costi, and Roberto Di Santo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501799k • Publication Date (Web): 28 Jan 2015
Downloaded from http://pubs.acs.org on February 3, 2015
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StructureꢀActivity Relationship of Pyrrolyl Diketo Acid
Derivatives as Dual Inhibitors of HIVꢀ1 Integrase and
Reverse Transcriptase Ribonuclease H Domain
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Giuliana Cuzzucoli Crucitti,† Mathieu Métifiot ‡ Luca Pescatori,† Antonella Messore,† Valentina Noemi
Madia,† Giovanni Pupo,† Francesco Saccoliti, † Luigi Scipione,† Silvano Tortorella,† Francesca
Esposito,∫ Angela Corona,∫ Marta Cadeddu,∫ Christophe Marchand,‡ Yves Pommier,‡ Enzo
Tramontano,∫ Roberta Costi,*,† and Roberto Di Santo†
† Dipartimento di Chimica e Tecnologie del Farmaco, Istituto PasteurꢀFondazione Cenci Bolognetti,
“Sapienza” Università di Roma, Italy, ‡Laboratory of Molecular Pharmacology, Center for Cancer
Research, National Cancer Institute, National Institutes of Health, Building 37, Room 5068, Bethesda,
Maryland 20892ꢀ4255, ∫Department of Life and Environmental Sciences, University of Cagliari, Italy
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
KEYWORDS: HIV, integrase, reverse transcriptase, ribonuclease H, dual inhibitors, pyrrole,
diketoacid.
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ABSTRACT. The development of HIVꢀ1 dual inhibitors is a highly innovative approach aimed to
reduce drug toxic side effects as well as therapeutic costs. HIVꢀ1 integrase (IN) and reverse
transcriptaseꢀassociated ribonuclease H (RNase H) are both selective targets for HIVꢀ1 chemotherapy
and the identification of dual IN/RNase H inhibitors is an attractive strategy for new drug development.
We synthesized newly pyrrolyl derivatives that exhibited good potency against IN and a moderate
inhibition of the RNase H function of RT, confirming the possibility to develop dual HIVꢀ1 IN/RNase H
inhibitors and obtaining new information for the further development of more effective dual HIVꢀ1
inhibitors.
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INTRODUCTION
The development of dualꢀaction drugs is a prosiming approach to ameliorate drugꢀdrug interactions,
reduce toxic side effects still suppressing viral resistance selection.1,2,3,4 Among dualꢀaction drugs, dual
inhibitors are single compounds which are able to inhibit two enzyme activities. Several reports have
shown that dual inhibitors may have a role in the treatment of different diseases such as Alzheimer,5
Parkinson,6 inflammation7 and cancer.1,8,9 This approach had been attempted also in the virological
arena aiming to inhibit rhinovirus replication.10 Recently, tropolones,11,12,13 madurahydroxylactone14 and
2ꢀhydroxyisoquinolinꢀ1,3(2H,4H)ꢀdiones15,16 have been reported to act as dual inhibitors against HIVꢀ1,
targeting viral integrase (IN) and reverse transcriptase (RT)ꢀ ribonuclease H (RNase H) activities.
HIVꢀ1 IN is the viral enzyme responsible for the integration of the proviral dsDNA into the cell host
chromosome through two coordinated enzyme functions, both accomplished by the same active site.17
In the first reaction, termed 3’ꢀend processing (3’ꢀP), IN removes the two terminal nucleotides (GT)
from each 3’ꢀend of the dsDNA.17 In the second reaction, termed strandꢀtransfer (ST), IN catalyzes a
nucleophilic attack by the free 3’ꢀOH of the viral processed DNA to the target chromosomal DNA,
resulting in covalent joining of the two molecules. Several classes of integrase inhibitors have been
identified;18 among these the diketoacids (DKAs) showed greatest promise and the first DKA bioisoster,
raltegravir (1), has been approved in 2008 for HIVꢀ1 therapy.19
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HIVꢀ1 IN belongs to the functionally diverse superfamily of DDE(D) nucleotidyltransferases, whose
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other notable members include RNaseH and MuA, Tn5, Mos1 transposases as well.17 The active sites of
these enzymes typically contain three essential carboxylates that coordinate a pair of divalent metal
cations (usually Mg2+). Thus, chelating inhibitors can be active across several classes of viral metalꢀ
dependent enzymes and chelation has been successfully used in drug design, also of dual inhibitors.20 In
particular, DKAs have been reported to chelate the divalent cations in the IN active site17,18 and notably,
DKAs originally developed against HIVꢀ1 IN have been also reported to inhibit the HIVꢀ1 RNase H.21,22
The HIVꢀ1 RTꢀassociated RNase H function hydrolizes the RNA strand of the replicative intermediate
RNA:DNA hybrid and, hence, is essential for viral replication.23 Even though several compounds have
been recently identified to inhibit this RT activity,24,25,26,27,28,29,30 up to today no RNase H inhibitor has
been approved for HIV therapy. Therefore, this viral function is a very attractive target for drug
development.
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The first DKA IN inhibitor later described also as RNase H inhibitor was the 4ꢀ[5ꢀ
(benzoylamino)thienꢀ2ꢀyl]ꢀ2,4ꢀdioxobutanoic acid (BTDBA, 2) discovered by Merck.21 Recently we
reported that 6ꢀ[1ꢀ(4ꢀfluorophenyl)methylꢀ1Hpyrrolꢀ2ꢀyl)]ꢀ2,4ꢀdioxoꢀ5ꢀhexenoic acid ethyl ester (RDS
1643, 3) inhibited the HIVꢀ1 RTꢀassociated RNase H function in biochemical assays with an IC50 value
of 8 ꢀM, the HIVꢀ1 IN ST (the IC50 value was 98 ꢀM) and blocked the HIVꢀ1 replication in cell based
assays with an EC50 value of <0.2 ꢀM (Chart 1).22 Starting from these observations, more recently we
designed a small library of 3 analogues with the aim to obtain dual IN/RNase H HIVꢀ1 inhibitors and
found compounds active at micromolar concetration against RNase H and low nanomolar IC50 values
against IN in recombinant assays.31 The best dual inhibition was found for compounds showing a diketo
ester group and fluorine atoms as substituents on the benzyl portion, as exemplified by compound 4
(Chart 1, IC50,IN = 0.6 ꢀM, IC50,RH = 3.0 ꢀM EC50 = 2 ꢀM). Further, we obtained similar results with a
quinolonyl diketo acid series, in which less marked dual activity was found, having in the best case
IC50,IN = 26.2 ꢀM, IC50,RH = 2.4 ꢀM and EC50 = 3.6 ꢀM (compound 5).32
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Herein we present the design, synthesis and biological evaluation of new compounds related to 3, to
better define the structureꢀactivity relationship (SAR) within the most interesting pyrrolyl diketo acid
series. All the newly designed pyrrolyl DKA both ester 6 and acid derivatives 7 are shown in Chart 2.
Basically, starting from 3 we fixed the pyrrole ring and the DKA chain while wider tranformations
included one or more of the following modifications: i) introduction of aromatic substituent in position
4 of the pyrrole ring; ii) shift of the diketo hexenoic chain from 2 to 3 position of the pyrrole moiety; iii)
shortening of the diketo hexenoic branch into a diketo butanoic group; iv) introduction of alkyl or aryl
group replacing the fluorobenzyl moiety; v) replacement of carboxylic function with a triazole ring; vi)
introduction of alkyl group within the DKA branch; vii) replacement of keto group of DKA moiety with
NH2 function. Notably, among the compounds described in this paper, 7k has been the first DKA
derivative reported by Merck as selective ST IN inhibitor.33 We decided to include this compound in
this study to define its properties as dual inhibitor of IN and RNase H.
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Chart 1. Selective Inhibitor of IN Enzyme (1), first Described Dual IN/RNase H Inhibitor (2), and
Recently Discovered Dual IN/RNase H Inhibitors 3ꢀ5
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Chart 2. Newly Designed Pyrrolyl DKA Derivatives 6aꢀl and 7aꢀm
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RESULT AND DISCUSSION
Chemistry. The synthesis of derivatives 6a-l and 7a-m is outlined in Schemes 1ꢀ4. Derivatives 6a-d
and 7a-d were synthesized according to the pathway described in the Scheme 1. The acetyl or propionyl
pyrrole intermediates 9a-c were obtained by two different procedures: 1) the alkylation with 4ꢀ
fluorobenzyl bromide in alkaline medium (K2CO3) of 1ꢀ(4ꢀphenylꢀ1Hꢀpyrrolꢀ3ꢀyl)ethanone34 or
derivatives 8, achieved via tolueneꢀ4ꢀsulphonylmethyl isocyanide (TosMIC) reaction from (E)ꢀ1ꢀ
phenylpentꢀ1ꢀenꢀ3ꢀone,35 2) termal transposition of the acetyl chain of 2ꢀacetylꢀ1ꢀ[(4ꢀ
fluorophenyl)methyl]pyrrole36 from 2ꢀ to 3ꢀ position of the pyrrole ring in the presence of CF3COOH.
Derivatives 9a-c were condensed in turn with diethyl oxalate in the presence of sodium ethoxide to
provide the diketobutanoic ethyl esters 6a-c. Compound 6a was used as substrate to provide i) 6d,
obtained by reacting the enol 6a with ammonium acetate in acid medium (CH3COOH) following a
known procedure reported for DKA derivarives37, and ii) 7a, achieved by hydrolysis of ester 6a in the
presence of 6 N NaOH. The last conditions have been also used to obtain 7b-c starting from 6b-c,
respectively. A sligthly different condition has been used to obtain 7d, as previously decribed.37
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Scheme 1. Synthetic Route to Pyrrolyl DKAs 6a-d and 7a-da
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Reagents and conditions: (i) TosMIC, NaH, Et2O/DMSO, room temp, 1 h; (ii) Trifluoroacetic acid,
80 °C, 24 h; (iii) 4ꢀFꢀbenzyl bromide, K2CO3, DMF, 100 °C, 24 h; (iv) diethyl oxalate, C2H5ONa, THF,
room temp, 2 h; (v) CH3COONH4, benzene, glacial acetic acid, reflux, 20 h; (vi) 1 N NaOH,
THF/CH3OH, room temp, 1 h.
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Derivatives 6e-h and 7e-h were obtained according to Scheme 2. The pyrroles 10 and 11 were used as
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starting materials to obtain the key intermediates 12e-h. Compound 10 was obtained by alkylation of
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commercially available 4ꢀiodoꢀ2ꢀformylpyrrole in alkaline medium (K2CO3) with 4ꢀFꢀbenzyl bromide.
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Scheme 2. Synthetic Route to Pyrrolyl DKAs 6e-h and 7e-ha
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Reagents and conditions: (i) alkylating agent, K2CO3, DMF, 100 °C, 24 h; (ii) phenylboronic acid,
Cs2CO3, P(tꢀBut)3, Pd2(dba)3, dioxane, 80 °C, 24 h; (iii) 1) DMF, 1,2ꢀdichloroethane dry, oxalyl
chloride, 0 °C, 15 min, room temp, 15 min; 2) 4ꢀFꢀbenzoyl chloride, AlCl3, room temp, 4 h; (iv)
phenylboronic acid, copper (II) acetate anhydrous, pyridine/NMP 1:1, MW: 60 Watt, 120 °C, 6 min; (v)
acetone, 4 N NaOH, room temp, 24 h; (vi) diethyl oxalate, C2H5ONa, THF, room temp, 2 h; (vii) 1 N
NaOH, THF/CH3OH, room temp, 1 h.
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Derivative 11 was obtained starting form pyrrole that underwent to two step oneꢀpot reaction
comprising i) formylation in the presence of oxalylchloride and DMF, and ii) FriedelꢀCraft reaction with
4ꢀFꢀbenzoyl chloride. Thus, 10 and 11 were alkylated with the appropriate alkyl halide to furnish 12e
and 12g, or arylated on nitrogen atom of pyrrole ring to give 12f, or finally coupled by a Suzuki reaction
to obtain 12h, respectively. The formylpyrroles 12e-h were condensed with acetone in alkaline medium
(4 N NaOH) to obtain α,βꢀinsaturated ketones 13e-h. The last compounds were in turn condensed with
diethyl oxalate in the presence of sodium ethoxide to provide the diketobutanoic ethyl esters 6e-h that
were finally hydrolyzed by reaction with 1 N NaOH to give the corresponding carboxylic acids 7e-h.
The synthetic pathway to obtain derivatives 6i,j and 7i,j is outlined in Scheme 3. The enones 17i,j
were obtained starting from 15 and 16, respectively. Pyrrole 15 has been achieved starting form pyrroleꢀ
2ꢀcarboxaldehyde that was alkylated in alkaline medium (K2CO3) to obtain 14. The termal transposition
of the formyl chain from position 2 to 3 of the pyrrole ring in the presence of trifluoro acetic acid, led to
derivative 15. In a parallel pathway (3E,5E)ꢀ6ꢀphenylhexaꢀ3,5ꢀdienꢀ2ꢀone38 underwent to ring closure
by reacting with TosMIC, giving the pyrrole derivatives 16. Interestingly, the attack of the TosMIC
reagent was specific on the 5,6 double bond of the starting dienone. Afterwards, derivative 15 was
condensed with acetone affording 17i; conversely, compound 16 was converted into 17j by reaction of
4ꢀFꢀbenzyl bromide in alkaline medium (K2CO3). Finally, intermediates 17i,j were converted into esters
6i,j by condensation with diethyl oxalate and then converted to acids 7i,j by alkaline hydrolysis.
Derivatives 6k,l and 7k,l were obtained according to the pathway described in Scheme 4. The
iodination of 2ꢀacetylꢀ1ꢀ[(4ꢀfluorophenyl)methyl]pyrrole in presence of Nꢀiodosuccinimide (NIS)
afforded derivative 18, which underwent to a Suzuki coupling reaction to give 19. Intermediates 19 and
2ꢀacetylꢀ1ꢀ[(4ꢀfluorophenyl)methyl]pyrrole were converted into the diketobutanoic acid derivatives 7k,l
through the ethyl esters 6k,l, according to the condensation/hydrolysis procedure described above.
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Scheme 3. Synthetic Route to Pyrrolyl DKAs 6i,j and 7i,ja
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Reagents and conditions: (i) 4ꢀFꢀbenzyl bromide, K2CO3, DMF, 100 °C, 24 h; (ii) Trifluoroacetic
acid, 80 °C, 24 h; (iii) acetone, 4 N NaOH, room temp, 24 h; (iv) Et2O/DMSO, NaH, TosMIC, room
temp, 1 h; (v) 4ꢀFꢀbenzyl bromide, K2CO3, DMF, 100 °C, 24 h; (vi) diethyl oxalate, C2H5ONa, THF,
room temp, 2 h; (vii) 1 N NaOH, THF/CH3OH, room temp, 1 h.
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Scheme 4. Synthetic Route to Pyrrolyl DKAs 6k,l and 7k,la
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Reagents and conditions: (i) NIS, acetone, ꢀ78 °C, 96 h; (ii) phenylboronic acid, Cs2CO3, P(tꢀBut)3,
Pd2(dba)3, dioxane, 80 °C, 24 h; (iii) diethyl oxalate, C2H5ONa, THF, room temp, 2 h; (iv) 1ꢀtritylꢀ1Hꢀ
[1,2,4]triazoleꢀ3ꢀcarboxylic acid ethyl ester,39 nꢀBuLi, THF, from ꢀ78 °C to 0 °C, 3.5 h; (v) 3 M HCl
sol, 1,4ꢀdioxane, 60 °C, 30 min; (vi) 1 N NaOH, THF/CH3OH, room temp, 1 h.
Finally, 2ꢀacetylꢀ1ꢀ[(4ꢀfluorophenyl)methyl]pyrrole was subjected to a condensation reaction in
presence of 1ꢀtritylꢀ1Hꢀ[1,2,4]triazoleꢀ3ꢀcarboxylic acid ethyl ester39 to afford the triazoleꢀprotected
derivative 20, which was deprotected by hydrolysis in the presence of 3M HCl to obtain the triazole
derivative 7m.
Evaluation of biological activities. All newly synthesized compounds 6a-l and 7a-m were tested in
vitro in enzyme assays against both recombinant RNase H and IN. The IC50 values obtained for each
compound in the inhibition of both the IN ST reaction and HIVꢀ1 RTꢀassociated RNase H function were
plotted against each other in correlation plots (Figure 1). In these plots, single dots correspond to single
compounds. The compounds are distributed around two perpendicular axes crossing the IN IC50 axis (X
axis) at 1 ꢀM and the RNase H IC50 axis (Y axis) at 10 ꢀM (bolded crosshair in the center of each
graph, Figure 1).
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Figure 1. Scatter plot for the inhibition of RNase H and IN enzymes. A. Compounds are categorized
according to their acidic or ester function. B. Compounds are categorized according to the nature of
their R substitution. C. Compounds are categorized according to the nature of their R1 substitution. D.
Compounds are categorized according to the nature of their R2 substitution. Compounds with one IC50
value missing such as 6d have been left out of the plot and compounds with IC50 values above 111 ꢀM
have been arbitrary positioned at the 100 ꢀM value.
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These two axes splice the graph into four quarts corresponding to RNase H/IN dual inhibitors (lower
left quarter), RNase Hꢀselective inhibitors (lower right quarter), IN selective inhibitors (upper left
quarter) and inhibitors of lower potency (upper right quarter). As seen on Figure 1, these graphs do not
show any particular correlation between RNase H and IN inhibition.
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The newly synthesized pyrrolyl derivatives 6a-l and 7a-m exhibited good potency of inhibition when
tested on the HIVꢀ1 IN ST (Table 1). In general, as reported previously,31,32 the acid derivatives 7a-m
were endowed of the highest potency showing IC50 values in the range of 26ꢀ0.019 ꢀM. In our assay
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Merck compound 7k was confirmed as potent IN ST inhibitor showing IC50 = 0.057 ꢀM (literature data
IC50 = 0.08 ꢀM).33 As seen on Figure 1A, 80% of the acid compounds (red dots) are distributed in the
left half of the graph, suggesting the acid function is critical for IN inhibition but not critical for RNAse
H inhibition. Among them, 7 compounds (7a,c,d,e,h,k,l) proven subꢀmicromolar activity (IC50 value
were in the range of 66 ꢀ 19 nM), while two derivatives (7b,i) can be considered less active to almost
inactive (IC50 values were 111 and 26 ꢀM, respectively). The most active compound was the 4ꢀ
phenylpyrrole derivative 7l with an IC50 value of 19 nM, three time less active than 1 in inhibiting of the
ST reaction of IN. Interestingly, even though not all the synthetized analogues were tested on the HIVꢀ1
3’ꢀP activity, results showed that the newly synthesized acids were selective inhibitors of the ST step,
with the IC50 values on the 3’ꢀP step 2ꢀ3 orders of magnitude higher with respect to the ones obtained on
ST, thus confirming that the DKAs are selective ST inhibitors (data not shown).
The newly synthesized pyrrolyl DKA derivatives 6a-l and 7a-m can be divided into two classes: the
diketobutanoic (6a-d,k,l and 7a-d,k,l) and the diketohexenoic (6e-j and 7e-j) derivatives. In the
diketobutanoic structure of derivatives 7a-d, 7k-l, two main differences involving the substitution of the
pyrrole ring emerged: the diketobutanoic chain can be linked on the pyrrole ring into two different
positions (2ꢀ or 3ꢀposition), along with a phenyl ring, that can be substituted in position 4 of the pyrrole
ring. Both two variables did not influence the IN inhibitory activity. Only the phenyl substitution at
position 4 of the pyrrole ring (R substituent) seems to favor slightly IN vs. RNaseH inhibition with a
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Journal of Medicinal Chemistry
majority of compounds bearing this substitution distributed in the two left quarters of the correlation
graphs (green dots, Figure 1B). In fact, from a comparison of the inhibition data of
diketobutanoic/diketohexenoic 7a/7l and 7c/7k, which are characterized respectively by the presence of
the phenyl ring and by its absence, a correspondence in the orders of magnitude of their IC50 values was
observed. Moreover, 7a and 7c linked the diketobutanoic chain in position 3 of the pyrrole ring, while
7k and 7l in 2ꢀposition of the same ring. All these compounds were characterized by similar potencies
(IC50 values of 22 nM, 24 nM, 57 nM and 19 nM, respectively). The 4ꢀfluoroꢀbenzoyl substitution at
position 4 of the pyrrole ring (R substituent) does not seem to favor the inhibition of either enzymes
(orange dots, Figure 1B).
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Other three modifications involved exclusively the diketobutanoic chain: (1) the substitution of the
enolic OH with a NH2 (7d), (2) the introduction of a methyl group in 3ꢀposition (7b), and (3) the
substitution of the carboxylic acid function with its bioisoster triazole ring (7m). The NH2 (7d) and
triazole (7m) derivatives were 2ꢀfold less active with respect to their OH (7a) and COOH (7h)
counterparts, respectively (IC50 values were 43 nM, 110 nM, 22 nM and 57 nM, respectively). On the
contrary, the 3ꢀmethylbutenoate derivative (7b) completely lost its ability to inhibit IN enzyme (IC50
values of > 111 ꢀM). It appears that IN seems to tolerate a wide variety of substitutions at the R1
position, including the absence of substituent, with a majority of compounds with such substitutions
distributed in the two left quarters of the correlation graphs (Figure 1C). No preferential inhibition
pattern can be observed for compounds with substitutions at the R2 position on the correlation plot
presented in Figure 1 D.
When the two series of diketobutanoic and the diketohexenoic esters and acids were tested on the
HIVꢀ1 RTꢀassociated RNase H function in biochemical assays, the most active derivatives were the
diketohexenoic ester 6f and the diketobutanoic acid 7d, with IC50 value of 1.8 ꢀM, and 2 ꢀM,
respectively. Interestingly, both compounds were more active then the references 3 and 4. Noteworty,
the structure of 6f is related to the references 3 since both are pyrrolyl diketohexenoic derivatives, but
6f bring a phenyl ring on nitrogen replacing the 4ꢀFꢀbenzyl group of 3 and a 4ꢀFꢀbenzoyl moiety linked
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in 4 position of the pyrrole ring. In the matter of compound 7d, it is a 3ꢀpyrrolylꢀdiketobutanoic acid
derivative characterized by the presence of an amine function that replaces the enole OH in position 3 of
the diketobutanoic chain. Since 7d is a carboxylic acid, its stronger inhibition towards IN than RNase H
function was expected; conversely, the ester 6f is the best dual inhibitor IN/RNase H of this series. From
a first analysis of the results we can state that i) as known, the acid function confer a better inhibitory
activity on IN, ii) the ester function is amenable for inhibition of RNase H function of RT, confirming
the results recently reported by as by the means of docking studies and mutagenesis experiments,40 and
iii) contemporaneously, the ester function is necessary for a dual inhibition IN/RNase H.
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59
60
Among the ester derivatives (6a-l), the diketohexenoic compounds (6e-j, IC50 values in the range of
1.8 ꢀ 55 ꢀM) were able to inhibit the HIVꢀ1 RNase H function with a slightly higher potency then the
diketobutanoic counterpart (6a-d,k,l, IC50 values in the range of 6 ꢀ 72 ꢀM). Differently, when the acid
derivatives (7a-m) were tested this difference was not observed.
In both the diketohexenoic and the diketobutanoic ester and acid derivatives, the presence of a phenyl
substituent in position 4 of the pyrrole ring influenced the inhibitory activity. In fact, the 4ꢀphenyl
substituted diketobutanoic ester and acid 6a/7a (IC50 values of 10 ꢀM, 7.5 ꢀM, respectively) and
diketohexenoic 6j/7j (IC50 values of 3.0 ꢀM, 7.0 ꢀM, respectively) were consistently more active then
the unsubstituted counterparts 6c/7c and 6i/7i (IC50 values of > 100 ꢀM, 41 ꢀM, 55 ꢀM and 69 ꢀM
respectively).
In general, when the DKA chain was shifted from 2ꢀ to 3ꢀposition, a 2ꢀ4 fold increase in potency of
inhibition was observed (compare 6h, 7h and 7l with 6j, 7j and 7a: IC50 values of 13.4 ꢀM and 23 ꢀM
14 ꢀM 3 ꢀM, 7 ꢀM, 7.5 ꢀM, respectively), with the sole exception of 6a and 6l, that showed
comparable potency (IC50 values of 10 ꢀM and 6 ꢀM, respectively).
The substitution of the enolic OH on the diketobutanoic chain (6a/7a) with a NH2 group (6d/7d) in
the ester series, lead to a 7ꢀfold decrease of the potency of RNase H inhibition (6d, IC50 values of 72
ꢀM) with respect to the unmodified counterpart (6a, IC50 value of 10 ꢀM); while within the acid series,
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Journal of Medicinal Chemistry
the NH2 derivative improved its potency of inhibition (7d, IC50 value of 2 ꢀM; 7a, IC50 value of 7.5
ꢀM). The introduction of a methyl group in position 3 of the diketobutanoic chain (7b) reduced the
inhibition (7b and 7a IC50 values of 64 ꢀM and 7.5 ꢀM, respectively) likewise the substitution of the
1
2
3
4
5
6
7
8
9
carboxylic acid function with its bioisoster triazole (7a and 7m, IC50 values of 7.5 ꢀM and 26 ꢀM,
respectively).
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Among all the newly synthesized derivatives, the 2ꢀpyrrolyldiketohexenoic ester 6f, athough not very
potent, emerged as dual inhibitor showing similar IC50 values against both IN enzyme and RNase H
function of RT (1.2 ꢀM and 1.8 ꢀM, respectively). This compound retains the ester function that is
demonstrated necessary for the dual inhibiton31 and is characterized by a phenyl ring on nitrogen
replacing the 4ꢀFꢀbenzyl group of 3 and by a 4ꢀFꢀbenzoyl moiety linked in 4 position of the pyrrole ring.
Cell-based assays. Among the newly synthesized pyrrolyl DKA derivatives 6a-l and 7a-m, seven
derivatives (6a,c,l and 7a,c-d,l) were characterized by a good antiꢀHIV activity showing a EC50 values
in the subꢀmicromolar concentration (EC50 in the range of 0.56 ꢀ 0.9 ꢀM) and a good selectivity index
(SI). Three compounds, the 6k-j and 7k derivatives, showed an antiꢀHIV activity in the range of the
micromolar concentration (EC50 in the range of 1 ꢀ 4.3 ꢀM), while 10 compounds were less asctive or
inactive (6d-i and 7b,e-f,i,m, EC50 in the range of 17.2 ꢀ > 50 ꢀM). In general, all the compounds had a
low citotoxicity index (CC50 > 50 ꢀM). Compound 7c, characterized by the diketobutanoic chain in 3ꢀ
position of the pyrrole ring, showed the best antiviral efficacy on HIVꢀ1 infected cell (EC50 = 0.58 ꢀM)
and a low citotoxicity (CC50 > 50 ꢀM, SI > 86). The best dual inhibitor derivative 6f that showed
activity at micromolar concentration in biochemical assays, was 20 times less potent in cellꢀbased
assays.
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Table 1. Cytotoxicity, Enzymatic, and Antiviral Activities of Compounds 6a-l and 7a-m
1
2
3
4
5
6
7
8
9
activity in
enzyme
assay, IC50
antiviral activity
a
d
e
Cpd
6a
6b
6c
6d
6e
6f
R
R1
H
R2
X
RHb
10
ꢀ
STc
0.42
ꢀ
EC50
0.56
ꢀ
CC50
11
SIf
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Ph
Ph
H
OH
OH
OH
Et
Et
Et
19.6
Me
H
ꢀ
ꢀ
>100 1.6
72 NT
>100 4.3
0.90
19
>50
>50
>50
>50
>50
ꢀ
>55
Ph
H
NH2 Et
ꢀ
4ꢀFBzg Bnh
ꢀ
ꢀ
ꢀ
Et
Et
Et
Et
Et
Et
19
ꢀ
4ꢀFBz
4ꢀFBz
Ph
Ph
1.8
28
1.2
20
ꢀ
Me
>333 48
ꢀ
6g
6h
6i
4ꢀFBn ꢀ
13.4
55
2.5
ꢀ
ꢀ
H
ꢀ
ꢀ
90
50
>50
26.9
33
ꢀ
Ph
ꢀ
ꢀ
3.0
>21
0.51
0.79
4.3
1.2
0.70
6.3
27
6
6j
H
ꢀ
ꢀ
COOEt 21
COOEt 6.0
6k
6l
Ph
ꢀ
ꢀ
3.9
>50
>50
>50
>50
ꢀ
Ph
H
OH
OH
OH
NH2
ꢀ
H
H
H
H
H
H
H
H
H
H
7.5
64
0.022 0.66
>75
ꢀ
7a
7b
7c
7d
7e
7f
Ph
CH3
H
>50
>111
H
41
0.024 0.58
0.043 0.63
0.063 >50
>86
>79
ꢀ
Ph
H
2.0
7.5
100
20
4ꢀFBz
4ꢀFBz
4ꢀFBz
Ph
Bn
Ph
Me
ꢀ
0.59
ꢀ
>50
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
7g
7h
7i
4ꢀFBn ꢀ
23
0.066
26
ꢀ
ꢀ
ꢀ
H
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
69
>50
17.2
>50
>50
28
ꢀ
Ph
7.0
0.73
>2.9
28
>72
7j
7ki
H
COOH 54
COOH 14
0.057 1.0
0.019 0.7
Ph
>50
7l
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Journal of Medicinal Chemistry
H
ꢀ
ꢀ
Trj
26
0.11
20.4
>50
ꢀ
7m
1
1
2
3
0.007 0.016
>250 >15000
4
5
6
7
8
9
10
11
12
3.2
8
1.9
98
>50
<0.2
2
2
>50
>50
>250
>25
3
3
0.60
2.4
4
26.2
3.6
>50
>13.8
5
aInhibitory concentration 50% (ꢀM) determined from dose response curves. Experiments performed
b
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c
against HIVꢀ1 RTꢀassociated RNase H activity. Experiments performed against HIVꢀ1 IN ST activity.
dEffective concentration 50% (ꢀM). eCytotoxic concentration 50% (ꢀM). fSI = CC50/EC50. gBz, benzoyl.
hBn, benzyl. iSee also reference 33. Tr, triazolyl.
j
CONCLUSION
The development of HIVꢀ1 dual inhibitors is a highly innovative approach aimed to reduce drug toxic
side effects and therapeutic costs as well. Since HIVꢀ1 IN and RNase H are both selective targets for
HIVꢀ1 chemotherapy, the identification of dual IN/RNase H inhibitors is an attractive strategy for new
drug development. The newly synthesized pyrrolyl derivatives 6a-l and 7a-m exhibited good potency
against IN and a moderate inhibition of the RNase H function of RT. In general, comparing the
inhibition data among the ester and the acid derivatives a different behaviour was observed. As
expected, the acid derivatives showed an higher potency of IN inhibition with respect to the
corresponding esters, while the latter have been often found more potent than the corresponding acids in
inhibiting the RNase H function of RT enzyme. Notably, compound 6f, although was not very potent on
HIVꢀinfected cell, showed a good correlation between HIVꢀ1 IN and RNase H inhibition. It is
characterized by a diketoester function, a phenyl ring on nitrogen and a 4ꢀFꢀbenzoyl moiety linked in 4
position of the pyrrole ring. We can state that although the acid function confer a better inhibitory
activity on IN an ester function is amenable for inhibition of RNase H function of RT and as the
conseguence the ester function is necessary for a dual inhibition IN/RNase H. This basic chemical
features should be considered for development of further more potent dual inhibitors. Overally, the data
reported in this work confirm the possibility to develop dual HIVꢀ1 IN/RNase H inhibitors and give new
information for the further development of effective dual HIVꢀ1 inhibitors.
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1
2
3
EXPERIMENTAL SECTION
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5
6
7
8
Chemistry. General. Melting points were determined on a Bobby Stuart Scientific SMP1 melting
point apparatus and are uncorrected. Compounds purity were always >95% determined by high pressure
liquid chromatography (HPLC). HPLC analysis were carried out with a Shimadzu LCꢀ10AD VP CTOꢀ
10AC VP. Column used was generally Discovery Bio Wide Pore C18 (10 cm X 4.6 mm, 3 ꢁm).
Infrared (IR) spectra were recorded on a PerkinꢀElmer Spectrumꢀone spectrophotometer. 1H NMR
spectra were recorded at 400 MHz on a Broker AC 400 Ultrashield 10 spectrophotometer (400 MHz).
Dimethylsulfoxideꢀd6 99.9% (code 44,139ꢀ2) and deuterochloroform 98.8% (code 41,675ꢀ4) of isotopic
purity (Aldrich) were used. Column chromatographies were performed on silica gel (Merck; 70ꢀ230
mesh) column or alluminium oxide (Sigmaꢀaldrich; 150 mesh) column. All compounds were routinely
checked on TLC by using aluminiumꢀbaked silica gel plates (Fluka DCꢀAlufolien Kieselgel 60 F254) or
alluminium oxide (Fluka DCꢀAlufolien Aluminium oxide). Developed plates were visualized by UV
light. Solvents were reagent grade and, when necessary, were purified and dried by standard methods.
Concentration of solutions after reactions and extractions involved the use of rotary evaporator (Büchi)
operating at a reduced pressure (ca. 20 Torr). Organic solutions were dried over anhydrous sodium
sulfate (Merck). All reactions were carried out under nitrogen; all solvents were freshly distilled under
nitrogen and stored over molecular sieves for at least 3 h prior to use.
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Microwave Irradiation Experiments. Microwave reactions were conduced using a CEM Discover
system unit (CEM. Corp., Matthews, NC). The machine consists of a continuous focused microwaveꢀ
power delivery system with operator selectable power output from 0 to 300 W. The temperature of the
contents of the vessel was monitored using a calibrated infrared temperature control mounted under the
reaction vessel. All experiments were performed using a stirring option whereby the contents of the
vessel are stirred by means of a rotating magnetic plate located below the floor of the microwave cavity
and a Teflonꢀcoated magnetic stir bar in the vessel.
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Journal of Medicinal Chemistry
General procedure A (GP-A): Synthesis of pyrrole nucleus. A solution of α,βꢀunsaturated ketone
(5.42 mmol) and tolueneꢀ4ꢀsulfonylmethylisocyanide (1.16 g, 5.96 mmol, 1.1 eq) dissolved in a mixture
of anhydrous dimethyl sulfoxide/ethyl ether (14:30 mL) was added dropwise into a wellꢀstirred
suspension of sodium hydride (60% in paraffine oil; 0.48 g, 11.93 mmol, 2.2 eq) in dry ethyl ether (30
mL) under argon atmosphere. After the addition the mixture was stirred at room temperature for 1 hour.
The reaction was treated with water and extracted with ethyl acetate. The organic layer was washed with
brine, dried over anhydrous sodium sulfate and concentrated under vacuum. The crude product was
purified by chromatography on aluminum oxide (chloroform as eluent) to afford the pure product. Yield
(%), melting point (°C), recrystallization solvent, IR, 1H NMR are reported for each compound.
General procedure B (GP-B): Alkylation of the pyrrole nitrogen. A mixture of the appropriate
pyrrole (1.1 mmol), alkylating agent (3.3 mmol), and anhydrous K2CO3 (210 mg, 1.5 mmol) in dry
DMF (10 mL) was stirred at 100 °C for 2 h. Then the mixture was cooled, treated with water (40 mL)
and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium
sulfate and concentrated under vacuum. The crude product was purified by chromatography on silica gel
to afford the pure product. Chromatography eluent, yield (%), melting point (°C), recrystallization
solvent, IR, 1H NMR are reported for each compound.
1
2
3
4
5
6
7
8
9
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59
60
General Procedure C (GP-C): Condensation of pyrrole carboxaldehyde with acetone. The proper
pyrrole carboxaldehyde (0.075mol) was dissolved in 250 mL of acetone. To this mixture was added 4 N
NaOH (110 mL) and the reaction was stirred at room temperature for 24 h. After this period water (300
mL) and ethyl acetate (250 mL) were added. The organic layer was separated, washed with water (2 x
100 mL), dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude product
was purified by column chromatography on silica gel to obtain pure products. Chromatography eluent,
yield (%), melting point (°C), recrystallization solvent, IR, 1H NMR are reported for each compound.
General procedure (GP-D): Acetyl Transposition. A mixture of opportune α acetylꢀsubstituted
pyrrole (1.23 mmol) in trifluoroacetic acid (5 mL), was heated at 80 °C for 20 h. After this period the
reaction was quenched with water (30 mL) and extracted with ethyl acetate (2 x 50 mL). The organic
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layers were collected, dried over sodium sulfate, filtered and evaporated under vacuum. The crude
product was purified by chromatography on silica gel (chloroform as eluent) to afford pure product as a
1
2
3
4
5
6
1
brown oils. Yield (%), melting point (°C), recrystallization solvent, IR, H NMR are reported for each
7
8
compound.
9
General Procedure e (GP-E): Suzuki reaction. Pd2(dba)3 (0.1 g, 1.7 mmol) was added ino a well
stirred mixture of appropriate 4ꢀiodopyrrole (1.7 mmol), phenylboronic acid (0.85 g, 7.0 mmol), Cs2CO3
(0.665 g, 2.0 mmol) and P(tꢀBut)3 in dioxane (20 mL). The reaction was stirred at 80 °C for 24 h under
argon atmosphere. Then the reaction was cooled to room temperature, filtered and washed with dioxane.
The organic layer was evaporated under vacuum. The raw material was extracted with water (50 mL)
and ethyl acetate (50 mL). The organic phase was separated, dried over sodium sulfate, filtered and
evaporated under vacuum. The raw material was purified by silica gel chromatography.
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50
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53
54
55
56
57
58
59
60
1
Chromatography eluent, yield (%), melting point (°C), recrystallization solvent, IR, H NMR are
reported for each compound.
General Procedure F (GP-F): Synthesis of Diketo Esters. Freshly prepared sodium ethoxide (390
mg, 5.5 mmol) was added into a wellꢀstirred mixture of the appropriate acetyl derivative (2.7 mmol) and
diethyl oxalate (790 mg, 5.4 mmol) in anhydrous THF (2.7 mL) under nitrogen atmosphere. The
mixture was stirred at room temperature for 2 h, and then was poured into nꢀhexane (50 mL). The
collected precipitate was vigorously stirred for 30 min in 1 N HCl (50 mL). The yellow solid that
formed was filtered, washed with water, and dried under IR lamp to afford the pure diketo esters. Yield
(%), melting point (°C), IR, 1H NMR are reported for each compound.
General Procedure G (GP-G): Synthesis of diketo acids. A mixture of 1 N NaOH (6.5 mL) and the
appropriate ester (1.3 mmol) in 1:1 THF/methanol (12 mL) was stirred at room temperature for 40 min
and then poured onto crushed ice. The aqueous layer was treated with 1 N HCl until pH 3 (pH 7 for 1d),
and the solid that formed was collected by filtration, then washed with water and dryed under warming
lamp to afford pure acids. Yield (%), melting point (°C), IR, 1H NMR are reported for each compound.
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Journal of Medicinal Chemistry
1-(4-Phenyl-1H-pyrrol-3-yl)propan-1-one (8).41 Compound 8 was prepared from (E)ꢀ1ꢀphenylpentꢀ
1ꢀenꢀ3ꢀone35 by means GPꢀA. 79% as a yellow solid; 169ꢀ170 °C; toluene. Anal. (C13H13NO) C, H, N.
1-(1-(4-Fluorobenzyl)-4-phenyl-1H-pyrrol-3-yl)ethanone (9a). Compound 9a was prepared from 1ꢀ
(4ꢀphenylꢀ1Hꢀpyrrolꢀ3ꢀyl)ethanone34 by means GPꢀB, using 4ꢀfluorobenzyl bromide as alkylatig agent.
Chloroform; 100% as brown oil; IR ν 1705 (C=O ketone) cmꢀ1; NMR (DMSO d6) δ 2.26 (s, 3H, CH3),
5.04 (s, 2H, CH2), 6.63 (s, 1H, pyrrole αꢀproton), 7.06 (m, 2H, benzyl H), 7.19 (m, 2H, benzyl H), 7.25ꢀ
1
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7.41 (m, 6H, benzene H and pyrrole αꢀproton). Anal. (C19H16FNO) C, H, N, F.
1-(1-(4-Fluorobenzyl)-4-phenyl-1H-pyrrol-3-yl)propan-1-one (9b). Compound 9b was prepared
from 8 by means GPꢀB, using 4ꢀfluorobenzyl bromide as alkylating agent. Chloroform/acetate 50:1;
1
66% as brown oil.; IR ν 1656 (C=O ketone) cmꢀ1. H NMR (DMSO d6) δ 1.04 (t, 3H, J = 8 Hz,
CH2CH3), 2.75 (q, 2H, J = 8 Hz, CH2CH3), 5.18 (s, 2H, CH2), 7.07 (d, 1H, J = 2.2 Hz, pyrrole C5ꢀH),
7.2ꢀ7.3 (m, 3H, benzene H), 7.32 (t, 2H, benzyl H), 7.4 (m, 2H, benzene H), 7.47 (m, 2H, benzyl H),
7.87 (d, 1H, J = 2 Hz, pyrrole C2ꢀH). Anal. (C20H18FNO) C, H, N, F.
1-(1-(4-Fluorobenzyl)-1H-pyrrol-3-yl)ethanone (9c). Compound 9c was prepared from 1ꢀ(1ꢀ(4ꢀ
fluorobenzyl)ꢀ1Hꢀpyrrolꢀ2ꢀyl)ethanone36 by means GPꢀD. 50% as brown oil; IR ν 1655 (C=O ketone)
cmꢀ1. 1H NMR (CDCl3) δ 2.37 (s, 3H, CH3), 5.03 (s, 2H, CH2), 6.60ꢀ6.63 (m, 2H, pyrrole C4ꢀH and C5ꢀ
H), 7.03 (t, 2H,benzene H), 7.12 (m, 2H, benzene H), 7.28 (t, 1H, J = 2.0 Hz, pyrrole C2ꢀH,). Anal.
(C13H14FNO) C, H, N, F.
1-(4-Fluorobenzyl)-4-iodo-1H-pyrrole-2-carboxaldehyde (10). Compound 10 was prepared from
commercially available 4ꢀiodopyrroleꢀ2ꢀcarboxaldehyde by means GPꢀB, using 4ꢀfluorobenzyl bromide
1
as alkylatig agent. Chloroform/nꢀhexane 4:1; 39% as brown oil; IR ν 1651 (C=O) cmꢀ1. H NMR
(CDCl3) δ 5.47 (s, 2H), 6.9ꢀ7.0 (m, 4H, pyrrole α−proton, pyrrole β−proton and benzene H), 7.02 (m,
2H, benzene H), 9.48 (s, 1H, CHO). Anal. (C12H9FINO) C, H, N, F, I.
4-(4-Fluorobenzoyl)-1H-pyrrole-2-carboxaldehyde (11). To a well stirred solution of DMF (3.9
mL, 50 mmol) in 1,2ꢀdichloroethane (100 mL) refrigerated in ice bath was added drop wise a solution of
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oxalyl chloride (6.35 g, 50 mmol) in 1,2 dichloroethane (100 mL) in a period of 15 minutes. After
addiction, the suspension was stirred at room temperature for 15 minutes. After this time the reaction
mixture was refrigerated in ice bath, and treated with a solution of pyrrole (49.9 mmol) in 1,2ꢀ
dichloroethane (100 mL). The mixture was stirred for 15 minutes at room temperature and then treated
with AlCl3 (14.6 g, 109 mmol) and 4ꢀfluoro benzoyl chloride (50 mmol). The reaction was maintained
at room temperature for 4 h. After this period the reaction was quenched with ice and water, and
extracted with ethyl acetate, dried over sodium sulfate, filtered and evaporated under vacuum. The crude
product was purified with chromatography on aluminum oxide (1:1 ethyl acetateꢀchloroform as eluent)
to afford 5 as yellow solid. 85%; 110ꢀ111°C; benzene/cyclohexane; IR ν 2900 (enole), 1660 (C=O
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ketone) 1640 (C=O) cmꢀ1. H NMR: (CDCl3) δ 7.1ꢀ7.4 (m, 3H, benzene H and pyrrole βꢀproton), 7.45
(s, 1H, pyrrole α−proton), 7.8ꢀ7.9 (m, 2H, benzene H), 9.60 (s, 1H, CHO), 12 (sb, 1H, NH). Anal.
(C12H8FNO2) C, H, N, F.
1-Benzyl-4-(4-fluorobenzoyl)-1H-pyrrole-2-carbaldehyde (12e). Compound 12e was prepared
from 11 by means GPꢀB, using benzyl bromide as alkylatig agent. Acetate/nꢀhexane 1:2; 34% as brown
oil; IR ν 1672 (C=O aldehyde) 1638 (C=O ketone) cmꢀ1. 1H NMR (CDCl3) δ 5.62 (s, 2H, CH2), 7.1ꢀ7.2
(m, 4H, benzyl H and pyrrole β−proton), 7.3ꢀ7.4 (m, 4H, benzyl H and benzoyl H), 7.58 (s, 1H, pyrrole
α−proton), 7.8ꢀ7.9 (m, 2H, benzoyl H), 9.62 (s, 1H, CHO). Anal. (C19H14FNO2) C, H, N, F.
4-(4-Fluorobenzoyl)-1-phenyl-1H-pyrrole-2-carbaldehyde (12f). Compound 11, phenylboronic
acid, pyridine, and copper (II) acetate anhydrous were dissolved in NMP (2.4ml) in a MW reactor tube
and leave to react at 60 Watt, 120 °C for 6 min. After this period the reaction was quenched with water,
and extracted with ethyl acetate (5 x 20ml), washed with water (5 x 20ml), dried over sodium sulfate,
filtered and evaporated under vacuum. The crude product was purified with chromatography on silica
gel (1:1 ethyl acetateꢀhexane as eluent) to afford 12f as brown solid (63% yield). 130ꢀ131°C.
benzene/cyclohexane; IR ν 1680 (C=O aldehyde) 1632 (C=O ketone) cmꢀ1. 1H NMR (CDCl3) δ 7.21 (t,
2H, benzoyl H), 7.4ꢀ7.5 (m, 2H, benzene H), 7.5ꢀ7.6 (m, 4H, benzene H and pyrrole β−proton), 7.67 (d,
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Journal of Medicinal Chemistry
1H, J = 2 Hz, pyrrole α−proton), 7.9ꢀ8.0 (m, 2H, benzoyl H), 9.68 (s, 1H, CHO). Anal. (C18H12FNO2)
C, H, N, F.
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1-Methyl-4-(4-fluorobenzoyl)-1H-pyrrole-2-carboxaldehyde (12g). Compound 12g was prepared
from 11 by means GPꢀB, using iodo methane as alkylatig agent. Chloroform; 90% as grey solid; 115ꢀ
116°C; benzene/cyclohexane; IR ν 1660 (C=O aldehyde) 1640 (C=O ketone) cmꢀ1. 1H NMR (CDCl3) δ
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4.03 (s, 3H, NꢀCH3), 7.2 (m, 2H, benzoyl H), 7.4 (d, 1H, pyrrole β−proton), 7.5 (s, 1H,
pyrrole α−proton), 7.8ꢀ7.9 (m, 2H, benzoyl H), 9.60 (s, 1H, CHO). Anal. (C13H10FNO2) C, H, N, F.
1-(4-Fluorobenzyl)-4-phenyl-1H-pyrrole-2-carbaldehyde (12h). Compound 12h was prepared
from 10 by means GPꢀE. 1:1 ethyl acetateꢀhexane as eluent; 95% as brown oil; IR ν 1642 (C=O
aldehyde) cmꢀ1. 1H NMR (CDCl3) δ 5.54 (s, 2H, CH2), 6.82 (d, 2H, J = 7.0 Hz, benzene H), 6.91 (t, 1H,
J = 7.0 Hz, benzene H), 6.99 (t, 2H, benzyl H), 7.16ꢀ7.24 (m, 4H, pyrrole β−proton, pyrrole α−proton,
benzyl H), 7.48 (d, 2H, J = 7 Hz, benzene H), 9.58 (s, 1H, CHO). Anal. (C18H14FNO) C, H, N, F.
4-(1-Benzyl-4-(4-fluorobenzoyl)-1H-pyrrol-2-yl)but-3-en-2-one (13e). Compound 13e was
prepared from 12e by means GPꢀC. 71% as yellow solid; 94ꢀ95 °C; benzene/cyclohexane; IR ν 1675
1
(C=O ketone) 1637 (C=O ketone) cmꢀ1. H NMR (CDCl3) δ 2.26 (s, 3H, CH3), 5.29 (s, 2H, CH2), 6.60
(d, 1H, J = 16 Hz, butenone C3ꢀH), 7.1ꢀ7.2 (m, 5H, butenone C4ꢀH, benzyl H and pyrrole β−proton),
7.3ꢀ7.4 (m, 4H, benzyl H and benzoyl H), 7.46 (d, 1H, J = 2 Hz, pyrrole αꢀproton), 7.90 (m, 2H,
benzoyl H). Anal. (C22H18FNO2) C, H, N, F.
4-(1-Phenyl-4-(4-fluorobenzoyl)-1H-pyrrol-2-yl)but-3-en-2-one (13f). Compound 13f was
prepared from 12f by means GPꢀC. 30% as yellow solid; 145ꢀ146 °C; benzene/cyclohexane; IR ν 1680
1
(C=O ketone) 1634 (C=O ketone) cmꢀ1. H NMR (CDCl3) δ 2.24 (s, 3H, CH3), 6.56 (d, 1H, J = 16 Hz,
butenone C3ꢀH), 7.19 (t, 2H, benzoyl H), 7.24 (d, 1H, J = 16 Hz, butenone C4ꢀH), 7.35 (d, 1H, J = 2
Hz, pyrrole β−proton), 7.38 (d, 1H, J = 2 Hz, pyrrole αꢀproton), 7.5ꢀ7.6 (m, 5H, benzene H), 7.94 (m,
2H, benzoyl H). Anal. (C21H16FNO2) C, H, N, F.
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4-(1-Methyl-4-(4-fluorobenzoyl)-1H-pyrrol-2-yl)but-3-en-2-one (13g). Compound 13g was
prepared from 12g by means GPꢀC. 70 % as yellow solid; 117ꢀ118 °C; benzene/cyclohexane; IR ν 1660
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(C=O ketone) 1640 (C=O ketone) cmꢀ1. H NMR (CDCl3) δ 2.32 (s, 3H, CH3), 3.78 (s, 3H, NꢀCH3),
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6.62 (d, 1H, J = 16 Hz, butenone C3ꢀH), 7.1ꢀ7.2 (m, 3H, benzene H and pyrrole β−proton), 7.35 (d, 1H,
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J = 3.7 Hz, pyrrole αꢀproton), 7.43 (d, 1H, J = 16 Hz, butenone C4ꢀH), 7.8ꢀ7.9 (m, 2H, benzene H).
Anal. (C16H14FNO2) C, H, N, F.
4-(1-(4-Fluorobenzyl)-4-phenyl-1H-pyrrol-2-yl)but-3-en-2-one (13h). Compound 13h was
prepared from 12h by means GPꢀC. 15% as yellow solid; 160ꢀ161°C; benzene/cyclohexane; IR ν 1604
1
(C=O ketone) cmꢀ1. H NMR (CDCl3) δ 2.28 (s, 3H, CH3), 5.24 (s, 2H, CH2), 6.57 (d, 1H, J = 16 Hz,
butenone C3ꢀH), 7.0ꢀ7.1 (m, 5H, pyrrole β−proton and benzyl H), 7.18 (d, 1H, J = 2 Hz,
pyrrole α−proton,), 7.24 (t, 1H, J = 7 Hz, benzene H), 7.3ꢀ7.4 (m, 3H, butenone C4ꢀH and benzene H),
7.5ꢀ7.6 (m, 2H, benzene H). Anal. (C21H18FNO) C, H, N, F.
1-(4-Fluorobenzyl)-1H-pyrrole-2-carboxaldehyde (14). Compound 14 was prepared from
commercially available pyrroleꢀ2ꢀcarboxaldehyde by means GPꢀB, using 4ꢀfluorobenzyl bromide as
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alkylating agent. Chloroform; 80% as brown oil; IR ν 1640 (C=O) cmꢀ1. H NMR (CDCl3) δ 5.54 (s,
2H, CH2), 6.30 (t, 1H, J = 4 Hz, pyrrole C4ꢀH), 6.9ꢀ7.0 (m, 4H, benzene H), 7.15 (d, 1H, J = 4 Hz,
pyrrole C3ꢀH), 7.17 (d, 1H, J = 4 Hz, pyrrole C5ꢀH), 9.57 (s, 1H, CHO). Anal. (C12H10FNO) C, H, N, F.
1-(4-Fluorobenzyl)-1H-pyrrole-3-carboxaldehyde (10). Compound 10 was prepared from 9 by
means GPꢀD. 55% as brown oil; IR ν 1640 (C=O aldehyde) cmꢀ1. 1H NMR (CDCl3) δ 5.08 (s, 2H, CH2),
6.6 (s, 1H, pyrrole C4ꢀH), 6.7 (s, 1H, pyrrole Cꢀ2H), 7.0ꢀ7.2 (m, 4H, benzene H), 7.31 (s, 1H, pyrrole
C2ꢀH), 9.75 (s, 1H, CHO). Anal. (C12H10FNO) C, H, N, F.
4-(4-Phenyl-1H-pyrrol-3-yl)but-3-en-2-one (16).42 Compound 16 was prepared from (3E,5E)ꢀ6ꢀ
phenylhexaꢀ3,5ꢀdienꢀ2ꢀone38 by means GPꢀA. 56% as brown solid; toluene; IR ν 1640 (C=O ketone)
cmꢀ1. Anal. (C14H13NO) C, H, N.
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4-(1-(4-Fluorobenzyl)-1H-pyrrol-3-yl)but-3-en-2-one (17i). Compound 17i was prepared from 15
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by means GPꢀC. 70% as brown oil; IR ν 1655 (C=O ketone) cmꢀ1. H NMR (CDCl3) δ 2.34 (s, 3H,
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CH3), 5.07 (s, 2H, CH2), 6.43ꢀ6.48 (m, 2H, pyrrole C4ꢀH and butenone C3ꢀH), 6.71 (s, 1H, pyrrole C2ꢀ
H), 7.0 (s, 1H, pyrrole C5ꢀH), 7.06ꢀ7.12 (m, 2H, benzene H), 7.15ꢀ7.18 (m, 2H, benzene H), 7.49 (d,
1H, butenone C4ꢀH, J = 16 Hz). Anal. (C15H14FNO) C, H, N, F.
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4-(1-(4-Fluorobenzyl)-4-phenyl-1H-pyrrol-3-yl)but-3-en-2-one (17j). Compound 17j was prepared
from 16 by means GPꢀB, using 4ꢀfluorobenzyl bromide as alkylating agent. Chloroform; 63% as brown
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oil; IR ν 1640 (C=O) cmꢀ1. H NMR (CDCl3) δ 2.44 (s, 3H, CH3), 5.07 (s, 2H, CH2), 6.82 (s, 1H, J =
16.3 Hz, butenone C3ꢀH), 6.90 (s, 1H, ), 6.99ꢀ7.54 (m, 11H, pyrrole C2ꢀH, pyrrole C5ꢀH, benzene H
and benzyl H), 7.80 (s, 1H, J = 16.3 Hz, butenone C4ꢀH). Anal. (C21H18FNO) C, H, N, F.
1-(1-(4-Fluorobenzyl)-4-iodo-1H-pyrrol-2-yl)ethanone (18). A mixture of 1ꢀ(1ꢀ(4ꢀfluorobenzyl)ꢀ
1Hꢀpyrrolꢀ2ꢀyl)ethanone36 (4g, 18.4 mmol) in acetone dry (100 mL) was cooled at ꢀ78 °C, NIS (4.98 g,
22.1 mmol) was added. The reaction was stirred and the temperature was slowly increased to 25 °C in a
period of 96 h. After this period the mixture was evaporated, ethyl acetate (50 mL) and NaHCO3(aq) (50
mL) were added. The organic phase was separated, dried over sodium sulfate, filtered and evaporated
under vacuum. The raw material was purified with a column chromatography on silica gel (1:10 ethyl
acetateꢀhexane as eluent) to afford 18 as white solid with a yield of 40%. 81ꢀ82 °C; nꢁhexane; IR ν 1640
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(C=O ketone) cmꢀ1. H NMR (CDCl3) δ 2.40 (s, 3H, CH3), 5.50 (s, 2H, CH2), 6.92 (d, 1H, J = 2.0 Hz,
pyrrole C3ꢀH), 7.00 (t, 2H, benzene H), 7.07 (d, 1H, J = 2.0 Hz, pyrrole C5ꢀH), 7.1ꢀ7.2 (m, 2H, benzene
H). Anal. (C13H11FINO) C, H, N, F, I.
1-(1-(4-Fluorobenzyl)-4-phenyl-1H-pyrrol-2-yl)ethanone (19). Compound 19 was prepared from 18
by means GPꢀE. 1:7 ethyl acetateꢀhexane as eluent. 43% as colourless oil; IR ν 1650 (C=O ketone) cmꢀ
1
1. H NMR (CDCl3) δ 2.48 (s, 3H, CH3), 5.58 (s, 2H, CH2), 6.97ꢀ7.02 (t, 2H, benzyl H), 7.14ꢀ7.27 (m,
5H, benzene H, pyrrole C3ꢀH and pyrrole C5ꢀH), 7.35ꢀ7.39 (t, 2H, benzyl H), 7.51 (d, 2H,benzene H).
Anal. (C18H21FNO) C, H, N, F.
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1-(1-(4-Fluorobenzyl)-1H-pyrrol-2-yl)-3-hydroxy-3-(1-trityl-1H-1,2,4-triazol-3-yl)prop-2-en-1-
one (20). A solution of 1ꢀ[1ꢀ(4ꢀfluorobenzyl)ꢀ1Hꢀpyrrolꢀ2ꢀyl]ethanone36 (1 g, 4.6 mmol) in THF
anhydrus (5 ml) was thermostated at ꢀ32 °C, LHMDS (9.2 mL) was added and the reaction was stirred
at the some temperature for 2 h. A solution of 1ꢀtritylꢀ1Hꢀ[1,2,4]triazoleꢀ3ꢀcarboxylic acid ethyl ester39
(2 g, 5.3 mmol) in THF anhydrus (18 mL) was added dropwise to the solution thermostated at ꢀ32 °C.
After the addiction, the reaction mixture was stirred for 1.5 h at room temperature. The reaction was
pored into 1 N HCl (100 mL), and extract with ethyl acetate. The organic phase was separated, washed
with water, dried over sodium sulfate, filtered and evaporated under vacuum obtaining 2.6 g of crude
product as light yellow solid. The raw material was purified by recristallization from
benzene/cyclohexane obtaining 1.84 g of pure 20. 54%; 110ꢀ112 °C; benzene/cyclohexane; IR ν 2954
(OH enole), 1626 (C=O ketone) cmꢀ1. 1H NMR (CDCl3) δ 5.62 (s, 2H, CH2), 6.3 (t, 1H, pyrrole C4ꢀH),
6.9ꢀ7.0 (m, 4H, butenoate C3ꢀH, pyrrole C3ꢀH, benzyl H), 7.1ꢀ7.2 (m, 9H, benzyl H, pyrrole C5ꢀH and
benzene H), 7.3ꢀ7.4 (m, 9H, benzene H), 8.01 (s, 1H, triazole H), 15 (br s, 1H, OH enole). Anal.
(C35H27FN4O2) C, H, N, F.
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Ethyl
4-(1-(4-fluorobenzyl)-4-phenyl-1H-pyrrol-3-yl)-2-hydroxy-4-oxobut-2-enoate
(6a).
Compound 6a was prepared from 9a by means GPꢀF. 88%; 111ꢀ112°C; benzene; IR ν 2900 (OH enole),
1
1720 (C=O ester), 1620 (C=O ketone) cmꢀ1. H NMR (CDCl3) δ 1.29 (t, 3H, CH3CH2), 4.25 (q, 2H,
CH2CH3), 5.06 (s, 2H, CH2 benzyl), 6.48 (s, 1H, butenoate C3ꢀH), 6.67 (d, 1H, J = 1.5 Hz, pyrrole C5ꢀ
H), 7.02ꢀ7.46 (m, 10H, pyrrole C2ꢀH, benzene H and benzyl H), 15 (br s, 1H, OH enole). Anal.
(C23H20FNO4) C, H, N, F.
Ethyl
4-(1-(4-fluorobenzyl)-4-phenyl-1H-pyrrol-3-yl)-2-hydroxy-3-methyl-4-oxobut-2-enoate
(6b). Compound 6b was prepared from 9b by means GPꢀF. 6b was extract with ethyl acetate. The
organic phase was separated, washed with brine, dried over sodium sulfate, filtered and evaporated
under vacuum obtaining a crude product that was purified with a column chromatography on silica gel
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(ethyl acetate/nꢀhexane 1:2); 30% as yellow oil; IR ν 1730 (C=O ester), 1650 (C=O ketone) cmꢀ1. H
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Journal of Medicinal Chemistry
NMR (DMSO d6) δ 1.14 (d, 3H, J = 7 Hz, CH3), 1.34 (t, 3H, J = 7.5 Hz, CH3CH2), 4.33 (q, 2H, J = 7.5
Hz, CH2CH3), 5.25 (s, 2H, CH2), 7.14 (d, 1H, J = 1.9 Hz, pyrrole C2ꢀH) 7.22ꢀ7.51 (m, 9H, benzene H
and benzyl H), 8.08 (s, 1H, J = 1.9 Hz, pyrrole C5ꢀH), 14 (br s, 1H, enole). Anal. (C24H22FNO4) C, H,
N, F.
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Ethyl 4-(1-(4-fluorobenzyl)-1H-pyrrol-3-yl)-2-hydroxy-4-oxobut-2-enoate (6c). Compound 6c was
prepared from 9c by means GPꢀF. 93% as yellow solid; 63ꢀ65 °C; ligroine; IR ν 3500ꢀ2500 (OH enole),
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1726 (C=O ester), 1633 (C=O ketone) cmꢀ1. H NMR (DMSO d6) δ 1.28 (t, 3H, J = 7 Hz, CH3CH2),
4.27 (q, 2H, J = 7 Hz, CH2CH3), 5.17 (s, 2H, CH2), 6.62 (t, 1H, pyrrole C4ꢀH), 6.73 (s, 1H, butenoate
C3ꢀH), 7.02 (t, 1H, pyrrole C5ꢀH), 7.18 (t, 2H, benzene H), 7.36 (dd, 2H, benzene H), 8.07 (s, 1H,
pyrrole C2ꢀH), 15 (br s, 1H, enole). Anal. (C17H18FNO4) C, H, N, F.
Ethyl 2-amino-4-(1-(4-fluorobenzyl)-4-phenyl-1H-pyrrol-3-yl)-4-oxobut-2-enoate (6d). To a stirred
mixture of 6a (1 g, 2.5 mmol), ammonium acetate (0.22 g, 2.9 mmol) in benzene (30 mL), was added
acetic acid glacial (0.2 ml, 3.9 mmol). The reaction was stirred at reflux for 20 h with a DeanꢀStark
apparatus. After this period the reaction was cooled to room temp and washed with a saturated solution
of NaHCO3 (50 mL). The organic layer was separated, dried over sodium sulfate, filtered and
evaporated under vacuum. The raw material was purified with chromatography on aluminum oxide
(chloroform as eluent) to afford 6d as yellow solid with an yield of 50%. 135ꢀ136°C. benzene; IR ν
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3500 (NH2), 1720 (C=O ester), 1620 (C=O ketone) cmꢀ1. H NMR (CDCl3) δ 1.27 (t, 3H, CH3CH2),
4.25 (q, 2H, CH3CH2), 5.08 (s, 2H, CH2), 6.13 (s, 1H, butenoate C3ꢀH), 6.67 (d, 1H, J = 1.5 Hz, pyrrole
C5ꢀH), 7.0ꢀ7.6 (m, 10H, benzene H, benzyl H and pyrrole C2ꢀH), 9 (br s, 2H, NH2). Anal.
(C23H21FN2O2) C, H, N, F.
Ethyl 6-(1-benzyl-4-(4-fluorobenzoyl)-1H-pyrrol-2-yl)-2-hydroxy-4-oxohexa-2,5-dienoate (6e).
Compound 6e was prepared from 13e by means GPꢀF. 62% as yellow solid; 154ꢀ155°C;
benzene/cyclohexane; IR ν 1730 (C=O ester), 1636 (C=O ketone) cmꢀ1. 1H NMR (Acetone d6) δ 1.34 (t,
3H, CH3CH2), 4.32 (q, 2H, CH2CH3), 5.60 (s, 2H, CH2), 6.86 (d, 1H, J = 15.6 Hz, hexanoate C5ꢀH),
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7.2ꢀ7.5 (m, 9H, J = 1.6 Hz, benzene H, pyrrole βꢀproton, benzoyl H and hexanoate C3ꢀH), 7.72 (d, 1H,
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J = 15.6 Hz, hexanoate C6ꢀH), 7.90 (d, 1H, J = 1.6 Hz, pyrrole αꢀproton), 7.9ꢀ8.0 (m, 2H, benzoyl H),
14 (bs, 1H, enole). Anal. (C26H22FNO5) C, H, N, F.
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Ethyl 6-(4-(4-fluorobenzoyl)-1-phenyl-1H-pyrrol-2-yl)-2-hydroxy-4-oxohexa-2,5-dienoate (6f).
Compound 6f was prepared from 13f by means GPꢀF. 80% as yellow solid; 129ꢀ130°C;
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benzene/cyclohexane; IR ν 3500 (OH enole), 1720 (C=O ester), 159 (C=O ketone) cmꢀ1. H NMR
(CD3OD) δ 1.36 (t, 3H, J = 7.5 Hz, CH3CH2), 4.31 (q, 2H, J = 7.5 Hz, CH2CH3), 7.26ꢀ7.48 (m, 3H,
benzoyl H and hexanoate C5ꢀH), 7.39 (d, 1H, J = 15.6 Hz, hexanoate C6ꢀH), 7.43ꢀ7.48 (m, 3H, benzene
H and hexanoate C3ꢀH), 7.58ꢀ7.62 (m, 4H, benzene H and pyrrole βꢀproton), 7.72 (s, 1H, pyrrole αꢀ
proton), 7.97ꢀ8.01 (m, 2H, benzoyl H), 14 (bs, 1H, OH enole). Anal. (C25H20FNO5) C, H, N, F.
Ethyl 6-(4-(4-fluorobenzoyl)-1-methyl-1H-pyrrol-2-yl)-2-hydroxy-4-oxohexa-2,5-dienoate (6g).
Compound 6g was prepared from 13g by means GPꢀF. 83% as yellow solid; 166ꢀ167°C;
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benzene/cyclohexane; IR ν 2900 (OH enole), 1720 (C=O ester), 1650 (C=O ketone) cmꢀ. H NMR
(CDCl3) δ 1.39 (t, 3H, J = 7.5 Hz, CH3CH2), 3.81 (s, 3H, NꢀCH3), 4.39 (q, 2H, J = 7.5 Hz, CH2CH3),
6.45 (s, 1H, hexanoate C3ꢀH), 6.50 (d, 1H, J = 15.4 Hz, hexanoate C5ꢀH), 7.12ꢀ7.20 (m, 3H, benzene H
and pyrrole βꢀproton), 7.36ꢀ39 (m, 1H, pyrrole αꢀproton), 7.63 (d, 1H, J = 15.4 Hz, hexanoate C6ꢀH),
7.83ꢀ7.91 (m, 2H, benzene H), 15 (bs, 1H, enole). Anal. (C20H18FNO5) C, H, N, F.
Ethyl 6-(1-(4-fluorobenzyl)-4-phenyl-1H-pyrrol-2-yl)-2-hydroxy-4-oxohexa-2,5-dienoate (6h).
Compound 6h was prepared from 13h by means GPꢀF. 92% as red solid; 169ꢀ170 °C; benzene; IR ν
2900 (OH enole), 1723 (C=O ester), 1602 (C=O ketone) cmꢀ1. 1H NMR (Acetone d6) δ 1.35 (t, 3H, J =
7.5 Hz, CH3CH2), 4.32 (q, 2H, J = 7.5 Hz, CH2CH3), 5.53 (s, 2H, CH2), 6.46 (s, 1H, hexanoate C3ꢀH),
6.74 (d, 1H, J = 16 Hz, hexanoate C5ꢀH), 7.13ꢀ7.24 (m, 3H, benzyl H and benzene H), 7.30ꢀ39 (m, 4H,
benzyl H, hexanoate C6ꢀH and pyrrole βꢀproton), 7.43 (s, 1H, pyrrole αꢀproton), 7.65 (d, 2H, benzene
H), 7.7ꢀ7.8 (m, 2H, benzene H), 15 (bs, 1H, enole). Anal. (C25H22FNO4) C, H, N, F.
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Journal of Medicinal Chemistry
Ester 6-(1-(4-fluorobenzyl)-1H-pyrrol-3-yl)-2-hydroxy-4-oxohexa-2,5-dienoate (6i). Compound 6i
was prepared from 17i by means GPꢀF. 41% as yellow solid; 88ꢀ90 °C; ligroine; IR ν 3400 (OH enole),
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1725 (C=O ester), 1625 (C=O ketone) cmꢀ1. H NMR (CDCl3) δ 1.44 (t, 3H, J = 7 Hz, CH3CH2), 4.41
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(q, 2H, J = 7 Hz, CH2CH3), 5.08 (s, 2H, CH2), 6.39 (s, 1H, J = 16 Hz, hexanoate C5ꢀH), 6.5ꢀ6.6 (m, 2H,
pyrrole C4ꢀH and hexanoate C3ꢀH), 6.7 (t, 1H, pyrrole C5ꢀH), 7.0 (t, 1H, pyrrole C2ꢀH), 7.08ꢀ7.20 (m,
4H, benzene H), 7.75 (s, 1H, J = 16 Hz, hexanoate C6ꢀH), 14 (br s, 1H, enole). Anal. (C19H18FNO4) C,
H, N, F.
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Ethyl 6-(1-(4-fluorobenzyl)-4-phenyl-1H-pyrrol-3-yl)-2-hydroxy-4-oxohexa-2,5-dienoate (6j).
Compound 6j was prepared from 17j by means GPꢀF. 56% as yellow solid; 121ꢀ122°C; cyclohexane;
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IR ν 2900 (OH enole), 1720 (C=O ester), 1620 (C=O ketone) cmꢀ1. H NMR (CDCl3) δ 1.43 (t, 3H,
CH3CH2), 4.40 (q, 2H, CH2CH3), 5.09 (s, 2H, CH2), 6.73 (s, 1H, hexanoate C3ꢀH) 6.87 (d, 1H, J = 16.6
Hz, hexanoate C5ꢀH), 7.01 (s, 1H, pyrrole C5ꢀH), 7.08ꢀ7.54 (m, 10H, pyrrole C2ꢀH, benzene H and
benzyl H), 7.74 (d, 1H, hexanoate C6ꢀH), 15 (bs, 1H, enole). Anal. (C25H21FNO4) C, H, N, F.
Ethyl 4-(1-(4-fluorobenzyl)-1H-pyrrol-2-yl)-2-hydroxy-4-oxobut-2-enoate (6k). Compound 6k
was prepared from 1ꢀ(1ꢀ(4ꢀfluorobenzyl)ꢀ1Hꢀpyrrolꢀ2ꢀyl)ethanone36 by means GPꢀF. 41% as yellow
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solid; 88ꢀ89°C; ligroine; IR ν 2900 (OH enole), 1720 (C=O ester), 1620 (C=O ketone) cmꢀ1. H NMR
(DMSO d6) δ 1.26 (t, 3H, J = 7.5 Hz, CH3CH2), 4.25 (q, 2H, J = 7.5 Hz, CH2CH3), 5.59 (s, 2H, CH2),
6.32 (dd, 1H, J = 2.5 Hz, J = 3.5 Hz, pyrrole C4ꢀH), 6.84 (s, 1H, butenoate C3ꢀH), 7.0ꢀ7.2 (m, 4H,
benzene H), 7.43 (d, 1H, J = 3.5 Hz, pyrrole C3ꢀH), 7.53 (s, 1H, pyrrole C5ꢀH), 14 (br s, 1H, enole).
Anal. (C17H16FNO4) C, H, N, F.
Ethyl
4-(1-(4-fluorobenzyl)-4-phenyl-1H-pyrrol-2-yl)-2-hydroxy-4-oxobut-2-enoate
(6l).
Compound 6l was prepared from 19 by means GPꢀF. 95% as yellow solid; 102ꢀ103 °C;
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benzene/cyclohexane; IR ν 3400 (OH enole), 1720 (C=O ester), 1620 (C=O ketone) cmꢀ1. H NMR
(DMSO d6) δ 1.34 (t, 3H, J = 7.5 Hz, CH3CH2), 4.33 (q, 2H, J = 7.5 Hz, CH2CH3), 5.68 (s, 2H, CH2),
7.06 (s, 1H, butenoate C3ꢀH), 7.17ꢀ7.31 (m, 5H, benzene H and benzyl H), 7.40ꢀ7.44 (m, 2H, benzene
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