Cinnamoyl compounds as simple molecules that inhibit p300 histone acetyltransferase

Article abstract of DOI:10.1021/jm060943s

Cinnamoly compounds 1a-c and 2a-d were designed, synthesized, and in vitro tested as p300 inhibitors. At different degrees, all tested compounds were proven to inactivate p300, particularly, derivative 2c was the most active inhibitor, also showing high specificity for p300 as compared to other histone acetyltransferases. Most notably, 2c showed anti-acetylase activity in mammalian cells. These compounds represent a new class of synthetic inhibitors of p300, characterized by simple chemical structures.

Full text of DOI:10.1021/jm060943s

J. Med. Chem. 2007, 50, 1973-1977
1973
Brief Articles
Cinnamoyl Compounds as Simple Molecules that Inhibit p300 Histone Acetyltransferase
,†
†
†
†
‡
§
‡
Roberta Costi,* Roberto Di Santo, Marino Artico, Gaetano Miele, Paola Valentini, Ettore Novellino, and Anna Cereseto
Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, UniVersit a` di Roma “La Sapienza”, P.le A. Moro 5,
I-00185 Roma, Italy, Laboratorio di Biologia Molecolare Scuola Normale Superiore di Pisa, Piazza dei CaValieri 7, I-56126 Pisa, Italy, and
Dipartimento di Chimica Farmaceutica e Tossicologica, UniVersit a` di Napoli “Federico II”, Via D. Montesano 49, I-80131 Napoli, Italy
ReceiVed August 4, 2006
Cinnamoly compounds 1a-c and 2a-d were designed, synthesized, and in vitro tested as p300 inhibitors.
At different degrees, all tested compounds were proven to inactivate p300, particularly, derivative 2c was
the most active inhibitor, also showing high specificity for p300 as compared to other histone acetyltrans-
ferases. Most notably, 2c showed anti-acetylase activity in mammalian cells. These compounds represent a
new class of synthetic inhibitors of p300, characterized by simple chemical structures.
Introduction
of cellular phenomena including cell cycle control, differentia-
tion, and apoptosis. Mutations in p300 enzyme have been
6
DNA is a charged polymer that is highly packaged in the
nucleus of eukaryotic cells. This extreme compaction is achieved
through association of DNA with a set of basic histone proteins
to form a structure known as chromatin. The fundamental repeat
unit of chromatin is the nucleosome, in which 146 base pairs
of DNA are wound around a histone octamer comprising two
copies of each histones H2A, H2B, H3, and H4.1,2 Nucleosomes
are in turn folded into progressively higher-order structures.
Though apparently repressive, the precise organization of
chromatin is essential for replication, repair, recombination, and
chromosomal segregation. Modification in the chromatin orga-
nization modulates the expression of underlying genes. The
dynamic changes in the chromatin structure are brought about
by post-translational modifications of the amino terminal tails
of the histones and the ATP-dependent chromatin remodeling.
Specific amino acids within the histone tails are the sites of a
variety of modifications including phosphorylation, acetylation,
proven to be associated with certain cancers and other human
disease processes. Therefore, these enzymes could be consid-
7
ered useful targets for a novel approach in chemotherapy. During
the past decade, significant progress has been made in the field
of HDAC inhibitors as antineoplastic agents, and some of these
compounds are already in clinical trial as anticancer drugs.8
Conversely, specific inhibitors for p300 were not identified until
recently.
The aim of the present work was the identification of novel
anti-p300 agents that could be useful as potential lead molecules
for anticancer as well as antiviral drug discovery. The anti-
p300 agents so far identified are (i) the natural products
9
10
11
garcinol, anacardic acid, and curcumin and (ii) the synthetic
12
derivative Lys-CoA, a lysine analog of HAT substrate acetyl-
CoA (Figure 1).
In particular, a screening of plant extracts from Curcuma
longa rhizome led to the discovery of curcumin as a potent and
3
methylation, ADP-ribosylation, and ubiquination. Among these
modifications, acetylation has been most widely studied in the
context of gene expression.
1
1
specific inhibitor of p300. Interestingly, in the course of our
studies aimed at the discovery of antiviral agents targeted to
HIV-1 integrase, we reported a group of curcumin-like deriva-
tives characterized by a 3,4-dihydroxycinnamoyl pharmaco-
The dynamic equilibrium between acetylation and deacetyl-
ation is maintained by the activity of hystone acetyltransferases
a
13
(
HATs ) and deacetylases (HDACs) that regulate the expression
phore. Based on this preliminary evidence and because the
of the genome. Specifically, HATs function enzymatically by
transferring an acetyl group from acetyl-coenzyme A to the
studies on structure-activity relationships (SARs) in the field
of anti-p300 agents are still limited and only a few Lys-CoA
1
4
ꢀ
-amino group of certain lysine side chains within a histone’s
analogs have been described, we set out to identify new
synthetic polyhydroxylated aromatic derivatives related to
curcumin, garcinol, and anacardic acid as p300 inhibitors. The
results of this study may represent a groundwork for the
development of novel anti-p300 agents as potential leading
molecules for anticancer as well as antiviral drug discovery.
4
basic N-terminal tail region. HATs are divided into five families
including the GNAT family, the MYST group, p300/CBP
HATs, the general transcription factor, and the nuclear hormone-
5
related HATs. p300 is a ubiquitously expressed global tran-
scriptional coactivator that has critical roles in a wide variety
An examination of the chemical structures of these natural
products led us to identify some structural features that
characterize these compounds: (i) a R,γ-diketo group; (ii) a
cinnamoyl moiety; (iii) a catechol ring; and (iv) a salicylic acid
portion. Therefore, we decided to test the activity against p300
of cinnamoyl compounds, such as 1a (related to curcumin), 2a,
and 2d (cyclohexanone derivatives), previously reported by us
in the course of our studies aimed at the discovery of antiviral
*
To whom correspondence should be addressed. Phone: +39-6-
4
9913996. Fax: +39-6-49913150. E-mail: roberta.costi@uniroma1.it.
†
Dipartimento di Studi Farmaceutici.
Scuola Normale Superiore di Pisa.
Dipartimento di Chimica Farmaceutica e Tossicologica.
Abbreviations: HAT, histone acetyl transferase; HDAC, histone
‡
§
a
deacetylase; GNAT, Gcn5-related N-acetyltransferase; P300/CBP, p300/
CREB binding protein; SAR, structure-activity relationship; H2B-EYFP,
H2B-enhanced yellow fluorescent protein; FCS-DMEM, fetal calf serum-
Dulbecco’s modified eagle’s medium.
1
3
agents targeted to HIV-1 integrase. In fact, 1a, 2a, and 2d
1
0.1021/jm060943s CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/10/2007

1
974 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Brief Articles
Scheme 2a
a
Reagents and conditions: (a) cyclohexanone, montmorillonite K-10, 5
min, 100 W, 100 °C. Yields: 2a, 75%; 2b, 50%; 2c, 89%; 2d, 75%.
and reacting less reactive methyls with the appropriate aldehyde
using 1,2,3,4-tetrahydroquinoline as a catalyst. Notably, the
synthesis of derivative 1b has already been reported in very
1
6
low yields (<9%) by Subaraju in three steps involving
protection/deprotection procedures of carboxylic groups. How-
ever, we obtained 1b in higher yields (50%) by the application
of the Babu method to 5-formylsalicylic acid in the three steps,
one-pot synthesis (Scheme 1).
Figure 1. Structures of p300 inhibitors reported in literature.
Bis-arylidene derivatives 2a-d were synthesized by conden-
sation of cyclohexanone with the appropriate benzaldehyde
(Scheme 2). A new procedure that did not require the prelimi-
nary protection of the OH groups was developed. In particular,
a dispersion of cyclohexanone and the substituted benzaldehyde
in montmorillonite K-10, was submitted to microwave-assisted
heating (100 °C, 100 W) for 5 min. Interestingly, montmoril-
lonite K-10 was used in this reaction as both an environmentally
benign solid support and a heterogeneous acid catalyst. This
procedure allowed (i) increased yields of these condensations
1
3
if compared to those previously reported, (ii) a reduction in
the synthetic pathway from three steps to one step, and (iii) a
minimization of the reaction time.
Figure 2. Structures of the cinnamoyl derivatives 1a-c and 2a-d
Evaluation of Biological Activities. The p300 inhibitory
activities of the newly synthesized cinnamolyl compounds 1a-c
reported in the present study and tested as p300 inhibitors.
Scheme 1a
17
and 2a-d were tested in an in vitro acetylation assay using
recombinant histones (H1, H2A, H2B, H3, and H4) and the
HAT domain of p300 (Figure 3A).
The inhibitory activity of each compound was tested, with
concentration ranging from 25 µM to 400 µM (data not shown),
or starting with 0.19 µM for the derivative 2c, to determine the
IC50 value (Figure 3C and Table 1). Figure 3B shows the histone
acetylation levels following incubation with p300 in the presence
of scalar amounts of the derivative 2c.
a
Reagents and conditions: (a) B(OH)3 DMF, 100 °C, 5 min; (b)
arylaldehydes, 1,2,3,4-tetrahydroquinoline, AcOH, DMF, 100 °C, 4 h; (c)
AcOH, room temp, 1 h. Yields for the three-steps, one-pot synthesis: 1a,14
1
0%; 1b, 50%; and 1c, 65%.
Compounds 1a-c and 2a-d could be divided into the
following: (i) curcumin derivatives with different substituents
on the aromatic moieties (1a-c) and (ii) 2,6-bis-arylidene
cyclohexanone derivatives (2a-d). In general, compounds 1a-c
and 2a-d showed good activities against p300, with IC50 values
ranging from 5 to 233 µM (Table 1). Derivative 2c was the
most potent compound of these series (IC50 ) 5 µM), being
six times more potent than Lys-CoA used as a reference drug.
Surprisingly, in our assays, curcumin was inactive at concentra-
tions up to 400 µM. Due to this result, we tested a new stock
of commercial curcumin (Fluka) after a further chromatography
share some of the above chemical features such as (i) the R,γ-
diketo group (1a), (ii) the cinnamoyl moiety (1a, 2a, 2d), and
(
iii) the catechol ring (1a, 2a). Moreover, as a preliminary SAR
study, we designed and synthesized salicylic derivatives 1b and
b and compounds 1c and 2c that are characterized by the
2
presence in the ortho position to OH groups by lipophilic and
withdrawing bromine atoms. Compounds 1a-c and 2a-d
(
Figure 2) were tested in in vitro assays for their inhibitory
activities against p300. To different extent, all synthesized
molecules inhibited p300 enzymatic activity. The most active
compound, 2c, was selective for p300 as compared to other
HATs and, most notably, was cell permeable, as demonstrated
by decreased histones acetylation.
1
purification and H NMR identification and tested with both
HAT domain as well as the full length p300 enzyme.18 In spite
of this, the inactivity of curcumin was confirmed.
Interestingly, the curcumin derivatives 1a-c were potent p300
inhibitors showing IC50 values from 21 to 46 µM, comparable
to that found for Lys-CoA used as a reference drug in the same
experiment (IC50 ) 30 µM). The most active derivative among
this group of molecules was 1b, which was characterized by
salicylic groups (1.4 times more potent than Lys-CoA). Re-
placement of the carboxylic function with a bromine or hydroxyl
groups led to 1c and 1a, which were 1.5 and 2 times less potent
than parent derivative 1b, respectively. In general, the activities
Results and Discussion
Chemistry. Synthesis of derivatives 1a-c and 2a-d is
outlined in Schemes 1 and 2. The curcumin analogs 1a-c were
synthesized according to the Babu and Rajasekharan method
(
Scheme 1).15
The fundamental step in this reaction is the protection of the
active methylene group by reacting with acetylacetone in the
presence of boric acid to get acetylacetone-boric acid complex

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1975
Figure 3. Inhibitory effects of compounds 1a-c and 2a-d on p300 activity. (A) Upper panel: autoradiography of a gel showing acetylated
1
4
histones following incubation with p300 and C acetyl-CoA in the presence of 400 µM of each indicated derivative, Lys-CoA (last lane) or DMSO
first lane). Lower panel: Coomassie blue staining of the same gel showing the total amounts of histones. (B) Upper panel: autoradiography of a
gel showing acetylated histones following incubation with p300 in the presence of the indicated concentration of 2c or DMSO alone (first lane).
C) Dose response curves obtained by densitometric analysis of the levels of histone acetylation mediated by p300 in the presence of 1a-c and
a-d. The graph summarizes the results obtained from three independent experiments.
(
(
2
Table 1. Inhibitory Activity of Compounds 1a-c and 2a-d against
p300 Enzyme
cmpd
R
IC50a
1
1
1
a
b
c
OH
COOH
Br
OCH3
OH
COOH
Br
46 ( 3.9
21 ( 8.7
33 ( 5.2
curcumin
>400
2
2
2
2
a
b
c
233 ( 120
168 ( 12
5 ( 1.3
111 ( 45
30 ( 1.6
Figure 4. Inhibitory activities of derivatives 2c (5 µM) and 1b (21
µM) on different HATs were tested in in vitro assays using equal molar
amounts of p300, PCAF, and GCN5. HAT activity for each enzyme is
expressed as percent variation as compared to that of the DMSO-treated
sample. The graph summarizes the mean densitometric values from
three independent experiments (mean ( standard error).
d
OCH3
Lys-CoA
a
Inhibitory concentration of 50% (µM) determined from dose-response
curves. Data represent the mean values of at least three separate experiments.
opposite results were found when the same groups were
introduced in the 3 position of benzene rings in the 2,6-bis-
arylidene cyclohexanone series (2a, IC50 ) 233 µM; 2b, IC50
in this series decreased if the electron-withdrawing groups
(COOH, Br) were replaced by electron-donor groups (OH,
)
168 µM).
OCH3). The following order, depending on substituents in the
Derivatives 2c and 1b, which showed the highest inhibitory
3
>
-positions of the aromatic rings, was observed: COOH > Br
activity, were additionally tested on PCAF and GCN5, both
belonging to a different class of HAT factors. The assays were
performed using concentrations of 2c and 1b corresponding to
the IC50 values formerly determined against p300 (5 µM and
OH > OCH3.
The cyclohexanone derivatives 2a-d were active against
p300 as well. The IC50 values obtained in the enzyme assays
ranged from 5 to 233 µM. The activities of compounds 2a-d
decreased based on the substituents in the 3-positions of the
aromatic rings in the following order: Br > OCH3 > COOH
2
1 µM for 2c and 1b, respectively; Table 1). As expected, the
activity of p300 was reduced to 50% with both compounds,
while the same concentration of derivative 2c showed no effect
on PCAF (100%) and only partial inactivation of GCN5 (70%),
indicating a selective inhibition of p300 activity. Conversely,
>
OH. In conclusion, the replacement of hydrophilic groups
COOH, OH) with the lipophilic ones (Br, OCH3) in the 2a-d
(
series led to increased anti-p300 activities. In particular, the
highest potency was obtained with the introduction of the
lipophilic and electron-withdrawing bromine atom on the
cinnamoyl portion.
1
b is partially active on PCAF (68%) and shows on GCN5
(38%) the same efficacy as for p300, indicating that this
compound is active on HATs other than p300 (Figure 4).
Several previously described HAT inhibitors, such as Lys-
CoA, are not cell permeable and cannot thus be used for in
vivo studies. Therefore, we have tested for its anti-acetylase
activity in cell culture system derivatives 1b and 2c, which
showed the most potent inhibitory effect against p300. HeLa
cells stably expressing fluorescent H2B histones (HeLasH2B-
EYFP) were treated with various concentrations of 2c and 1b
and subsequently immunostained with antibodies against acety-
lated H3 histones. The fluorescent H2B histones were used as
internal control to monitor protein expression levels. Derivative
1b at concentrations up to 200 µM did not alter either the H3
acetylation levels or the H2B protein expression. This experi-
ment led us to hypothesize that 1b is not cellular permeable
(data not shown). Conversely, we found that at 20 µM and 40
The preliminary SARs in the series of cyclohexanone
derivatives (compounds 2a-d) were different if compared with
those found in the curcumin series (compounds 1a-c and
curcumin). A direct comparison among the two series led to
the following conclusions: (i) compounds 2a-d were generally
less potent than 1a-c derivatives, showing IC50 values from
1
8
1
11 to 233 µM, with the exception of 2c, which was the most
potent derivative described in this work (IC50 ) 5 µM); (ii)
introduction of bromine atoms in the 3 position of the benzene
rings gave derivatives 1c and 2c, which were both endowed
with good activities; and (iii) introduction of OH or COOH
groups in the same positions within the curcumin series gave
compounds 1a and 1b, which showed good anti-p300 potency;

1
976 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Brief Articles
of the nuclear lamina in HeLa cells not expressing H2B-EYFP
(data not shown).
Conclusions
In conclusion, herein we reported a new class of small
synthetic molecules that inhibited the p300 activities in in vitro
assays. In particular, we described the discovery of cinnamoyl
compounds 1a-c and 2a-d as inhibitors of the p300 enzyme.
Among them, derivative 2c was proven the most potent anti-
p300 agent, which was six times more active than Lys-CoA
used as a reference drug and with a high selectivity for p300,
as demonstrated by comparative assays performed with different
HATs belonging to another family of enzymes. Most notably,
derivative 2c was active in mammalian cells, as demonstrated
by the downregulation of histone H3 acetylation. For all the
above-mentioned reasons, derivative 2c might be considered a
lead compound for further studies in this field.
Extensive SARs, as well as molecular modeling studies, are
ongoing to increase the knowledge within these series of p300
inhibitors. Due to the vital role of p300 in the reversible
processes of acetylation of histones and other cellular proteins,
the development of these inhibitors might result in novel
approaches to antitumor and antiviral chemotherapies.
Experimental Section
Chemistry. General. Melting points were determined with a
B u¨ chi 530 capillary apparatus and are uncorrected. Infrared (IR)
1
spectra were recorded on a Spectrum-one spectrophotometer. H
NMR spectra were recorded on a Bruker AC 400 spectrometer,
Figure 5. (A) HeLasH2B-EYFP cells treated with derivative 2c (20
µM or 40 µM) or DMSO were immunostained with antibodies anti-
acetyl H3 and analyzed with appropriate wavelengths to visualize
acetylated H3 or H2B-EYFP total protein levels. (B) The percent
inhibition of histone H3 acetylation was obtained by measuring the
mean fluorescence intensity with anti-acetyl H3 antibodies relative to
the mean fluorescence intensity values of H2B-EYFP from the same
cells. The graph summarizes data obtained from three independent
experiments. Mean and standard error were derived analyzing 150 cells
in each experiment.
4
using tetramethylsilane (Me Si) as an internal standard. All
1
compounds were routinely checked by TLC and H NMR. TLC
was performed by using aluminum-baked silica gel plates (Fluka
F254) and aluminum-baked aluminum oxide plates (Fluka F254).
Concentration of solutions after reactions and extractions involved
the use of a rotatory evaporator operating at a reduced pressure of
approximately 20 Torr. Organic solutions were dried over anhydrous
sodium sulfate. The microwave reactions were performed in a
Discover CEM, which produced controlled irradiation with a power
of 0-300 W.
µM of 2c the levels of H3 acetylation decreased, while the H2B
expression remained unaltered, indicating specificity of anti-
acetylase treatment (Figure 5A).
Concentrations lower than 20 µM did not have any effect on
the acetylation levels, while over 40 µM cell toxicity was
observed as indicated by decreased H2B-EYFP fluorescence
Syntheses. Specific examples presented below illustrate general
synthetic procedures.
1,7-Bis(3-bromo-4-hydroxyphenyl)-1,6-heptadiene-3,5-dione
(
1c). A solution of 3-bromo-4-hydroxybenzaldehyde (1.0 g, 5.0
mmol) and acetylacetone (250 mg, 2.5 mmol) in N,N′-dimethyl-
formamide (0.5 mL) was treated with boric acid (490 mg, 8.0
mmol), and the mixture was heated at 100 °C for 5 min. After this
time, a solution of 1,2,3,4-tetrahydroquinoline (0.5 mL, 530 mg,
(
data not shown). Interestingly, the effect of 2c is not homo-
geneous in cell culture. Indeed, a high percentage of cells
estimated around 24%) showed no detectable H3 acetylation
even though the H2B expression remained unaltered (Figure
A, cell in the upper-center in the middle panels as a representa-
4.0 mmol) and acetic acid (0.15 mL) in N,N′-dimethylformamide
(
(0.5 mL) was added. The resulting mixture was heated at 100 °C
for 1.5 h, then cooled, diluted with 20% acetic acid (25 mL), and
stirred at room temperature for 1 h. The precipitate that formed
was extracted with ethyl acetate (3 × 50 mL), and the organic
extracts were collected, washed with brine (3 × 100 mL), and dried.
Evaporation of the solvent gave crude product, which was chro-
matographed on a silica gel column (chloroform/methanol, 20:1,
as eluent) to obtain pure 1c (760 mg, 65% yield); mp 175-176 °C
5
tive image). Finally, we observed that 2c determined an overall
increase of H2B-EYFP fluorescence intensity. This observation
is indicative of decreased histone acetylation that results in
chromatin condensation. This effect was visualized by increased
fluorescence of the exogenously expressed histones as previously
reported in similar cellular systems.1 To quantify the different
levels of histone H3 acetylation, the average fluorescence
intensity obtained from the immunostaining with antibodies
against acetylated H3 was measured and normalized with values
obtained in the same cells with fluorescent histones H2B-EYFP.
Results summarized in Figure 5B indicate that the acetylation
levels were 30% reduced in cells treated with 20 µM of 2c,
and a reduction higher than 50% was observed using 40 µM as
compared to that of DMSO control cells.
9,20
4
(isopropanol/isopropyl ether). Anal. (C19H14BrO ) C, H, Br. This
procedure was used for the synthesis of compounds 1b starting
from 5-formylsalicylic acid. 1b: 50%; mp >270 °C (dioxane). Anal.
(C H O ) C, H.
21 16 8
2
,6-Bis(3-bromo-4-hydroxybenzylidene)cyclohexanone (2c).
3-Bromo-4-hydroxybenzaldehyde (300 mg, 1.5 mmol) was dis-
solved in MeOH and treated with montmorillonite K-10 (600 mg).
Evaporation of the solvent gave a dispersion that was placed in a
5
mL glass tube and treated with cyclohexanone (75 mg, 0.75
mmol). The vessel was sealed with a septum and placed into the
microwave cavity. Microwave irradiation of 100 W was used, the
temperature being ramped from room temperature to 100 °C. Once
Similar results were obtained in parallel where the H3
acetylation level was normalized with the level of expression

Brief Articles
00 °C was reached, the reaction mixture was held at this
temperature for 5 min. The reaction vessel was opened, and the
mixture was diluted with methanol and filtered. Evaporation of the
solvent gave crude product, which was chromatographed on a silica
gel column (chloroform/methanol, 20:1, as eluent) to obtain pure
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1977
1
(10) Balasubramanyam, K.; Swaminathan, V.; Ranganathan, A.; Kundu,
T. K. Small molecule modulators of histone acetyltransferase p300.
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(
2
c (310 mg, 89% yield); mp 201-202 °C (isopropanol/water). Anal.
20 2 3
(C H16Br O ) C, H, Br. This procedure was used for the synthesis
of compounds 2a, 2b, and 2d starting from 3,4-dihydroxybenzal-
dehyde, 5-formylsalicylic acid, and 4-hydroxy-3-methoxybenzal-
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2
79, 51163-51171.
(
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H. 2d: 75%, 179-181 °C, and acetic acid.
18 7
H O ) C,
Biological Assays. Acetylation Assay To Test the Efficacy of
Curcumin Derivatives. To test the efficacy of derivatives 1a-c
and 2a-d, the catalytic activity of p300 has been measured by an
in vitro assay as previously reported.17
Acetylation Assays To Test the Efficacy of the Curcumin
Derivatives in Mammalian Cells. HeLa cells, stably transfected
with histones H2B fused to EYFP,20 were cultured in 10% FCS
DMEM. Histone H3 acetylation was analyzed as described in
Supporting Information.
9
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Acknowledgment. This project was supported by Ministero
della Sanit a` , Istituto Superiore di Sanit a` , “Programma Nazionale
di Ricerca sull’AIDS” (Grant No. 30F.19 and 40F.25), Italian
MIUR (PRIN 2006), and Fondazione Cassa di Risparmio di
Pisa (Grant No. 120/06).
(
Supporting Information Available: Spectroscopic data for
derivatives 1b,c and 2a-d, elemental analyses for derivatives 1b,c
and 2b,c, and biological assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
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