Evaluation of new dihydrophthalazine-appended 2,4-diaminopyrimidines against Bacillus anthracis: Improved syntheses using a new pincer complex

Article abstract of DOI:10.3390/molecules20047222

The synthesis and evaluation of ten new dihydrophthalazine-appended 2,4-diaminopyrimidines as potential drugs to treat Bacillus anthracis is reported. An improved synthesis utilizing a new pincer catalyst, dichlorobis[1-(dicyclohexylphosphanyl)-piperidine]palladium(II), allows the final Heck coupling to be performed at 90°C using triethylamine as the base. These milder conditions have been used to achieve improved yields for new and previously reported substrates with functional groups that degrade or react at the normal 140°C reaction temperature. An analytical protocol for separating the S and R enantiomers of two of the most active compounds is also disclosed. Finally, the X-ray structure for the most active enantiomer of the lead compound, (S)-RAB1, is given.

Full text of DOI:10.3390/molecules20047222

Molecules 2015, 20, 7222-7244; doi:10.3390/molecules20047222
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Evaluation of New Dihydrophthalazine-Appended
,4-Diaminopyrimidines against Bacillus anthracis: Improved
Syntheses Using a New Pincer Complex
2
1
1
1
2
Nagendra Prasad Muddala , Baskar Nammalwar , Subhashini Selvaraju , Christina R. Bourne ,
3
1,
1
3
Mary Henry , Richard A. Bunce *, K. Darrell Berlin , Esther W. Barrow and
William W. Barrow 3
1
Department of Chemistry, Oklahoma State University, 107 Physical Sciences, Stillwater,
OK 74078, USA; E-Mails: muddala@okstate.edu (N.P.M.); baskar@okstate.edu (B.N.);
subhas@okstate.edu (S.S.); kdb@okstate.edu (K.D.B.)
2
Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway,
Norman, OK 73019, USA; E-Mail: cbourne@ou.edu
3
Department of Veterinary Pathobiology, Oklahoma State University, 250 McElroy Hall, Stillwater,
OK 74078, USA; E-Mails: henrym@okstate.edu (M.H.); esther.barrow@okstate.edu (E.W.B.);
bill.barrow@okstate.edu (W.W.B.)
*
Author to whom correspondence should be addressed; E-Mail: rab@okstate.edu;
Tel.: +1-405-744-5952; Fax: +1-405-744-6007.
Academic Editor: Derek J. McPhee
Received: 20 March 2015 / Accepted: 15 April 2015 / Published: 21 April 2015
Abstract: The synthesis and evaluation of ten new dihydrophthalazine-appended
2
,4-diaminopyrimidines as potential drugs to treat Bacillus anthracis is reported. An
improved synthesis utilizing a new pincer catalyst, dichlorobis[1-(dicyclohexylphosphanyl)-
piperidine]palladium(II), allows the final Heck coupling to be performed at 90 °C using
triethylamine as the base. These milder conditions have been used to achieve improved
yields for new and previously reported substrates with functional groups that degrade or
react at the normal 140 °C reaction temperature. An analytical protocol for separating the
S and R enantiomers of two of the most active compounds is also disclosed. Finally, the
X-ray structure for the most active enantiomer of the lead compound, (S)-RAB1, is given.

Molecules 2015, 20
7223
Keywords: 2,4-diaminopyrimidine antifolates; dihydrofolate reductase (DHFR);
Bacillus anthracis; pincer complexes; Heck coupling
1
. Introduction
Bacillus anthracis, a Gram-positive, non-motile bacterium distributed as spores, is the etiologic
agent responsible for deadly anthrax in both humans and animals [1,2]. The Center for Disease Control
and Prevention (CDC) lists B. anthracis spores as a category A agent which can spread to individuals
via gastrointestinal, cutaneous, injection, or inhalation routes and is considered a serious bioweapon
threat [3–5]. Due to the worldwide emergence of antibiotic resistance, these pathogens can be
engineered against current available drugs for mass destruction purposes [6–9]. Thus, there is an
imminent need to develop new antibiotics to counteract this organism in case of a bioterror event [10].
In the last 50 years, inhibition of dihydrofolate reductase enzyme (DHFR) in the folate pathway has
been a focus for the development of various antibacterial drugs [11,12]. With the advent of the drug
trimethoprim (TMP), which demonstrated higher inhibition (5 log10) of bacterial DHFR compared to
mammalian DHFR, many researchers targeted these enzymes to develop medicinal agents with better
pharmacokinetics [11].
Some bacteria have proven resistant to TMP due to encoded differences in the chromosomal DHFR
sequence, which makes it an ineffective drug. B. anthracis is one such bacterium, which presumably
has innate resistance to TMP due to the poor binding affinity of the drug with DHFR. Due to the
increased concern regarding bioterrorism and engineered drug-resistant B. anthracis strains, a new
method to treat this bacterium has been sought. Over the last five years, our research group
has developed substituted dihydrophthalazine-appended 2,4-diaminopyrimidines (DAP inhibitors) as
modified TMP derivatives for effective inhibition of this organism [13–15]. These structures have
shown inhibition of B. anthracis at 0.5–2 μg/mL concentrations. In pursuit of identifying a drug with
enhanced activity and bioavailability, we previously developed first and second generation DAP
1
inhibitors. In the current project, a family of compounds with changes at the R position of the ring
system has been prepared and studied (see Figure 1). It was observed earlier that any alteration of R1,
at the C-1 stereocenter of the dihydrophthalazine unit, tended to modulate interaction of the protein
surface with the surrounding solvent, and thus, the inhibitory activity [13,15]. Alternatively, changes at
2
3
4
the R , R and R positions on the ring disrupted the compound orientation in the binding pocket,
1
which resulted in attenuated potency [14,16]. Numerous compounds with changes at the R position
were synthesized and evaluated, but derivatives with sensitive functional groups at this position proved
challenging to prepare. Such groups often underwent decomposition at high temperatures leading to
an increased impurity profile and decreased yields of the targeted products. Hence, isolation and
purification remained tedious with respect to these substrates. To overcome this difficulty, a highly
active, reliable, versatile, functional group tolerant catalyst, which could perform the final Heck coupling
reaction at lower temperature was sought.

Molecules 2015, 20
7224
enhances biological activity
with small alkene, alkane
or aromatic groups
stability of functional group
controls Heck coupling
sensitive functional
group
NH2
O
R1
N 1
NH2
N
R1
O
I
N
N
N 1
N
N
R4
OR2
H N
2
N
H N
2
OCH3
OR3
OCH3
substitution decreases
biological activity
larger substitutents
decrease biological
activity
Figure 1. Modification of DAP inhibitors.
Over the past decade, a number of palladium pincer complexes with high temperature and moisture
tolerance have been used in various Heck couplings [17–19]. These pincer complexes have played
an important role in cross-coupling aryl/alkyl halides with alkenes to create new C-C bonds [20]. The
recent development of pincer complexes has permitted milder reaction conditions, better substrate
scope, and lower catalyst loading to afford cleaner products in higher yields. Frech and coworkers have
synthesized dichlorobis[1-(dicyclohexylphosphanyl)piperidine]palladium(II) (Pd pincer-II, see Figure 2),
which performs arylations of olefins using a wide variety of substrates, including activated, unactivated
and deactivated systems, as well as heterocyclic aryl bromides and various substituted olefins [21,22].
This aminophosphine palladium complex offers a superior conversion rate by forming palladium
nanoparticles and functions as a homogeneous catalyst. Hence, we chose the Pd pincer-II over the
Pd pincer-I [18,23,24] catalyst to carry out Heck couplings at lower temperatures compared with
conventional palladium catalysts, which generally required 120–140 °C.
Pd(OAc)2
(Ph P) PdCl
3 2 2
palladium acetate
bis(triphenylphosphine)
palladium(II) dichloride
N
P
Cl
Pd
Cl
N
P
HN
NH
P
N
P
Pd
Cl
N
2
2
{2,6-bis[(di-1-piperidinylphosphino)amino]- dichloro{bis[1-(dicyclohexylphosphanyl)-
phenyl}palladium(II) chloride
piperidine]}palladium(II)
Pd pincer-I
Pd pincer-II
Figure 2. Pd catalysts for Heck coupling.

Molecules 2015, 20
7225
2
. Results and Discussion
2
.1. Chemistry
1
To address the issue of functional group tolerance at R , the newly developed pincer catalyst
Pd pincer-II) [21,22] was evaluated along with the previously reported commercial Pd pincer-I
catalyst [18,23,24], Pd(OAc) , and (PPh PdCl . Experiments involving the coupling of 1 with 2a to
(
2
3
)
2
2
give 3a using various catalysts, bases, and solvents at different temperatures are summarized in Table 1.
Table 1. Reaction optimization.
NH2
N
O
NH2
N
O
I
N
N
N
Pd catalyst
N
N
N
base, solvent
H N
2
OCH3
H N
2
OCH3
OCH3
OCH3
1
2a
3a
b
Catalyst (mol %)
Pd(OAc) (5.0)
Solvent Base a
Temp (°C)
Time (h)
24
24
24
18
12
18
8
Yield (%)
2
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMSO
DMSO
NEP
NEP
TEA
NEP
NEP
TEA
TEA
90
90
35
22
20
81
85
67
(
(
PPh
PPh
3
)
2
PdCl
PdCl
2
(5.0)
(5.0)
3
)
2
2
90
Pd pincer-I (0.06)
Pd pincer-II (0.054)
Pd pincer-I (0.06)
Pd pincer-II (0.054)
Pd pincer-I (0.06)
Pd pincer-II (0.054)
Pd pincer-I (0.06)
Pd pincer-II (0.054)
Pd pincer-I (0.06)
Pd pincer-II (0.054)
Pd pincer-I (0.06)
Pd pincer-II (0.054)
Pd pincer-I (0.06)
Pd pincer-II (0.054)
Pd pincer-I (0.06)
Pd pincer-II (0.054)
Pd pincer-I (0.06)
Pd pincer-II (0.054)
140
140
90
c
90
88
K
2
CO
3
3
90
24
24
24
24
18
18
24
24
24
24
24
24
24
24
NR
NR
6
K
2
CO
90
NaOAc
NaOAc
TEA
90
90
NR
72
120
120
110
110
90
TEA
74
dioxane TEA
dioxane TEA
NR
NR
NR
NR
12
MeCN
MeCN
NMP
NMP
THF
NEP
NEP
NEP
NEP
NEP
NEP
90
130
130
140
140
20
NR
NR
THF
a
c
NMP = N-methylpyrrolidinone; NEP = N-ethylpiperidine; TEA = triethylamine; b NR = no reaction;
Optimized conditions.
The results revealed that 0.054 mol % of the Pd pincer-II complex gave high yields of the coupled
product using TEA as the base in DMF at 90 °C. By comparison, other promoters required greater
loading (up to 5 mol %), a stronger base (NEP) and higher temperature (140 °C) [18,25]. Although the
Pd pincer-I complex catalyzed the coupling at a loading of 0.06 mol % using TEA at 90 °C, the reaction
did not undergo complete conversion, even after extended reaction times. For all catalysts, the choice

Molecules 2015, 20
7226
CO
of base and solvent proved crucial. For example, the reaction did not proceed with bases such K
2
3
or NaOAc, or with solvents such as dioxane, THF, MeCN, or NMP. Moderate conversions were obtained
in DMF and DMSO using NEP, but cross coupling of iodide 1 with the substituted alkene at the R1
position competed with the desired coupling process.
The synthesis of our DAP inhibitors 3a–r is outlined in Scheme 1. The overall structures are
comprised of three rings: namely, a 2,4-diaminopyrimidine, linked by a one-carbon bridge to a central
dimethoxyaromatic ring, which in turn, is tethered via an acrylamide chain to N-2 of a C-1-substituted
dihydrophthalazine. Precursor 1, consisting of a 2,4-diaminopyrimidine and a dimethoxybenzyl ring,
was synthesized by a two-step sequence using a previously reported strategy [15]. The substituted
dihydrophthalazines 2 were obtained by addition of organolithium/magnesium reagents to phthalazine (4)
in THF to generate racemic adducts 5a–p, which were N-acylated with acryloyl chloride and TEA to
furnish 2a–p [26]. Precursors 2q and 2r were generated via a two-step process involving Bi(OTf)
promoted hydrolysis of tert-butyl ester 2p in benzene to give acid 6, followed by re-esterification with
ethyl and methyl alcohol, respectively, using catalytic Bi(OTf) [13]. Final Heck coupling of 1 and 2a–r
using Pd pincer-II then delivered the desired targets 3a–r (Table 2).
3
3
NH2
O
R
1
NH2
O
R1
I
N
N
N
Pd catalyst
N
N
N
base, solvent
H N
2
N
OCH3
H N
2
N
OCH3
OCH3
OCH3
1
R = a-r defined
1
2 a-r
in Table 2
3 a-r
R1
R1
Cl
O
1
N
N
R -M
HN
O
N
N
solvent
N
TEA, DCM
4
M = MgBr or Li
5 a-r
2 a-r
O
O
OH
5
-mol% (Bi(OTf)3
C H
5-mol% (Bi(OTf)3
N
2
p
N
EtOH
2q
2r
6
6
MeOH
6
Scheme 1. Synthesis of DAP inhibitors.
After the optimized conditions were identified, the method was employed to install a broad range of
1
groups at R to validate the reaction scope. A selection of alkenyl- (3a–c), alkyl- (3d–e), cycloalkyl-
(
3f–h), heteroaryl- (3j–o) and ester- (3p–r) substituted derivatives was prepared (see Table 2). For all
cases, the reactants underwent coupling at lower temperature (90 °C) with milder base (TEA) and in
shorter times (8–12 h) compared to the standard conditions (140 °C, NEP, 16 h). Compounds 3a, d, f,
j, k, p, q, r were previously obtained in lower yields under the standard protocol, which made the
purification more tedious.

Molecules 2015, 20
7227
Table 2. Product yields.
Cpd
R1
CH=C(CH
CH -CH=CH
CH=CH
CH CH
n-C
Time (h) (h)
Yield (%)
88
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
3
)
2
8
8
2
2
81
2
8
85
a
2
2
CH
3
8
86
5
H
11
12
10
10
10
12
8
90
c-C
c-C
c-C
3
4
5
H
5
H
7
H
9
84
g
h
i
87
92
C(CH
3
)
3
83
j
furan-2-yl
90
k
l
thiophen-2-yl
8
81
1-methylindol-2-yl
benzofuran-2-yl
benzothiophen-2-yl
benzothiazol-2-yl
10
8
75
m
n
o
80
10
10
8
78
72
p
q
r
CH
CH
CH
2
2
2
CO
CO
CO
2
2
2
C(CH
3
)
3
83
CH
2
CH
3
8
80
CH
3
8
82
a
This compound has been previously designated as RAB1 [6].
2
.2. Enantiomer Resolutions for (±)-3a and (±)-3d and X-ray Studies of (S)-3d
1
The addition of R at C-1 of the dihydrophthalazine ring creates a stereocenter, and thus, generates
a racemic mixture of the target molecules. Co-crystallization of racemic 3a and 3d with the B. anthracis
DHFR exhibited a preference for binding the (S)-enantiomers, which was revealed by X-ray studies;
the (R)-isomers were not observed [6,27]. Previously, Chiral Technologies (West Chester, PA, USA)
was engaged to resolve the enantiomers of 3a and 3d. The separation of 50 mg of material was
®
accomplished under supercritical conditions with a 4.6 mm × 100 mm Chiralpak ADH column (5 µm
particle size) using 40% EtOH/0.2% Et
2
NH/60% CO as the mobile phase at 35 °C [28]. For the
2
current study, a new in-house method was developed to resolve these enantiomers under non-supercritical
®
conditions using a 10 mm × 250 mm Chiralpak IA preparative HPLC column (5 µm). This column has
®
greater stability than the Chiralpak ADH column, tolerates more solvents, and permits separation of a
wider range of compounds. In the current work, the Chiralpak® IA column, eluted with
MeCN/MeOH/Et NH (50:50:0.1) at 23 °C, permitted the resolution of 20 mg of the target molecule
2
per run with 99% purity. The separation yielded the (S)- and (R)-isomers, in sufficient quantities for
evaluation against bacterial targets and also for generating crystals suitable for X-ray diffraction studies.
The X-ray structure of (S)-3d, previously designated as (S)-RAB1, is shown in Figure 3. Figure 3A
shows a single molecule of the compound and Figure 3B shows the arrangement of molecules within
the unit cell. The single molecule shows an angular structure, which minimizes steric interactions between
functional groups on the three rings. In the unit cell, the dihydrophthalazine and dimethoxyaromatic
subunits from two molecules align head-to-tail to maximize two π-stacking interactions between the
electron-poor acrylamide in one molecule and the electron-rich dimethoxyaromatic ring of the other.

Molecules 2015, 20
7228
The 2,4-diaminopyrimidine rings position themselves above and below the cage created by this
stacking interaction. Additionally, the two proximal structures align to take advantage of intermolecular
H-bonding interactions between the acrylamide carbonyl of one molecule and the C-4 amino group
of the 2,4-diaminopyrimidine of the other. Finally, as elemental analysis has suggested, there are
2
.5 water molecules in the unit cell and two of these are observed to be associated with the highly
polar diaminopyrimidine rings.
(
A)
(
B)
Figure 3. X-ray structure of (S)-3d [(S)-RAB1]: (A) Single molecule; (B) Unit cell
.3. Biology
The biological activities for the new subset of DAP-based inhibitors are summarized in Table 3.
2
These activities were evaluated employing whole cell bacterial cultures using the standard Clinical
Laboratory Standards Institute guidelines. The values are reported in minimum inhibitory concentrations
(
MIC) in μg/mL [29]. The enzymatic activities were evaluated using purified B. anthracis DHFR
protein in a standardized assay yielding the concentration required to inhibit the half maximal enzyme
activity rates. This IC50 value (data not given) was used in combination with the substrate affinity of
the DHFR enzyme, in this case the K
M
for dihydrofolate, to derive the inhibition constants (K ) reported
i
in Table 3.

Molecules 2015, 20
7229
values for B. anthracis DHFR.
Table 3. MIC values of the inhibitors against B. anthracis and K
i
MIC (μ/mL)
K (nM ± SEM)
i
Cpd
R1
B. anthracis
B. anthracis DHFR
2631
(
R)-3a *
(R)-CH=C(CH
3
)
2
8
(
S)-3a *
RS)-3b
RS)-3c
R)-3d *
S)-3d *
(S)-CH=C(CH
CH -CH=CH
CH=CH
(R)-CH CHCH
(S)-CH CHCH
n-C
3
)
2
0.5
4.4 ± 0.2
8.2 ± 0.3
3.2 ± 0.2
>2870
(
2
2
4
(
2
1–2
(
2
3
8
(
2
3
0.25–0.5
5.0 ± 0.5
5.5 ± 0.2
5.5 ± 0.2
5.2 ± 0.2
6.4 ± 0.2
87.6 ± 0.9
48.8 ± 0.5
111.6 ± 0.9
66.9 ± 0.5
(
RS)-3e
RS)-3g
RS)-3h
5
H
11
4
4
(
c-C
4
5
H
7
9
(
c-C
H
4
(
RS)-3i
RS)-3l
C(CH
3
)
3
8
(
1-methylindol-2-yl
benzofur-2-yl
>32
>32
>32
>32
(
RS)-3m
RS)-3n
RS)-3o
(
benzothiophen-2-yl
benzothiazol-2-yl
(
*
Data obtained from earlier work for comparison [27].
Comparison of the single crystal X-ray structure of (S)-3d (Figure 3A) with its pose in the DHFR
binding site (Figure 4A) shows a very similar conformation. Thus, the enzyme permits the substrate to
adopt a low energy conformation when bound with the protein, similar to its preferred solid-state
1
minimum. The R group at the C-1 dihydrophthalazine stereocenter serves as an interface between the
substrate-binding pocket and the surrounding solvent. Figure 4B,C demonstrate the position of the R1
group of (S)-3d ((S)-RAB1) within the binding pocket of the B. anthracis dihydrofolate reductase
(
BaDHFR) protein and its proximity to the surrounding medium.
1
Earlier studies indicated that R interacts effectively with surrounding amino acid residues in
B. anthracis DHFR such as Lys33, Thr35, Leu41, Pro56, and Arg58 [15,27]. Derivatives with smaller
1
groups at R , such as (RS)-3a–h, adopted planar conformations within the binding pocket that have
more optimal interactions with the guanidinium moiety of Arg58, and hence we observed increased
activity for these compounds. Additionally, compounds containing alkenes (RS)-3a-c have a restricted
motion within the pocket, and this also leads to higher activity.
1
Larger aryl groups at the R position were previously prepared and studied by Nammalwar and
co-workers [13,15]. It was surmised that aryl substituents protruded into solvent space outside the
binding pocket, resulting in lesser inhibitory properties. A similar situation was presumed for
compounds 3l-o. These derivatives exhibited lower potency and this was attributed to the larger size
1
and the spatial orientation of R groups, which forced the molecules to move outside the binding
1
pocket. These observations demonstrated that the protrusion of more hydrophobic R groups beyond
the confines of the protein binding site should be energetically unfavorable and render these
compounds as weaker inhibitors.

Molecules 2015, 20
7230
Figure 4. The inhibitor series adopts a low energy conformation prior to binding, and retains
this conformation when buried in the DHFR substrate-binding pocket. (A) Superposition of
S)-3d [(S)-RAB1] from single crystal analysis (cyan) with that observed when bound by
(
the BaDHFR protein (yellow). Note the highly similar conformation, with differences
in torsional rotation to optimize the DAP ring placement, and the induced bend of the
dihydrophthalazine from planar (cyan) to 107° to 117° (yellow), critical for interactions
with the protein site [6]; (B) View of (S)-RAB1 bound in the BaDHFR binding site, with
the protein van der Waals surface colored by electrostatic potential; (C) Highly buried
position of (S)-RAB1 in the BaDHFR binding site, with part of the protein van der Waals
surface shown by outline to permit visualization of the inhibitor (NADPH also visible,
green). Note the single crystal (cyan) version of (S)-RAB1 adopts a highly similar orientation
even in the absence of the DHFR protein. Both (B) and (C) illustrate the limited volume for
1
R modifications, with favorable occupancy by up to one ring structure, but extension
beyond this results in protrusion into the solvent region.
. Experimental Section
3
3.1. General Information
Commercial anhydrous N,N-dimethylformamide (DMF) and dimethylsulfoxide (DMSO) were
stored under dry nitrogen and transferred by syringe into reactions when needed. Tetrahydrofuran

Molecules 2015, 20
7231
(
THF) was dried over potassium hydroxide pellets and distilled from lithium aluminium hydride prior
to use. Other commercial reagents [bismuth(III) triflate (Bi(OTf) ), butyllithium (n- and t-BuLi),
3
magnesium sulfate (MgSO
4
), triethylamine (TEA)] and solvents (acetonitrile (MeCN),
dichloromethane (DCM), ethyl acetate (EtOAc), hexanes, methanol (MeOH)) were used as received.
Aqueous wash solutions (saturated sodium chloride (NaCl) and ammonium chloride (NH
4
Cl)) were
used in work-up procedures.
All reactions were run under dry nitrogen in oven-dried glassware. Reactions were monitored by
thin layer chromatography (TLC) on silica gel GF plates (Analtech, No. 21521, Newark, DE, USA)
and visualized using a hand-held UV lamp. Preparative column chromatography was carried out on
6
3–200 µm silica gel (Sorbent Technologies, Norcross, GA, USA). Melting points were determined
using a 200 W MEL-TEMP apparatus (Cambridge, MA, USA) and were uncorrected. FT-IR spectra
1
13
were run as DCM solutions between NaCl disks. Unless otherwise indicated, H and C-NMR spectra
were measured at 400 MHz and 100 MHz, respectively, in the indicated solvent. Chemical shifts (δ)
are referenced to internal (CH
3
)
4
Si and coupling constants (J) are given in Hz. Elemental analyses
(
±0.4%) were performed by Atlantic Microlabs, Inc. (Norcross, GA, USA).
3
.2. 2,4-Diamino-5-(5-iodo-3,4-dimethoxybenzyl)pyrimidine (1)
This compound was prepared from 5-iodovanillin in 70% yield on a 0.40-mol scale according to the
literature procedure [15], mp 217–218 °C (lit. [15] mp 217–218 °C). The spectral data matched those
reported [15].
3
.3. Preparation of Phthalazin-2(1H)-yl]-2-propen-1-ones 2a–r
(±)-1-[1-(2-Methyl-1-propen-1-yl)phthalazin-2(1H)-yl]-2-propen-1-one (2a). This compound was
prepared from 4 and 2-methyl-1-propenylmagnesium bromide in 62% yield on a 15.4-mmol scale
according to the literature procedure [15]. The spectral data matched those reported [15].
(±)-1-(1-Allylphthalazin-2(1H)-yl)-2-propen-1-one (2b). A stirred solution of 4 (2.50 g, 19.2 mmol) in
dry THF (60 mL) was treated dropwise with a solution of allylmagnesium bromide (12.4 mL, 24.9 mmol,
2
1
M in THF) for 30 min at 0 °C. The reaction mixture was warmed to room temperature and stirred for
h. After completion, the reaction mixture was treated with saturated NH Cl (50 mL) and extracted
4
with EtOAc (3 × 100 mL). The combined organic extracts were washed with saturated NaCl (50 mL),
dried (MgSO ), filtered, and concentrated under vacuum to give 3b as a dark brown, viscous liquid.
4
The crude 5b was dissolved in DCM (70 mL) and TEA (5.87 mL, 41.7 mmol) was added, followed by
dropwise addition of acryloyl chloride (1.35 mL, 16.7 mmol) at 0 °C. The reaction mixture was stirred
at this temperature for 2 h. The mixture was then treated with saturated NaCl (100 mL), the organic
layer was separated, and the aqueous layer was extracted with DCM (2 × 50 mL). The combined extracts
were washed with saturated NaCl (50 mL), dried (MgSO
4
), filtered, and concentrated to afford the
crude product. This material was purified on a silica gel column eluted with hexanes–EtOAc (7:3) to
−1 1
afford 2b (2.47 g, 57%) as a viscous, yellow liquid. IR: 1662 cm ; H-NMR (CDCl
.43 (td, J = 7.4, 1.4 Hz, 1H), 7.35 (td, J = 7.6, 1.2 Hz, 1H), 7.30 (dd, J = 17.1, 10.5 Hz, 1H), 7.28 (d,
J = 7.6, 0.6 Hz, 1H), 7.15 (dd, J = 7.6, 0.6 Hz, 1H), 6.42 (dd, J = 17.1, 2.1 Hz, 1H), 5.91 (t, J = 6.4 Hz,
3
): δ 7.60 (s, 1H),
7

Molecules 2015, 20
7232
1
4
1
H), 5.78 (dd, J = 10.5, 2.1 Hz, 1H), 5.68 (ddt, J = 17.1, 10.1, 7.3 Hz, 1H), 4.98 (dm, J = 10.2 Hz, 1H),
1
3
.90 (dm, J = 17.0 Hz, 1H), 2.41 (m, 2H); C-NMR (CDCl
3
): δ 166.3, 142.2, 133.2, 132.7, 131.4,
28.4, 128.1, 127.1, 126.6, 125.6, 123.9, 118.7, 51.1, 39.8.
(
±)-1-(1-Vinylphthalazin-2(1H)-yl)-2-propen-1-one (2c). This compound was prepared via the procedure
described for 2b using 4 (2.50 g, 19.2 mmol) and vinylmagnesium bromide (11.5 mL, 23.0 mmol,
.0 M in THF) in dry THF (60 mL) to afford 3c, followed by acylation using TEA (5.3 mL, 37.8 mmol)
and acryloyl chloride (1.20 mL, 15.1 mmol) in DCM (70 mL) to give 2c (2.36 g, 58%) as a colorless,
2
−1 1
viscous liquid. IR: 1665 cm ; H-NMR (CDCl
3
): δ 7.59 (s, 1H), 7.45 (td, J = 7.4, 0.8 Hz, 1H), 7.40–7.28
(
(
m, 3H), 7.21 (dd, J = 7.6, 0.4 Hz, 1H), 6.51 (dd, J = 17.4, 2.1 Hz, 1H), 6.37 (d, J = 4.9 Hz, 1H), 5.81
1
3
m, 2H), 5.10 (dd, J = 10.3, 0.4 Hz, 1H), 4.86 (d, J = 17.4 Hz, 1H); C-NMR (CDCl
34.7, 131.9, 131.7, 128.8, 128.4, 126.9, 126.8, 125.9, 123.8, 116.2, 53.0.
3
): δ 166.2, 141.7,
1
(±)-1-(1-Propylphthalazin-2(1H)-yl)-2-propen-1-one (2d). This compound was prepared in 70% yield
from 4 (2.00 g, 15.4 mmol) and propylmagnesium chloride (8.45 mL, 16.9 mmol, 2.0 M in ether) in
dry THF (50 mL) to afford 5d, followed by acylation using TEA (1.82 g, 2.50 mL, 18.0 mmol) and
acryloyl chloride (1.40 g, 1.26 mL, 15.5 mmol) in DCM (70 mL) to afford 2d according to the
literature procedure [15]. The spectral data matched those reported [15].
(±)-1-(1-Pentylphthalazin-2(1H)-yl)-2-propen-1-one (2e). This compound was prepared via the procedure
described for 2b from 4 (2.50 g, 19.2 mmol) and n-pentylmagnesium bromide (18.4 mL, 18.4 mmol,
1
3
M in THF) in dry THF (60 mL) to afford 5e, followed by acylation using TEA (3.27 g, 4.51 mL,
2.4 mmol) and acryloyl chloride (1.05 mL, 12.9 mmol) in DCM (70 mL) to give 2e (3.05 g, 62%) as
−1 1
an off-white solid, mp 52–54 °C. IR: 1664 cm ; H-NMR (CDCl
3
): δ 7.61 (s, 1H), 7.42 (td, J = 7.4,
.1 Hz, 1H), 7.33 (m, 2H), 7.26 (d, J = 7.0 Hz, 1H), 7.15 (dd, J = 7.4, 0.7 Hz, 1H), 6.47 (dd, J = 17.2,
.4 Hz, 1H), 5.83 (t, J = 6.7 Hz, 1H), 5.77 (dd, J = 10.5, 2.4 Hz, 1H), 1.63 (m, 2H), 1.21 (m, 6H), 0.81
1
2
1
3
(
t, J = 6.6 Hz, 3H); C-NMR (CDCl
3
): δ 166.1, 142.3, 134.0, 131.3, 128.2, 127.9, 127.1, 126.4, 125.6,
1
23.8, 51.2, 34.9, 31.4, 24.4, 22.3, 13.8.
(
±)-1-(1-Cyclopropylphthalazin-2(1H)-yl)-2-propen-1-one (2f). This compound was prepared in 87%
yield from 4 (2.00 g, 15.4 mmol) and cyclopropylmagnesium chloride (33.8 mL, 16.9 mmol, 0.5 M in
THF) in dry THF (50 mL) to give 5f, followed by acylation using TEA (1.86 g, 2.56 mL, 18.4 mmol)
and acryloyl chloride (1.39 g, 1.25 mL, 15.4 mmol) in DCM (70 mL) to afford 2f according to the
literature procedure [13]. The spectral data matched those reported [13].
(±)-1-(1-Cyclobutylphthalazin-2(1H)-yl)-2-propen-1-one (2g). This compound was prepared via the
procedure described for 2b using 4 (2.50 g, 19.2 mmol) and cyclobutylmagnesium bromide (24.9 mL,
2
4
2
1
4.9 mmol, 1 M in THF) in dry THF (60 mL) to give 5g, followed by acylation using TEA (3.25 g,
.48 mL, 32.2 mmol) and acryloyl chloride (1.16 g, 1.04 mL, 12.8 mmol) in DCM (70 mL) to afford
−1 1
g (3.04 g, 66%) as a yellow liquid. IR: 1663 cm ; H-NMR (CDCl
3
): δ 7.61 (s, 1H), 7.44 (td, J = 7.4,
.5 Hz, 1H), 7.35 (td, J = 7.4, 1.5 Hz, 1H), 7.31 (dd, J = 17.2, 10.5 Hz, 1H), 7.29 (m, 1H), 7.16 (dd,
J = 7.5, 0.7 Hz, 1H), 6.48 (dd, J = 17.2, 2.0 Hz, 1H), 5.83 (d, J = 8.4 Hz, 1H), 5.77 (dd, J = 10.5,

Molecules 2015, 20
7233
1
3
2
.0 Hz, 1H), 2.60 (sextet, J = 8.4 Hz, 1H), 1.98 (m, 1H), 1.90–1.73 (m, 2H), 1.67 (m, 3H); C-NMR
(
CDCl ): δ 166.5, 143.8, 132.6, 131.3, 128.2, 128.0, 127.1, 126.5, 125.6, 124.0, 54.5, 40.2, 25.6, 24.9, 17.6.
3
(±)-1-(1-Cyclopentylphthalazin-2(1H)-yl)-2-propen-1-one (2h). This compound was prepared via the
procedure described for 2b from 4 (2.50 g, 19.2 mmol) and cyclopentylmagnesium bromide (24.9 mL,
2
5
2
4.9 mmol, 1 M in THF) in dry THF (60 mL) to give 5h, followed by acylation using TEA (3.78 g,
.20 mL, 37.4 mmol) and acryloyl chloride (1.35 g, 1.21 mL, 14.9 mmol) in DCM (70 mL) to afford
−
1 1
h (2.73 g, 56%) as a viscous, colorless liquid. IR: 1663 cm ; H-NMR (CDCl ): δ 7.69 (s, 1H), 7.43
3
(
td, J = 7.4, 1.4 Hz, 1H), 7.35 (td, J = 7.4, 1.4 Hz, 1H), 7.32 (dd, J = 17.2, 10.5 Hz, 1H), 7.28 (dd,
J = 7.4, 1.4 Hz, 1H), 7.18 (dd, J = 7.4, 0.5 Hz, 1H), 6.46 (dd, J = 17.2, 2.1 Hz, 1H), 5.80 (d, J = 8.6 Hz,
H), 5.77 (dd, J = 10.5, 2.1 Hz, 1H), 2.12 (m, 1H), 1.61 (m, 3H), 1.42 (m, 4H), 1.23 (m, 1H);
13C-NMR (CDCl
): δ 166.3, 143.6, 133.5, 131.2, 128.3, 128.0, 127.1, 126.8, 125.4, 124.2, 54.1, 45.2,
9.3, 28.7, 24.3, 24.2.
1
3
2
(±)-1-(1-(tert-Butyl)phthalazin-2(1H)-yl)-2-propen-1-one (2i). A stirred solution of 4 (2.00 g, 17.6 mmol)
in 50 mL of dry THF was treated dropwise with a solution of t-BuLi (16.9 mL, 16.9 mmol, 1.0 M in
heptanes) over a period of 15 min at −78 °C. The reaction was stirred at this temperature for 1 h and
then slowly warmed to room temperature and stirred for an additional 30 min. The reaction mixture
was added to cold saturated NH
4
Cl (50 mL) and extracted with EtOAc (3 × 50 mL). The combined
), filtered, and concentrated
organic extracts were washed with saturated NaCl (50 mL), dried (MgSO
4
under vacuum to give 5i as a dark brown liquid. The crude product was dissolved in DCM (100 mL),
and TEA (4.00 g, 5.50 mL, 39.6 mmol) was added, followed by dropwise addition of acryloyl chloride
(1.44 g, 1.29 mL, 15.9 mmol) at 0 °C. The reaction mixture was stirred at this temperature for 2 h. The
mixture was then added to saturated NaCl (100 mL), the organic layer was separated, and the aqueous
layer was extracted with DCM (2 × 50 mL). The combined extracts were washed with saturated NaCl
(
50 mL), dried (MgSO
4
), filtered, and concentrated to afford the crude product. The crude product was
purified on a silica gel column eluted with hexanes–EtOAc (7:3) to afford 2i as a viscous, yellow
−
1 1
liquid (2.79 g, 75%). IR: 1666 cm ; H-NMR (CDCl
.37 (td, J = 7.4, 1.4 Hz, 1H), 7.34 (dd, J = 17.1, 10.1 Hz, 1H), 7.29 (dd, J = 7.4, 0.8 Hz, 1H), 7.19 (d,
J = 7.8 Hz, 1H), 6.43 (dd, J = 17.1, 2.3 Hz, 1H), 5.78 (s, 1H), 5.75 (dd, J = 10.1, 2.3 Hz, 1H), 0.86 (s,
3
): δ 7.65 (s, 1H), 7.46 (td, J = 7.4, 1.4 Hz, 1H),
7
1
3
9H); C-NMR (CDCl
8.1, 39.4, 26.6, 25.5.
3
): δ 167.0, 144.6, 133.4, 131.0, 130.6, 128.6, 128.2, 128.0, 127.4, 125.4, 125.1,
5
(
±)-1-(1-(Furan-2-yl)phthalazin-2(1H)-yl)-2-propen-1-one (2j). This compound was prepared in 72%
yield from 4 (2.00 g, 15.4 mmol), furan-2-yllithium [from furan (1.20 g, 17.6 mmol) and n-BuLi (7.30 mL,
1
8.3 mmol, 2.5 M in hexanes)] in dry THF (50 mL) to give 5j, followed by acylation using TEA (2.37 g,
.26 mL, 23.5 mmol) and acryloyl chloride (1.59 g, 1.43 mL, 17.6 mmol) in DCM (70 mL) to afford 2j
3
according to the literature procedure [13]. The spectral data matched those reported [13].
(
±)-1-[1-(Thiophen-2-yl)phthalazin-2(1H)-yl]-2-propen-1-one (2k). This compound was prepared in
0% yield from 4 (2.00 g, 15.4 mmol), thiophen-2-ylmagnesium bromide [from 2-bromothiophene
1.77 g, 1.69 mL, 21.0 mmol) and magnesium (0.69 g, 28.4 mmol)] in dry THF (50 mL) to give 5k.
The crude 5k was acylated using TEA (2.80 g, 3.86 mL, 27.7 mmol) and acryloyl chloride (1.90 g,
6
(

Molecules 2015, 20
7234
1
.71 mL, 21.0 mmol) in DCM (70 mL) to afford 2k according to the literature procedure [13]. The
spectral data matched those reported [13].
(±)-1-[1-(1-Methyl-1H-indol-2-yl)phthalazin-2(1H)-yl]-2-propen-1-one (2l). To a stirred solution of
1
1
-methylindole (1.50 g, 11.4 mmol) in dry THF (25 mL) was added dropwise n-BuLi (6.86 mL,
7.2 mmol, 2.5 M in hexanes) over a period of 30 min at −78 °C. The solution was warmed to −25 °C,
and stirring was continued at this temperature for 1 h. The reaction mixture was cooled to −78 °C, and
a solution of 4 (1.48 g, 11.4 mmol) in dry THF (20 mL) was added dropwise over 30 min. The reaction
mixture was stirred at this temperature for 2 h. The mixture was poured into saturated NH
and extracted with EtOAc (3 × 50 mL). The combined organic extracts were washed with saturated
NaCl (50 mL), dried (MgSO ), filtered, and concentrated under vacuum to give 5l as a light yellow
4
Cl (100 mL)
4
liquid. The crude product 5l was dissolved in DCM (100 mL), and TEA (2.08 g, 2.87 mL, 20.6 mmol)
was added, followed by dropwise addition of acryloyl chloride (0.81 g, 0.73 mL, 8.95 mmol) at 0 °C.
The reaction mixture was stirred at this temperature for an additional 2 h. The aqueous layer was added
to saturated NaCl (50 mL), and the organic layer was separated. The aqueous layer was extracted with
DCM (2 × 30 mL), and the combined organic extracts were washed with saturated NaCl (50 mL),
dried (MgSO
4
), filtered, and concentrated to afford the crude product. The product was purified on
a silica gel column eluted with hexanes–EtOAc (7:3) to afford 2l (3.01 g, 62%) as a light yellow solid,
−1 1
mp 69–71 °C. IR: 1657 cm ; H-NMR (CDCl
3
): δ 7.79 (s, 1H), 7.42–7.34 (complex, 5H), 7.22 (dd,
J = 17.2, 10.5 Hz, 1H), 7.18 (s, 1H), 7.16 (m, 2H), 7.00 (t, J = 7.6 Hz, 1H), 6.45 (dd, J = 7.2, 2.0 Hz,
1
3
1
1
1
H), 5.89 (s, 1H), 5.75 (dd, J = 10.5, 2.0 Hz, 1H), 3.99 (s, 3H); C-NMR (CDCl ): δ 166.0, 143.3,
3
38.9, 137.4, 132.3, 132.1, 129.0, 128.6, 127.2, 127.0, 126.7, 126.0, 123.7, 122.0, 120.5, 119.6, 109.6,
03.3, 46.6, 30.6.
(
±)-1-[1-(Benzofuran-2-yl)phthalazin-2(1H)-yl]-2-propen-1-one (2m). The compound was prepared
using benzofuran-2-yllithium [from benzofuran (2.00 g, 16.9 mmol) and n-BuLi (6.8 mL, 17.0 mmol,
.5 M in hexanes)] and 4 (2.20 g, 16.9 mmol) in dry THF (75 mL) to give 5m. The crude 5m was
acylated using TEA (3.05 g, 4.20 mL, 30.2 mmol) and acryloyl chloride (1.09 g, 0.97 mL, 12.0 mmol)
2
−1
in DCM (120 mL) to afford 2m (2.37 g, 51%) as a light yellow solid, mp 55–57 °C. IR: 1657 cm ;
1H-NMR (CDCl
): δ 7.84 (d, J = 7.8 Hz, 1H), 7.63 (dd, J = 7.8, 0.6 Hz, 1H), 7.56 (d, J = 8.2 Hz, 1H),
.48–7.40 (complex, 3H), 7.36 (td, J = 7.5, 1.2 Hz, 2H), 7.27 (t, J = 7.5 Hz, 1H), 7.23 (s, 1H), 7.23
d, J = 7.5 Hz, 1H), 6.54 (dd, J = 17.1, 2.1 Hz, 1H), 5.84 (dd, J = 10.4, 2.0 Hz, 1H), 5.02 (s, 1H);
13C-NMR (CDCl
): δ 166.4, 154.9, 151.1, 141.8, 131.7, 131.0, 139.1, 138.1, 137.7, 126.5, 126.3,
26.0, 125.4, 123.6, 123.3, 121.5, 111.5, 108.0, 42.0.
3
7
(
3
1
(±)-1-[1-(Benzo[b]thiophen-2-yl)phthalazin-2(1H)-yl]-2-propen-1-one (2n). The compound was prepared
using benzothiophen-2-yllithium [from benzothiophene (2.00 g, 14.9 mmol) and n-BuLi (6.0 mL,
1
5
1
1
7
5.0 mmol, 2.5 M in hexanes)] and 4 (1.94 g, 14.9 mmol) in dry THF (75 mL) to give 5n. The crude
n was acylated using TEA (2.86 g, 3.94 mL, 28.3 mmol) and acryloyl chloride (1.02 g, 0.92 mL,
1.3 mmol) in DCM (120 mL) to afford 2n (2.94 g, 62%) as a light yellow solid, mp 69–71 °C. IR:
−1 1
663 cm ; H-NMR (CDCl
3
): δ 7.86 (m, 1H), 7.81 (m, 2H), 7.70 (s, 1H), 7.51 (t, J = 7.6 Hz, 1H),
.46–7.36 (complex, 5H), 7.32 (d, J = 7.2 Hz, 1H), 6.54 (dd, J = 17.2, 1.8 Hz, 1H), 5.87 (dd, J = 10.3,

Molecules 2015, 20
7235
1
3
1
.8 Hz, 1H), 5.05 (s, 1H); C-NMR (CDCl
3
): δ 166.4, 146.4, 139.8, 139.7, 139.0, 131.8, 131.7, 129.2,
1
28.2, 126.6, 126.1, 125.8 (2C), 125.7, 124.6, 124.4, 124.2, 122.2, 42.2.
(±)-1-[1-(Benzo[d]thiazol-2-yl)phthalazin-2(1H)-yl]-2-propen-1-one (2o). The compound was prepared
using benzothiazol-2-yllithium [from (2.00 g, 14.8 mmol) and n-BuLi (6.5 mL, 16.3 mmol, 2.5 M in
hexanes)] and 4 (1.92 g, 14.8 mmol) in dry THF (75 mL) to give 5o. The crude 5o was acylated using
TEA (2.86 g, 3.94 mL, 28.3 mmol) and acryloyl chloride (1.02 g, 0.92 mL, 11.3 mmol) in DCM
−
1 1
(
120 mL) to afford 5o (3.20 g, 68%) as a light yellow solid, mp 68–70 °C. IR: 1663 cm ; H-NMR
CDCl ): δ 7.93 (d, J = 8.2 Hz, 1H), 7.75 (dt, J = 8.0, 0.6 Hz, 1H), 7.70 (s, 1H), 7.56 (d, J = 7.6 Hz,
H), 7.51 (td, J = 7.4, 1.4 Hz, 1H), 7.45–7.27 (complex, 6H), 6.56 (dd, J = 17.4, 2.0 Hz, 1H), 5.87 (dd,
(
3
1
1
3
J = 10.5, 2.0 Hz, 1H); C-NMR (CDCl
3
): δ 169.5, 166.6, 153.0, 141.5, 135.2, 132.2, 130.3, 129.9,
1
29.3, 127.7, 126.5, 126.3, 125.9, 125.2, 123.6, 123.2, 121.5, 53.2.
(
±)-t-Butyl 2-(2-acryloylphthalazin-2(1H)-yl)acetate (2p). This compound was prepared in 87% yield
from tert-butyl acetate (2.67 g, 3.08 mL, 23.0 mmol), n-BuLi (7.7 mL, 19.3 mmol, 2.5 M in hexanes),
4
(2.99 g, 23.0 mmol), TEA (1.86 g, 2.56 mL, 18.4 mmol) and acryloyl chloride (1.39 g, 1.25 mL,
5.4 mmol) according to the literature procedure [13]. The spectral data matched those reported [13].
1
(
±)-2-(2-Acryloylpthalazin-2(1H)-yl)acetic acid (6). This compound was prepared in 94% yield from
p (1.50 g, 5.00 mmol), and Bi(OTf) (0.164 g, 0.25 mmol, 5 mol %) in benzene (25 mL) according to
the literature procedure [13]. The spectral data matched those reported [13].
2
3
(
±)-Ethyl 2-(2-acryloylphthalazin-2(1H)-yl)acetate (2q). This compound was prepared in 95% yield
from 6 (1.00 g, 4.10 mmol), and Bi(OTf) (0.134 g, 0.20 mmol, 5 mol %) in ethanol (25 mL) according
to the literature procedure [13]. The spectral data matched those reported [13].
3
(
±)-Methyl 2-(2-acryloylphthalazin-2(1H)-yl)acetate (2r). This compound was prepared in 95% yield
from 6 (1.00 g, 4.10 mmol), Bi(OTf) (0.134 g, 0.20 mmol, 5 mol %) in CH OH (25 mL) according to
the literature procedure [13]. The spectral data matched those reported [13].
3
3
3
.4. Preparation of Drug Candidates 3a–r
(
±)-(E)-3-{5-[(2,4-Diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl}-1-[1-(2-methyl-1-propen-1-
yl)phthalazin-2(1H)yl]-2-propen-1-one (3a). To a stirred solution of 1 (1.00 g, 2.59 mmol) in dry DMF
10 mL) was added a solution of 2a (0.684 g, 2.85 mmol) in DMF (2 mL), followed by TEA (0.313 g,
.431 mL, 3.10 mmol), and the Pd pincer-II catalyst (1 mg, 0.0014 mmol). The reaction was heated
(
0
at 90 °C for 8 h and then cooled using an ice bath. The product was purified by directly pouring the
crude reaction mixture onto a 50 cm × 2.5 cm silica gel chromatography column slurry packed with
DCM. Impurities were eluted using DCM, and the final product was collected using DCM/MeOH/TEA
(
95:4:1). Evaporation of the solvent gave a yellow solid, which was dried under high vacuum for 2 h.
MeOH (5 mL) was added to dissolve the crude product, followed by ether (10 mL), and the mixture
was cooled for 4 h to crystallize the product. The product was filtered and dried under vacuum to
afford 3a as a yellow solid (1.18, 88%). The melting point and spectral data matched those in the
literature [15].

Molecules 2015, 20
7236
(
±)-(E)-1-(1-Allylphthalazin-2(1H)-yl)-3-{5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxy-
phenyl}-2-propen-1-one (3b). This compound was prepared as above using 1 (2.00 g, 5.18 mmol), 2b
1.55 g, 6.73 mmol), TEA (0.680 g, 0.937 mL, 6.73 mmol), and the Pd pincer-II catalyst (2 mg,
(
0
3
1
.0028) in dry DMF (15 mL) to give 3b (2.03 g, 81%) as a white solid, mp 215–217 °C. IR: 3418,
−1 1
123, 3122, 1657, 1641, 1598, 1564 cm ; H-NMR (DMSO-d
6
): δ 7.91 (s, 1H), 7.86 (d, J = 16.2 Hz,
H), 7.62 (d, J = 16.2 Hz, 1H), 7.60 (s, 1H), 7.56–7.37 (complex, 4H), 7.24 (s, 1H), 6.99 (s, 1H), 6.19
(
br s, 2H), 5.95 (t, J = 6.3 Hz, 1H), 5.74 (br s, 2H), 5.66 (ddt, J = 17.4, 10.5, 6.3 Hz, 1H), 4.94 (dm,
J = 10.5 Hz, 1H), 4.86 (dm, J = 17.4 Hz, 1H), 3.79 (s, 3H), 3.74 (s, 3H), 3.60 (s, 2H), 2.36 (m, 2H);
13C-NMR (DMSO-d
): δ 165.6, 162.3, 162.2, 155.8, 152.5, 146.0, 142.5, 136.6, 133.2, 132.9, 131.7,
28.3, 127.8, 126.6, 126.0, 123.7, 118.4, 118.3, 117.9, 114.8, 105.7, 60.8, 55.7, 54.9, 50.4, 32.4
1 aromatic/alkene C unresolved); Anal. Calcd for C27 ·1.3 H O: C, 63.84; H, 6.07; N, 16.54.
Found: C, 63.84; H, 5.69; N, 16.28.
6
1
(
H
28
N O
6 3
2
(±)-(E)-3-{5-[(2,4-Diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl}-1-(1-vinylphthalazin-2(1H)-yl)-
2-propen-1-one (3c). This compound was prepared as above using 1 (1.60 g, 4.15 mmol), 2c (1.14 g,
5
.38 mmol), TEA (0.543 g, 0.750 mL, 5.38 mmol) and the Pd pincer-II catalyst (1.5 mg, 0.0021 mmol)
in dry DMF (15 mL) to give 3c (1.65 g, 85%) as a white solid, mp 210–212 °C. IR: 3354, 3169, 1638,
−
1 1
1
1
593, 1567 cm ; H-NMR (DMSO-d
6
): δ 7.91 (s, 1H), 7.89 (d, J = 16.0 Hz, 1H), 7.68 (d, J = 16.0 Hz,
H), 7.60–7.44 (complex, 5H), 7.32 (d, J = 1.3 Hz, 1H), 7.25 (br s, 2H), 7.04 (d, J = 1.4, 1H), 6.73 (br
s, 2H), 6.40 (d, J = 4.5 Hz, 1H), 5.80 (ddd, J = 1.5, 10.2, 4.5 Hz, 1H), 5.08 (d, J = 10.2 Hz, 1H), 4.78
1
3
(
d, J = 17.0 Hz, 1H), 3.81 (s, 3H), 3.76 (s, 3H), 3.65 (s, 2H); C-NMR (DMSO-d
6
): δ 165.6, 163.2,
57.7, 152.6, 146.8, 146.3, 142.1, 136.9, 135.4, 135.0, 132.0, 131.7, 128.6, 127.9, 126.9, 126.3, 123.5,
18.8, 117.9, 115.3, 115.0, 107.6, 60.8, 55.9, 52.5, 31.9; Anal. Calcd for C26 ·4.6 H O·0.1
OH: C, 56.21; H, 5.32; N, 15.01. Found: C, 56.25; H, 4.99; N, 15.04.
1
1
26
H N
6
O
3
2
C
2
H
5
(±)-(E)-3-{5-[(2,4-Diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl}-1-(1-propylphthalazin-2(1H)-yl)-
2-propen-1-one (3d). This compound was prepared as above using 1 (1.00 g, 2.59 mmol), 2d (0.701 g,
3.10 mmol), TEA (0.313 g, 0.432 mL, 3.10 mmol), and the Pd pincer-II catalyst (1 mg, 0.0014 mmol)
in dry DMF (15 mL) to give 3d (1.08 g, 86%) as a white solid. The melting point and spectral data
matched those in the literature [15].
(
±)-(E)-3-{5-[(2,4-Diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl}-1-(1-pentylphthalazin-2(1H)-
yl)-2-propen-1-one (3e). This compound was prepared as above using 1 (1.60 g, 4.15 mmol), 2e (1.39 g,
.35 mmol), TEA (0.500 g, 0.689 mL, 4.95 mmol), and the Pd pincer-II catalyst (1.5 mg, 0.0021 mmol)
in dry DMF (15 mL) to give 3e (1.91 g, 90%) as a white solid, mp 212–213 °C. IR: 3363, 3173, 1638,
5
−1 1
1
1
1
3
590, 1560 cm ; H-NMR (DMSO-d ): δ 7.92 (s, 1H), 7.86 (d, J = 16.4 Hz, 1H), 7.64 (d, J = 16.4 Hz,
6
H), 7.54 (s, 1H), 7.52 (m, 2H), 7.43 (td, J = 7.4, 0.8 Hz, 1H), 7.38 (d, J = 1.6 Hz, 1H), 7.28 (d, J = 1.6 Hz,
H), 7.08 (br s, 2H), 7.02 (d, J = 1.5 Hz, 1H), 6.58 (br s, 2H), 5.82 (t, J = 6.7 Hz, 1H), 3.79 (s, 3H),
13
.74 (s, 3H), 3.63 (s, 2H), 1.53 (m, 2H), 1.16 (m, 6H) 0.77 (t, J = 6.6 Hz, 3H); C-NMR (DMSO-d ):
6
δ 165.6, 163.1, 158.3, 152.6, 148.2, 146.2, 142.8, 136.5, 135.2, 133.6, 131.7, 128.3, 127.9, 126.5,
26.1, 123.6, 118.7, 118.0, 114.9, 107.3, 60.8, 55.8, 50.5, 34.5, 32.0, 30.9, 24.0, 21.9, 13.8; Anal. Calcd
·3.9 H O·0.3 C OH: C, 59.94; H, 6.42; N, 14.21. Found: C, 59.61; H, 6.42; N, 14.21.
1
for C29H N O
34 6 3
2
H
2 5

Molecules 2015, 20
7237
(
±)-(E)-1-(1-Cyclopropylphthalazin-2(1H)-yl)-3-{5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-
dimethoxyphenyl}-2-propen-1-one (3f). This compound was prepared as above using 1 (1.00 g,
.59 mmol), 2f (0.645 g, 2.80 mmol), TEA (0.313 g, 0.432 mL, 3.10 mmol), and the Pd pincer-II
2
catalyst (1 mg, 0.0014 mmol) in dry DMF (15 mL) to give 3f (1.05 g, 84%) as a white solid. The
melting point and spectral data matched those in the literature [13].
(
±)-(E)-1-(1-Cyclobutylphthalazin-2(1H)-yl)-3-{5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-
dimethoxyphenyl}-2-propen-1-one (3g). This compound was prepared as above using 1 (1.50 g,
.89 mmol), 2g (1.21 g, 5.04 mmol), TEA (0.510 g, 0.702 mL, 5.05 mmol), and the Pd pincer-II
catalyst (1.5 mg, 0.0021 mmol) in dry DMF (15 mL) to give 3g (1.68 g, 87%) as a white solid, mp
3
−
1 1
1
25–127 °C. IR: 3397, 3272, 1642, 1605, 1562 cm ; H-NMR (DMSO-d
J = 16.0 Hz, 1H), 7.64 (d, J = 16.0 Hz, 1H), 7.59 (s, 1H), 7.52 (m, 2H), 7.44 (m, 2H), 7.25 (s, 1H),
.00 (s, 1H), 6.39 (br s, 2H), 5.92 (br s, 2H), 5.86 (d, J = 8.2 Hz, 1H), 3.79 (s, 3H), 3.74 (s, 3H), 3.60
6
): δ 7.93 (s, 1H), 7.85 (d,
7
1
3
(
s, 2H), 2.54 (sextet, J = 8.2 Hz, 1H), 1.84 (m, 3H), 1.63 (m, 3H); C-NMR (DMSO-d
6
): δ 165.9,
62.4, 161.4, 154.0, 152.5, 146.0, 143.3, 136.6, 136.3, 132.1, 131.7, 128.3, 127.8, 126.6, 126.1, 123.8,
18.4, 117.9, 114.8, 106.1, 60.8, 55.8, 53.8, 32.3, 25.1, 24.2, 17.1; Anal. Calcd for C28 ·1.6
O: C, 60.27; H, 6.31; N, 14.84. Found: C, 60.08; H, 6.07; N, 15.01.
1
1
H
30
N O
6 3
H
2
(
±)-(E)-1-(1-Cyclopentylphthalazin-2(1H)-yl)-3-{5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-
dimethoxyphenyl}-2-propen-1-one (3h). This compound was prepared as above using 1 (1.50 g,
.89 mmol), 2h (1.28 g, 5.04 mmol), TEA (0.510 g, 0.702 mL, 5.05 mmol), and the Pd pincer-II
catalyst (1.5 mg, 0.0021 mmol) in dry DMF (15 mL) to give 3h (1.83 g, 92%) as a white solid, mp
3
−
1 1
1
7
2
2
1
1
6
5
90–192 °C. IR: 3338, 3174, 1637, 1594, 1560 cm ; H-NMR (DMSO-d , 400 MHz): δ 7.96 (s, 1H),
6
.81 (d, J = 16.1 Hz, 1H), 7.60 (d, J = 16.1 Hz, 1H), 7.52 (s, 1H), 7.49 (m, 1H), 7.42 (d, J = 7.4 Hz,
H), 7.38 (d, J = 7.4, 1H), 7.23 (d, J = 1.6 Hz, 1H), 6.97 (d, J = 1.6 Hz, 1H), 6.86 (br s, 2H), 6.37 (br s,
H), 5.74 (d, J = 8.4 Hz, 1H), 3.76 (s, 3H), 3.70 (s, 3H), 3.58 (s, 2H), 2.10 (sextet, J = 8.4 Hz, 1H),
1
3
.50 (m, 3H), 1.36 (m, 3H), 1.22 (m, 2H); C-NMR (DMSO-d
6
): δ 165.4, 162.9, 159.2, 152.6, 149.7,
46.1, 144.1, 136.6, 135.5, 133.1, 131.6, 128.3, 127.9, 126.9, 125.9, 124.1, 118.6, 118.0, 114.9, 107.0,
0.8, 55.8, 53.4, 44.6, 32.1, 29.0, 28.2, 24.1, 24.0; Anal. Calcd for C29
.84; N, 14.55. Found: C, 60.19; H, 5.79; N, 14.57.
H N
32 6
O
3
·3.5 H O: C, 60.32; H,
2
(
±)-(E)-1-(1-tert-Butylphthalazin-2(1H)-yl)-3-{5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-
dimethoxyphenyl}-2-propen-1-one (3i). This compound was prepared as above using 1 (2.20 g,
.70 mmol), 2i (1.77 g, 7.32 mmol), TEA (0.748 g, 1.03 mL, 7.41 mmol), and the Pd pincer-II catalyst
2 mg, 0.0028 mmol) in dry DMF (15 mL) to give 3i (2.36 g, 83%) as a white solid, mp 228–230 °C.
5
(
−1 1
IR: 3354, 3174, 1637, 1590, 1561 cm ; H-NMR (DMSO-d
6
): δ 7.97 (s, 1H), 7.84 (d, J = 16.0 Hz,
H), 7.71 (d, J = 16.0 Hz, 1H), 7.56 (td, J = 7.5, 1.6 Hz, 1H), 7.53 (m, 2H), 7.48 (td, J = 7.4, 1.2 Hz,
H), 7.46 (br s, 2H), 7.39 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 1.5 Hz, 1H), 7.02 (d, J = 2.0 Hz, 1H), 6.92
1
1
13
(
br s, 2H), 5.79 (s, 1H), 3.80 (s, 3H), 3.73 (s, 3H), 3.64 (s, 2H), 0.80 (s, 9H); C-NMR (DMSO-d
δ 166.7, 163.4, 156.8, 152.6, 146.2, 145.1, 144.7, 136.3, 134.6, 131.3, 129.8, 128.5, 128.4, 128.0,
25.5, 125.1, 118.9, 118.4, 114.9, 107.9, 60.8, 53.7, 55.9, 39.0, 31.8, 26.4 (3C); Anal. Calcd for
·2.4 H O·1.1 C OH: C, 56.50; H, 5.96; N, 14.21. Found: C, 56.31; H, 5.74; N, 13.93.
6
):
1
C
28
H
32
N
6
O
3
2
2
H
5

Molecules 2015, 20
7238
(±)-(E)-3-{5-[(2,4-Diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl}-1-[1-(furan-2-yl)phthalazin-
2
(1H)-yl]-2-propen-1-one (3j). This compound was prepared as above using 1 (1.00 g, 2.59 mmol), 2j
(
0.785 g, 3.12 mmol), TEA (0.313 g, 0.432 mL, 3.10 mmol), and the Pd pincer-II catalyst (1 mg,
.0014 mmol) in dry DMF (15 mL) to give 3j (1.19 g, 90%) as a white solid. The melting point and
spectral data matched those in the literature [13].
0
(
±)-(E)-3-{5-[(2,4-Diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl}-1-[1-(thiophen-2-
yl)phthalazin-2(1H)-yl]-2-propen-1-one (3k). This compound was prepared as above using 1 (1.00 g,
.59 mmol), 2k (0.836 g, 3.12 mmol), TEA (0.313 g, 0.432 mL, 3.10 mmol), and the Pd pincer-II
2
catalyst (1 mg, 0.0014 mmol) in dry DMF (15 mL) to give 3k (1.10 g, 81%) as a white solid. The
melting point and spectral data matched those in the literature [13].
(
±)-(E)-3-{5-[(2,4-Diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl}-1-[1-(1-methyl-1H-indol-2-
yl)phthalazin-2(1H)-yl]-2-propen-1-one (3l). This compound was prepared as above using 1 (1.30 g,
.37 mmol), 2l (1.27 g, 4.04 mmol), TEA (0.440 g, 0.60 mL, 4.36 mmol), and the Pd pincer-II catalyst
1.3 mg, 0.0018 mmol) in dry DMF (15 mL) to give 3l (1.45 g, 75%) as a white solid, mp 178–180 °C.
3
(
−1 1
IR: 3345, 3150, 1634, 1602, 1562 cm ; H-NMR (DMSO-d
6
): δ 8.12 (s, 1H), 7.86 (d, J = 16.0 Hz,
H), 7.63 (d, J = 16.0 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.55–7.48 (complex, 4H), 7.44 (d, J = 7.2 Hz,
H), 7.36 (d, J = 7.8 Hz, 1H), 7.27 (d, J = 1.5 Hz, 1H), 7.26 (s, 1H), 7.11 (td, J = 7.4, 0.8 Hz, 1H), 7.02
1
1
(
br s, 2H), 7.01 (d, J = 1.5 Hz, 1H), 6.95 (td, J = 7.4, 0.8 Hz, 1H), 6.52 (br s, 2H), 5.92 (s, 1H), 4.05 (s,
13
3
1
1
H), 3.78 (s, 3H), 3.71 (s, 3H), 3.61 (s, 2H); C-NMR (DMSO-d ): δ 165.2, 163.0, 158.5, 152.6,
6
48.5, 146.2, 142.8, 140.3, 137.2, 136.6, 135.3, 132.3, 128.7, 127.8, 126.8, 126.7, 123.1, 121.6, 120.0,
19.4, 118.7, 117.8, 115.1, 110.1, 107.2, 100.9, 60.8, 55.8, 46.3, 32.0, 30.5 (2 aromatic C unresolved);
Anal. Calcd for C33
H
31
N
7
O
3
·4.1 H O: C, 61.70; H, 5.28; N, 14.92. Found: C, 61.75; H, 5.13; N, 15.25.
2
(
±)-(E)-1-[1-(Benzofuran-2-yl)phthalazin-2(1H)-yl]-3-{5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-
dimethoxyphenyl}-2-propen-1-one (3m). This compound was prepared as above using 1 (1.80 g,
.66 mmol), 2m (1.83 g, 6.06 mmol), TEA (0.615 g, 0.85 mL, 6.06 mmol), and the Pd pincer-II
catalyst (2 mg, 0.0028 mmol) in dry DMF (15 mL) to give 3m (2.09 1.51 g, 80%) as a light yellow
4
−1
1
solid, mp 222–224 °C. IR: 3345, 3150, 1634, 1602, 1562 cm ; H-NMR (DMSO-d ): δ 7.83 (d,
6
J = 16.0 Hz, 1H), 7.83 (m, 2H), 7.73 (d, J = 16.0 Hz, 1H), 7.73 (obscured, 1H), 7.61 (s, 1H), 7.58 (t,
J = 7.4 Hz, 1H), 7.53 (s, 1H), 7.52–7.42 (complex, 4H), 7.33 (t, J = 7.4 Hz, 1H), 7.17 (d, J = 1.7 Hz,
1
3
1
1
1
H), 7.05 (d, J = 1.7 Hz, 1H), 6.18 (br s, 2H), 5.68 (br s, 2H), 5.05 (s, 1H), 3.78 (s, 3H), 3.76 (s, 3H),
1
3
.60 (s, 2H); C-NMR (DMSO-d
6
): δ 166.1, 162.3, 162.1, 155.5, 154.4, 152.5, 150.5, 146.1, 141.2,
37.6, 136.6, 132.1, 131.3, 128.5, 127.8, 127.7, 126.8, 126.0, 125.7, 123.6, 123.2, 122.1, 119.2, 117.8,
15.2, 111.6, 108.7, 105.8, 60.7, 55.8, 42.0, 32.4; Anal. Calcd for C32
4.92. Found: C, 68.30; H, 4.95; N, 14.92.
H
28
N
6
4
O : C, 68.56; H, 5.03; N,
(±)-(E)-1-[1-(Benzo[b]thiophen-2-yl)phthalazin-2(1H)-yl]-3-{5-[(2,4-diaminopyrimidin-5-yl)methyl]-
2,3-dimethoxyphenyl}-2-propen-1-one (3n). This compound was prepared as above using 1 (1.60 g,
4.15 mmol), 2n (1.71 g, 5.38 mmol), TEA (0.543 g, 0.75 mL, 5.38 mmol), and the Pd pincer-II catalyst
(
1.5 mg, 0.0021 mmol) in dry DMF (15 mL) to give 3n (1.86 g, 78%) as a yellow solid, mp 256–258 °C.
−1 1
IR: 3347, 3180, 1637, 1601, 1559 cm ; H-NMR (DMSO-d ): δ 8.27 (d, J = 6.4 Hz, 1H), 8.10–7.30
6

Molecules 2015, 20
7239
(
complex, 10H), 7.15 (s, 2H), 6.18 (br s, 2H), 5.76 (s, 1H), 5.65 (br s, 2H), 5.06 (s, 2H), 3.84 (s, 3H),
1
3
3
1
1
.80 (s, 3H), 3.67 (s, 2H); C-NMR (DMSO-d
6
): δ 165.9, 162.9, 158.8, 152.6, 149.1, 146.3, 144.5,
42.5, 139.1, 138.5, 137.6, 135.4, 132.4, 131.5, 129.3, 127.7, 127.3, 126.5, 124.6, 124.5, 123.8, 123.5,
22.4 (2C), 118.8, 117.6, 115.1, 107.1, 60.8, 55.8, 49.6, 32.0; Anal. Calcd for C32 S·2.1 H O:
H
28
N
6
O
3
2
C, 62.55; H, 5.18; N, 13.14. Found: C, 62.72; H, 4.91; N, 13.06.
(±)-(E)-1-[1-(Benzo[d]thiazol-2-yl)phthalazin-2(1H)-yl]-3-{5-[(2,4-diaminopyrimidin-5-yl)methyl]-
2
,3-dimethoxyphenyl}-2-propen-1-one (3o). This compound was prepared as above using 1 (1.50 g,
.89 mmol), 2o (1.61 g, 5.04 mmol), TEA (0.510 g, 0.70 mL, 5.05 mmol), and the Pd pincer-II catalyst
3
(
1 mg, 0.0014 mmol) in dry DMF (15 mL) to give 3o (1.61 g, 72%) as a white solid, mp 145–147 °C.
−1 1
IR: 3339, 3202, 1660, 1614, 1564 cm ; H-NMR (DMSO-d
6
): δ 8.04 (s, 1H), 8.03 (obscured dm, 1H),
7
1
7
.95 (d, J = 16.0 Hz, 1H), 7.91 (dd, J = 7.4, 0.8 Hz, 1H), 7.74 (d, J = 16.0 Hz, 1H), 7.63 (td, J = 7.4,
.2 Hz, 1H), 7.60 (m, 2H), 7.54 (td, J = 7.4, 0.8 Hz, 1H), 7.47 (td, J = 7.4, 0.8 Hz, 1H), 7.45 (s, 1H),
.40 (td, J = 7.4, 1.2 Hz, 1H), 7.34 (d, J = 1.6 Hz, 1H), 7.04 (d, J = 1.6 Hz, 1H), 6.66 (br s, 2H), 6.17
13
(
br s, 2H), 5.77 (s, 1H), 3.81 (s, 3H), 3.75 (s, 3H), 3.63 (s, 2H); C-NMR (DMSO-d
6
): δ 170.1, 168.1,
62.6, 160.3, 152.5, 152.2, 151.9, 146.3, 141.9, 137.8, 136.0, 134.7, 132.3, 130.1, 129.5, 127.8, 127.6,
26.7, 126.4, 125.5, 123.2, 122.8, 122.4, 118.6, 117.3, 115.2, 106.5, 60.8, 55.8, 52.5, 32.2; Anal. Calcd
S·3.0 H O·0.5 C OH: C, 58.70; H, 5.12; N, 14.08. Found: C, 58.49; H, 4.81; N, 14.15.
1
1
for C31
H N O
27 7 3
2
H
2 5
(±)-tert-Butyl (E)-2-[2-(3-{5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl}acryloyl)-1,2-
dihydrophthalazin-1-yl]acetate (3p). This compound was prepared as above using 1 (1.00 g, 2.59 mmol),
2
p (0.935 g, 3.12 mmol), TEA (0.313 g, 0.432 mL, 3.10 mmol), and the Pd pincer-II catalyst (1 mg,
0.0014 mmol) in dry DMF (15 mL) to give 3p (1.20 g, 83%) as a white solid. The melting point and
spectral data matched those in the literature [13].
(
±)-Ethyl
(E)-2-[2-(3-{5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl}acryloyl)-1,2-
dihydrophthalazin-1-yl]acetate (3q). This compound was prepared as above using 1 (1.00 g, 2.59 mmol),
2
q (0.849 g, 3.12 mmol), TEA (0.313 g, 0.432 mL, 3.10 mmol), and the Pd pincer-II catalyst (1 mg,
0.0014 mmol) in dry DMF (15 mL) to give 3q (1.10 g, 80%) as a white solid. The melting point and
spectral data matched those in the literature [13].
(±)-Methyl
(E)-2-[2-(3-{5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl}acryloyl)-1,2-
dihydrophthalazin-1-yl-acetate (3r). This compound was prepared as above using 1 (1.00 g, 2.59 mmol),
2
r (0.805 g, 3.12 mmol), TEA (0.313 g, 0.432 mL, 3.10 mmol), and the Pd pincer-II catalyst (1 mg,
.0014 mmol) in dry DMF (15 mL) to give 3r (1.10 g, 82%) as a white solid. The melting point and
0
spectral data matched those in the literature [13].
3
.5. Enantiomer Resolutions for (±)-3a and (±)-3d
Chiral HPLC enantiomer separations for (±)-3a and (±)-3d were performed using a Waters 600 series
pump in conjunction with a Waters Model 2487 (Milford, MA, USA). The Empower 3 software
Orlando, FL, USA) was used for instrument control, data acquisition, and data analysis. A Chiralpak®
IA column (10 mm × 250 mm id) from Chiral Technologies (West Chester, PA, USA) was used for all
(

Molecules 2015, 20
7240
of the analyses. The mobile phase consisted of MeCN–MeOH–Et
2
NH (50:50:0.1), and the samples
were dissolved in the mobile phase. All HPLC separations were performed at 25 °C with 3 mL/min
flow rate, while detection was monitored at 230 nm. For semi-preparative analyses, a 5 mL sample with
a concentration of 2 mg/mL was injected. In both cases the (S) enantiomer eluted first.
3
.6. X-ray Structure for (S,E)-3-{5-[(2,4-Diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl}-1-(1-
propylphthalazin-2(1H)-yl)-2-propen-1-one [(S)-(3d), (S)-RAB1]
A colorless, plate-shaped crystal of dimensions 0.340 × 0.140 × 0.050 mm was selected for structural
analysis. Intensity data for this compound were collected using a diffractometer with a Bruker APEX
ccd area detector [30,31] and graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). The sample
was cooled to 100(2) K. Cell parameters were determined from a non-linear least squares fit
of 2159 peaks in the range 2.50° < θ < 24.72°. A total of 18456 data were measured in the range
1
.312 < θ < 25.998° using φ and ω oscillation frames. The data were corrected for absorption by the
empirical method [32] giving minimum and maximum transmission factors of 0.971 and 0.996.
The data were merged to form a set of 9928 independent data with R(int) = 0.0596 and a coverage
of 99.9%.
The triclinic space group P1 was determined by statistical tests and verified by subsequent
refinement. The structure was solved by direct methods and refined by full-matrix least-squares
2
methods on F [33]. The positions of hydrogens bonded to carbons and nitrogens were initially
determined by geometry and were refined using a riding model. Hydrogens bonded to oxygens were
located on a difference map, and their positions were refined with a riding model. Non-hydrogen atoms
were refined with anisotropic displacement parameters. Hydrogen atom displacement parameters were
set to 1.2 (1.5 for methyl) times the isotropic equivalent displacement parameters of the bonded atoms.
2
A total of 676 parameters were refined against 9 restraints and 9928 data to give wR(F ) = 0.1912 and
2
2
2
2
+ 2F 2
]/3. The final R(F)
S = 0.968 for weights of w = 1/[σ (F ) + (0.0960P) ], where P = [F
o
c
was 0.0665 for the 5384 observed, [F > 4σ(F)], data. The largest shift/s.u. was 0.001 in the final
refinement cycle. The final difference map had maxima and minima of 0.486 and −0.417 e/Å3,
respectively. The absolute structure was determined by refinement of the Flack parameter [34]. The
three polar axis restraints were taken from Flack and Schwarzenbach [35].
3
.7. Biological Potency Measurements
Measurements of the MIC and the K utilized a racemic mixture, unless otherwise noted, of each
i
compound, as described earlier [8,13,27,36]. In brief, MIC values were based on standardized cultures
of B. anthracis Sterne strain as prescribed by the CLSI [29]. Evaluation of growth utilized
spectrophotometric values of turbidity at 600 nm and on visual inspection for assessment of bacterial
growth. The lowest concentration that yielded no growth after 18 h incubation was assigned as the
MIC. Evaluation of the enzymatic activity and inhibition utilized purified DHFR protein cloned from
B. anthracis Sterne strain and expressed recombinantly in E. coli BL21 (DE3) cells. The reaction was
reconstituted, including the NADPH co-factor, and was initiated by the addition of the dihydrofolate
substrate. The reaction was carried out at 30 °C, and the linear rate was monitored for 2.8 min. These
rates were plotted as a function of inhibitor concentration, and the 50% activity point was calculated

Molecules 2015, 20
7241
using a 4-parameter curve fit. These IC50 values were converted to K
i
values using the Cheng-Prusoff
equation [37].
4
. Conclusions
A newly developed protocol, employing dichlorobis[1-(dicyclohexylphosphanyl)piperidine]-
palladium(II) (Pd pincer-II), allowed for the Heck synthesis of potential drugs 3a–r under milder
conditions, which gave higher yields for substrates with sensitive/reactive functional groups. The
catalyst was highly active and enabled the reaction to be performed at much lower temperatures and in
shorter reaction times with low catalyst loading. Single crystal analysis of the most active enantiomer
of the lead compound revealed an orientation in the unit cell highly similar to that observed for this
isomer seated in the DHFR protein binding site. This could be a highly favorable property in terms of
the energetics of binding, as it seems that this inhibitor requires only limited re-orientation to conform
to the optimum position needed for binding to the enzyme substrate site. Biological studies
1
demonstrated that derivatives with smaller groups at R showed greater activity, with planar groups
such as allyl (3b) and vinyl (3c) exhibiting the highest potency both in inhibition of B. anthracis
culture growth and in the DHFR enzymatic assay. On the other hand, larger heteroaromatic
substituents, such as those in 3l–o, tempered the inhibition of the whole cell growth. These results
reinforced previous findings on the role of size and nature of functional groups that could fit into the
binding pocket. An in-house separation protocol for resolution of the S and R enantiomers was
developed for both 3a and 3d. Screening indicated that the S isomers produced greater inhibition at
both the whole cell growth and the enzymatic level with the DHFR protein.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/04/7222/s1.
Electronic Supplementary Information (ESI) available: CCDC 1052963 contains supplementary
crystallographic data for (S)-RAB1 [(S)-3d], see doi:10.3390/molecules20047222. These data can be
obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12
Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: deposit@ccdc.cam.ac.uk).
Acknowledgments
The authors gratefully acknowledge support of this work by the National Institutes of Allergy and
Infectious Diseases (R01-AI090685) of the NIH/NIAID and the Sitlington Chair in Infectious
Diseases, both to W.W.B. NSF (BIR-9512269), the Oklahoma State Regents for Higher Education, the
W. M. Keck Foundation, Conoco, Inc, and the College of Arts and Sciences at Oklahoma State
University provided funding for the NMR spectrometers of the Oklahoma State-Wide Shared NMR
Facility. NSF (CHE-0130835) and the University of Oklahoma provided funds to purchase of the
X-ray instrument and computers at the University of Oklahoma Chemical Crystallography Laboratory.
We are also pleased to acknowledge Douglas R. Powell (University of Oklahoma, Norman, OK, USA)
for his skill and effort in acquiring the X-ray structure. Finally, the authors wish to thank Christian M.

Molecules 2015, 20
7242
Frech (Zürich University of Applied Sciences, Universität Zürich, Zürich, Switzerland) for a generous
sample of dichlorobis[1-(dicyclohexylphosphanyl)piperidine]palladium(II) (the Pd pincer-II) catalyst.
Author Contributions
N.P.M., B.N., R.A.B. and K.D.B. performed the compound synthesis work. S.S. performed the
separation of enantiomers using chiral HPLC. C.R.B., M.H., E.W.B. and W.W.B. performed the
biological screening. R.A.B. and C.R.B. wrote the paper, but all authors read and approved the final
version of the manuscript before submission.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
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(