Inhibitor design to target a unique feature in the folate pocket of Staphylococcus aureus dihydrofolate reductase

Article abstract of DOI:10.1016/j.ejmech.2020.112412

Staphylococcus aureus (Sa) is a serious concern due to increasing resistance to antibiotics. The bacterial dihydrofolate reductase enzyme is effectively inhibited by trimethoprim, a compound with antibacterial activity. Previously, we reported a trimethoprim derivative containing an acryloyl linker and a dihydophthalazine moiety demonstrating increased potency against S. aureus. We have expanded this series and assessed in vitro enzyme inhibition (K_i) and whole cell growth inhibition properties (MIC). Modifications were focused at a chiral carbon within the phthalazine heterocycle, as well as simultaneous modification at positions on the dihydrophthalazine. MIC values increased from 0.0626–0.5 μg/mL into the 0.5–1 μg/mL range when the edge positions were modified with either methyl or methoxy groups. Changes at the chiral carbon affected K_i measurements but with little impact on MIC values. Our structural data revealed accommodation of predominantly the S-enantiomer of the inhibitors within the folate-binding pocket. Longer modifications at the chiral carbon, such as p-methylbenzyl, protrude from the pocket into solvent and result in poorer K_i values, as do modifications with greater torsional freedom, such as 1-ethylpropyl. The most efficacious K_i was 0.7 ± 0.3 nM, obtained with a cyclopropyl derivative containing dimethoxy modifications at the dihydrophthalazine edge. The co-crystal structure revealed an alternative placement of the phthalazine moiety into a shallow surface at the edge of the site that can accommodate either enantiomer of the inhibitor. The current design, therefore, highlights how to engineer specific placement of the inhibitor within this alternative pocket, which in turn maximizes the enzyme inhibitory properties of racemic mixtures.

Full text of DOI:10.1016/j.ejmech.2020.112412

Journal Pre-proof
Inhibitor design to target a unique feature in the folate pocket of Staphylococcus
aureus dihydrofolate reductase
N. Prasad Muddala, John C. White, Baskar Nammalwar, Ian Pratt, Leonard M.
Thomas, Richard A. Bunce, K. Darrell Berlin, Christina R. Bourne
PII:
S0223-5234(20)30383-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112412
EJMECH 112412
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 February 2020
Revised Date: 28 April 2020
Accepted Date: 28 April 2020
Please cite this article as: N.P. Muddala, J.C. White, B. Nammalwar, I. Pratt, L.M. Thomas, R.A.
Bunce, K.D. Berlin, C.R. Bourne, Inhibitor design to target a unique feature in the folate pocket of
Staphylococcus aureus dihydrofolate reductase, European Journal of Medicinal Chemistry (2020), doi:
https://doi.org/10.1016/j.ejmech.2020.112412.
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Inhibitor design to target a unique feature in the folate pocket of
Staphylococcus aureus dihydrofolate reductase
N. Prasad Muddala1,¶,#a, John C. White2,¶, Baskar Nammalwar1,#b, Ian Pratt2,
Leonard M. Thomas2, Richard A. Bunce1, K. Darrell Berlin1, and Christina R.
Bourne2*
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8
1: Department of Chemistry, 107 Physical Sciences I, Oklahoma State University,
Stillwater, OK 74078
9
10 2: Department of Chemistry and Biochemistry, 101 Stephenson Parkway, University of
11 Oklahoma, Norman, OK 73019
12
13 ¶Co-first authors with equivalent contributions, order determined alphabetically
14 *Corresponding author: cbourne@ou.edu +1 (405) 325-5348
15
16 #aCurrent Address: Impec Labs, Plot No. 5-5-35/87/1, 401, Prasanthi Nagar, IDA
17 Kukatpally, Hyderabad-500072, India
18 #bCurrent Address: Forge Therapeutics, 10578 Science Center Drive, Suite 205, San
19 Diego, California-92121, USA
20
21 Running Title: Targeting SaDHFR inhibitors to a unique surface pocket
22
23 Keywords: Dihydrofolate reductase, 2,4-diaminopyrimidine, Staphylococcal aureus,
24 antibacterial, enzyme inhibition, folate pathway, Heck coupling, alkyl
25 dihydrophthalazines
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31
1

32 Abstract
33 Staphylococcus aureus (Sa) is a serious concern due to increasing resistance to
34 antibiotics. The bacterial dihydrofolate reductase enzyme is effectively inhibited by
35 trimethoprim, a compound with antibacterial activity. Previously, we reported a
36 trimethoprim derivative containing an acryloyl linker and a dihydophthalazine moiety
37 demonstrating increased potency against S. aureus. We have expanded this series and
38 assessed in vitro enzyme inhibition (Ki) and whole cell growth inhibition properties
39 (MIC). Modifications were focused at a chiral carbon within the phthalazine heterocycle,
40 as well as simultaneous modification at positions on the dihydrophthalazine. MIC values
41 increased from 0.0626 – 0.5 µg/mL to the 0.5 – 1 µg/mL range when the edge positions
42 were modified with methyl or methoxy groups. Changes at the chiral carbon affected Ki
43 measurements but with little impact on MIC values. Our structural data revealed
44 accommodation by only the S-enantiomer of the inhibitors within the folate-binding
45 pocket. Longer modifications at the chiral carbon, such as p-methylbenzyl, would
46 protrude from the pocket into solvent and result in poorer Ki values, as do modifications
47 with greater torsional freedom, such as 1-ethylpropyl. The most efficacious Ki was 0.7 ±
48 0.3 nM, obtained with a cyclopropyl derivative containing dimethoxy modifications at the
49 dihydrophthalazine edge. The co-crystal structure revealed an alternative placement of
50 the phthalazine moiety into a shallow surface at the edge of the site that can
51 accommodate either enantiomer of the inhibitor. The current design, therefore,
52 highlights how to engineer specific placement of the inhibitor within this alternative
53 pocket, which in turn maximizes the enzyme inhibitory properties.
54
2

55 1. Introduction
56 The emergence of antibiotic resistance negatively impacts human life and causes a
57 substantial financial burden to society [1, 2]. Staphylococcus aureus, a gram-positive
58 pathogen, contributes substantially to serious human infections. Recent increases in S.
59 aureus infections have resulted from both a rise in nosocomial cases, including
60 endocarditis and growth on implanted devices, as well as through an increased
61 prevalence of skin and soft tissue infections. A recent study utilized whole genome
62 sequencing methods to track public exposure to pathogens and discovered high levels
63 of drug-resistant S. aureus in public spaces [3]. In 2017, the Centers for Disease
64 Control reported that 119,000 people in the US suffered from bloodstream infections
65 caused by both drug resistant and sensitive strains of this organism; of these, 20,000
66 individuals succumbed to the infection [4]. Furthermore, a sharp spike in S. aureus
67 infections has been associated with increased intravenous opioid abuse (4). Overall,
68 this highlights the continuing need for control measures to combat S. aureus infections.
69
70 The bacterial biosynthetic folate pathway has provided important strategic advances for
71 controlling bacterial growth, for which the synthetic compound trimethoprim (TMP) is a
72 gold standard inhibitor [5]. Mutations conferring resistance to trimethoprim are partially
73 responsible for the increased treatment failures of S. aureus infections [6-8], as are
74 additional mobile isoforms of DHFR containing mutations rendering them less
75 susceptible to TMP [9]. The folate pathway is also of interest due to the unusual
76 synergy of TMP-inhibited dihydrofolate reductase (DHFR) enzyme when combined with
77 one of the sulfa drug classes that inhibit the dihydroneopterin synthase enzyme. The
78 cause of this unique and highly potent synergy has recently been defined as “mutual
79 potentiation” that arises from stagnated metabolic flux of the precursor substrates [10].
80 Given the many positive benefits from DHFR inhibition, the development of next
81 generation antifolates continues to be heavily pursued [11, 12].
82
83 Inhibitors of DHFR are typically substrate mimetics that mediate competitive inhibition
84 with respect to the dihydrofolate. While these have wide medical applications targeting
85 mammalian DHFR [for example, 13-15], the current work is focused on compounds
3

86 specific for bacterial versions of DHFR. Considered “non-classical” due to the lack of
87 glutamylation, many rely on replacement of the native pterin heterocycle with a 2,4-
88 diaminopyrimidine (DAP) ring [11, 12, 16, 17]. One such highly potent DAP-containing
89 DHFR inhibitor is Iclaprim, originally developed by Basilea Pharmaceutica (Switzerland).
90 Despite multiple clinical trials and FDA applications for treatment of acute bacterial skin
91 and skin structure infections and hospital acquired bacterial pneumonia, it is currently
92 no longer in development [18, 19]. These reports indicate levels of hepatotoxicity,
93 although the extent of this is unclear.
Other successful DAP-containing DHFR
94 inhibitors include a collection of 7-aryl-2,4-diaminoquinazolines, in particular Rx101005,
95 developed by Trius Therapeutics and recently acquired by Merck via Cubist
96 Pharmaceuticals (San Diego, CA).
This compound has potent in vivo anti-
97 Staphylococcal activity and is highly bioavailable, but its development has been
98 discontinued [20]. A similar result was obtained for AR-709, which was developed by
99 Evolva (Switzerland) but is no longer listed as an active project [21, 22]. Another
100
compound, Emmacin, was identified through a diversity synthesis project sponsored by
AstraZeneca and Pfizer and is highly potent for MRSA; its current status is unknown
[23]. There is on-going active development of propargyl-linked trimethoprim derivatives
that have demonstrated potent activity against a wide spectrum of MRSA isolates [9,
24]. For this series, the linker extending beyond the DAP ring is longer and rigid,
allowing the compounds to explore unique positions within the main folate pocket that
impact the co-factor NADPH placement.
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The series of inhibitors in the current work were derived from compounds originally
designed by Basilea that led to the development of Iclaprim. Our earlier studies
demonstrated potent MIC values (≤ 0.0625-0.125 ug/mL) for MRSA and VRSA, which
are approximately 64-times more potent than the standard of care TMP-sulfa drug
combination [6]. These inhibitor molecules are based on trimethoprim, but are extended
from the central dimethoxyphenyl ring via an acryloyl linker with an appended
dihydrophthalazine system. The acryloyl linker allows rotational freedom of the
phthalazine moiety relative to the remaining 2,4-diaminopyrimidine (DAP) trimethoprim-
like structure. Structure determinations confirmed the exquisite conformational fit of the
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dihydrophthalazine ring system to the distal portion of the DHFR binding site from S.
aureus, as well as B. anthracis and E. faecalis [6, 25, 26]. These studies revealed an
unusual plasticity in the S. aureus DHFR site, delineated by the dihydrophthalazine
docked both in the canonical deeper folate binding pocket, as well as on a more
surface-exposed shallow cavity at the distal portion of the binding site [6].
2. Results and discussion
2.1 Design of the novel Staphylococcus aureus dihydrofolate reductase inhibitors
To further explore this phenomenon, we have now tested additional derivatives that vary
at the chiral center of the dihydrophthalazine, and in addition, have characterized the
impact of derivatization of the distal phthalazine edge with methyl or methoxy groups.
The moieties at the dihydrophthalazine chiral center have subtle influences on inhibition,
such that as they extend in length from the dihydrophthalazine, the inhibition in general
is reduced. This is likely due to surface exposure as they protrude outward from the
folate pocket. However, additional space occupied laterally along the binding site by
such modifications is favored, possibly due to additional contacts along the rim of the
binding pocket. Interestingly, derivatization of C6 and C7 of the dihydrophthalazine with
OCH3 or CH3 greatly impacted the whole cell inhibition (MIC values). This may be due
to a more limited ability to diffuse across cell membranes and thus interact with the
DHFR target.
In general, completed crystal structures support these findings.
Surprisingly, however, one derivative was able to completely occupy the shallow
surface pocket unique to S. aureus DHFR. This inhibitor structure combined a smaller
cyclopropyl at the chiral center of the dihydrophthalazine and bulkier methoxy additions
at the dihydrophthalazine edge. It remains to be seen if this inhibitor would interact and
inhibit other DHFR enzymes from bacteria that do not have this shallow surface.
Additional structures provide insights on the hydration of the empty folate-binding
pocket, with highly ordered and conserved water molecules demarking the polar
interaction sites with inhibitors. Further optimizing interactions at these polar sites
would provide additional improvement of inhibitors. The current inhibitor design clearly
outlines how to manipulate occupancy of the traditional deeper folate-binding pocket
versus the shallower surface pocket unique to S. aureus.
2.2 Chemistry
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Compounds were varied at the chiral stereocenter (Fig. 1, “R3” and Table S1), as this
position was previously identified as impacting the inhibitory profile of other bacterial
DHFR enzymes [25, 26].
Additional new modifications of methyl or methoxy
substituents at the distal positions of the dihydrophthalazine moiety were also explored
(Fig. 1, “R1, R2”). A series of racemic compounds was synthesized as shown in
Scheme I. The structural motif of these desired targets was achieved by synthesizing
two separate ring systems: the (i) 2,4-diaminopyrimidine ring (Scheme I) and the (ii)
substituted dihydrophthalazine rings (Scheme II).
The 2,4-diaminopyrimidine ring was generated from 5-iodovanillin derivative 2, as
previously described [27]. The 2,4-diaminopyrimidine ring construction involved the
preparation of 3-morpholinopropionitirile (1) reacting with 2 and NaOEt using DMSO as
solvent to obtain an adduct that later underwent cyclization in the presence of
PhNH2.HCl, guanidine hydrochloride and NaOEt in EtOH, to achieve the desired 3 as
shown in Scheme I.
Scheme I
50 oC
O
NH
CN
O
N
CN
1
I
I
(CH3O)2SO2
O
O
K2CO3 / DMF
120 oC
OH
OCH3
OCH3
OCH3
2
NH2
N
1. NaOEt / DMSO, 72 oC
I
N
2
1
2. PhNH2.HCl / EtOH, 78 oC
H2N
OCH3
3. (H2N)2C=NH.HCl / NaOMe
EtOH, 78 oC
OCH3
3
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The phthalazine moieties for the desired targets were obtained commercially or by
constructing substituted phthalazine heterocycles using a previously published
procedure.[28, 29] These substituted phthalazines (4 and 5) were subjected to
treatment with organomagnesium reagents in THF under anhydrous conditions to
provide racemic intermediates (7 and 8) in Scheme II. These intermediates were further
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subjected to N-acylation using acryloyl chloride and triethylamine in DCM to obtain the
H/methyl/methoxy 1,2-dihydrophthalazine derivatives (9 and 10). Coupling of the
acrylamides 9 and 10 with 2,4-diaminopyrimidine derivatives 3 was achieved via a Heck
reaction in the presence of Pd(OAc)2 and N-ethylpiperidine to afford the desired drug
candidates (12 and 13) in yields of 80-90% as shown in Scheme II [28-30].
Scheme II
O
R3
Cl
R3
R3MgBr
6 a-j
R1
R2
R1
R1
R2
O
N
N
HN
N
N
N
THF, 0 o
C
Et3N, DCM
R2
7 a-j (R1 = R2 = -OCH3)
8 a-f (R1 = R2 = -CH3)
4 (R1 = R2 = -OCH3)
5 (R1 = R2 = -CH3)
9 a-j (R1 = R2 = -OCH3)
10 a-f (R1 = R2 = -CH3)
NH2
O
R3
NH2
N
O
R3
Pd(OAc)2
1-Ethylpiperidine, DMF
120 o
I
R1
R2
R1
R2
N
N
N
N
N
H2N
N
OCH3
OCH3
H2N
N
OCH3
OCH3
C
9 a-j (R1 = R2 = -OCH3)
10 a-f (R1 = R2 = -CH3)
3
11 a-q (R1 = R2 = H)*
12 a-j (R1 = R2 = -OCH3)
13 a-f (R1 = R2 = -CH3)
177
Inhibitor
Name
11a*
11b*
11c*
11d*
11e*
11f*
Inhibitor
Name
12a
12b
12c
12d
12e
12f
R1 = R2
R3
R1 = R2
R3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
propyl
isopropyl
trifluoropropyl
isobutyl
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
-OCH3
ethyl
propyl
cyclopropyl
vinyl
isobutenyl
phenyl
o-tolyl
isobutenyl
1-ethylpropyl
cyclohexyl
phenyl
11g*
11h*
11i*
12g
12h
12i
o-ethylphenyl
o-tolyl
p-tolyl
-OCH3 o-methoxyphenyl
-OCH3 p-methoxyphenyl
11j*
12j
11k*
11l*
13a
13b
13c
13d
13e
13f
3,5-dimethylphenyl
m-fluorophenyl
p-fluorophenyl
benzyl
p-methylbenzyl
p-methoxybenzyl
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
ethyl
propyl
cyclopropyl
vinyl
isobutenyl
phenyl
11m*
11n*
11o*
11p*
11q*
p-trifluoromethoxybenzyl
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*
The synthesis of 11a-q is reported in our previous publication24
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3. Biological evaluation
3.1 Efficacy of inhibitors with SaDHFR enzyme activity and S. aureus growth
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The potency of each compound was evaluated for in vitro inhibition of the target DHFR
enzyme, and cell-based for its ability to prevent the growth of S. aureus cultures. In
general, trends of potency were correlated in both these assays.
For ease of
interpretation, the compounds are grouped based on the properties of the modifications
at position R3.
The group of alkyl-based modifications at R3 included ethyl, propyl, isopropyl,
cyclohexyl, trifluoropropyl, isobutyl, isobutenyl, cyclopropyl, vinyl, and 1-ethylpropyl (Fig.
1). The most efficacious modifications for in vitro enzyme inhibition were the cyclohexyl
(11g) and cyclopropyl (12c) moieties, followed by isopropyl (11b), propyl (11a), and
trifluoropropyl (11c), and with only slightly less potency when isobutyl (11d) or
isobutenyl (11e) were installed. This is a distinctly different preference profile from that
of B. anthracis DHFR, where the isobutyl and isobutenyl modifications were equally the
most potent [31]. Finally, 1-ethylpropyl (11f) installed at R3 was the least effective at
mediating enzyme inhibition among this series. When the torsional freedom of this
group is compared to, for example, the most potent inhibitor with a cyclohexyl moiety
(11g), it is clear that restricting the torsional freedom of the moiety at this position
results in better inhibition. When tested for whole cell inhibition of S. aureus cell growth,
variations in compound potency become less obvious (Fig. 1).
However,
dihydrophthalazine derivatization at R1 and R2 negatively impacts the MIC values by 2-
to 4-times, with the dimethoxy modification producing the least favorable inhibition. For
example, when R3 = propyl, the Ki values resulting from enzyme inhibition are
essentially the same, regardless of the dihydrophthalazine ring modification (1.2 ± 0.1
nM with R1 = R2 = H (11a), 1.1 ± 0.7 nM with R1 = R2 = OCH3 (12b), and 1.2 ± 0.5 nM
with R1 = R2 = CH3 (13b)). However, the MIC values are 0.0625-0.25 μg/mL for R1 = R2
= H (11a), 1 μg/mL for R1 = R2 = OCH3 (12b), and 0.5-1 μg/mL for R1 = R2 = CH3 (13b).
Overall, this trend is consistently independent of the modification at the R3 position,
indicating that the changes in potency must be due to solubility and membrane
permeability of these derivatives rather than direct inhibition at the target enzyme.
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The exception to this trend is the compound with R1 = R2 = OCH3 and R3 = cyclopropyl
(12c). It is strikingly potent in vitro, with the lowest Ki value at 0.7 ± 0.3 nM. The reason
for this improved target inhibition became clear when structural studies were completed
(below). However, this marked improvement was not realized at the whole cell level,
with MIC values remaining at the highest value for this series at 1 μg/mL.
The next class of R3 derivatives contains an aromatic moiety, and positions around this
ring were modified. These modifications were extended to also assess hydrophobic
versus polar substitutions. Overall, the placement of an aromatic moiety at position R3
resulted in a very potent inhibitor. While the unsubstituted phenyl modification was
among the most efficacious in vitro, modifications appending a methyl group in the
orthro, or fluorine in the para or meta positions had essentially no impact on the Ki
value. Alteration of the para position with methyl or dimethyl groups placed in
equivalent meta positions had worse inhibition in vitro. However, the p-methyl addition
(11j) had a surprisingly low MIC value, at 0.046-0.187 μg/mL. This was equivalent to
the other lowest MIC value (0.938-0.1875 μg/mL) in this series (11b, R3 = isopropyl).
The trend of equivalent Ki values, and yet increasing MIC values with
dihydrophthalazine derivatization at R1 = R2, is also evident with R3 = aromatic moieties.
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The final class of modifications was based on the success of aromatic substituents, but
extended the length of the R3 dihydrophthalazine heterocycle by one carbon atom,
generating R3 = benzyl derivatives (11n-q). This series is among the least effective,
and so was not included in the R1 = R2 derivatization. Extension from the ring structure
in the para position revealed a preference for a more polar p-methoxybenzyl group
(11p, Ki 1.2 ± 0.4) in comparison to a nonpolar p-methylbenzyl moiety (11o, Ki 2.3 ± 0.4
nM), which was the least potent in vitro among all the tested compounds. This class
appears to delineate the limit to modifications at this position, likely due to impinging on
the protein:solvent boundary as the R3 modifications become longer. This is also
reflected in the whole cell phenotypic assay, where the MIC value range is intermediate
with values of 0.25-1 μg/mL.
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3.2 Binding poses of selected inhibitors in the folate pocket of SaDHFR
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To assist in rationalizing results with this inhibitor series, we carried out crystallographic
studies of SaDHFR co-crystallized with the co-factor NADPH and saturated with
racemic inhibitor compounds (crystallographic data statistics are given in Table S2).
These efforts resulted in complexed structures for four inhibitors with R1 = R2 = H,
where R3 was 1-ethylpropyl (11f), p-tolyl (11j), 3,5-dimethylphenyl (11k) and benzyl
(11n), and two with R1 = R2 = OCH3, where R3 was cyclopropyl (12c) and p-
methoxyphenyl (12j). In an attempted X-ray structure determination with R1 = R2 = H
and R3 = p-methoxybenzyl (11p), the folate pocket was discovered to be void of the
inhibitor. This provided a fortuitous opportunity to compare the hydration of the empty
folate pocket with those systems with the inhibitor-complexed structures.
The structure of the diaminopyrimidine (DAP) ring in the current inhibitor series is
conserved from the compound trimethoprim, as are the contacting residues (Fig. 2
)
[32]. In particular, this portion of the binding site requires specific hydrogen bonds
formed with substrate, with inhibitor, or with water molecules. An acidic residue at
position 27 (Asp in Sa) forms hydrogen bonds to nitrogen atoms in the pterin of
dihydrofolate or in the diaminopyrimidine of an inhibitor. There are additional hydrogen
bonds between this amino moiety and the side chain oxygen of Thr111, as well as the
main chain carbonyls of Leu5, Val6, and Phe92. The absence of an inhibitor in one of
the crystal structures allows examination of the hydration of the folate pocket under
these crystallization conditions. Multiple water molecules bind within the empty folate
pocket and maintain a network that must be disrupted to allow substrate or inhibitor
access. Specific waters are positioned to satisfy polar interactions previously noted to
be critical to pterin or DAP binding (25,28). This pattern is extended by placement of a
water molecule at the central face, and thus between the nitrogen atoms of the
pyrimidine ring, which are typical of trimethoprim-based inhibitors and serve as a
mimetic of the substrate nitrogen-containing dihydropteridin heterocycle.
Other
hydrogen bonds and interactions conserved in this area of the binding site are directed
at the tetrahydropteridin-derived nitrogen that is reduced in the catalytic cycle. This
nitrogen atom can form bonds with the main chain carbonyl oxygen of residue Phe92,
as well as with atoms in the NADPH co-factor.
Elemental analyses of the final
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compounds 12a-j and 13a-f revealed a strong tendency to retain water, perhaps
mimicking the polar interactions of the DAP ring within the DHFR folate pocket.
The only other polar interaction between SaDHFR and the inhibitors was between a
methoxy group extending from the central ring, analogous to that found in trimethoprim,
with the side chain of Ser49 (Fig. 2B). The remaining contacts are all hydrophobic, and
as was previously noted, rely on shape complementarity to interact with the inhibitors [6,
30]. The closest approach of the inhibitor to atoms of the protein are carbon-carbon
atoms in the 3.5 Å to 4 Å range of residues Leu28 and Leu54, with Phe92 protruding
upwards from the base of the binding site and forming a surface that supports the
acryloyl linker. The dihydrophthalazine heterocycle occupies a groove along the
protein’s substrate pocket and makes van der Waals contact and hydrophobic
interactions with small hydrophobic residues or the aliphatic portions of longer side
chain residues. In particular, amino acids Leu28, Lys29, Val31, Lys32, Leu54 and
Pro55 line the crevice that conforms to the inhibitor shape (Fig. 2B, C). The aromatic
portion of the dihydrophthalazine moiety is adjacent to the polar regions of side chains
Asn56 and Arg57. The latter is a source of a persistently strained steric clash in all
inhibitor-bound structures from these series.
Co-crystals with inhibitors containing R3 = 1-ethylpropyl (11f, orange), p-tolyl (11j, light
green), 3,5-dimethylphenyl (12k, teal), and benzyl (11n, yellow) variations revealed a
remarkably conserved fit to the SaDHFR binding site (Fig. 2C). Each of these
compounds contain predominantly the
S-enantiomer, with minimal R-form visible for 1-
ethylpropyl and -tolyl derivatives. Given the low occupancy of these systems, they are
p
not modeled into the crystal structures (see Fig. S1). All S-enantiomers occupy a space
created by the aliphatic portions of lysine residues 29 and 32, with Leu28 and Val31
also in close proximity. The phenyl rings of p-tolyl and 3,5-dimethylphenyl occupy the
exact same position. The 1-ethylpropyl, which has relatively poor inhibitory properties,
had diffuse density (see Fig. S1 for the density from omit maps), consistent with higher
torsional freedom perhaps leading to the observed weaker inhibition. Its position closely
agrees with that of the benzyl derivative. It is clear that any extensions beyond this
11

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benzyl moiety would protrude from the binding site into solvent. This result defines the
limit of what can be accommodated at the R3 position of this inhibitor series.
These structures suggest that the complementarity of binding could be enhanced by
modifying the aromatic portion of the dihydrophthalazine ring with a polar group or by
including a hydrogen bond acceptor to interact with Arg57 (e.g., see the polar surface
coloring used in Fig. 2B). Other derivatives incorporate methyl or methoxy groups at
this distal position of the phthalazine (R1 = R2). Guided by the potency measurements,
only the methoxy-modified inhibitors were included in crystallization trials.
Co-
crystallization with R3 = -methoxyphenyl (12j) revealed a poorer fit of the phthalazine
p
into the crevice, with a 0.7 Å translation of this moiety upwards and out of the site (Fig.
3A, magenta). However, the R3 group aligned perfectly with the p-tolyl (11j, light green)
modification despite the change at the dihydrophthalazine edge. It seems likely,
therefore, that simultaneous modifications at R1 and R2 are not ideal, as the lower
portion of the phthalazine is already at the limits of what will fit in the binding site when
only a hydrogen atom is present.
A previous structure determination of SaDHFR with a propyl-derivative of the current
compound (R3 = propyl, R1 = R2 = H; 11a) series revealed conformational flexibility
inherent in the acryloyl-based linker of the inhibitors, which allowed the
dihydrophthalazine moiety to rotate into an alternate conformation [6]. The majority of
the current structures do not contain convincing electron density to allow modeling into
this shallow surface cavity. However, the exception is the R1 = R2 = OCH3 derivatives
of the dihydrophthalazine combined with an R3 = cyclopropyl (12c), which surprisingly
exhibits full occupancy of this alternative conformation. In this binding mode, the R-
enantiomer of the R3 modifications is favored, although the S-enantiomer still retains
some electron density (approx. 60% is in the
R state, with 40% in the S state, see Table
S2). This non-canonical conformation is significantly less buried than the other
inhibitors and is surrounded by hydrophobic residues Ile50, the aliphatic portion of
Lys52, Leu54 underneath the dihydrophthalazine, and Pro55 (Fig. 3B, cyan). The
methoxy groups at R1 and R2 do not appear to make any polar interactions, even with
12

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ordered water molecules. Instead, they appear to provide a bulk that strains the fit
within the canonical folate pocket. When the R3 group is large, as with the
methoxyphenyl modification (12j Fig. 3, magenta), this strain in the canonical site is
p-
,
tolerated to allow a more favored placement of R3. However, the smaller cyclopropyl
moiety occupies a completely solvent-exposed position, seemingly to favor the binding
of the dihydrophthalazine into the less-strained non-canonical site (12c, Fig. 3B, cyan).
Over-filling of the folate pocket, therefore, is key to accessing the non-canonical shallow
surface uniquely found in SaDHFR, and its complete occupancy has not been noted for
any other inhibitors. Binding of inhibitors in this arrangement has a benefit of lessening
the enantiomeric preference of the site, which would remove an eventual need
downstream for the purification of racemic mixtures.
4. Conclusions
We have synthesized and evaluated methyl- and methoxy- dihydrophthalazine-
appended DAP inhibitors for their ability to inhibit the DHFR enzyme and the whole cell
growth of S. aureus. This series extends previous work with the S. aureus organism
and reveals conservation of MIC values at or below 0.25 µg/mL, approximately 10-fold
lower than the parent trimethoprim antibacterial [6]. The aryl groups appended to the
dihydrophthalazine appear to hamper the permeability of the molecule, thus increasing
the MIC values for these derivatives.
Important conclusions can be taken from the variations in inhibitors tested by defining a
steric limit to modifications of the scaffold. For example, extensions beyond a benzyl
moiety at R3 result in poorer efficacy and likely distort the enzyme, precluding packing
into a crystal form as observed for the co-crystallization attempt with R3 =
p-
methoxybenzyl (R1 = R2 = H, 11p). This allowed comparison of the site of hydration
under the same crystallization conditions as that co-crystallized with inhibitors. The
DAP ring is well suited to substitute for the observed conserved water network, likely
driving the favorable interaction with all such DAP-containing inhibitors. Estimates of
hydration effects in inhibitor binding can be up to 4.4 kcal/mol per contact [33]. In the
current series, the strength and specificity of these interactions is likely highly important,
13

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anchoring the scaffold in the site while alternative interactions are possible with the
dihydrophthalazine moiety.
Structure determinations of co-crystallized SaDHFR with saturated racemic inhibitor
solutions consistently yield weak density at the distal end of the dihydrophthalazine
scaffold (Fig. S1). Interestingly, this seems to also be the case for the native folate
substrate. Recent studies on time-resolved catalysis by the well-characterized DHFR
from E. coli found similarly weak or diffuse electron density at this region of the pocket
[34]. This, again, reinforces the importance of the polar interactions with water
molecules, pterin heterocycles, or DAP ring structures to the binding energy and overall
ordering within the pocket.
The previous observation of an alternate binding surface, found specifically in SaDHFR,
has been confirmed in the current work [6]. The appending of additional bulk at the
distal edge of the dihydrophthalazine creates a strained fit within the folate pocket, as
seen in structure 12j (R1 = R2 = OCH3; R3 =
apparently tolerated to gain favorable placement of the relatively hydrophobic R3
moiety. When -methoxyphenyl at R3 is changed to a smaller cyclopropyl moiety (12c),
p-methoxyphenyl). However, this strain is
p
the energetics balance with the strain imparted by the R1 and R2 methoxy groups. In
this situation, the inhibitor is found to completely occupy the alternate binding site by
rotating at the linker and placing the dihydrophthalazine on a hydrophobic ledge within
the binding site. Furthermore, in this binding mode there is no observed preference for
enantiomers at the chiral R3 position. This novel insight then outlines the inhibitor
design needed to maintain inhibitor potency while targeting this unique feature of the
SaDHFR folate pocket.
5. Experimental
5.1 General methods
Commercial reagents were used directly as received. All reactions were performed
under nitrogen in oven-dried glassware. All Grignard reagents were purchased from
Sigma Aldrich. Commercial anhydrous (DMF) and dimethyl sulfoxide (DMSO) were
stored under dry nitrogen and transferred by syringe when needed. Tetrahydrofuran
14

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(THF) was dried over potassium hydroxide pellets and distilled from lithium aluminum
hydride prior to use. Reactions were monitored by thin layer chromatography (TLC) on
silica gel GF plates (Analtech, No. 21521) and visualized using a hand-held UV lamp.
Preparative column chromatography was carried out on silica gel (Sorbent
Technologies, 63-200 mesh) mixed with 0.5-1% UV-active phosphor (Sorbent
Technologies, No. UV-05). Melting points were determined using a MEL-TEMP
apparatus and were uncorrected. FT-IR spectra were run as dichloromethane solutions
1
13
on NaCl disks. H and C NMR spectra were measured at 400 MHz and 100 MHz or
300 MHz and 75 MHz, respectively, in the indicated solvent unless specified. Chemical
shifts (δ) are referenced to internal (CH3)4Si and coupling constants (J) are given in Hz.
Elemental analyses (± 0.4%) were performed by Atlantic Microlabs, Inc., Norcross, GA
30071.
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433
434
5.1.1. 3-Morpholinopropionitrile (1): This compound was prepared on a 0.47 mol scale
according to the literature procedure [27, 35]. The product was distilled at 88-90 oC/0.5
o
mm Hg (lit [35, 36] bp 149 C/20 mm Hg) to give
1
(38.2 g, 95%) as a colorless liquid.
3.72 (t, 4H, = 4.7 Hz), 2.68 (t, 2H, = 6.8
= 4.7 Hz); 13C NMR (75 MHz):
118.6, 66.7,
IR: 2253 cm-1; 1H NMR (CDCl3, 400 MHz):
δ
J
J
Hz), 2.52 (t, 2H,
53.6, 53.0, 15.7.
J
= 7.0 Hz), 2.50 (t, 4H,
J
δ
5.1.2. 5-Iodo-3,4-dimethoxybenzaldehyde (2): This compound was prepared on a
0.27-mol scale using the method of Nimgirawath [37]. The crude product was
o
recrystallized (4:1 ethanol:water) to give
(lit [37] mp 71-72 oC). IR: 2832, 2730, 2693 cm-1; 1H NMR (CDCl3, 300 MHz):
1H), 7.85 (d, 1H, = 1.7 Hz), 7.41 (d, 1H, = 1.7 Hz), 3.93 (s, 3H), 3.92 (s, 3H); C
NMR (75 MHz): 189.7, 154.2, 153.0, 134.7, 133.9, 111.0, 92.1, 60.7, 56.1.
2
(25.2 g, 96%) as a white solid, mp 71-72 C
δ
9.83 (s,
13
J
J
δ
5.1.3. 2,4-Diamino-5-(5-iodo-3,4-dimethoxybenzyl)pyrimidine (
of Roth et al. [38] was modified. To a stirred solution of (6.92 g, 54.1 mmol) in DMSO
(20 mL), NaOMe (0.29 g, 5.40 mmol) was added and heated at 70-72 C. A pre-
heated solution of (12.2 g, 41.8 mmol) in DMSO (15 mL) was added to the reaction
mixture dropwise over a period of 15 min and the reaction was heated for an additional
45 min. The crude reaction mixture was poured into cold ice water (50 mL) and
extracted with DCM (3 100 mL). The combined organic layers were washed with
3): The general method
1
o
2
×
satd. NaCl (100 mL), dried (MgSO4) and concentrated under vacuum to give 3-
morpholino-2-(5-iodo-3,4-dimethoxybenzyl)acrylonitrile (90%) as a dark red oil. The
15

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crude material was further dissolved in ethanol (75 mL), followed by addition of aniline
hydrochloride (6.76 g, 52.2 mmol), and refluxed for 1 h. During the reflux, guanidine
hydrochloride (9.55 g, 100 mmol) and sodium methoxide (9.00 g, 167 mmol) were
added and the reflux was continued for an additional 3 h. The reaction mixture was
o
then concentrated to 1/3rd volume and cooled to 0 C for 30 min. Addition of ice-cold
water (40 mL) and stirring resulted in an off-white product as a precipitate. The
resulting crude product was filtered, washed and recrystallized (EtOH:H2O (4:1)) to give
o
1
3
(9.68 g, 60%) as a tan solid, mp 217-218 C. IR: 3467, 3315, 3140, 1638 cm-1; H
NMR (DMSO-
Hz), 6.16 (br s, 2H), 5.77 (br s, 2H), 3.77 (s, 3H), 3.66 (s, 3H), 3.54 (s, 2H); C NMR
(DMSO- 6, 75 MHz): 162.4, 162.1, 156.0, 152.0, 146.3, 138.9, 129.1, 113.8, 105.2,
92.4, 59.8, 55.8, 31.7.
d6, 300 MHz): δ 7.57 (s, 1H), 7.14 (d, 1H, J = 1.8 Hz), 6.98 (d, 1H, J = 1.8
13
d
δ
5.2
5.2.1. (±)-1-(6,7-Dimethoxy-1-ethylphthalazin-2(1H)-yl)prop-2-en-1-one (9a): To a
stirred solution of 6,7-dimethoxyphthalazine ( , 2.00 g, 16.3 mmol) [39] in dry THF (60
.
4
o
mL) at 0 C, ethylmagnesium bromide (6a,12.6 mL, 12.6 mmol) was added dropwise
over a period of 30 min. Stirring was continued at 0 C for 30 min and continued at
o
room temperature for 1 h. The reaction mixture was quenched with NH4Cl (50 mL) and
extracted with EtOAc (3 × 100 mL). The organic layer was washed with satd. NaCl (30
mL), dried (MgSO4) and concentrated under vacuum to give the crude (±)-1,2-dihydro-1-
ethyl-6,7-dimethoxyphthalazine (7a) as a viscous oil (90%). The material was taken to
the next step without further purification.
To a stirred cooled solution of 7a in DCM (150 mL), TEA (15.8 mL, 2.21 mmol)
was added, followed by acryloyl chloride (0.95 mL, 11.6 mmol), and the reaction was
stirred for 2 h. The reaction mixture was quenched with water (50 mL) and extracted
with DCM (3 × 30 mL). The combined extracts were washed with satd. NaCl (50 mL),
dried (MgSO4) and concentrated under vacuum. The residue was purified on a column
chromatography using silica gel with EtOAc:hexanes (3:7) to give 9a (2.05 g, 57%) as a
1
viscous, yellow oil. IR: 2839, 1659, 1614 cm-1; H NMR (CDCl3, 300 MHz):
1H), 7.31 (dd,
δ
7.52 (s,
= 17.2, 1.9
= 6.6 Hz, 1H), 3.93 (s, 3H), 3.91 (s,
166.2, 151.8,
J = 17.2, 10.2 Hz, 1H), 6.79 (s, 1H), 6.67 (s, 1H), 6.46 (dd, J
Hz, 1H), 5.77 (dd,
J
= 10.2, 1.9 Hz, 1H), 5.74 (t, J
13
3H), 1.69 (m, 2H), 0.83 (t,
J
= 7.4 Hz, 3H); C NMR (CDCl3, 75 MHz):
δ
148.6, 144.2, 128.1, 127.4, 127.3, 116.9, 109.2, 108.3, 56.12, 56.07, 52.1, 28.3, 9.6.
5.2.2. (±)-1-(6,7-Dimethoxy-1-propylphthalazin-2(1H)-yl)prop-2-en-1-one
(9b): This
compound was prepared using the same procedure as for 9a above. Yield: 2.03 g
o
1
(67%) as an off-white solid, mp 58-60 C; IR: 2843, 1659, 1614 cm-1; H NMR (CDCl3,
300 MHz): 7.53 (s, 1H), 7.29 (dd, = 17.2, 10.2 Hz, 1H), 6.78 (s, 1H), 6.67 (s, 1H),
6.46 (d,
δ
J
J
= 17.2 Hz, 1H), 5.78 (overlapping d,
J = 10.1 Hz, 1H and t, J = 6.5 Hz, 1H),
16

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3.93 (s, 3H), 3.91 (s, 3H), 1.61 (m, 2H), 1.27 (sextet,
3H); 13C NMR (CDCl3, 75 MHz):
166.3, 151.9, 148.6, 142.5, 128.2, 128.0, 127.3,
116.9, 109.2, 108.4, 56.2, 56.1, 50.9, 37.5, 18.5, 13.9.
J = 7.3 Hz, 2H), 0.84 (t, J = 7.3 Hz,
δ
5.2.3.
(±)-1-(1-Cyclopropyl-6,7-dimethoxyphthalazin-2(1H)-yl)prop-2-en-1-one (9c):
This compound was prepared using the same procedure as above. Yield: 2.48 g (66%)
1
as a colorless viscous, yellow oil; IR: 2843, 1658, 1613 cm-1; H NMR (CDCl3, 300
MHz):
(dd,
(s, 3H), 3.92 (s, 3H), 1.17 (m, 1H), 0.58 (m, 1H), 0.43 (m, 2H), 0.32 (m, 1H); C NMR
(CDCl3, 75 MHz): 166.4, 151.8, 148.7, 143.4, 128.0, 127.3, 126.5, 117.0, 109.2,
108.1, 56.1, 56.0, 53.5, 16.6, 3.5, 2.3.
δ
7.58 (s, 1H), 7.35 (dd,
J
= 17.1, 10.5 Hz, 1H), 6.81 (s, 1H), 6.67 (s, 1H), 6.47
J
= 17.1, 2.0 Hz, 1H), 5.79 (dd,
J
= 10.5, 2.0 Hz, 1H), 5.46 (d, = 7.8 Hz, 1H), 3.94
J
13
δ
5.2.4.
compound was prepared using the same procedure as above. Yield: 2.07 g (58%) as a
colorless oil; IR: 2852, 1661, 1614 cm-1; 1H NMR (CDCl3, 300 MHz):
7.51 (s, 1H), 7.31
(dd, = 17.3, 10.4 Hz, 1H), 6.79 (s, 1H), 6.71 (s, 1H), 6.49 (dd, = 17.3, 1.6 Hz, 1H),
6.31 (d, = 5.1 Hz, 1H), 5.82 (m, 1H), 5.79 (d, = 10.2 Hz, 1H), 5.11 (d, = 10.2 Hz,
1H), 4.89 (d,
= 17.0 Hz, 1H), 3.93 (s, 3H), 3.92 (s, 3H); 13C NMR (CDCl3, 75 MHz):
(±)-1-(6,7-Dimethoxy-1-vinylphthalazin-2(1H)-yl)prop-2-en-1-one (9d):
This
δ
J
J
J
J
J
J
δ
166.3, 152.1, 149.0, 141.7, 134.7, 128.6, 127.1, 125.6, 117.0, 116.3, 109.4, 108.5,
56.23, 56.16, 52.7.
5.2.5. (±)-1-(6,7-Dimethoxy-1-(2-methylprop-1-en-1-yl)phthalazin-2(1H)-yl)prop-2-en-1-
one (9e): This compound was prepared using the same procedure as above. Yield:
1
1.95 g (62%) as an off-white solid, mp 52-54 °C; IR: 2836, 1660, 1609 cm-1; H NMR
(CDCl3, 300 MHz):
1H), 6.45 (dd, = 17.0, 1.5 Hz, 1H), 6.44 (d,
1H), 5.24 (d, = 9.7 Hz, 1H), 3.92 (s, 3H), 3.90 (s, 3H), 2.02 (s, 3H), 1.65 (s, 3H); C
NMR (CDCl3, 75 MHz): 166.0, 152.2, 148.7, 141.7, 134.1, 128.2, 128.0, 127.4, 122.3,
δ
7.52 (s, 1H), 7.39 (dd,
J
= 17.0, 10.4 Hz, 1H), 6.77 (s, 1H), 6.60 (s,
J
J
= 9.7 Hz, 1H), 5.75 (dd, = 10.4, 1.5 Hz,
J
13
J
δ
116.3, 108.6, 108.4, 56.1 (2C), 49.5, 25.7, 18.6.
5.2.6. (±)-1-(6,7-Dimethoxy-1-phenylphthalazin-2(1H)-yl)prop-2-en-1-one (9f):
This
compound was prepared using the same procedure as above. Yield: 1.85 g (56%) as a
viscous, colorless oil; IR: 2835, 1660, 1609 cm-1; 1H NMR (CDCl3, 300 MHz):
7.58 (s,
1H), 7.31 (dd, = 17.2, 10.5 Hz, 1H), 7.26-7.17 (complex, 5H), 6.92 (s, 1H), 6.83 (s,
1H), 6.70 (s, 1H), 6.46 (dd, = 17.5, 2.0 Hz, 1H), 5.76 (dd, = 10.5, 2.1 Hz, 1H), 3.92
(s, 3H), 3.87 (s, 3H); 13C NMR (CDCl3, 75 MHz):
166.5, 152.3, 149.0, 141.8, 141.1,
δ
J
J
J
δ
128.6, 127.8, 127.3, 126.5, 116.8, 109.7, 108.4, 56.20, 56.16, 53.8 (two aromatic C
unresolved).
17

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523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
5.2.7.
(±)-1-(6,7-Dimethoxy-1-(2-methylphenyl)phthalazin-2(1H)-yl)prop-2-en-1-one
(9g): This compound was prepared using the same procedure as above. Yield: 2.04 g
(58%) as a white solid, mp 73-75 °C; IR: 2835, 1662, 1616 cm-1; H NMR (CDCl3, 300
MHz): 7.51 (s, 1H), 7.32 (dd, = 17.2, 10.6 Hz, 1H), 7.18 (dd, = 7.4, 1.6 Hz, 1H),
7.15-7.01 (complex, 3H), 6.90 (s, 1H), 6.78 (s, 1H), 6.54 (s, 1H), 6.38 (dd, = 17.2, 2.1
Hz, 1H), 5.71 (dd,
= 10.5, 2.2 Hz, 1H), 3.89 (s, 3H), 3.81 (s, 3H), 2.73 (s, 3H); 13C
NMR (CDCl3, 75 MHz): 166.4, 152.3, 148.7, 142.3, 140.0, 132.7, 130.7, 128.3, 128.1,
1
δ
J
J
J
J
δ
127.7, 127.6, 127.5, 126.8, 115.4, 108.8, 108.7, 56.1, 56.0, 52.0, 20.1.
5.2.8. (±)-1-(1-(2-Ethylphenyl)-6,7-dimethoxyphthalazin-2(1H)-yl)prop-2-en-1-one (9h):
This compound was prepared using the same procedure as above. Yield: 1.90 g (53%)
as a white solid, mp 69-71 °C; IR: 2830, 1663, 1616 cm-1; 1H NMR (CDCl3, 300 MHz):
δ
7.52 (s, 1H), 7.32 (dd,
J
= 17.2, 10.5 Hz, 1H), 7.19 (td, 2H,
= 7.8, 1.1 Hz), 7.03 (td, = 7.4, 1.6 Hz, 1H), 6.97 (s, 1H), 6.78 (s, 1H), 6.58 (s, 1H),
= 17.2, 2.1 Hz, 1H), 5.70 (dd, = 10.5, 2.1 Hz, 1H), 3.89 (s, 3H), 3.80 (s,
= 7.4 Hz, 3H); C NMR (CDCl3, 75 MHz):
J = 7.8, 1.1 Hz), 7.15 (td, 1H,
J
J
6.38 (dd,
J
J
13
3H), 3.18 (m, 2H), 1.41 (t,
J
δ 166.4, 152.3,
148.6, 141.4, 139.9, 128.6, 128.1, 127.84, 127.78, 127.6, 126.5, 115.5, 113.5, 109.1,
108.7, 56.1, 55.9, 51.5, 25.2, 15.6 (one aromatic C was unresolved).
5.2.9.
(±)-1-(6,7-Dimethoxy-1-(2-methoxyphenyl)phthalazin-2(1H)-yl)prop-2-en-1-one
(9i): This compound was prepared using the same procedure as above. Yield: 2.03 g
(55%) as a white solid, mp 71-72 °C; IR: 2836, 1664, 1616 cm-1; H NMR (CDCl3, 300
MHz): 7.51 (s, 1H), 7.39 (dd, = 17.2, 10.5 Hz, 1H), 7.21-7.13 (complex, 3H), 7.04 (s,
1H), 6.86 (d, = 8.2 Hz, 1H), 6.81 (t, = 7.4 Hz, 1H), 6.72 (s, 1H), 6.40 (dd, = 17.2,
2.0 Hz, 1H), 5.76 (dd,
= 10.5, 2.2 Hz, 1H), 3.96 (s, 3H), 3.86 (s, 6H); 13C NMR (CDCl3,
75 MHz): 166.3, 154.9, 152.0, 148.6, 140.6, 132.1, 128.9, 128.4, 128.0, 127.4, 126.6,
1
δ
J
J
J
J
J
δ
121.3, 115.6, 111.3, 109.4, 108.5, 56.1, 55.9, 55.8, 49.9.
5.2.10. (±)-1-(6,7-Dimethoxy-1-(4-methoxyphenyl)phthalazin-2(1H)-yl)prop-2-en-1-one
(
9j): This compound was prepared using the same procedure as above. Yield: 2.10 g
(59%) as a yellow solid, mp 55-56 °C; IR: 2836, 1659, 1609 cm-1; 1H NMR (CDCl3, 300
MHz): 7.58 (s, 1H), 7.28 (dd, = 17.2, 10.5 Hz, 1H), 7.14 (d, = 9.0 Hz, 2H), 6.89 (s,
1H), 6.84 (s, 1H), 6.77 (d, = 9.0 Hz, 2H), 6.67 (s, 1H), 6.45 (dd, = 17.2, 2.2 Hz, 1H),
δ
J
J
J
J
5.75 (dd,
75 MHz):
J
= 10.5, 2.2 Hz, 1H), 3.93 (s, 3H), 3.87 (s, 3H), 3.73 (s, 3H); 13C NMR (CDCl3,
166.5, 159.1, 152.4, 149.0, 141.8, 133.6, 128.8, 128.5, 127.4, 126.8, 116.9,
δ
113.9, 109.6, 108.3, 56.21, 56.20, 55.3, 53.2.
5.3.
5.3.1.
(±)-1-(1-Ethyl-6,7-dimethylphthalazin-2(1H)-yl)prop-2-en-1-one (10a
): This
compound was prepared with dimethylphthalazine (
5) [39] using the same procedure as
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for 9a above. Yield: 2.29 g (60%) as a viscous, colorless oil; IR: 1663, 1619 cm-1; H
NMR (CDCl3, 300 MHz): 7.52 (s, 1H), 7.32 (dd, = 17.2, 10.5 Hz, 1H), 7.02 (s, 1H),
6.91 (s, 1H), 6.45 (dd, = 17.2, 2.3 Hz, 1H), 5.75 (dd, = 10.5, 2.3 Hz, 1H), 5.74 (t,
7.1 Hz, 1H), 2.29 (s, 3H), 2.26 (s, 3H), 1.63 (m, 2H), 0.81 (t,
= 7.5 Hz, 3H); 13C NMR
(CDCl3, 75 MHz): 166.3, 142.6, 140.7, 136.4, 131.4, 128.0, 127.7, 127.4, 126.8,
δ
J
J
J
J =
J
δ
121.9, 52.3, 28.4, 20.1, 19.5, 9.5.
5.3.2. (±)-1-(6,7-Dimethyl-1-propylphthalazin-2(1H)-yl)prop-2-en-1-one (10b): This
compound was prepared using the same procedure as above. Yield: 2.26 g (70%) as
an off-white solid, mp 62-64 oC; IR: 1663, 1619 cm-1; 1H NMR (CDCl3, 300 MHz):
δ
7.55
= 17.2,
J = 10.5, 2.3 Hz, 1H), 2.29 (s, 3H), 2.27
(s, 1H), 7.30 (dd,
2.3 Hz, 1H), 5.77 (t,
(s, 3H), 1.59 (m, 2H), 1.25 (sextet,
(CDCl3, 75 MHz): 166.2, 142.8, 140.8, 136.4, 131.9, 128.1, 127.6, 127.4, 126.8,
J
= 17.2, 10.5 Hz, 1H), 7.04 (s, 1H), 6.92 (s, 1H), 6.43 (dd,
= 6.7 Hz, 1H), 5.75 (dd,
= 7.3 Hz, 2H), 0.85 (t,
J
J
13
J
J = 7.4 Hz, 3H); C NMR
δ
121.8, 51.0, 37.6, 20.1, 19.5, 18.3, 13.9.
5.3.3. (±)-1-(1-Cyclopropyl-6,7-dimethylphthalazin-2(1H)-yl)prop-2-en-1-one (10c): This
compound was prepared using the same procedure as above. Yield: 2.61 g (65%) as a
yellow oil; IR: 1666, 1619 cm-1; 1H NMR (CDCl3, 300 MHz):
δ
7.60 (s, 1H), 7.33 (dd,
J
=
17.2, 10.5 Hz, 1H), 7.06 (s, 1H), 6.92 (s, 1H), 6.45 (dd,
= 10.5, 2.0 Hz, 1H), 5.42 (d,
0.60 (m, 1H), 0.41 (m, 2H), 0.30 (m, 1H); 13C NMR (CDCl3, 75 MHz):
141.0, 136.7, 130.7, 128.2, 127.7, 127.5, 126.7, 122.0, 53.8, 20.2, 19.6, 16.8, 3.8, 2.4.
J
= 17.2, 2.0 Hz, 1H), 5.77 (dd,
J
J
= 8.2 Hz, 1H), 2.31 (s, 3H), 2.28 (s, 3H), 1.16 (m, 1H),
166.6, 142.9,
δ
5.3.4.
(±)-1-(6,7-Dimethyl-1-vinylphthalazin-2(1H)-yl)prop-2-en-1-one (10d):
This
1.85 g (61%) as a
7.52 (s, 1H), 7.32
compound was prepared using the same procedure as above. Yield
viscous, yellow oil; IR: 1664, 1619 cm-1; 1H NMR (CDCl3, 300 MHz):
:
δ
(dd,
6.30 (d,
Hz, 1H), 4.88 (d,
166.3, 142.1, 141.2, 137.0, 135.1, 129.7, 128.7, 128.0, 127.2, 127.0, 121.7, 115.9,
53.0, 20.2, 19.6.
J
= 17.2, 10.5 Hz, 1H), 7.06 (s, 1H), 6.98 (s, 1H), 6.51 (dd,
= 4.7 Hz, 1H), 5.80 (m, 1H), 5.79 (dd, = 10.5, 1.9 Hz, 1H), 5.08 (d,
= 16.8 Hz, 1H), 2.30 (s, 3H), 2.28 (s, 3H); 13C NMR (CDCl3, 75 MHz):
J
= 17.2, 1.9 Hz, 1H),
J
J
J
= 9.2
J
δ
5.3.5.
(±)-1-(6,7-Dimethyl-1-(2-methylprop-1-en-1-yl)phthalazin-2(1H)-yl)prop-2-en-1-
one (10e): This compound was prepared using the same procedure as above. Yield:
2.71 g (66%) as an off-white solid, mp 51-53 °C; IR: 1666, 1619 cm-1; H NMR (CDCl3,
1
300 MHz):
6.45 (dd,
1H), 5.24 (d,
δ
7.52 (s, 1H), 7.29 (dd,
= 17.3, 2.0 Hz, 1H), 6.43 (d,
= 10.0 Hz, 1H), 2.27 (s, 3H), 2.25 (s, 3H), 2.02 (s, 3H), 1.63 (s, 3H); 13C
J
= 17.2, 10.5 Hz, 1H), 7.02 (s, 1H), 6.88 (s, 1H),
J
J
= 10.0 Hz, 1H), 5.99 (dd, = 10.5, 2.0 Hz,
J
J
19

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NMR (CDCl3, 75 MHz): δ 166.0, 141.9, 141.3, 136.4, 133.9, 132.2, 128.0, 127.5, 127.2,
126.9, 122.4, 121.0, 49.7, 25.7, 20.1, 19.5, 18.6.
5.3.6. (±)-1-(6,7-Dimethyl-1-phenylphthalazin-2(1H)-yl)prop-2-en-1-one (10f): This
compound was prepared using the same procedure as above: Yield: 2.10 g (58%) as a
o
1
white solid, mp 69-70 C; IR: 1663, 1618 cm-1; H NMR (CDCl3, 300 MHz):
δ
7.59 (s,
= 17.2, 10.5 Hz, 1H), 7.26-7.15 (complex, 5H), 7.10 (s, 1H), 7.00 (s,
1H), 6.88 (s, 1H), 6.45 (dd, = 17.2, 1.9 Hz, 1H), 5.76 (dd, = 10.5, 1.9 Hz, 1H), 2.27
(s, 3H), 2.26 (s, 3H); 13C NMR (CDCl3, 75 MHz):
166.4, 142.0, 141.54, 141.50, 136.9,
1H), 7.32 (dd,
J
J
J
δ
130.7, 128.6 (2C), 128.3, 127.7, 127.3, 127.0, 121.4, 54.2, 20.2, 19.5 (one aromatic C
unresolved).
5.4.
5.4.1. Synthesis of Derivatives 11a-q
.
The preparation of these compounds was
previously reported [27].
5.4.2. (±)-(E)-3-(5-((2,4-Diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)-1-(1-ethyl-
6,7-dimethoxyphthalazin-2(1H)-yl)prop-2-en-1-one (12a): To stirred solution of 2,4-
diamino-5-(5-iodo-3,4-dimethoxybenzyl)pyrimidine (
(25 mL), (±)-1-(6,7-dimethoxy-1-ethylphthalazin-2(1H)-yl)prop-2-en-1-one (9a) (2.23 g,
7.76 mmol), palladium acetate (0.143 g, 0.64 mmol), and -ethylpiperidine (2.67 mL,
3) (2.50 g, 6.47 mmol) in dry DMF
N
o
19.4 mmol) were added at heated at 120 C for 12 h. After completion, the reaction
mixture was poured into ice-cold water (40 mL) and extracted EtOAc (4 × 20 mL). The
organic layers were combined, washed with satd. NaCl (50 mL), dried (MgSO4) and
concentrated under vacuum to give the crude product. The dark brown residue was
purified by silica gel chromatography eluted with MeOH:DCM:TEA by silica gel
chromatography (5:94:1) to furnish a yellow solid. This solid was further purified by
recrystallization from MeOH:Et2O (2:3) to give 12a (2.02 g, 59%) as a white solid, mp
1
215-217 °C. IR: 3424, 3334, 3145, 3105, 2839, 1657, 1638, 1600 cm-1; H NMR
(DMSO-d6, 300 MHz):
1H), 7.59 (s, 1H), 7.24 (d,
1H), 6.27 (br s, 2H), 5.81 (br s, 2H), 5.75 (t,
3.79 (s, 3H), 3.73 (s, 3H), 3.59 (s, 2H), 1.60 (m, 2H), 0.74 (t,
(DMSO-d6, 75 MHz): 165.5, 162.2, 161.8, 154.9, 152.4, 152.6, 148.2, 145.9, 142.5,
δ
7.85 (d,
= 1.5 Hz, 1H), 7.11 (s, 1H), 7.05 (s, 1H), 6.98 (d,
= 6.3 Hz, 1H), 3.83 (s, 3H), 3.80 (s, 3H),
= 7.4 Hz, 3H); 13C NMR
J
= 16.0 Hz, 1H), 7.78 (s, 1H), 7.63 (d,
J
= 16.0 Hz,
J = 1.5 Hz,
J
J
J
δ
136.4, 136.1, 127.8, 126.8, 118.2, 118.0, 116.5, 114.6, 109.8, 109.0, 105.8, 60.7, 55.7,
55.6, 55.5, 51.3, 32.3, 27.8, 9.3. Anal. Calcd for C28H32N6O5·1.0 H2O: C, 61.08; H,
6.22; N, 15.26. Found: C, 61.16; H, 6.01; N, 15.11.
5.4.3.
(±)-(E)-3-(5-((2,4-Diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)-1-(6,7-
dimethoxy-1-propylphthalazin-2(1H)-yl)prop-2-en-1-one (12b): This compound was
20

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673
prepared using the same procedure as for 12a above. Yield: 2.18 g (62%) as a white
1
solid, mp 228-230 °C; IR: 3356, 3222, 3160, 2838, 1662, 1633, 1606 cm-1; H NMR
(DMSO-
1H), 7.58 (s, 1H), 7.38 (s, 1H), 7.14 (s, 1H), 7.05 (s, 1H), 7.01 (s, 1H), 6.88 (br s, 2H),
6.40 (br s, 2H), 5.81 (t, = 6.4 Hz, 1H), 3.84 (s, 3H), 3.80 (s, 6H), 3.75 (s, 3H), 3.63 (s,
2H), 1.53 (m, 2H), 1.19 (sextet, = 7.5 Hz, 2H), 0.82 (t,
= 7.4 Hz, 3H); 13C NMR
(DMSO- 6, 101 MHz): 164.9, 162.3, 152.0, 151.1, 147.7, 145.5, 142.3, 135.6, 134.9,
127.4, 126.8, 118.0, 117.6, 115.9, 114.2, 109.2, 108.6, 106.4, 60.2, 55.3, 55.2, 55.1,
49.5, 36.5, 31.5, 17.3, 13.2 (two aromatic C unresolved). Anal. Calcd for
C29H34N6O5·4.5 H2O: C, 55.49; H, 6.31; N, 13.39. Found: C, 55.89; H, 6.00; N, 13.75.
d6, 400 MHz): δ 7.86 (d, J = 16.1 Hz, 1H), 7.82 (s, 1H), 7.64 (d, J = 16.1 Hz,
J
J
J
d
δ
5.4.4.
(±)-(E)-1-(1-Cyclopropyl-6,7-dimethoxyphthalazin-2(1H)-yl)-3-(5-((2,4-
12c): This
diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)prop-2-en-1-one
(
compound was prepared using the same procedure as above. Yield: 1.05 g (50%) as a
1
white solid, mp 125-127 °C; IR: 3363, 3213, 3170, 2836, 1650, 1635, 1605 cm-1; H
NMR (DMSO-
1H), 7.57 (s, 1H), 7.26 (s, 1H), 7.14 (s, 1H), 7.10 (s, 1H), 7.00 (s, 1H), 6.73 (br s, 2H),
6.25 (br s, 2H), 5.39 (d, = 8.2 Hz, 1H), 3.83 (s, 3H), 3.80 (s, 3H), 3.79 (s, 3H), 3.74 (s,
3H), 3.61 (s, 2H), 1.08 (m, 1H), 0.48 (m, 2H), 0.40 (m, 1H), 0.31 (m, 1H); 13C NMR
(DMSO- 6, 75 MHz): 165.8, 162.7, 159.9, 152.6, 151.8, 151.1, 148.4, 146.1, 143.0,
d6, 300 MHz): δ 7.84 (s, 1H and d, J = 16.0 Hz, 1H), 7.65 (d, J = 16.0 Hz,
J
d
δ
136.3, 135.7, 128.0, 126.5, 118.6, 118.3, 116.7, 114.8, 110.0, 109.0, 106.7, 60.8, 55.9,
55.8, 55.7, 53.0, 32.2, 16.6, 3.8, 2.0. Anal. Calcd for C29H32N6O5·2.9 H2O: C, 58.36; H,
5.93; N, 14.08. Found: C, 58.15; H, 5.72; N, 13.69.
5.4.5.
dimethoxy-1-vinylphthalazin-2(1H)-yl)prop-2-en-1-one (12d):
prepared using the same procedure as above. Yield: 1.14 g (52%) as a white solid, mp
(±)-(E)-3-(5-((2,4-Diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)-1-(6,7-
This compound was
1
210-212 °C; IR: 3341, 3158, 3082, 2842, 1667, 1637, 1629 cm-1; H NMR (DMSO-d6
300 MHz): 7.85 (d, = 16.2 Hz, 1H), 7.75 (s, 1H), 7.66 (d, = 16.2 Hz, 1H), 7.62 (br s,
2H), 7.54 (s, 1H), 7.31 (s, 1H), 7.13 (br s, 4H), 7.02 (s, 1H), 6.29 (d, = 3.5 Hz, 1H),
= 16.8, 1.1 Hz, 1H), 3.81 (s,
165.5,
,
δ
J
J
J
5.76 (m, 1H), 5.03 (dd,
J
= 10.1, 1.1 Hz, 1H), 4.77 (dd, J
13
3H), 3.78 (2s, 6H), 3.73 (s, 3H), 3.63 (s, 2H); C NMR (DMSO-d6, 75 MHz):
δ
163.6, 156.1, 152.7, 151.9, 148.5, 146.3, 146.0, 142.2, 136.5, 135.4, 134.4, 128.0,
125.3, 118.9, 118.1, 116.4, 115.0, 114.9, 110.0, 109.3, 108.2, 60.8, 55.89, 55.85, 55.7,
52.1, 31.8. Anal. Calcd for C28H30N6O5·4.1 H2O: C, 54.67; H, 5.92; N, 13.84. Found: C,
54.88; H, 5.72; N, 13.84.
5.4.6.
(±)-(E)-3-(5-((2,4-Diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)-1-(6,7-
dimethoxy-1-(2-methylprop-1-en-1-yl)phthalazin-2(1H)-yl)prop-2-en-1-one (12e): This
compound was prepared using the same procedure as above. Yield: 1.45 g (56%) as a
21

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yellow solid, mp 212-213 °C; IR: 3466, 3324, 3150, 3100, 2838, 1643, 1598 cm-1; H
NMR (DMSO- 6, 400 MHz): 7.80 (overlapping s, 1H and d, = 15.4 Hz, 1H), 7.58
(overlapping s, 1H and d, = 15.4 Hz, 1H), 7.22 (s, 1H), 7.13 (s, 1H), 6.98 (s, 1H), 6.87
(s, 1H), 6.43 (d, = 9.7 Hz, 1H), 6.18 (br s, 2H), 5.74 (br s, 2H), 5.19 (d, = 9.7 Hz,
1H), 3.82 (s, 3H), 3.80 (2s, 6H), 3.74 (s, 3H), 3.60 (s, 2H), 1.97 (s, 3H), 1.60 (s, 3H); 13C
NMR (DMSO 6, 101 MHz): 164.6, 161.7, 161.6, 155.2, 151.9, 151.4, 147.8, 145.4,
d
δ
J
J
J
J
d
δ
141.7, 136.0, 135.8, 132.7, 127.3, 126.8, 121.8, 117.7, 117.5, 115.5, 114.1, 108.7,
108.4, 105.1, 60.2, 55.2, 55.1, 48.2, 31.8, 24.8, 17.9 (one OCH3 unresolved). Anal.
Calcd for C30H34N6O5·1.5 H2O: C, 61.53; H, 6.37; N, 14.35. Found: C, 61.27; H, 6.32;
N, 14.43.
5.4.7.
(±)-(E)-3-(5-((2,4-Diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)-1-(6,7-
dimethoxy-1-phenylphthalazin-2(1H)-yl)prop-2-en-1-one (12f): This compound was
prepared using the same procedure as above. Yield: 1.74 g (58%) as a white solid, mp
190-192 °C; IR: 3455, 3385, 3339, 3183, 2839, 1654, 1612 cm-1; H NMR (DMSO-d6
,
1
300 MHz):
1H), 7.31-7.19 (complex, 7H), 7.17 (s, 1H), 6.98 (d,
(br s, 2H), 5.72 (br s, 2H), 3.82 (s, 3H), 3.80 (s, 3H), 3.78 (s, 3H), 3.73 (s, 3H), 3.59 (s,
δ
7.86 (s, 1H), 7.85 (d,
J
= 16.0 Hz, 1H), 7.68 (d,
J = 16.0 Hz, 1H), 7.60 (s,
J
= 16.0 Hz, 1H), 6.94 (s, 1H), 6.17
13
2H); C NMR (DMSO-d6, 75 MHz): δ 165.7, 162.3, 162.1, 155.8, 152.5, 152.1, 148.5,
146.0, 142.3, 141.6, 136.8, 136.6, 128.5, 127.7, 127.4, 126.3, 126.2, 118.3, 117.8,
116.1, 114.8, 110.2, 109.3, 105.7, 60.8, 55.9, 55.72, 55.67, 53.2, 32.4. Anal. Calcd for
C32H32N6O5·0.7 H2O: C, 64.79; H, 5.67; N, 14.17. Found: C, 64.75; H, 5.67; N, 14.08.
5.4.8.
(±)-(E)-3-(5-((2,4-Diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)-1-(6,7-
12g): This
dimethoxy-1-(2-methylphenyl)phthalazin-2(1H)-yl)prop-2-en-1-one
(
compound was prepared using the same procedure as above. Yield: 2.10 (55%) as a
1
yellow solid, mp 179-180 °C; IR: 3466, 3330, 3099, 2835, 1669, 1643, 1603 cm-1; H
NMR (DMSO-d6, 300 MHz):
δ 7.84 (s, 1H), 7.79 (d, J = 16.0 Hz, 1H), 7.64 (d, J = 16.0
Hz, 1H), 7.60 (s, 1H), 7.25 (s, 1H), 7.19 (s, 1H), 7.18-7.03 (complex, 4H), 6.97 (s, 1H),
6.90 (s, 1H), 6.73 (s, 1H), 6.21 (br s, 2H), 5.77 (br s, 2H), 3.80 (s, 3H), 3.77 (s, 3H), 3.75
(s, 3H), 3.70 (s, 3H), 3.59 (s, 2H), 2.73 (s, 3H); 13C NMR (DMSO-d6, 75 MHz):
δ 165.6,
162.21, 162.19, 155.5, 152.5, 152.0, 148.4, 146.0, 142.8, 140.7, 136.5, 133.9, 130.4,
127.8, 127.3, 126.9, 126.7, 118.2, 118.0, 115.2, 114.7, 109.7, 109.0, 105.8, 60.8, 55.72,
55.68, 51.1, 32.4, 19.7 (1 aromatic C and 1 OCH3 were unresolved). Anal. Calcd for
C33H34N6O5·1.7 H2O: C, 63.39; H, 5.98; N, 13.44. Found: C, 63.52; H, 5.68; N, 13.69.
5.4.9.
(±)-(E)-3-(5-((2,4-Diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)-1-(1-(2-
ethylphenyl)-6,7-dimethoxyphthalazin-2(1H)-yl)prop-2-en-1-one (12h): This compound
was prepared using the same procedure as above. Yield: 1.29 g (56%) as a white solid,
mp 147-149 °C; IR: 3455, 3328, 3101, 2834, 1669, 1647, 1606 cm-1; H NMR (DMSO-
1
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741
742
743
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745
746
747
748
749
750
751
752
753
d6, 400 MHz): δ 7.82 (s, 1H), 7.79 (d, J = 16.1 Hz, 1H), 7.65 (d, J = 16.1 Hz, 1H), 7.60
(s, 1H), 7.24 (s, 1H), 7.24-7.02 (complex, 4H), 7.18 (s, 1H), 6.98 (s, 1H), 6.97 (s, 1H),
6.73 (s, 1H), 6.17 (br s, 2H), 5.73 (br s, 2H), 3.80 (s, 3H), 3.77 (s, 3H), 3.73 (s, 3H), 3.70
(s, 3H), 3.59 (s, 2H), 3.16 (m, 2H), 1.36 (t,
MHz): 165.4, 162.2, 162.1, 155.7, 152.4, 151.8, 148.3, 145.9, 141.9, 140.7, 139.5,
J d6, 75
= 7.5 Hz, 3H); 13C NMR (DMSO-
δ
136.5, 136.4, 128.3, 127.7, 127.5, 127.2, 126.9, 126.3, 118.1, 118.0, 115.1, 114.6,
109.6, 109.0, 105.6, 60.7, 55.62, 55.57, 55.5, 50.6, 32.3, 24.4, 15.3. Anal. Calcd for
C34H36N6O5·3.0 H2O: C, 58.70; H, 5.12; N, 14.08. Found: C, 58.49; H, 4.81; N, 14.51.
5.4.10.
(±)-(E)-3-(5-((2,4-Diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)-1-(6,7-
12i): This
dimethoxy-1-(2-methoxyphenyl)phthalazin-2(1H)-yl)prop-2-en-1-one
(
compound was prepared using the same procedure as above. Yield: 1.55 g (58%) as a
1
yellow solid, mp 257-259 °C; IR: 3486, 3376, 3177, 2833, 1659, 1606 cm-1; H NMR
(DMSO-
1H), 7.62 (s, 1H), 7.29 (s, 1H), 7.19 (t,
(s, 1H), 7.08 (dd, = 7.8, 1.2 Hz, 1H), 7.01 (d,
6.81 (t, = 7.8 Hz, 1H), 6.20 (br s, 2H), 5.75 (br s, 2H), 3.95 (s, 3H), 3.81 (s, 3H), 3.78
(s, 3H), 3.77 (s, 3H), 3.71 (s, 3H), 3.61 (s, 2H); 13C NMR (DMSO-
6, 75 MHz): 165.6,
d
6, 300 MHz):
δ
7.82 (s, 1H), 7.81 (d,
= 7.8 Hz, 1H), 7.15 (s, 1H), 7.10 (s, 1H), 7.09
= 7.8 Hz, 1H), 6.99 (d, J = 1.5 Hz, 1H),
J = 16.0 Hz, 1H), 7.74 (d, J = 16.0 Hz,
J
J
J
J
d
δ
162.3, 162.2, 155.8, 154.5, 152.5, 151.7, 148.3, 146.0, 141.0, 136.7, 136.6, 131.8,
128.8, 127.7, 126.8, 125.5, 120.0, 118.2, 117.9, 115.3, 114.7, 111.4, 109.5, 109.1,
105.8, 60.8, 55.9, 55.72, 55.68, 55.0, 52.6, 32.4. Anal. Calcd for C33H34N6O6·1.0 H2O:
C, 63.05; H, 5.77; N, 13.37. Found: C, 63.02; H, 5.84; N, 13.21.
5.4.11.
(±)-(E)-3-(5-((2,4-Diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)-1-(6,7-
12j): This
dimethoxy-1-(4-methoxyphenyl)phthalazin-2(1H)-yl)prop-2-en-1-one
(
compound was prepared using the same procedure as above. Yield: 1.70 g (60%) as a
pale yellow solid, mp 223-224 °C; IR: 3476, 3393, 3317, 3184, 2839, 1657, 1609 cm-1;
1H NMR (DMSO-d6, 300 MHz):
δ
7.87 (d,
16.0 Hz, 1H), 7.61 (s, 1H), 7.26 (s, 1H), 7.18 (s, 1H), 7.17 (s, 1H), 7.13 (d,
2H), 6.99 (s, 1H), 6.90 (s, 1H), 6.83 (d, = 8.6 Hz, 2H), 6.20 (br s, 2H), 5.76 (br s, 2H),
3.81 (2s, 6H), 3.79 (s, 3H), 3.73 (s, 3H), 3.68 (s, 3H), 3.60 (s, 2H); 13C NMR (DMSO-d6
75 MHz): 165.7, 162.3, 162.2, 158.5, 155.8, 152.5, 152.1, 148.5, 146.0, 142.3, 136.7,
J
= 16.0 Hz, 1H), 7.87 (s, 1H), 7.67 (d,
J =
J
= 8.6 Hz,
J
,
δ
136.6, 133.8, 127.8, 127.7, 126.6, 118.3, 117.9, 116.2, 114.7, 113.8, 110.1, 109.2,
105.8, 60.8, 55.9, 55.73, 55.68, 55.0, 52.6, 32.4. Anal. Calcd for C33H34N6O6·0.4 H2O:
C, 64.15; H, 5.68; N, 13.60. Found: C, 64.38; H, 5.67; N, 13.61.
5.5.
5.5.1. (±)-(E)-3-(5-((2,4-Diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)-1-(1-ethyl-
6,7-dimethylphthalazin-2(1H)-yl)prop-2-en-1-one (13a): This compound was prepared
23

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759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
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775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
using the same procedure as for 12a above. Yield: 1.45 g (56%) as a white solid, mp
210-212 °C. IR: 3425, 3350, 3171, 1667, 1634, 1605 cm-1; H NMR (DMSO-
MHz): 7.84 (d, = 16.0 Hz, 1H), 7.80 (s, 1H), 7.63 (d, = 16.0 Hz, 1H), 7.57 (s, 1H),
7.25 (s, 2H), 7.15 (s, 1H), 7.00 (s, 1H), 6.58 (br s, 2H), 6.11 (br s, 2H), 5.68 (t, = 6.3
Hz, 1H), 3.79 (s, 3H), 3.73 (s, 3H), 3.60 (s, 2H), 2.27 (s, 3H), 2.25 (s, 3H), 1.57 (m, 2H),
0.72 (t, 6, 75 MHz): 164.9, 162.0, 159.9, 151.9,
= 7.5 Hz, 3H); 13C NMR (DMSO-
d6, 300
1
δ
J
J
J
J
d
δ
145.4, 142.0, 140.0, 135.7 (2C), 135.6, 135.3, 130.2, 127.3, 126.9, 126.2, 121.0, 117.9,
117.5, 114.2, 105.8, 60.2, 55.2, 50.9, 31.6, 27.4, 19.1, 18.5, 8.7. Anal. Calcd for
.
C28H32N6O3 2.0 H2O: C, 62.67; H, 6.76; N, 15.66. Found: C, 62.66; H, 6.36; N, 15.53.
5.5.2.
dimethyl-1-propylphthalazin-2(1H)-yl)prop-2-en-1-one
prepared using the same procedure as above. Yield: 1.52 g (52%) as a white solid, mp
(±)-(E)-3-(5-((2,4-Diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)-1-(6,7-
(
13b): The compound was
1
228-230 °C; IR: 3354, 3164, 1667, 1638, 1607 cm-1; H NMR (DMSO-
7.82 (s, 1H), 7.77 (d, = 16.0 Hz, 1H), 7.62 (d, = 16.0 Hz, 1H), 7.57 (s, 1H), 7.25 (s,
1H), 7.23 (d, = 1.6 Hz, 1H), 7.14 (s, 1H), 6.99 (d,
(br s, 2H), 5.74 (t,
2.24 (s, 3H), 1.48 (m, 2H), 1.16 (sextet,
NMR (DMSO- 6, 75 MHz):
d6, 300 MHz): δ
J
J
J
J
= 1.5 Hz, 1H), 6.39 (br s, 2H), 5.93
= 6.5 Hz, 1H), 3.78 (s, 3H), 3.73 (s, 3H), 3.59 (s, 2H), 2.27 (s, 3H),
J
13
J
= 7.5 Hz, 2H), 0.80 (t, J = 7.4 Hz, 3H); C
d
δ
164.9, 161.9, 160.4, 152.8, 151.9, 145.4, 142.2, 140.1,
135.8, 135.7, 135.5, 130.7, 127.3, 126.8, 126.2, 120.9, 117.8, 117.5, 114.1, 105.6, 60.2,
55.2, 49.6, 36.5, 31.7, 19.1, 18.5, 17.2, 13.1. Anal. Calcd for C29H34N6O3·1.7 H2O: C,
63.88; H, 6.91; N, 15.41 Found: C, 63.72; H, 6.59; N, 15.02.
5.5.3.
(±)-(E)-1-(1-Cyclopropyl-6,7-dimethylphthalazin-2(1H)-yl)-3-(5-((2,4-
13c): This
diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)prop-2-en-1-one
(
compound was prepared using the same procedure as above. Yield: 1.48 g (51%) as a
1
white solid, mp 125-127 °C; IR: 3357, 3165, 1660, 1646, 1608, 1596 cm-1; H NMR
(DMSO-d6, 300 MHz):
1H), 7.58 (s, 1H), 7.25 (m, 2H), 7.19 (s, 1H), 7.00 (d,
6.22 (br s, 2H), 5.34 (d, = 8.2 Hz, 1H), 3.79 (s, 3H), 3.74 (s, 3H), 3.61 (s, 2H), 2.27 (s,
3H), 2.25 (s, 3H), 1.07 (m, 1H), 0.50 (m, 1H), 0.39 (m, 2H), 0.31 (m, 1H); 13C NMR
(DMSO-d6, 75 MHz): 165.8, 162.7, 160.1, 152.5, 151.5, 146.1, 143.0, 140.8, 136.44,
δ
7.87 (s, 1H), 7.84 (d,
J
= 16.0 Hz, 1H), 7.65 (d,
J = 16.0 Hz,
J
= 1.4 Hz, 1H), 6.69 (br s, 2H),
J
δ
136.41, 135.8, 130.4, 127.9, 127.5, 126.7, 121.6, 118.5, 118.2, 114.8, 106.6, 60.8, 55.8,
53.2, 32.2, 19.7, 19.1, 16.8, 3.8, 2.0. Anal. Calcd for C29H32N6O3·1.8 H2O: C, 63.91; H,
6.58; N, 15.42. Found: C, 63.70; H, 6.19; N, 15.13.
5.5.4.
(±)-(E)-3-(5-((2,4-Diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)-1-(6,7-
This compound was
dimethyl-1-vinylphthalazin-2(1H)-yl)prop-2-en-1-one (13d):
prepared using the same procedure as above. Yield: 1.25 g (54%) as a white solid, mp
1
215-217 °C; IR: 3358, 3178, 3071, 1662, 1631, 1595 cm-1; H NMR (DMSO-d6, 400
24

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800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
MHz):
7.27 (s, 2H), 7.24 (s, 1H), 7.01 (s, 1H), 6.65 (br s, 2H), 6.28 (d,
s, 2H), 5.77 (ddd, = 15.9, 10.2, 4.7 Hz, 1H), 5.04 (d, = 10.1 Hz, 1H), 4.78 (d,
Hz, 1H), 3.80 (s, 3H), 3.75 (s, 3H), 3.61 (s, 2H), 2.28 (s, 3H), 2.26 (s, 3H); 13C NMR
(DMSO- 6, 101 MHz): 164.9, 162.0, 159.6, 151.9, 151.1, 145.5, 141.5, 140.4, 136.13,
δ
7.87 (d,
J
= 16.1 Hz, 1H), 7.80 (s, 1H), 7.66 (d,
J
= 16.1 Hz, 1H), 7.58 (s, 1H),
= 4.7 Hz, 1H), 6.23 (br
= 16.6
J
J
J
J
d
δ
136.06, 135.3, 134.9, 128.7, 127.2, 127.1, 126.4, 120.7, 117.9, 117.3, 114.4, 114.3,
.
105.9, 60.2, 55.2, 51.7, 31.6, 19.1, 18.5. Anal. Calcd for C28H30N6O3 2.1 H2O: C, 62.70;
H, 6.43; N, 15.67. Found: C, 62.57; H, 6.09; N, 15.52.
5.5.5.
(±)-(E)-3-(5-((2,4-Diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)-1-(6,7-
This
dimethyl-1-(2-methylprop-1-en-1-yl)phthalazin-2(1H)-yl)prop-2-en-1-one (13e):
compound was prepared using the same procedure as above. Yield: 1.26 g (58%) as a
1
yellow solid, mp 212-213 °C; IR: 3425, 3360, 3192, 1668, 1636, 1596 cm-1; H NMR
(DMSO-
16.1 Hz, 1H), 7.24 (s, 1H), 7.23 (s, 1H), 7.05 (s, 1H), 6.99 (s, 1H), 6.40 (d,
1H), 6.29 (br s, 2H), 5.84 (br s, 2H), 5.18 (d, = 9.8 Hz, 1H), 3.79 (s, 3H), 3.74 (s, 3H),
3.60 (s, 2H), 2.25 (s, 3H), 2.23 (s, 3H), 1.96 (s, 3H), 1.59 (s, 3H); C NMR (DMSO-d6
101 MHz): 165.2, 162.3, 161.8, 154.9, 152.5, 146.0, 142.1, 141.2, 136.5, 136.4,
d6, 400 MHz):
δ
7.84 (d,
J
= 16.1 Hz, 1H), 7.81 (s, 1H), 7.60 (s, 1H), 7.59 (d,
J =
J
= 9.8 Hz,
J
13
,
δ
136.3, 133.0, 131.5, 127.9, 127.0, 122.5, 120.9, 118.3, 118.0, 114.7, 105.9, 60.8, 55.7,
49.0, 32.4, 25.3, 19.6, 19.6, 18.4 (1 aromatic C was unresolved). Anal. Calcd for
C30H34N6O3·1.0 H2O: C, 66.16; H, 6.66; N, 15.43. Found: C, 66.12; H, 6.48; N, 15.38.
5.5.6.
dimethyl-1-phenylphthalazin-2(1H)-yl)prop-2-en-1-one (13f):
prepared using the same procedure as above. Yield: 1.17 g (55%) as a white solid, mp
(±)-(E)-3-(5-((2,4-Diaminopyrimidin-5-yl)methyl)-2,3-dimethoxyphenyl)-1-(6,7-
This compound was
1
190-192 °C; IR: 3425, 3385, 3178, 1650, 1638, 1607, 1595 cm-1; H NMR (DMSO-d6
400 MHz): 7.88 (s, 1H), 7.85 (d, = 16.1 Hz, 1H), 7.69 (d, = 16.1 Hz, 1H), 7.58 (s,
1H), 7.35-7.17 (complex, 8H), 7.00 (s, 1H), 6.87 (s, 1H), 6.51 (br s, 2H), 6.04 (br s, 2H),
,
δ
J
J
13
3.79 (s, 3H), 3.72 (s, 3H), 3.60 (s, 2H), 2.25 (s, 3H), 2.24 (s, 3H); C NMR (DMSO-d6
,
101 MHz): 165.7, 162.5, 160.8, 152.9, 152.5, 146.1, 142.1, 142.0, 141.4, 136.9,
δ
136.7, 136.1, 130.6, 128.6, 128.0, 127.7, 127.4, 127.1, 126.1, 120.8, 118.5, 117.8,
114.9, 106.3, 60.8, 55.7, 53.7, 32.2, 19.7, 19.0. Anal. Calcd for C32H32N6O3·3.5 H2O: C,
62.83; H, 6.43; N, 13.74. Found: C, 62.72; H, 6.19; N, 13.56.
828
829
830
831
832
5.6 Assessment of inhibition
Measurements of the whole cell inhibition (MIC) and the enzymatic inhibition (Ki) utilized
a racemic mixture of each inhibitor and followed previously published procedures [6,
30].
25

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835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
In brief, MIC values were based on standardized cultures of S. aureus strain 29213 as
prescribed by the CLSI [40]. 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
previously cloned from S. aureus and expressed recombinantly in E. coli BL21 (DE3)
cells. The enzymatic reaction was reconstituted, including the NADPH co-factor and
varied concentrations of inhibitor diluted from a 10 mM stock in DMSO, with initiation of
the reaction by addition of the dihydrofolate substrate. The reaction was carried out at
o
30 C and monitored for 2.8 min, during which time the rate was linear. These rates
were plotted as a function of inhibitor concentration, and the 50% activity point was
calculated using a 4-parameter curve fit (Prism 6.0d). The IC50 values were converted to
Ki values using the Cheng-Prusoff equation and the previously measured KM value [6,
41].
5.7 Crystallization and structure determination
851
852
853
854
855
856
857
858
859
860
Methods closely followed previously published procedures [6, 30]. The 6×His affinity
tag was removed by digestion with thrombin, further purified using size exclusion
chromatography, and concentrated to 12-15 mg/mL for crystallization. Solid racemic
compound was added to saturation directly to the protein solution, followed by NADPH
at a final concentration of 1 mM. After 2 h of incubation at room temperature, samples
were centrifuged to remove excess saturated inhibitor and subjected to crystallization.
Hanging drop vapor diffusion was carried out using 2 µL of protein solutions mixed with
2 µL of well solution containing 0.1 MES, pH 6.2-6.4, 0.1-0.2 M sodium acetate, and 18-
25% polyethylene glycol 6000. Crystals typically grew to usable sizes within 1 week
when incubated at room temperature.
861
862
Data were collected from crystals cryoprotected with 15% glycerol in mother liquor and
saturated with inhibitor. Data collection was carried out at the University of Oklahoma
26

863
864
865
866
867
868
869
Macromolecular Crystallography Laboratory using a Rigaku RU3HR generator coupled
with a Raxis 4++ image plate detector, or a Rigaku MicroMax 007HF generator coupled
with a Dectris Pilatus 200K silicon pixel detector. Data from the Raxis 4++ detector were
indexed and scaled using d*TREK v 9.7 [42], while those from the Dectris Pilatus were
indexed and scaled using HKL3000 [43]. All structures were solved by molecular
substitution with PDB ID 3M08 [6]. Refinement and rebuilding of the structures were
carried out using the programs Phenix and Coot [44, 45].
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
Conflicts of Interest
The authors declare no competing interests.
Author Contributions
Chemical synthesis and analyses were carried out in the Chemistry Department at
Oklahoma State University by N.P.M., B.N., R.A.B, and K.D.B. Remaining studies were
carried out in the Chemistry and Biochemistry Department at the University of
Oklahoma. Protein purification and crystallization was carried out by J.C.W. and
C.R.B.; structure solution and analysis was carried out by J.C.W., L.M.T. and C.R.B.
MIC determinations were made by C.R.B., and enzyme inhibition was measured by I.P.
and C.R.B. All authors have approved the submitted form of this manuscript.
Acknowledgments
We gratefully acknowledge support of this work with start-up funds from the University
of Oklahoma to C.R.B, and in the early stages from the National Institutes of Allergy and
Infectious Diseases (R01-AI090685). The chemical syntheses were enabled by funding
from NSF (BIR-9512269), the Oklahoma State Regents for Higher Education, the W. M.
Keck Foundation, and Conoco, Inc., which included funding for a 300 MHz NMR
spectrometer of the Oklahoma Statewide shared NMR facility. The authors also wish to
thank the OSU College of Arts and Sciences for funds to purchase new FT-IR
instruments and a new 400 MHz NMR spectrometer in the Chemistry Department. The
OU Biomolecular Structure Core is supported in part by an Institutional Development
Award (IDeA) from the National Institute of General Medical Sciences of the National
27

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898
899
Institutes of Health (Award P20GM103640), the National Science Foundation (Award
0922269), and the University of Oklahoma Department of Chemistry and Biochemistry.
Funders had no involvement in the study design, collection, analysis and interpretation,
or writing of this report.
References
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