Synthesis and structure-activity relationships of thieno[2,3-d]pyrimidines as atypical protein kinase C inhibitors to control retinal vascular permeability and cytokine-induced edema

Article abstract of DOI:10.1016/j.bmc.2020.115480

Studies demonstrate that small molecule targeting of atypical protein kinase C (aPKC) may provide an effective means to control vascular permeability, prevent edema, and reduce inflammation providing novel and important alternatives to anti-VEGF therapies for certain blinding eye diseases. Based on a literature tricyclic thieno[2,3-d]pyrimidine lead (1), an ATP-competitive inhibitor of the aPKC iota (ι) and aPKC zeta (ζ) isoforms, we have synthesized a small series of compounds in 1–2 steps from a readily available chloro intermediate. A single pyridine congener was also made using 2D NMR to assign regiochemistry. Within the parent pyrimidine series, a range of potencies was observed against aPKCζ whereas the pyridine congener was inactive. Selected compounds were also tested for their effect toward VEGF-induced permeability in BREC cells. The most potent of these (7l) was further assayed against the aPKCι isoform and showed a favorable selectivity profile against a panel of 31 kinases, including kinases from the AGC superfamily, with a focus on PKC isoforms and kinases previously shown to affect permeability. Further testing of 7l in a luciferase assay in HEK293 cells showed an ability to prevent TNF-α induced NFκB activation while not having any effect on cell survival. Intravitreal administration of 7l to the eye yielded a complete reduction in permeability in a test to determine whether the compound could block VEGF- and TNFα-induced permeability across the retinal vasculature in a rat model. The compound in mice displayed good microsomal stability and in plasma moderate exposure (AUC and C_max), low clearance, a long half-life and high oral bioavailability. With IV dosing, higher levels were observed in the brain and eye relative to plasma, with highest levels in the eye by either IV or PO dosing. With a slow oral absorption profile, 7l accumulates in the eye to maintain a high concentration after dosing with higher levels than in plasma. Compound 7l may represent a class of aPKC inhibitors for further investigation.

Full text of DOI:10.1016/j.bmc.2020.115480

Journal Pre-proofs
Synthesis and structure-activity relationships of thieno[2,3-d]pyrimidines as
atypical protein kinase C inhibitors to control retinal vascular permeability
and cytokine-induced edema
Xuwen Liu, Michael W. Wilson, Kun Liu, Pil Lee, Larisa Yeomans, Susan E.
Hagen, Cheng-Mao Lin, Bo Wen, Duxin Sun, Andrew D. White, Hollis D.
Showalter, David A. Antonetti
PII:
DOI:
Reference:
S0968-0896(20)30300-X
https://doi.org/10.1016/j.bmc.2020.115480
BMC 115480
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
5 September 2019
25 March 2020
27 March 2020
Please cite this article as: Liu, X., Wilson, M.W., Liu, K., Lee, P., Yeomans, L., Hagen, S.E., Lin, C-M., Wen, B.,
Sun, D., White, A.D., Showalter, H.D., Antonetti, D.A., Synthesis and structure-activity relationships of
thieno[2,3-d]pyrimidines as atypical protein kinase C inhibitors to control retinal vascular permeability and
cytokine-induced edema, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.
2
020.115480
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Synthesis and structure-activity relationships of thieno[2,3-d]pyrimidines as atypical
protein kinase C inhibitors to control retinal vascular permeability and cytokine-
induced edema
Xuwen Liu, Michael W. Wilson,⊥,§ Kun Liu,⊥ Pil Lee,⊥ Larisa Yeomans, Susan E. Hagen,⊥ Cheng-
‡
,§
,§
,§
⊥
‡
,§
⊥
‡,#,*
∥
∥
⊥
Mao Lin, Bo Wen, Duxin Sun, Andrew D. White, Hollis D. Showalter and David A. Antonetti
‡
§
⊥
Department of Ophthalmology and Visual Sciences, Department of Medicinal Chemistry, Vahlteich
#
∥
Medicinal Chemistry Core, Department of Pharmaceutical Sciences, Department of Molecular and
Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA
*
Corresponding author.
E-mail address: dantonet@med.umich.edu
D.A. Antonetti)
(
Abstract

Studies demonstrate that small molecule targeting of atypical protein kinase C (aPKC) may provide an
effective means to control vascular permeability, prevent edema, and reduce inflammation providing
novel and important alternatives to anti-VEGF therapies for certain blinding eye diseases. Based on a
literature tricyclic thieno[2,3-d]pyrimidine lead (1), an ATP-competitive inhibitor of the aPKC and
aPKC isoforms, we have synthesized a small series of compounds in 1-2 steps from a readily available
chloro intermediate. A single pyridine congener was also made using 2D NMR to assign
regiochemistry. Within the parent pyrimidine series, a range of potencies was observed against aPKC
whereas the pyridine congener was inactive. Selected compounds were also tested for their effect
toward VEGF-induced permeability in BREC cells. The most potent of these (7l) was further assayed
against the aPKC isoform and showed a favorable selectivity profile against a panel of 31 kinases,
including kinases from the AGC superfamily, with a focus on PKC isoforms and kinases previously
shown to affect permeability. Further testing of 7l in a luciferase assay in HEK293 cells showed an
ability to prevent TNF-α induced NFκB activation while not having any effect on cell survival.
Intravitreal administration of 7l to the eye yielded a complete reduction in permeability in a test to
determine whether the compound could block VEGF- and TNFα-induced permeability across the retinal
vasculature in a rat model. The compound in mice displayed good microsomal stability and in plasma
moderate exposure (AUC and Cmax), low clearance, a long half-life and high oral bioavailability. With
IV dosing, higher levels were observed in the brain and eye relative to plasma, with highest levels in the
eye by either IV or PO dosing. With a slow oral absorption profile, 7l accumulates in the eye to maintain
a high concentration after dosing with higher levels than in plasma. Compound 7l may represent a class
of aPKC inhibitors for further investigation.
Key words: blinding eye diseases; atypical protein kinase C (aPKC); vascular permeability; vascular
endothelial growth factor (VEGF); tumor necrosis factor- (TNF); homology model; thieno[2,3-
d]pyrimidine inhibitors; SAR
1
. Introduction
1
Macular edema (ME) remains a leading cause of visual loss in a host of blinding eye diseases. In
the normal eye, tight control of fluid and solute flux across the retinal capillary barrier and retina
pigment epithelium (RPE) help to control the retinal environment. The accumulation of fluid leading to
ME may occur from changes in permeability across these barriers and contribute to vision loss in a

number of blinding eye diseases including diabetic retinopathy (DR), retinal vein occlusion,
hypertensive retinopathy, inflammatory disorders including uveitis syndromes, and intraocular tumors.
2
DR is the leading cause of blindness in working age adults, and changes in blood vessel permeability
and ME are clinical outcomes most closely associated with loss of visual acuity in DR.3,4
Changes in retinal vascular permeability may occur through a host of potential signaling pathways
driven by cytokines. Most prominently, increased vascular endothelial growth factor (VEGF)
contributes to ME and visual loss and therapies targeting VEGF (anti-VEGF) have profoundly improved
medical treatment for retinal diseases involving vascular permeability.4,5 However, about half of
patients with DR fail to respond to anti-VEGF therapy, potentially due to the contribution of additional
inflammatory factors that increase in DR and contribute to vascular permeability.
A number of inflammatory factors have been observed to be elevated in both patients with DR and
in animal models of DR that may account for increased vascular permeability. Gene deletion of the
intercellular adhesion molecule-1 (ICAM-1), or its leukocyte binding partner CD18, reduces leukostasis
6
in mouse models of DR. High dose aspirin, the cyclooxygenase (COX)-2 inhibitor, meloxicam, and a
soluble tumor necrosis factor- (TNF) receptor/Fc hybrid (etanercept) each normalize vascular
permeability, leukostasis and NFB activation suggesting TNF and COX contribute to diabetic
7
8
retinopathy. Gene deletion of TNF led to normalization of permeability at six months of diabetes.
Together, these animal models demonstrate a role for inflammatory cytokines in diabetic retinopathy.
Furthermore, in humans elevated interleukin (IL)-1 and TNF have been associated with proliferative
diabetic retinopathy9,10,11 and TNF with non-proliferative diabetic retinopathy. A measure of soluble
cytokines in 345 patients identified IL-6, IL-8 and chemokine, C-C motif, ligand (CCL)-2 (or MCP) as
elevated in patients with diabetic macular edema, proliferative diabetic retinopathy, and branch and
12
13
central retinal vein occlusions, all of which involve retinal blood vessel permeability. Finally, DR
1
4
patients demonstrate evidence of TNF activation in the plasma and intravenous, anti-TNF therapies
lead to significant improvement in visual acuity compared to placebo-treated eyes in a small cohort of
1
5
patients refractive to other treatments. Collectively, these data suggest that inflammation contributes to
retinal ME in diabetic retinopathy and other eye disease with increased retinal vascular permeability.
Novel therapies that prevent or restore barrier properties may provide benefit to patients, particularly
those not responsive to anti-VEGF therapy.

Studies from our laboratory and others have identified atypical protein kinase C (aPKC) as a
common downstream signaling mechanism in the control of vascular permeability. There are two aPKC
isoforms termed  and or  in mice), which are part of the larger PKC family. However, unlike
conventional PKC isoforms, the aPKC isoforms do not require calcium or diacylglycerol for activation.
PKC/ is required for the formation of cell polarity and gene deletion of aPKC (Prkcz) leads to
1
6
embryonic lethality. However, once polarity is established, inhibition of aPKC activities does not
17
disrupt polarity or the junctional complex. However, inhibition of aPKC using siRNA, expression of a
kinase dead form of PKC, which acts in a dominant manner, and peptide inhibition all prevent or
1
8
reduce VEGF-induced endothelial permeability. Inhibition of aPKC can also prevent permeability
induced by CCL2, TNF-α, and thrombin.19,20,21 These studies led to the discovery of a series of novel 2-
amino-3-carboxy-4-phenylthiophenes as specific aPKC inhibitors, which also blocked endothelial
permeability in cells and in vivo.18,22,23 The molecular targets of aPKC are not clear but in a model of
stroke, the ubiquitin ligase PDZRN3 regulates vascular permeability in a mechanism dependent on
aPKC activity.24
In addition to vascular permeability, published data strongly implicates aPKC in the regulation of
inflammation. Evidence from genetic loss-of-function studies suggests aPKC isoforms play a role in
2
5
innate immune function. Prkcz gene deletion yields developmentally normal mice but with severely
impaired NF-κB dependent gene transcription following either TNF-α or interleukin-1β treatment.26
2
7
ICAM-1 phosphorylation and leukocyte binding in response to TNF-α requires aPKC activity. Further,
inhibitor studies reveal aPKC also contributes to macrophage activation via nitric oxide synthase 2
(
NOS2) in experimentally induced uveitis.28
Recent data reveal that small molecule inhibition of aPKC, or expressing a kinase dead form of
aPKC under a Tie2 promoter, dramatically reduces retinal
permeability in response to two models of inflammation, both
ischemia reperfusion and intra-ocular co-injection of VEGF and
Expression of kinase dead aPKC under a Tie2 promoter, which
restricted endothelial and myeloid expression, inhibits ischemia
induced microglial expansion and granulocyte recruitment to the
reduces expression of an array of inflammatory factors including
dramatically compliment factor B.22 Collectively these studies
retinal
TNFα.22
has
reperfusion
retina, and
most
Fig. 1. Structure of
lead aPKC inhibitor
demonstrate

that small molecule targeting of aPKC may provide an effective means to control vascular permeability
and prevent edema induced by multiple vasoactive cytokines, and reduce inflammation providing novel
and important alternatives to anti-VEGF therapies for blinding eye diseases. However, none of the
2
3
series of phenylthiophenes discussed above was amenable to further development. Therefore, we
refocused medicinal chemistry efforts to a different scaffold.
To date, several atypical PKC inhibitors have been reported.29,30,31,32,33 These include a series of
thieno[2,3-d]pyrimidines that was synthesized and identified as ATP-competitive inhibitors.31,34 From
this work, compound 1 (CRT0066854; Fig. 1) was characterized by crystallographic and biochemical
methods, as well as phenotypic cell-based assays, as a potent and selective chemical tool to modulate
aPKC activity both enzymatically and in cells. The crystal structure with PKC shows the tricyclic core
of 1 buried deep in a lipophilic pocket with the basic aromatic sidechain projected towards solvent.
PKC and PKC share the exact same key amino acids that form the binding pocket (within 4.5 Å) for
compound 1. The sequence alignment of these isoforms (human) is shown in Fig. SM1, Supplemental
Information. There are differences between PKC and PKC out to 11Å of the pocket but these are
unlikely to impact selectivity differences.35 We reasoned that SAR changes toward the solvent front
would afford the opportunity to modulate properties and improve potency.
In this paper, we report on medicinal chemistry efforts to expand the structure-activity relationships
(SAR) of the thieno[2,3-
d]pyrimidine series toward
uncovering inhibitors with
increased enzyme potency,
selectivity, and efficacy in
selected functional assays to
assess their potential utility to
control vascular permeability and
cytokine-induced edema. Using
compound 1 as a lead, we
synthesized a small series of
analogues that was tested against
aPKC. A subset of the most
Scheme 1. Reagents and conditions. (i) 4M HCl in p-dioxane, RT, 19 h,
3%; (ii) Vilsmeier reagent, 1,2-DCE, 60 °C, 4 h, 98%; (iii) Method A:
TEA or Hunig’s base, EtOH, reflux, overnight, 25-84%; (iv) Method B: HCl
in p-dioxane, DCM, RT, overnight, 47-96%.
8

potent compounds was then evaluated in cellular assays and in vivo to determine their efficacy as
inhibitors of retinal endothelial permeability and NF-κB activation.
2
. Chemistry
The 4-(N-substituted)-2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine
analogues shown in Table 1 were synthesized in a one- to two-step process from a key 4-chloro
36
intermediate 5a as shown in Scheme 1. Thus, utilizing standard literature conditions, simple base-
catalyzed condensation of 5a in refluxing ethanol with 6 (piperidines, X = CH or piperazines, X = N) in
which bond forming was directed onto the ring nitrogen, or alternatively with related side chains 8 (R =
Boc) in which condensation occurred on a terminal primary amine, provided products 7a-c, e, f, h, j, k
and 9a, c (Method A). Adducts with N-Boc protecting groups (7c, f, h, k and 9a, c) were then
transformed to corresponding amines (7d, g, i, l and 8b, d, respectively) with anhydrous HCl (Method
B).The synthesis of 5a, utilizing a modification
3
7
of a known procedure, commenced with HCl-
catalyzed condensation of thiophene ester 2 with
isonicotinonitrile 3 to give pyrimidinone 4 in
8
3% yield. A small amount of ether side product
5
b also formed during this reaction.
Chlorination of 4 with Vilsmeier reagent utilized
3
8
a literature procedure to provide key 4-chloro
intermediate 5a in 98% yield. Our route to the 4-
(N-substituted)-2-(pyridin-4-yl)-5,6,7,8
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine
class of compounds offers an efficient and
convenient complement to literature procedures
that proceed via a sulfonate ester intermediate (5,
X = 2,4,6-triisopropylbenzenesulfonyl).31,34
Scheme 2. Reagents and conditions. (i) Hunig’s base,
ethyl 3-chloro-3-oxopropanoate, DCM, RT, 18 h, 80%; (ii)
NaH, THF, reflux, 15 h, 32%; (iii) 2N aq. NaOH, reflux, 5
h, 19%; (iv) PhP(O)Cl
2
, 180 °C, 4 h, 74%; (v) piperazine,
NMP, 200 °C, microwave, 1.5 h, 52% (13a) and 21%
(
(
14a); (vi) Boc
13b) and 84% (14b); (vii) pyridin-4-ylboronic acid,
, Na CO , 60% aq. DMF, 95 °C, 2 h, 68% (15a)
2
O, TEA, DMAP, MeOH, RT, 20 h, 75%
P(PPh
3
)
4
2
3
and 40% (16a); (viii) 4M HCl in dioxane, ether, RT,
overnight, 61% (15b) and 33% (16b).
The synthesis of deaza (pyridine) congener
(15b) of parent pyrimidine 7a utilized a sequence
similar to that outlined to make a series of interleukin-1 receptor-associated kinase (IRAK) inhibitors39

(Scheme 2). Accordingly, base-catalyzed acylation of ester 2 with ethyl 3-chloro-3-oxopropanoate
provided 10 in 80% yield. This was engaged in an intramolecular Dieckmann condensation to provide
tricyclic pyridone ester 11a in 32% yield, which was then refluxed in 2N aqueous NaOH to effect
hydrolysis and decarboxylation to give 11b in 19% yield. Heating 11b in neat phenylphosphonic
dichloride at 180 ºC provided key dichloro intermediate 12 in 74% yield. Microwave-assisted mono-
chloride displacement with excess piperazine in N-methyl-2-pyrrolidone at 200 °C gave a 2.5:1 mixture
of isomers (13a, 14a) that was chromatographically separated with each isomer N-Boc-derivatized to
provide 13b and14b, respectively, in a 18-39% two-step yield. Coupling of these with (pyridin-4-
yl)boronic acid was conducted under standard Suzuki conditions to provide isomeric products 15a
(68%) and 16a (40%), respectively. N-Boc deprotection on these was carried out with 4M HCl in p-
dioxane to give isomeric products 15b (61%) and 16b (33%), respectively, as hydrochloride salts.
All compounds were rigorously purified by
chromatography or crystallization, and their
assignments were supported by diagnostic
flash
structural
peaks in
1
the H NMR spectra and by mass
spectrometry. For regioisomers 15a and 16a,
structural
1
13
1
1
assignments were based on 1D H, C spectra,
2D H- H
1
13
NOESY and 2D H- C HSQC, HMBC
experiments. For compound 15a, the ,  and
in the same plane, hence the signal of the C-11
 rings are
proton at
downfield
rings for
pyridine
Fig. 2. NMR assignment of  ring
attachments for compounds 15a and 16a. A.
7
.34 ppm is deshielded, resulting in a
Structures of 15a and 16a with labeled
1
positions; B. Assigned H NMR peaks for -
shift. On the other hand, while the  and 
compound 16a are in the same plane, the 
pyridine and C-11 protons.
ring is rotated out of plane around the C-10,13 bond. This results in a shielding of the C-11 proton
giving an upfield shift at 6.39 ppm (Fig. 2). In addition, only in compound 16a are the C-14 and 22
protons in close enough proximity to show an observed cross peak in NOESY. Molecular models of the
regioisomers (Fig. SM2) and digital copies of 1D and 2D spectra are provided in Supplemental
Material.
3
. Kinase inhibition, modeling studies, and functional activity

One goal for our small designed library was to exchange the C-4 position 3-phenyl propane-1,2-
diamine moiety of compound 1 for substituents that would provide improved aqueous solubility with
such substituents derived from commercially available amines. A second goal was to evaluate the effect
on kinase activity of swapping the N-3 of the tricyclic core of 1 for carbon for one selected inhibitor.
All compounds were investigated for inhibition of commercially purchased recombinant full-length
human aPKC (Table 1). The IC50 values shown were determined by dose-response in a Kinase-Glo™
luminescent assay with the peptide CREBtide as substrate and are technically apparent IC50 values. Data
points are the average of two determinations for each reaction in triplicate. Selected compounds were
further evaluated in a cell-based assay for VEGF-induced primary bovine retinal endothelial cell
(
BREC) permeability. Compound 1, previously developed as an aPKC inhibitor,31,34 was used as a
positive control. It demonstrated an IC50 of 0.27 µM compared to the published value of 0.62 µM,
derived using the ADP Quest™ assay, and an EC50 of 3 µM in VEGF-induced BREC permeability.
Toward prosecuting the first goal, we focused first on selected C-4 piperazine analogues (X = N; 7a,
7
b, 7d) with different substituents off the basic distal piperazine nitrogen. Compound 7a (R = H) was
our most potent inhibitor of aPKC (IC50 = 0.012 M), but showed no effect toward VEGF-induced
permeability at 0.01 M. Basal permeability was increased at 1, 0.1, and 0.03 M, potentially
associated with cell death. Alkyl congeners 7b (R = CH
.413 M) were less potent. The next set of analogues looked at piperidine (X = CH) substitution
flanked by solubilizing R groups. A pattern of modest potency (IC50, 0.31 – 0.56 M) for compounds
g, 7i, 7j, and 7l with basic R substituents was observed. For 7e (R = OH), potency decreased markedly
IC50, 2.3 M) likely due to reduced solubility. Selected compounds within this subset were tested for
3
; IC50, 3.7 M) and 7d (R = CH
2
CH
2
NH
2
; IC50,
0
7
(
their effect toward VEGF-induced permeability. Alcohol 7e was unresponsive at 3 M and amines 7g
and 7j each displayed modest potency (EC50, 0.3 M), whereas the homologated congener 7l (R =
CH
9b) or amine (9d) spacer connecting the tricyclic core to a terminal piperazine or piperidine substituent,
respectively, showed poor or modest potency (IC50, 12.1, 0.71 M, respectively). Analogues 7b-l and
b,d are larger and may require more desolvation energy, reducing potency. Finally, altering the
pyrimidine ring of 7a to a pyridine (15b) resulted in a dramatic 95-fold loss of potency (IC50, 0.012 vs
.14 M,
2
NH ) showed a robust response (EC50, 0.071 M). Two additional analogues with a C-4 aminoethyl
2
(
9
1
Table I. In vitro activity of thieno[2,3-d]pyrimidines and C-ring deaza congeners.

No.
n
X
R
aPKC inhibition VEGF – induced
a
endothelial permeability
IC50±SEM (M)
b
(EC50, M)
13
1,34
-
-
-
-
-
-
-
-
-
2
0
-
-
-
-
H
CH
CH
0.27±0.03
0.012±0.01
3.7±0.25
0.42±0.03
2.4±0.06
0.38±0.05
0.36±0.09
0.56±0.04
0.52±0.11
11.06±1.97
0.70±0.08
1.15±0.04
16.23±1.89
3
4
n.e.c
7
7
7
7
7
7
7
7
9
9
1
1
a3
N
N
N
3
4
d
b
d
e
g
i
3
2
n.d.
n.d.
n.e.
0.3
n.d
0.3
0.071
n.d
n.d.
n.e.
n.d.
CH
2
NH
2
CH OH
CH NH
CH NHCH
CH N(CH
CH CH NH
N
CH
-
2
3
j
3
)
2
l
2
2
b
d
5b
6b
-
-
-
-
-
a
Kinase-Glo™ luminescent assay against 250 ng/mL PKCζ (full length), 30 μM CREBtide and 0.1 μM ATP
with a 5- to 6-point dose response curve to determine IC50. Values are the mean of at least n ≥3 and were
b
repeated in independent experiments. 5-point dose-response to determine EC50 by comparing inhibition of
VEGF-induced permeability relative to inhibition of VEGF response in the presence of inhibitor.
c
d
n.e., no effect. n.d., not done.

respectively) against the enzyme and similarly showed no effect toward VEGF-induced permeability at
M. The C-2 pyridine substituent of 15b is more basic relative to the pyrimidine 7a (see Table SM3,
1
Supplementary Material) and should improve interaction with the adjacent residue Val 333 (see Fig.
SM3 (B), Supplementary Material). However, this substituent is also more free to rotate around the C-
2
/pyridyl aryl-aryl bond compared to pyrimidine 7a, which may explain its loss in activity. The
regioisomer (16b) was predictably completely inactive.
To better understand relative enzyme
potencies, we docked compound 7a
(magenta) into a homology model of
aPKCand overlaid with compound 1
(
green) in the PKC crystal structure
3zh8.pdb; Fig. 3). Compound 1 is a
(
pseudo big ring with an intramolecular T-
shaped – stack likely stabilizing the U-
shaped conformation. Compound 7a by
contrast occupies less of the pocket but has
a lot fewer rotatable bonds, likely
Fig. 3. Docking of compound 7a (magenta) in the homology
model of aPKC in comparison with the crystal structure of
compound 1 (green) bound in aPKC (3zh8.pdb).
optimizing the ΔS term of binding. The
detailed ligand-protein interaction of 7a
with the homology model of aPKCis
shown in Fig. SM3 (B), Supplemental Material. As a reference, the ligand-protein interaction of
compound 1 with aPKC is shown in Fig. SM3 (A). The overall interaction of compound 7a with
aPKCis very similar to the interaction of compound 1 with aPKC. The tricycle of compound 7a is in a
hydrophobic pocket surrounded by Ile258, Gly259, Gly261, Tyr263, Val266, Tyr332, and Val333. The
compound is making several hydrogen bonds: most notably the distal nitrogen of the piperazine ring is
making hydrogen bonds with Asp337 as well as with Asp380 through a water molecule, and the
nitrogen in the pyridine ring with Val333. The N-1 nitrogen in the pyrimidine ring of the tricycle is in
close contact with Thr393 and the sulfur with Asp394. Compared to compound 1/aPKC interactions,
compound 7a binds to aPKC in a similar fashion with respect to hydrophobic and hydrogen bonding
interactions. Missing the benzyl group, which forms an internal T-shaped – stacking interaction with
the pyridine ring of compound 1, should not significantly affect activity. In fact, the loss of entropy in

compound 7a with two rotatable bonds compared to compound 1 with six rotatable bonds and
diminished desolvation energy can account for the improved activity of 7a.
Due to its activity toward VEGF-induced permeability, compound 7l was advanced to further
testing. Kinase testing against aPKC (IC50, 0.52 M; Table 1) was followed up with aPKC, the other
aPKC isoform, using the Kinase-
Glo™ luminescent assay. This showed
a 7.5-fold greater potency with an IC50
of 0.07 M. Six point dose–response
curves for both determinations are
shown in Fig. SM4, Supplementary
Material. Further, compound 7l was
tested in a radiometric kinase assay by
Fig. 4. A. BREC were grown to confluence on Transwell filters to
determine permeability of 70 kDa RITC–dextran tracer. BRECs were
treated for 2 h with the indicated concentration of 7l prior to 1 h
treatment with 50 ng/mL VEGF where indicated. Results are expressed
as the mean relative to the control, ±S.E.M. with analysis by one-way
ANOVA with Bonferroni post-test. **P < 0.001, ***P < 0.0002, and
a commercial vendor in a 9-point
dose-response assay against PKC at
an ATP concentration of 15 µM
*
***P < 0.0001. B. EC50 of 7l for inhibition of VEGF-induced
(previously determined Km) and PKC
permeability calculated from the data shown in A.
at 155 µM (Km). These assays
confirmed the inhibitory activity of 7l with an IC50 of 1.64 µM for PKCand 4.08 µM for PKC (see
Fig. SM5, Supplemental Material). The reduced potency against each kinase was unsurprising given the
high concentration of ATP used in the PKC assay and the fact that 7l is an ATP competitive inhibitor.
As shown in Table 1, compound 7l demonstrated robust activity toward inhibition of VEGF-induced
permeability of bovine retinal endothelial cells (BREC). Fig. 4A shows the response assay in BREC
and the ability of compound 7l to inhibit permeability over a 0.01 – 1.0 M range. From this data, an
EC50 of 0.071 µM was determined (Fig. 4B).
Additional screening of 7l was conducted against a panel of 31 commercial kinases including
kinases from the AGC superfamily with a focus on PKC isoforms and kinases previously shown to
affect permeability (Table SM1, Supplementary Material). Compound 7l at 5 M showed limited
activity against almost all tested kinases including PKCI, a conventional PKC isoform implicated in
4
0
endothelial permeability. However, 7l was effective at inhibiting PKA, the cAMP-activated kinase and

Rho-associated, Kinase II (ROCK-II). To further test PKA response, a three-point dose response to PKA
was conducted (Table SM2, Supplementary Material). This limited dose response reveals an IC50 to
PKA of ~0.5 µM, similar to that observed for aPKC. Previous studies suggest a role for Rho kinase in
VEGF induced permeability,41,42 while other studies fail to observe any effect of Rho kinase inhibition
4
3
on VEGF-induced permeability. To determine whether ROCK-II inhibition affects permeability in
retinal endothelial cells, the BREC permeability assay was performed in the presence of Y-27632, a well
characterized ROCK ATP-competitive inhibitor.44,45 As shown in Fig. SM6, Supplementary Material,
inhibition of Rho kinase activity at 10, 3 or 1 µM of Y-27632 failed to reduce VEGF-induced
permeability in BREC and, in fact, increased permeability at the highest dose. However,
immunoblotting of phospho-myosin light chain revealed the inhibitor was effective at blocking Rho
kinase activity. These data strongly suggest that the inhibitory effect of 7l on VEGF-induced
permeability in BREC is independent of Rho kinase.
The atypical PKC isoforms, and particularly PKC, have been implicated in inflammation through
modulating NFκB activity. Therefore, the ability of compound 7l to prevent TNF-α induced NFκB
activation was tested in HEK293 cells stably
transfected with the NFκB enhancer driving
luciferase as described previously.23 Fig. 5A
shows the dose response in the luciferase assay
to inhibition by 7l of TNF-α induced NFκB
activation over a 2.5 – 10 M range. These
Fig. 5. Compound 7l inhibits TNF-α induced NF-B
data compute to an EC50 of 6.65 µM (Fig. 5B)
for 7l. Furthermore, these doses of 7l and
TNFα had no effect on cell survival as assessed
in the WST-1 assay (Fig. SM7, Supplementary
Material).
activity in vitro. A. HEK293/NFκB-luc cells were treated for
2
h with the indicated concentration of 7l prior to a 5 h
treatment with 5 ng/mL TNF-α and luciferase reporter
activity was assayed. Results are expressed as the mean
relative to the control, ±S.E.M. with analysis by ANOVA
with Bonferroni post-test. ****P < 0.0001. B. EC50 of 7l
blocking TNF-α induced NF-B activity calculated from the
data shown in A.
To determine whether compound 7l blocks
retinal vascular permeability in vivo, we tested its ability to prevent VEGF- and TNFα-induced
permeability of fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) across the retinal
vasculature in Long-Evans rats. Intravitreal injection with VEGF and TNFα induced more than a three-
fold increase in accumulation of FITC-BSA in retina (Fig. 6). Administration of 1 or 3 μM of compound
7
l, with or without co-injection of VEGF and TNFα, yielded a dose-response reduction in VEGF/TNFα

induced permeability with 3 µM of 7l completely preventing VEGF- and TNFα-induced FITC-BSA
permeability.
As an assessment of toxicity, we completed measures
of retinal morphology and function after intraocular
injection of compound 7l. Compound was given to four
Long-Evans rats by intravitreal injection to a final
concentration of 3 µM and compared to vehicle controls at
3
-5 h post injection. A scotopic electroretinogram (ERG)
was completed as a measure of retinal function at light
Fig. 6. Compound 7l prevents VEGF and
TNF-α induced retinal vascular permeability
in vivo. Long-Evans rats were injected
intravitreally with 7l followed by receiving an
i.v. injection of fluorescein isothiocyanate
2
2
2
intensities of 0.1 cd.s/m , 1 cd.s/m , and 10 cd.s/m . No
differences were observed in A-wave or B-wave amplitude
at any of the light intensities tested (see Fig. SM8,
Supplementary Material). A-wave and B-wave latency were
nearly identical for vehicle and compound 7l (data not
shown). Furthermore, optical coherence tomography
volume scans centered on the optic disk, completed as
(
FITC)-bovine serum albumin (BSA). FITC-
BSA accumulation in the eye was quantified
as a measure of permeability and the results
are expressed as the mean relative to the
control, ±S.E.M. with analysis by ANOVA
with Bonferroni post-test. *P < 0.05, ***P <
0
.0002, and ****P < 0.0001.
2
2
previously described, revealed no difference in retinal
structure 5 h after compound injection compared to vehicle.
4
. Computed properties
A balance between lipophilicity and aqueous solubility are key parameters that influence success in a
standard oral, low-dose drug discovery program. Toward assessing drug-like properties relating to oral
absorption for our series of inhibitors, lipophilic ligand efficiency (LLE) and solubility forecast index
(SFI) calculators were utilized to derive computed values for compounds in Table 1. Using cLogD7.4 and
pIC50 data, LLE can be calculated which provides a measure of a compound’s lipophilicity associated
with potency.46,47 That, along with SFI calculations derived also from cLogD7.4 information, provide
valuable information regarding a compound’s potential to be a drug. The data are shown in Table SM3,
Supplemental Material, with equations for LLE and SFI calculations given in the footnotes. Computed
LLE values range from 1.2 – 4.9 vs aPKC, imbuing several compounds with favorable properties
relative to compound 1 (LLE of 3.4). With regard to aqueous solubility, clogD7.4 values together with the
48

number of aromatic rings in a given molecule provide a simple, useful guide of potential solubility of
particular molecules with values ≤ 6 predicting for good aqueous solubility. Utilizing the SFI calculator,
all compounds in Table SM3 except for 7b and 7e are predicted to have acceptable aqueous solubility
(SFI of 6.0 – 4.3) with each displaying a lower SFI than compound 1 (7.4). The calculation (4.9) for 7l
trihydrochloride is predictive of its thermodynamic aqueous solubility (10.1 mM) determined by a
previously described assay.49 Compound 1 trihydrohydrochloride, despite a slightly elevated SFI, has a
solubility of 23.1 mM in the same assay.
5
. Metabolism and PK studies
When incubated in vitro against mouse liver microsomes, compound 7l showed good stability
(t
1/2 >60 min). During the test, verapamil was employed as a positive control in order to ensure that the
microsomes and NADPH used in this study were active and incubation conditions were appropriate to
perform the study. Based on the observed degradation pattern, compound 7l was considered stable
enough for an in vivo mouse pharmacokinetic (PK) study. Our main objective was to determine if this
compound could be delivered to the eye and brain by PO administration. Table 2 summarizes the PK
parameters of 7l in the plasma of CD-1 mice using both intravenous (IV, 10 mg/kg dose) and oral (PO,
2
0 mg/kg dose) administration at multiple time points over a 24 h period. To determine levels in the eye
and brain, we measured 7l concentrations at 4 and 24 h. A complete compilation of measured
concentrations of 7l at each time point in these three compartments is shown in Table SM4,
Supplemental Material. In plasma, compound 7l demonstrated moderate exposure (AUC and Cmax) and
low clearance (CLobs). A long half-life (t1/2 2.8 h for IV and 7.8 h for PO) was observed along with high
oral bioavailability (81.7%). With IV dosing a brain/plasma ratio of 2.3 and an eye/plasma ratio of 2.9
was observed at 4 h. Higher levels of compound 7l were also found in the eye than in the brain at 4 h
(
IV: eye/brain ratio of 1.3; PO: eye/brain ratio of 2.0). Comparative concentration-time profiles for
compound 7I in mouse plasma, brain and eye are shown in Fig. SM9, Supplemental Material. With a
slow oral absorption profile (Tmax of 7 h) in mice, 7l accumulates in the eye to maintain a high
concentration (~375ng/g) until 24 h after dosing with levels seven times higher than in plasma. Our
studies also showed a good correlation between in vitro microsomal stability and in vivo PK profile with
each exhibiting a reasonably long half-life.

Table 2. Compound 7l pharmacokinetic parameters in plasma of CD-1 mice following IV and PO
administration
Route
Dose
C
max
T
max
AUCobs – tlcd
(h*ng/mL)
4373
t
1/2
CL
%F
(
mg/kg)
(ng/mL)
N/A
(h)
(h)
(mL/h/kg)
2279
IV
10
20
N/A
7.0
2.77
7.83
NA
PO
488
6762
NA
81.7
PK parameters were estimated using non-compartmental analysis with Phoenix/WINONLIN. Cmax = Maximum observed concentration.
max = Time to reach Cmax. AUC0-tldc = Area under the concentration-time curve from time zero to time of last detectable concentration.
Terminal elimination half-life (t½) was calculated based on data points (≥ 3) in the terminal phase with correlation of coefficient > 0.90. CL
T
=
Systemic clearance. %F = oral bioavailability.
6
. Conclusions
As part of a focused research campaign to identify small molecule atypical PKC inhibitors with a
potential therapeutic application to treat a number of macular edema-related blinding diseases, including
diabetic retinopathy, we have designed analogues of a lead thieno[2,3-d]pyrimidine scaffold (1)
previously synthesized and identified as an ATP-competitive inhibitor of the aPKC and aPKC
isoforms. Based on crystal structure information, we reasoned that SAR changes with less lipophilic
basic side chains projected toward the solvent front would afford an opportunity to modulate
physicochemical properties resulting in an improvement in enzyme potency, selectivity, and
metabolic/PK profile, which could pave the way for in vivo efficacy studies. Toward this end, we
synthesized a series of ten 4-(N-substituted)-2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidines in 1-2 steps from a readily available 4-chloro intermediate 5a, offering an efficient and
convenient complement to the literature method. We also synthesized a single deaza (pyridine)
congener 15b of 7a, the most potent compound of the parent series.
Within the parent pyrimidine series, maximal potency against aPKC was associated generally with
compounds of lower molecular weight (e.g., 7a, 7g, 9d) possessing a basic distal amine substituent and
higher lipophilic ligand efficiency (Table SM3, Supplementary Material). Compounds of higher

molecular weight (e.g., 9b) or without a basic distal amine (e.g., 7e) were less potent, possibly suffering
from poor solubility in the assay medium. As a whole, analogues adorned with C-4 basic piperidine
substituents showed potency superior to piperazine. Pyridine analogue 15b was surprisingly less active
than its pyrimidine congener 7a. The C-2 pyridine ring of 15b is more basic relative to that of 7a and
molecular modeling suggests the piperazine moiety is freer to rotate around the C-4 aryl-aryl bond
compared to 7a, which may explain the loss in activity.
For selected compounds that were tested for their effect toward VEGF-induced permeability in
BREC, most showed no effect or modest potency (EC50, 0.3 M). Compound 7l showed a surprisingly
robust response (EC50, 0.071 M). Its performance may represent some nuance to the assay that imparts
just the right balance of aqueous solubility and permeability. Computations on 7l (Table SM3,
Supplementary Material) show overall favorable drug-like properties. Further profiling of 7l show that
it has a favorable selectivity profile against a panel of 31 kinases including the kinases from the AGC
superfamily and kinases involved in permeability, but with potency against PKA and ROCK-II. The
well characterized Rho kinase inhibitor Y-27632 has no effect on reducing VEGF-induced permeability
in BREC cells, indicating Rho kinase does not contribute to the inhibitory effect of 7l in VEGF-induced
permeability in BREC.
Due to its kinase selectivity profile and potency toward inhibition of VEGF-induced permeability in
BREC, compound 7l was tested in a luciferase assay in HEK293 cells for its ability to prevent TNF-α
induced NFκB activation, showing an EC50 of 6.65 µM while not having any effect on cell survival in
the WST-1 assay. These data provided impetus for initial in vivo evaluation of 7l toward assessing its
efficacy in the eye. In a test to determine whether 7l could block VEGF- and TNFα-induced
permeability across the retinal vasculature in a rat model, we found that intravitreal administration of
compound 7l, with or without co-injection of VEGF and TNFα, yielded a complete reduction in
permeability at 3 µM. This finding raises the question of whether systemic administration of 7l can
produce a similar effect in vivo. Toward this end, we evaluated the stability of 7l in mouse liver
microsomes and carried out a mouse pharmacokinetic study. The results of such studies show that 7l
has good microsomal stability and in plasma moderate exposure (AUC and Cmax), low clearance, a long
half-life and high oral bioavailability. With IV dosing, higher levels of 7l were observed at 4 h in the
brain and eye relative to plasma, with highest levels in the eye by either IV or PO dosing. With a slow

oral absorption profile, 7l accumulates in the eye to maintain a high concentration for 24 h after dosing
with levels seven times higher than in plasma.
Potential limitations to the use of aPKC inhibitors clinically need to be considered. A specific
isoform of aPKCtermed PKM contributes to memory, specifically late-phase long-term potentiation
5
0
maintenance (reviewed ). Further, aPKC has been implicated in insulin-stimulated glucose
transport.51,52 As with all drug development studies, dose and toxicity are critical to assess the clinical
potential of any compound However, the eye represents a relatively contained environment that may
allow targeted intraocular delivery, which we have carried out in animal studies in our laboratory, and as
such may avoid potential complications of systematic administration.
In summary, from a focused SAR campaign we have identified a compound with good aPKC
inhibition, functional activity, and favorable drug-like properties, which may represent a class of aPKC
inhibitors for further investigation.
7
. Chemistry experimental
Materials and Methods. All starting monomers were obtained from commercial suppliers and
1
were used without further purification. Routine H NMR spectra were recorded at 400 or 500 MHz on a
Varian Inova 400 or 500 instrument, respectively, with chloroform-d, DMSO-d
6
, or methanol-d as
4
solvent. 13C NMR spectra were recorded on the 500 instrument at 126 MHz with chloroform-d as
solvent. Chemical shift values are recorded in  units (ppm). Mass spectra were recorded on a
Micromass TofSpec-2E Matrix-Assisted, Laser-Desorption, Time-of-Flight Mass Spectrometer in a
+
positive ESI mode (TOFES ) unless otherwise noted. High resolution mass spectrometry (HRMS)
analysis was performed on an Agilent Q-TOF system. Analytical HPLC was performed on an Agilent
1
100 series instrument with an Agilent Zorbax Eclipse Plus C18 (4.6 mm × 75 mm, 3.5 μm particle size)
column with the gradient 10% acetonitrile / water (1 min), 10−90% acetonitrile / water (6 min), and 90%
acetonitrile / water (2 min) flow = 1 mL/min. Thin-layer chromatography (TLC) was performed on silica
gel GHLF plates (250 microns) purchased from Analtech. Column chromatography was carried out in
the flash mode utilizing silica gel (220−240 mesh) purchased from Silicycle or a Teledyne ISCO
Combiflash RF with Redisep Gold RF columns. Extraction solutions were dried over sodium sulfate or
magnesium sulfate prior to concentration.

7
.1. 2-(Pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (4; Scheme 1).
The following is a modification of a literature preparation:37 To an ice-cold solution of 4M HCl in p-
dioxane (12 mL) in a sealed tube was added ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carboxylate (2; 1.02 g, 4.53 mmol). The stirred suspension was then treated with isonicotinonitrile (3;
6
13 mg, 5.9 mmol) followed by 4 mL of additional 4M HCl in p-dioxane. The tube was capped and the
suspension was placed in a sonicator bath for 1 h during which a brilliant yellow precipitate formed
while the temperature reached 40 °C. The suspension was stirred at room temperature for 16 h and then
sonicated again (2 h). TLC (ethyl acetate) showed complete consumption of starting thiophene with
clean generation of product and a trace of a higher R fluorescent impurity. The suspension was diluted
f
with 20 mL of ether and the solids were collected, washed well with ether and dried to leave 1.87 g of a
bright yellow cake-like solid. The solids were suspended in cold water followed by vigorous stirring to
leave a thick suspension that was treated dropwise with con. ammonium hydroxide until all yellow color
had discharged to provide an off-white suspension. The solids were collected, washed thoroughly with
water, and vacuum dried to leave an off-white solid that was suspended in ethyl acetate (~20 mL). The
suspension was sonicated for 5 min and then heated to reflux. The solids were collected by filtration
and then vacuum dried at 80 °C to leave 4 (1.06 g, 83%) as an insoluble, off-white powdery solid,
37
1
mp >320 °C (lit. mp 311 – 314 °C). H NMR (400 MHz, DMSO-d
6
) δ 8.54 (m, 2H), 8.13 (m, 2H),
2
.89 (s, 2H), 2.65 (s, 2H), 1.85 (q, J = 3.7, 3.3 Hz, 4H). TLC shows the presence of product plus a faint
trace of the higher R impurity. Further purification can be effected by DMF recrystallization. The above
f
ethyl acetate filtrate was concentrated to a solid that was triturated from 2-propanol. From this was
1
isolated ~15 mg of a pale yellow solid, mp 150 – 152 °C shown by H NMR and mass spectrometry to
1
be the 4-ethoxy imidate 5b. H NMR (500 MHz, DMSO-d
6
): δ 8.73 (d, J = 5.2 Hz, 2H), 8.28 – 8.23 (m,
2
3
H), 4.66 (q, J = 7.1 Hz, 2H), 2.92 (m, 2H), 2.84 (m, 2H), 1.83 (d, J = 5.9 Hz, 4H), 1.44 (t, J = 7.0 Hz,
H)..
7
.2. 4-Chloro-2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (5a; Scheme 1).
The following is a modification of a literature preparation:34 To a chilled solution of N,N-
dimethylformamide (0.64 mL) in 1,2-dichloroethane (8 mL) was added dropwise oxalyl chloride (4
mL). The solution was stirred for 5 min and the formed Vilsmeier reagent was then treated with
pyrimidinone 4 (1.06 g, 3.74 mmol) followed by 16 mL more 1,2-dichloroethane. The mixture was
heated at 60 °C for 4 h during which there was almost complete dissolution to a deep orange solution.
The cooled solution was concentrated to a solid residue that was ice-cooled and treated with ~35 mL of

sat. aq. NaHCO . The resultant suspension was stirred to homogeneity and the solids were collected,
3
washed well with water and then sparingly with 2-propanol, and vacuum dried to leave 5a (1.105 g,
1
9
2
8%) as a pale green solid, mp 194 – 195 °C. H NMR (400 MHz, DMSO-d
6
): δ 8.76 (m, 2H), 8.22 (m,
H), 3.02 (s, 2H), 2.90 (s, 2H), 1.85 (q, J = 4.0, 3.2 Hz, 4H). HRMS (ESI) m/z: Calcd for
+
[
M+H] 302.0513; Found: 302.0519.
7
.3. Method A: General procedure for the synthesis of 4-substituted-2-(pyridin-4-yl)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines. 4-Chloropyrimidine 5a in absolute ethanol (0.05
mmol/mL) was charged with 4-substituted- piperidine or piperazine (1.3 – 1.5 eq.) or piperazine (10 eq.)
and triethylamine or Hunig’s base (3 eq.), and the mixture was stirred overnight at reflux while being
monitored for disappearance of 5a by TLC (1:1 ethyl acetate : hexanes). The mixture was concentrated,
diluted with dichloromethane, and washed with 5% aq. NaHCO . The aqueous layer was further
3
extracted (2x) with dichloromethane or ethyl acetate. The combined organic extracts were washed with
brine, dried, and concentrated to a residue that was triturated either in acetonitrile or diethyl ether. The
solids were collected and washed with an appropriate solvent, or further purified by flash silica gel
chromatography (ethyl acetate : hexanes) followed by crystallization from ether / hexanes.
7
.3.1. 4-(Piperazin-1-yl)-2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (7a
trihydrochloride; Scheme 1). Following the general procedure, 198 mg of 5a, 10 eq. of piperazine, and
1
3
eq. of triethylamine at reflux for 17 h gave 7a (117 mg, 51%) as a yellow powder. H NMR (400 MHz,
DMSO-d
6
): δ 8.70 (d, J = 5.0 Hz, 2H), 8.22 (d, J = 5.1 Hz, 2H), 2.88 (s, 6H), 1.88 (s, 2H), 1.73 (s, 2H),
+
other peaks hidden under water peak. HRMS (ESI) m/z: Calcd. for [M+H] : 352.159; Found: 352.1603.
HPLC: rt 4.86 min (98% purity). TLC: R 0.37 (9:1 dichloromethane : methanol). To an ice-cold
f
solution of 36 mg of 7a free base in 3 mL of dichloromethane was added dropwise 0.21 mL of 1M HCl
in diethyl ether. The dark yellow suspension was stirred at 0 °C for 1 h and the solids were collected,
1
washed with ether, and dried to leave 43 mg (99%) of 7a trihydrochloride salt as a yellow solid. H
NMR (500 MHz, DMSO-d
6
): δ 9.63 (s, 2H), 8.96 (d, J = 5.8 Hz, 2H), 8.70 (d, J = 5.8 Hz, 2H), 3.74 (t, J
=
4.9 Hz, 4H), 3.28 (s, 4H), 2.94 (d, J = 5.7 Hz, 4H), 1.90 (tt, J = 8.0, 5.2 Hz, 2H), 1.81 – 1.67 (m, 2H).
+
HRMS (ESI) m/z: Calcd. for [M+H] , 352.159; Found, 352.1591. HPLC: rt 4.85 min (95% purity).
7
.3.2. 4-(4-Methylpiperazin-1-yl)-2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine
(7b; Scheme 1). Following the general procedure, 30 mg of 5a, 1.3 eq. of 1-methylpiperazine, and 3 eq.

1
of triethylamine at reflux for 20 h gave 7b (23 mg, 63%) as a pale yellow solid. H NMR (400 MHz,
DMSO-d ): δ 8.74 – 8.68 (m, 2H), 8.26 – 8.20 (m, 2H), 3.52 (s, 3H), 2.93 – 2.84 (m, 4H), 2.70 (s, 2H),
.63 – 2.50 (m, 2H), 2.37 (s, 2H), 2.07 (s, 1H), 1.89 (d, J = 6.6 Hz, 2H), 1.74 (s, 2H). HRMS (ESI) m/z:
6
2
+
Calcd. for [M+H] , 366.1747; Found, 366.1755. HPLC: rt 4.90 min (99% purity). TLC: R
dichloromethane : methanol).
f
0.23 (95 : 5
7
.3.3. tert-Butyl (2-(4-(2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)piperazin-1-yl)ethyl)carbamate (7c; Scheme 1). Following the general procedure, 50 mg of 5, 1.5 eq.
of tert-butyl (2-(piperazin-1-yl)ethyl)carbamate, and 3 eq. of Hunig’s base at reflux for 17 h gave 7c (39
1
mg, 48%) as a pale yellow powder. H NMR (400 MHz, chloroform-d): δ 8.70 (d, 2H), 8.29 (d, 2H),
5
2
4
.03 (br s, 1H), 3.52 (m, 4H), 3.29 – 3.21 (m, 2H), 2.96 – 2.89 (m, 4H), 2.66 (br s, 4H), 2.59 – 2.43 (m,
H), 1.99 – 1.94 (m, 2H), 1.84 – 1.77 (m, 2H), 1.45 (s, 9H). HRMS (ESI) m/z: Calcd. for [M+H]+,
95.246; Found, 495.254.
7
.3.4. 1-(2-(Pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)piperidin-4-ol (7e;
Scheme 1). Following the general procedure, 46 mg of 5a, 1.5 eq. of piperidin-4-ol, and 3 eq. of
1
triethylamine at reflux for 19 h gave 7e (47 mg, 84%) as a yellow solid. H NMR (400 MHz, DMSO-
6
d ): δ 8.75 – 8.69 (m, 2H), 8.27 – 8.21 (m, 2H), 4.79 (d, J = 4.2 Hz, 1H), 3.77 (d, J = 14.4 Hz, 4H), 3.19
(t, J = 11.5 Hz, 1H), 2.90 (dd, J = 12.7, 6.3 Hz, 4H), 1.90 (d, J = 6.3 Hz, 4H), 1.75 (s, 2H), 1.66 – 1.50
+
(
m, 2H). HRMS (ESI) m/z: Calcd. for [M+H] , 367.1587; Found, 367.1599. HPLC: rt 5.76 min (98%
purity). TLC: R 0.2 (95 : 5 dichloromethane : methanol).
f
7
.3.5. tert-Butyl (1-(2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)piperidin-4-yl)carbamate (7f; Scheme 1). Following the general procedure, 45 mg of 5a, 1.5 eq. of
tert-butyl piperidin-4-ylcarbamate, and 3 eq. of triethylamine at reflux for 20 h gave 7f (44 mg, 63%) as
1
a pale yellow solid. H NMR (500 MHz, chloroform-d): δ 8.72 (s, 2H), 8.30 (d, J = 5.0 Hz, 2H), 4.54 (br
s, 1H), 3.89 (d, J = 13.2 Hz, 2H), 3.74 (s, 1H), 3.16 (t, J = 12.3 Hz, 2H), 2.92 (dd, J = 10.6, 5.6 Hz, 4H),
2
.15 – 2.07 (m, 2H), 2.01 – 1.91 (m, 2H), 1.87 – 1.78 (m, 2H), 1.70 – 1.60 (m, 2H), 1.46 (s, 9H).
7
.3.6. tert-Butyl methyl(1-(2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)piperidin-4-yl)carbamate (7h; Scheme 1). Following the general procedure, 60 mg of 5a, 1.5 eq. of
tert- butyl methyl(piperidin-4-yl)carbamate, and 3 eq. of triethylamine at reflux for 20 h gave 7h (63

1
mg, 65%) as a pale white solid. H NMR (400 MHz, chloroform-d): δ 8.72 (d, J = 5.2 Hz, 2H), 8.34 –
.25 (m, 2H), 4.04 (d, J = 12.7 Hz, 2H), 3.10 (t, J = 12.6 Hz, 2H), 2.93 (dd, J = 13.6, 6.7 Hz, 4H), 2.79
s, 3H), 2.02 – 1.73 (m, 9H), 1.48 (s, 9H). TLC: R 0.4 (4 : 1 hexanes : acetone).
8
(
f
7
.3.7. N,N-Dimethyl-1-(2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)piperidin-4-amine (7j; Scheme 1). Following the general procedure, 60 mg of 5a, 1.5 eq. of N,N-
dimethylpiperidin-4-amine, and 3 eq. of triethylamine at reflux for 20 h gave a residue that was purified
by preparative plate silica gel chromatography (eluting with 13 : 1 : 0.01 dichloromethane : methanol :
1
triethylamine) to give 7j (20 mg, 25 %) as a yellow solid. H NMR (500 MHz, chloroform-d): δ 8.75 –
8
1
.67 (m, 2H), 8.33 – 8.25 (m, 2H), 3.98 (d, J = 13.0 Hz, 2H), 3.07 – 2.85 (m, 6H), 2.35 (s, 6H), 2.05 –
.99 (m, 2H), 1.95 (ddt, J = 8.6, 6.3, 4.0 Hz, 3H), 1.82 (dd, J = 7.3, 4.3 Hz, 2H), 1.71 (dd, J = 11.7, 3.7
+
Hz, 2H). HRMS (ESI) m/z: Calcd. for [M+H] , 394.206; Found, 394.2062. HPLC: rt 5.12 min (98%
purity). TLC: R 0.1 (95 : 5 dichloromethane : methanol).
f
7
.3.8. tert-Butyl ((1-(2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)piperidin-4-yl)methyl)carbamate (7k; Scheme 1). Following the general procedure, 1.0 g of 5, 1.5 eq.
of tert-butyl (piperidin-4-ylmethyl)carbamate, and 3 eq. of Hunig’s base at reflux for 17 h gave 7k (1.2
1
g, 76%) as a pale yellow powder. H NMR (400 MHz, chloroform-d): δ 8.71 (d, J = 5.5 Hz, 2H), 8.29
(d, 2H), 4.66 (s, 1H), 3.96 (d, J = 12.9 Hz, 2H), 3.15 – 2.72 (m, 8H), 1.98 - 1.59 (m, 9H), 1.49 (9H).
+
HRMS (ESI) m/z: Calcd. for [M+H] , 480.236; Found, 480.243. HPLC: rt 6.9 min (94.6% purity).
7
.3.9. tert-Butyl 4-(2-((2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)amino)ethyl)piperazine-1-carboxylate (9a; Scheme 1). Following the general procedure, 50 mg of 5,
1
9
–
2
.5 eq. of tert-butyl (2-(piperazin-1-yl)ethyl)carbamate, and 3 eq. of Hunig’s base at reflux for 20 h gave
1
a (25 mg, 31%) as a pale yellow powder. H NMR (400 MHz, DMSO-d
6
): δ 8.69 – 8.67 (m, 2H), 8.21
8.20 (m, 2H), 6.74 – 6.72 (m, 1H), 3.71 – 3.68 (m, 2H), 2.95 (s, 2H), 2.78 (s, 2H), 2.65 – 2.62 (m, 2H),
+
.44 – 2.42 (m, 8H), 1.84 – 1.83 (m, 4H), 1.38 (s, 9H). HRMS (ESI) m/z: Calcd. for [M+H] , 495.246;
Found, 495.254. HPLC: rt 5.35 min (97.1% purity).
7
.3.10. tert-Butyl 4-((2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-
yl)amino)piperidine-1-carboxylate (9c; Scheme 1). Following the general procedure, 50 mg of 5, 1.5 eq.
of tert-butyl 4-aminopiperidine-1-carboxylate, and 3 eq. of Hunig’s base at reflux for 20 h gave 9c (43

1
mg, 56%) as a pale yellow powder. H NMR (400 MHz, chloroform-d): δ 8.71 (m, 2H), 8.25 (m, 2H),
.24 (d, J = 7.2 Hz,1H), 4.73 – 4.65 (m, 1H), 4.48 – 4.44 (m, 1H), 4.15 – 3.98 (m, 2H), 3.10 (t, J = 12.2
5
Hz, 2H), 3.03 – 2.76 (m, 5H), 2.19 (d, J = 12.7 Hz, 2H), 1.93 (dd, J = 11.6, 6.2 Hz, 4H), 1.49 (s, 11H).
+
HRMS (ESI) m/z: Calcd. for [M+H] , 466.219; Found, 466.227. HPLC: rt 7.01 min.
7
.4. Method B. General procedure for Boc deprotection of 4-substituted-2-(pyridin-4-yl)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines. A room temperature solution of Boc intermediate in
anhydrous dichloromethane (0.03 mmol/mL) was treated with 20 eq. of HCl in p-dioxane (4N).
Consumption of starting material was monitored by TLC (1:1 ethyl acetate : hexanes). After stirring
overnight, the mixture was concentrated and the residue was triturated in ether and collected. The solids
were washed with ether and dried to provide the hydrochloride salt.
7
.4.1. 2-(4-(2-(Pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)piperazin-1-
yl)ethan-1-amine trihydrochloride (7d; Scheme 1). Following the general procedure, 30 mg of 7c gave
1
7
2
3
d trihydrochloride salt (22 mg, 92%) as a yellow solid. H NMR (400 MHz, DMSO-d
6
): δ 11.82 (br s,
H), 9.84 (br s, 2H), 8.99 (d, J = 6.0 Hz, 2H), 8.75 (d, J = 5.9 Hz, 2H), 8.54 (s, 1H), 8.35 (s, 1H), 4.12 –
.98 (m, 2H), 3.82- 3.10 (m, 10H), 2.96 (m, 4H), 2.01 – 1.77 (m, 4H). HRMS (ESI) m/z: Calcd .for
+
[
M+H] , 395.194; Found, 395.201. HPLC: rt 4.50 min (99% purity).
7
.4.2. 1-(2-(Pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)piperidin-4-amine
trihydrochloride (7g; Scheme 1). Following the general procedure, 14 mg of 7f gave 7g trihydrochloride
salt (14.5 mg, 100%) as a crude yellow solid, 74% pure by HPLC. The solid was purified by silica gel
preparative plate chromatography, eluting with 9 : 1 : 0.1 dichloromethane : methanol : trimethylamine
1
to leave 7g (5.1 mg, 47%). H NMR of trihydrochloride (500 MHz, DMSO-d
6
) δ 8.93 (brs, 2H), 8.62 (s,
2
H), 8.13 (d, J = 4.8 Hz, 3H), 4.02 (d, J = 13.3 Hz, 2H), 3.33 (m, 2H), 3.16 (t, J = 12.6 Hz, 2H), 2.94 (d,
J = 5.4 Hz, 5H), 2.08 (d, J = 12.5 Hz, 2H), 1.97 – 1.87 (m, 2H), 1.76 (d, J = 9.1 Hz, 4H). HRMS
+
(
ESI) m/z: Calcd. for [M+H] , 366.1747; Found, 366.1759. HPLC: rt 5.02 min (92% purity).
7
4
.4.3. N-Methyl-1-(2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)piperidin-
-amine trihydrochloride (7i; Scheme 1). Following the general procedure, 24 mg of 7h gave 7i
1
trihydrochloride salt (19.4 mg, 86%) as a yellow solid. H NMR (500 MHz, DMSO-d
6
): δ 8.92 (br s,
3
H), 8.58 (s, 2H), 4.06 (d, J = 13.4 Hz, 2H), 3.11 (t, J = 12.7 Hz, 2H), 2.95 (d, J = 6.3 Hz, 4H), 2.58 (s,

4
H), 2.17 (d, J = 12.4 Hz, 2H), 1.92 (t, J = 6.1 Hz, 2H), 1.77 (s, 5H). HRMS (ESI) m/z: Calcd. for
+
[
M+H] , 380.1903; Found, 380.1910. HPLC: rt 5.08 min (96% purity). R
f
0.44 (4 : 1 dichloromethane :
methanol).
7
.4.4. (1-(2-(Pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)piperidin-4-
yl)methanamine trihydrochloride (7l; Scheme 1). Following the general procedure, 1.2 g of 7k gave 7l
1
trihydrochloride salt (1.0 g, 96%) as a yellow solid. H NMR (400 MHz, DMSO-d
6
): δ 8.98 (d, 2H),
8
6
.74 (d, 2H), 8.15 (br s, 3H), 4.22 – 3.95 (m, 3H), 3.13 – 2.87 (m, 3H), 2.77 (s, 4H), 2.05 – 1.67 (m,
+
H), 1.50 -1.28 (m, 3H). HRMS (ESI) m/z: Calcd. for [M+H] , 380.183; Found, 380.190 HPLC: rt 4.76
min (95.9% purity).
7
4
.4.5. N-(2-(Piperazin-1-yl)ethyl)-2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-
-amine trihydrochloride (9b; Scheme 1). Following the general procedure, 35 mg of 9a gave 9b
1
trihydrochloride salt (12 mg, 75%) as a yellow solid. H NMR (400 MHz, DMSO-d
6
+ chloroform-d): δ
8
7
3
.95 (d, J = 5.9 Hz, 2H), 8.87 (d, J = 5.2 Hz, 2H), 7.27-7.22 (m, 2H), 4.20-3.90 (m, 4H), 3.57 -3.45 (m,
+
H), 3.06 (s, 4H), 2.83 (s, 2H), 1.84 (s, 4H). HRMS (ESI) m/z: Calcd. for [M+H] , 395.194; Found,
95.201. HPLC: rt 4.50 min (100 % purity).
7
.4.6. N-(Piperidin-4-yl)-2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine
trihydrochloride (9d; Scheme 1). Following the general procedure, 40 mg of 9c gave 9d trihydrochloride
1
salt (27 mg, 86%) as a yellow solid. H NMR (400 MHz, DMSO-d
6
): δ 8.94 (d, 2H), 8.67 (d, 2H), 4.69 –
4
.64 (m, 2H), 3.48 – 3.33 (m, 3H), 2.92 (s, 2H), 2.86 (s, 2H), 1.84 – 1.82 (m, 4H), 1.47 – 1.43 (m, 4H).
+
HRMS(ESI) m/z: Calcd. for [M+H] , 366.167 ; Found, 366.158. HPLC: rt 4.63 min (94.2% purity).
7
.5. Ethyl 2-(3-ethoxy-3-oxopropanamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (10;
Scheme 2). A 0 °C solution of ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (2; 4
g, 17.75 mmol) in dichloromethane (30 mL) was treated with N,N-diisopropylethylamine (3.82 ml, 21.3
mmol). Ethyl 3-chloro-3-oxopropanoate (2.94 g, 19.5 mmol) in dichloromethane (10mL) was then
added dropwise over 5 min at 0 °C. The resulting solution was stirred at 0 °C for 18 h, diluted with
water, and extracted with ethyl acetate (2x). The combined organic phase was washed with saturated
1
brine, dried, and concentrated to give 10 (4.8 g, 80%) as an oil. H NMR (400 MHz, DMSO-d
6
): δ 11.52
(s, 1H), 4.28 (q, J = 7.1 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 3.32 (s, 1H), 2.69 (s, 2H), 2.58 (s, 2H), 1.71

+
(
q, J = 3.7 Hz, 4H), 1.29 - 1.15(m, 6H). HRMS (ESI) m/z: Calcd. for [M+H] 340.114; Found 340.122.
HPLC: rt 8.6 min (100% purity).
7
.6. Ethyl 4-hydroxy-2-oxo-1,2,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-b]pyridine-3-carboxylate (11a;
Scheme 2). A suspension of hexane washed sodium hydride (940 mg of 60% dispersion, 23.51 mmol)
in tetrahydrofuran (100 mL) cooled to 0º C was treated portion-wise with ester 10 (3.8 g, 11.20 mmol)
and the resulting mixture was heated at reflux for 15 h under nitrogen. The cooled mixture was
concentrated, diluted sequentially with water (100mL) and 5 mL conc. HCl, and stirred for 1 h at room
temperature. The precipitated solid pellets were collected, washed with water and recrystallized from
1
abs. ethanol to give 11a (1.1g, 32%) as a brown solid following drying. H NMR (400 MHz,
chloroform-d): δ 4.48 (q, J = 7.1 Hz, 2H), 2.94 (d, J = 6.4 Hz, 2H), 2.69 (m, 2H), 1.84 (q, J = 8.2 Hz,
+
4
7
H), 1.48 (t, J = 7.1 Hz, 3H). HRMS (ESI) m/z: Calcd for [M+H] 294.079; Found: 294.07. HPLC: rt
.04 min (100% purity).
7
.7. 4-Hydroxy-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-2(1H)-one (11b; Scheme 2). Ester
1a (1.0 g, 3.41 mmol) was heated at reflux in 2 N aq. NaOH (50 mL) for 5 h. The mixture was filtered
1
hot, cooled to 0º C, and acidified to pH 1 with conc. HCl. The precipitate was collected and dried to
1
leave 11b (140 mg, 19%) as a brown solid. H NMR (400 MHz, DMSO-d
6
): δ 11.30 (s, 1H), 5.82 (s,
+
1
H), 2.80 (s, 2H), 2.63 (s, 2H), 1.76 – 1.70 (m, 4H). HRMS (ESI) m/z: Calcd for [M+H] 222.051;
Found: 222.058. HPLC: rt 4.14 min (90% purity).
7
.8. 2,4-Dichloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine (12; Scheme 2). A mixture of
pyridone 11b (140 mg, 0.63 mmol) and phenylphosphonic dichloride (5 mL; 1.23 g, 6.33 mmol) was
heated under argon at 180 °C for 4 h and then kept at room temperature overnight. The mixture was
poured into ice cold water and extracted with dichloromethane (2x). The combined organic phase was
washed with satd. brine, dried, and concentrated to a residue that was purified by flash chromatography
(
Isco Flash) with gradient elution from 0% to 30% ethyl acetate/ hexane followed by concentration of
1
product fractions to provide 12 (120 mg, 74%) as a yellow solid. H NMR (400 MHz, chloroform-d): δ
+
7
.19 (s 1H), 3.10 (s, 2H), 2.85 (s, 2H), 1.90 (s, 4H). HRMS (ESI) m/z: Calcd for [M+H] 257.983;
Found: 257.991.

7
.9. 2-Chloro-4-(piperazin-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine (13a; Scheme 2)
and 7.10. 4-chloro-2-(piperazin-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine (14a; Scheme
). A mixture of dichloride 12 (80 mg, 0.31 mmol), and piperazine (100 mg, 1.16 mmol) in N-methyl-2-
2
pyrrolidone (1 mL) was heated at 200 °C in a microwave reactor for 1.5 h. The mixture was diluted with
water, extracted with ~1:1 ethyl acetate : ether (2x), and the combined extracts were washed with satd.
brine and dried. Concentration left a solid showing two major spots by TLC (95:5 dichloromethane :
methanol). Purification by flash chromatography (Isco Flash) with gradient elution with 0% to 10%
methanol / dichloromethane and concentration of product fractions gave 13a (50 mg, 52%) and 14a (20
1
mg, 21%) that were carried directly into the next step. 13a: H NMR (400 MHz, chloroform-d): δ 6.78
1
(
s, 1H), 3.09 – 2.97 (m, 8H), 2.85 (m, 2H), 1.94 – 1.77 (m, 6H). 14a: H NMR (400 MHz, chloroform-
d): δ 6.59 (s, 1H), 3.58 – 3.52 (m, 3H), 3.03 – 2.98 (m, 5H), 2.76 (m, 2H), 1.95 – 1.84 (m, 6H).
7
.10. tert-Butyl 4-(2-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-4-yl)piperazine-1-
carboxylate (13b; Scheme 2). A stirred solution of 13a (25 mg, 0.08 mmol), triethylamine (8.2 mg,
.08 mmol,) and methanol (3 mL) was treated portion-wise over 5 min with di-tert-butyl dicarbonate (18
0
mg, 0.08 mmol) followed by 4-dimethylaminopyridine (10 mg, 0.08 mmol). The solution was stirred at
room temperature for 20 h and concentrated to a residue that was dissolved in dichloromethane. The
solution was washed sequentially with 0.5 M aq. HCl (2x), saturated aq. sodium carbonate, saturated
brine, dried, and concentrated to a residue that was filtered through flash silica gel, eluting with 7 : 3
1
ethyl acetate/ hexanes to provide 13b (25 mg, 75%) as a clear oil. H NMR (400 MHz, chloroform-d): δ
6
.78 (s, 1H), 3.02 –2.84 (m, 10H), 1.93 – 1.79 (m, 6H), 1.49 (s, 9H). HRMS (ESI) m/z: Calcd for
+
[
M+H] 409.143; Found: 409.151.
7
.11. tert-Butyl 4-(4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-2-yl)piperazine-1-
carboxylate (14b; Scheme 2). Reaction of 14a (45 mg, 0.15 mmol) with di-tert-butyl dicarbonate (32
mg, 0.15 mmol) and workup was carried out exactly as described for the synthesis of 13b above to
1
provide 14b (50 mg, 84%) as a clear oil. H NMR (400 MHz, chloroform-d): δ 6.59 (s, 1H), 3.54 (s,
8
H), 3.01 (br s, 2H), 2.76 (br s, 2H), 1.87 – 1.84 (m, 4H), 1.48 (s, 9H). HRMS (ESI) m/z: Calcd for
+
[
M+H] 409.143; Found: 409.151.
7
.12. tert-Butyl 4-(2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-4-yl)piperazine-1-
carboxylate (15a; Scheme 2). A stirred mixture of 13b (20 mg, 0.05 mmol), pyridin-4-ylboronic acid

(9.06 mg, 0.07 mmol), tetrakis(triphenylphosphine)palladium(0) (1.14 mg, 0.98 µmol) and sodium
carbonate (5.2 mg, 0.05 mmol) in 3 : 2 water : N,N-dimethylformamide (2.5 mL) was purged with argon
for 3 min and then heated under argon at 95 ºC for 2 h The cooled mixture was diluted with water and
extracted with ethyl acetate (2x). The combined extracts were washed with saturated brine, dried, and
concentrated to a residue that was purified by flash silica gel chromatography with gradient elution from
0
% to 10% methanol / dichloromethane. Product fractions were combined and concentrated to leave 15a
1
(
20 mg, 68%). H NMR (500 MHz, chloroform-d): δ 8.72 (s, 2H), 8.17 – 8.06 (m, 2H), 7.34 (d, J = 3.1
Hz, 1H), 4.92 (s, 2H), 4.09 (s, 1H), 3.20 (s, 5H), 3.11 – 3.03 (m, 2H), 2.97 – 2.89 (m, 2H), 1.94 (ddt, J =
.4, 6.3, 2.3 Hz, 2H), 1.91 – 1.82 (m, 2H), 1.50 (s, 9H).
13C NMR (126 MHz, chloroform-d): δ 163.35, 156.32, 154.71, 149.59, 149.34, 147.18, 139.07, 128.02,
8
1
27.74, 121.90, 107.22, 80.20, 52.94, 43.86, 42.90, 28.41, 26.46, 26.29, 22.97, 22.66. HRMS (ESI) m/z:
+
Calcd for [M+H] 451.209; Found: 451.216.
7
.13. 4-(Piperazin-1-yl)-2-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine
trihydrochloride (15b; Scheme 2). A solution of 15a (32 mg, 0.07 mmol) in diethyl ether (2 mL) was
treated with 4.0 M HCl in p-dioxane (1.0 mL, 4.0 mmol) and stirred overnight at room
temperature. Concentration left a solid that was triturated in ether, collected and dried to leave 15b
1
trihydrochloride salt (20 mg, 61%) as a yellow solid. H NMR (400 MHz, methanol-d
4
): δ 8.89 (d, J =
1
2.7 Hz, 4H), 7.92 (s, 1H), 3.52 (m, 8H), 3.16 (s, 2H), 2.98 (d, J = 6.5 Hz, 2H), 1.96 (m, 4H). HRMS
+
(
ESI) m/z: Calcd for [M+H] 351.157; Found: 351.164 HPLC: rt 4.1 min (92% purity).
7
.14. tert-Butyl 4-(4-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-2-yl)piperazine-1-
carboxylate (16a; Scheme 2). Reaction of 14b (50 mg, 0.12 mmol), pyridin-4-ylboronic acid (23 mg,
.18 mmol), tetrakis(triphenylphosphine)palladium(0) (6.28 mg, 5.44 µmol) and sodium carbonate (13
0
mg, 0.12 mmol) in 3:2 water:N,N-dimethylformamide (2.5 mL) followed by workup as described above
1
for the synthesis of 15a gave 16a (22 mg, 40 %). H NMR (500 MHz, chloroform-d): δ 8.75 (s, 2H),
7
6
7
.53 (s, 2H), 6.39 (s, 1H), 3.66 – 3.51 (m, 9H), 2.80 (t, J = 6.3 Hz, 2H), 1.92 – 1.73 (m, 4H), 1.61 (p, J =
.0 Hz, 2H), 1.49 (s, 9H). 13C NMR (126 MHz, chloroform-d): δ 154.76, 126.29, 104.93, 80.08, 77.25,
7.20, 76.99, 76.74, 45.50, 29.69, 28.41, 27.41, 25.80, 22.80, 22.66. HRMS (ESI) m/z: Calcd for
+
[
M+H] 451.209; Found: 451.216.

7
.15. 2-(Piperazin-1-yl)-4-(pyridin-4-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine
dihydrochloride (16b; Scheme 2). Reaction of 16a (20 mg, 0.04 mmol) and workup as described above
for the synthesis of 15b gave 16b dihydrochloride salt (10 mg, 33%) as a yellow solid. HRMS
+
(
ESI) m/z: Calcd for [M+H] 351.157; Found: 351.164. HPLC: rt 4.5 min (90% purity).
8
. Biology Experimental
1
Materials and Methods. (S)-3-phenyl-N -(2-(pyridin-4-yl)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)propane-1,2-diamine (compound 1), 95% pure by hplc,
was made according to the literature procedure.31 To make a solution of the compounds in Table 1,
each compound was reconstituted with DMSO at 10 mM and incubated at 30° C overnight to be used
the following day in kinase and cell permeability assays, or in vivo studies. The final concentration of
DMSO was 1% in the kinase assay and 0.1% in the cell permeability assay.
8
.1. Enzyme inhibition assay. Compounds of Table 1 were tested against commercially purchased
recombinant full-length human aPKC or PKC (SignalChem, British Columbia, Canada) with the
peptide substrate (CREBtide) measuring ATP depletion in order to determine inhibitor IC50 in vitro. The
Kinase-Glo™ luminescent assay (Promega, WI, USA) was used as per manufacturer instructions.
Briefly, compounds were serially diluted and incubated for 3 h at room temperature with 250 ng/mL
PKCζ and 30 μM CREBtide in the presence of 0.1 μM of ATP. Kinase reactions were performed in
solid white, V -bottom 96-well plates in a total volume of 50 μL. Data points are the average of two
determinations for each reaction in triplicate, and error bars are ± S.E.M. ATP depletion was measured
in kinase reactions using Veritas™ Microplate Luminometer (Promega, WI, USA) with a 5- or 6-point
dose-response curve to determine IC50. Compound 7l was tested in the Kinase-Glo™ luminescent assay
at 5µM, 10µM and 30µM and compared to DMSO vehicle. The compound 7l had no effect on ATP
levels at any dose tested. Alternatively, a commercial kinase IC50Profiler™ Service (Eurofins Pharma
Discovery Services, Celle l'Evescault, France) was used to determine the IC50 of the inhibitor using a 9-
33
point assay against full-length enzyme via incorporation of radioactivity from γ-[ P]-ATP into substrate
in the presence of the indicated compound concentrations. Curve fitting was performed using GraphPad
Prism sigmoidal dose-response (variable slope) software.

8
.2. Counter enzyme screening. A commercial radiometric kinase assay (Eurofins Pharma Discovery
Services, Dundee, United Kingdom or Celle l'Evescault, France) was used at 5 µM of compound 7l
against 31 full-length kinases of the AGC superfamily with a focus on PKC isoforms.
8
.3. TNF/VEGF permeability assay. Recombinant human VEGF and recombinant human TNFα were
purchased from R&D Systems (Minneapolis, MN, USA). Endothelial permeability response to VEGF
and the effective concentration of inhibitor that blocked permeability were measured using primary
bovine retinal endothelial cells (BREC) prepared as previously reported.53 BREC were grown on trans-
well filters and diffusive permeability to 70kda RITC dextran (Po) after VEGF addition was measured
as described previously.54 A 5-point dose-response was used to determine EC50 for inhibition of VEGF
induced permeability by comparing VEGF-induced permeability to the relative inhibition of the VEGF
response in the presence of inhibitor.
8
.4. TNF-induced NFB activation assay. HEK293 cells stably transfected with the NFκB enhancer
driving luciferase were treated with compound 7l for 2 h and then treated with 5 ng/mL TNFα.
Luciferase activity was measured by Veritas™ Microplate Luminometer with Luciferase Assay System
(
(
Promega,, WI, USA ) as described previously.23 Cell survival was assessed by the WST-1 assay
Roche Diagnostics GmbH, Mannheim, Germany).
8
.5. In vivo retinal vascular permeability assay. To determine whether a test compound blocks retinal
vascular permeability in vivo, its ability to prevent VEGF and TNFα induced permeability of fluorescein
isothiocyanate-bovine serum albumin (FITC-BSA) across the retinal vasculature was determined.
Briefly, Long-Evans rats were injected intravitreally with 15.6 ng or 46.8 ng of compound 7l
trihydrochloride in 2 µL of PBS with 0.1% BSA yielding an estimated final concentration of 1 μM or 3
μM with or without co-injection of VEGF (50 ng) and TNFα (10 ng) per eye and compared with vehicle
injection. After 3 h, rats received an intravenous injection of FITC-BSA at 2 μL/g body weight (equal to
2
00 mg/kg body weight). After 2 h, animals were perfused with warm saline for 2 min, retinas were
removed, dried and FITC-BSA was extracted. FITC-BSA was quantified on a spectrophotometer and
normalized to plasma levels measured pre-perfusion.
8
.6. Retinal function assay. A Celeris System (Diagnosys) was used to measure electroretinogram
(ERG) retinal response to light flash. Long-Evans rats were dark adapted overnight, anesthetized with

intraperitoneal injection of ketamine (50 mg/kg body weight) and xylazine (5 mg/kg body weight) and
compound 7l trihydrochloride was given by intravitreal injection to a final concentration of 3 µM as
described in Section 8.5 above. At 3-5 hours post injection, rats were anesthetized again for ERG tests.
Rats received a drop of 0.5% tropicamide to stimulate eye dilation and a drop of 0.5% proparacaine to
numb the eyes. A drop of 0.3% hypromellose (Genteal) was used to cushion the contact with the
2
electrode stimulators and ERG responses were recorded at 0.1, 1, and 10 cd.s/m intensity stimuli.
8
.7. Microsomal stability study. The in vitro study was performed in mice liver microsomes to evaluate
the susceptibility of compound 7l to cytochrome P450-mediated metabolism using pooled CD-1 mouse
liver microsomes (purchased from Xeno Tech). The test compound at 1 μM was incubated with 0.5
mg/mL liver microsomes and 1.7 mM cofactor -NADPH in 0.1 M phosphate buffer (pH = 7.4)
containing 3.3 mM MgCl at 37 °C. The DMSO concentration was less than 0.1% in the final incubation
2
system. At 0, 5, 10, 15, 30, 45, and 60 min of incubation, a 40 µL aliquot of reaction mixture was
withdrawn, and the reaction was quenched immediately by adding a 3-fold excess of cold acetonitrile
containing 100 ng/mL of internal standard for quantification. The incubation without the addition of
NADPH was used as negative control. Verapamil were incubated similarly as positive control substrate.
The collected fractions were centrifuged at 15000 rpm for 10 min to collect the supernatant for
LC−MS/MS analysis from which the amount of remaining test compound was determined. The natural
log of the amount of test compound remaining was plotted against time to determine its disappearance
rate and the half-life. The LC−MS/MS method consisted of a Shimadzu HPLC system and
chromatographic separation of test compounds was achieved using a Waters XBridge-C18 column (5
cm × 2.1 mm, 3.5 μm). An AB Sciex QTrap 5500 mass spectrometer equipped with an electrospray
ionization source (Applied Biosystems, Toronto, Canada) in the negative-ion multiple reaction
monitoring (MRM) mode was used for detection. The mobile phases utilized were 0.1% formic acid in
purified water (A) and 0.1% formic acid in acetonitrile (B). The gradient (B) was held at 2% (0-0.5
min), increased to 95% at 2.0 min, maintained at isocratic 95% B for 1.6 min, and then immediately
stepped back down to 2% for a 2 min re-equilibration. The flow rate was set at 0.4 mL/min, and 7l and
internal standard (IS; NSLS503) were detected on the mass spectrometer by using multiple reaction
monitoring transitions 380.1 → 309.1 m/z and 406.2 → 71.5 m/z, respectively. Mass spectrometry
parameter optimization was performed using an automated quantitative method provided by the
manufacturer.