Synthesis and evaluation of pyrimidoindole analogs in umbilical cord blood ex vivo expansion

Article abstract of DOI:10.1016/j.ejmech.2019.04.042

The scarcity of hematopoietic stem cells (HSCs)significantly hindered their clinical potentials. Umbilical cord blood (UCB)has become the leading source of HSCs for both research and clinical applications. But the low content of HSCs in a single UCB unit limited its use only to pediatric patients. Various cytokines and small molecules have demonstrated strong abilities in promoting HSC ex vivo expansion, of which UM171 is the newest and by far the most potent HSC ex vivo expansion agent. In this study, we synthesized 37 pyrimidoindole analogs and identified 6 compounds to be potent in promoting HSC ex vivo expansion. In particular, analog 11 was found to be the most effective in stimulating ex vivo expansion of UCB CD34⁺ cells and CD34⁺CD38^? cells. Initial data indicated that compound 11 promoted the absolute number of long term HSCs and inhibited their differentiation. UCB HSCs expanded with 11 retained adequate multi-lineage differentiation capacity. In addition, compound 11 is not cytotoxic at its test concentrations, suggesting that it merits further investigation for potential clinical applications.

Full text of DOI:10.1016/j.ejmech.2019.04.042

Accepted Manuscript
Synthesis and evaluation of pyrimidoindole analogs in umbilical cord blood ex vivo
expansion
Yue Feng, Xiao-Yang Xie, Yi-Qiu Yang, Yu-Tong Sun, Wen-Hui Ma, Peng-Jun Zhou,
Zi-Yao Li, Hui-Qiang Liu, Yi-Fei Wang, Yun-Sheng Huang
PII:
S0223-5234(19)30357-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.042
EJMECH 11277
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 January 2019
Revised Date: 15 April 2019
Accepted Date: 15 April 2019
Please cite this article as: Y. Feng, X.-Y. Xie, Y.-Q. Yang, Y.-T. Sun, W.-H. Ma, P.-J. Zhou, Z.-Y. Li, H.-
Q. Liu, Y.-F. Wang, Y.-S. Huang, Synthesis and evaluation of pyrimidoindole analogs in umbilical cord
blood ex vivo expansion, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.04.042.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Synthesis and evaluation of pyrimidoindole analogs in
umbilical cord blood ex vivo expansion
a,b,#
a,#
c
a
a
b
Yue Feng , Xiao-Yang Xie , Yi-Qiu Yang , Yu-Tong Sun , Wen-Hui Ma , Peng-Jun Zhou , Zi-Yao
b
b
b,*
a,*
Li , Hui-Qiang Liu , Yi-Fei Wang , Yun-Sheng Huang
a
School of Pharmacy, Guangdong Medical University, 1 Xincheng Ave, Songshan Lake Technology
Park, Dongguan, Guangdong 523808, China.
b
Institute of Biomedicine, College of Life Science & Technology, Jinan University, Guangzhou,
Guangdong 510632, China.
c
Department of Pharmacy, Zhaoqing Medical College, 6 Xijiang South Rd, Zhaoqing, Guangdong
5
26020, China.
#
Contributed equally as co-first authors.
*
Corresponding author: Yi-Fei Wang, Tel: +86-20-85223426, Email: twangyf@jnu.edu.cn
Corresponding author: Yun-Sheng Huang, Tel: +86-0769-22896547, Email: yshuang@gdmu.edu.cn
*

ACCEPTED MANUSCRIPT
Synthesis and evaluation of pyrimidoindole analogs in
umbilical cord blood ex vivo expansion
a,b,#
a,#
c
a
a
b
Yue Feng , Xiao-Yang Xie , Yi-Qiu Yang , Yu-Tong Sun , Wen-Hui Ma , Peng-Jun Zhou , Zi-Yao
b
b
b,*
a,*
Li , Hui-Qiang Liu , Yi-Fei Wang , Yun-Sheng Huang
a
School of Pharmacy, Guangdong Medical University, 1 Xincheng Ave, Songshan Lake Technology
Park, Dongguan, Guangdong 523808, China.
b
Institute of Biomedicine, College of Life Science & Technology, Jinan University, Guangzhou,
Guangdong 510632, China.
c
Department of Pharmacy, Zhaoqing Medical College, 6 Xijiang South Rd, Zhaoqing, Guangdong
5
26020, China.
#
Contributed equally as co-first authors.
*
Corresponding author: Yi-Fei Wang, Tel: +86-20-85223426, Email: twangyf@jnu.edu.cn
Corresponding author: Yun-Sheng Huang, Tel: +86-0769-22896547, Email: yshuang@gdmu.edu.cn
*
Abstract
The scarcity of hematopoietic stem cells (HSCs) significantly hindered their clinical
potentials. Umbilical cord blood (UCB) has become the leading source of HSCs for
both research and clinical applications. But the low content of HSCs in a single UCB
unit limited its use only to pediatric patients. Various cytokines and small molecules
have demonstrated strong abilities in promoting HSC ex vivo expansion, of which
UM171 is the newest and by far the most potent HSC ex vivo expansion agent. In this
study, we synthesized 37 pyrimidoindole analogs and identified 6 compounds to be
potent in promoting HSC ex vivo expansion. In particular, analog 11 was found to be
+
the most effective in stimulating ex vivo expansion of UCB CD34 cells and
+
-
CD34 CD38 cells. Initial data indicated that compound 11 promoted the absolute
number of long term HSCs and inhibited their differentiation. UCB HSCs expanded
with 11 retained adequate multi-lineage differentiation capacity. In addition,
compound 11 is not cytotoxic at its test concentrations, suggesting that it merits
further investigation for potential clinical applications.
1

ACCEPTED MANUSCRIPT
Keywords: Umbilical cord blood, HSC, ex vivo expansion, UM171, pyrimidoindole
1
. Introduction
Hematopoietic stem cells (HSCs) are primitive cells that are capable of
self-renewal and multi-lineage differentiation into a diverse range of different lineages
of hematopoietic cells throughout a human lifetime [1-3]. Since the pioneer work of
allogeneic bone marrow transplantation (hematopoietic stem cell transplantation or
HSCT) by Dr. Thomas for cancer treatment [4], HSCT has been widely used to treat
hematopoietic malignancies including acute myeloid leukemia and chronic myeloid
leukemia, genetic defects such as sickle cell anemia and thalassemia, autoimmune
diseases and certain solid cancers [5]. According to their sources, HSCs can be
classified into bone marrow, peripheral blood, and umbilical cord blood (UCB)
hematopoietic stem cells [6]. Until now, allogeneic bone marrow and mobilized
peripheral blood cells have been the most common sources of HSCs for clinical
HSCT. However, the lack of human leukocyte antigen (HLA) matching/compatible
donors remains to be an obstacle [7-8]. Cord blood, once discarded as a waste, now
becomes a valuable source of HSCs for transplantation due to its tolerance for HLA
mismatches, low incidence of chronic graft-versus host disease (GvHD), rapid
availability, low disease relapse rate, and low immunogenicity [9-12]. However, the
small quantity of HSCs obtained from a single human UCB blood unit makes it more
suitable for transplantation into small children and less successful for adult patients
due to delayed engraftment, increased infections, and high mortality rate [13]. Clinical
+
studies have concluded that the stem cell dose (measured as CD34 cell numbers) is
highly related to homing, engraftment and patient survival [14,15]. A minimum dose
6
+
of 2 x 10 CD34 cells/kg is recommended for a higher success of transplantation
5
+
[
16,17]. On average, a single UCB unit contains ~ 2 x 10 CD34 cells and thus is not
sufficient for an adult transplantation [15-18].
Ex vivo expansion of cord blood stem cells has become the leading solution to
achieve enough dose of HSCs and the main strategy is to proliferate the HSCs by
2

ACCEPTED MANUSCRIPT
promoting self-renewal and inhibiting/delaying differentiation [19]. In the past two
decades, extensive studies have shown that both internal and external biological
substances, such as transcription factors, signal transducers, anti-apoptotic proteins,
cell cycle regulators and epigenetic modifiers, are involved in controlling the balance
between self-renewal and differentiation processes of HSCs [14,20-22]; several
signaling pathways, such as the Wnt, HOX, Notch, Shh (Sonic hedgehog), FGF
(fibroblast growth factor), are implicated in the stimulation of hematopoiesis [7,14].
Though good expansion could be achieved by manipulation with these intrinsic
regulators, continuous activation by genetic stimulation may potentially lead to
malignancy, exhaustion and apoptosis [14,22]. Therefore, extrinsic modulators that
promote the HSC self-renewal mechanism are preferred for ex vivo expansion of
HSCs [7,14]. Many cytokines, such as SCF (stem cell factor) , TPO (thrombopoietin),
Flt3L (Fms-like tyrosine kinase 3 ligand), IL-3, 6, 11, GM-CSF (granulocyte
macrophage colony stimulating factor), and combinations of these, have been studied
in HSC ex vivo expansion, but proved to be unsuccessful due to usually leading to
HSC differentiation, exhaustion and progressive depletion of long-term repopulating
cells [14,20-24]. Further studies indicated that the combination of cytokines and some
extrinsic regulators, such as Wnt proteins, Notch-ligands, BMP-4 (bone morphogenic
protein-4), FGFs, IGFs (insulin like growth factors), Angptls (angiopoietin-like
proteins), etc. had effectively shown to promote HSC expansion [7,14,22,25]. In
addition, co-culture of HSCs with hematopoietic microenvironment cells to construct
a simulated 3D hematopoietic "niche" could initiate self-renewal [26-28]; co-culture
of HSCs with bone marrow-derived stromal cells, endothelial cells and mesenchymal
stem cells (MSCs) could promote HSC ex vivo expansion [29-32]. Unfortunately, the
aforementioned HSC ex vivo expansion methods are still not yet suitable for clinical
applications due to low capacity of expanded HSCs, delayed immune reconstitution,
increased incidence of viral infection, high failure of engraftment, batch to batch
variation, carcinogenesis concerns, high cost and uncontrollability [33-35].
In recent years, studies have demonstrated that some small molecules can be very
effective at promoting HSC ex vivo expansion [9,19,35,36], which offers clear
3

ACCEPTED MANUSCRIPT
advantages in terms of lower cost, ease of manipulation, and potential for more
precise control, making them more promising for clinical applications [37-39].
Among the representative small molecules in HSC ex vivo expansion (Figure S1 in
supporting information) are epigenetic modifying agents [17,35,40], GSK-3β
2
+
inhibitors [19,41], Cu chelators [14], transcription factor regulators [35,42-45], cell
cycle inhibitors [46,47], p38 MAPK inhibitors [48], dipeptidyl peptidase DPP-4
protease inhibitors [7,9], caspase inhibitors [19], and PGE2 agonists [49]. More
recently, several new small molecules, such SR1 and UM171, have been identified to
effectively promote HSC ex vivo expansion. SR1 (StemRegenin 1) is a purine
derivative and an AhR (aryl hydrocarbon receptor) antagonist that promotes ex vivo
+
expansion of UCB CD34 cells with a reported EC value of ~120 nM [50]. UM171,
50
a pyrimidoindole derivative, is the most recently discovered and to date the most
efficient HSC ex vivo expansion agent and is more potent than SR1 in promoting HSC
self-renewal with a reported effective concentration of ~18 nM [51]. UM171
+
-
promotes the proliferation of the primitive human CD34 CD45RA HSCs that have
been confirmed to be essential to multi-lineage hematopoietic regeneration and the
success of a HSC transplantation [7,9,51]; whereas SR1 is implicated to preferentially
+
+
expand the more mature human CD34 CD45RA HSCs that have short-term
repopulation potential [9,51]. These two agents could potentially complement each
other and lead to shorter culture time and promote both short term HSC (ST-HSC) and
long term HSC (LT-HSC) engraftments. Though the exact mechanism of action
(MOA) by which UM171 expands HSCs is still not clear, the primary results gained
from initial clinical studies showed benefits beyond faster engraftment with fewer
infectious complications and better HLA matching [52].
UM171 is a derivative of UM729 that also demonstrated good activity in
+
-
promoting ex vivo expansion of UCB CD34 CD45RA HSCs [51]. By substitution at
the 2 position with a benzyl moiety, conversion of the methoxycarbonyl group at the 7
position to a 2-methyl-2H-tetrazol-5-yl moiety and replacement of the
3
-(piperidin-1-yl)propyl)amino moiety with 4-aminocyclohexylamino group at the 4
position on the pyrimidoindole ring of UM729, the resultant analog UM171 is >10
4

ACCEPTED MANUSCRIPT
times more potent than the parent compound UM729. In a effort to further explore the
structure activity relationship of UM171, we synthesized 37 analogs by using various
substituents to replace the 4-aminocyclohexylamino moiety or altering the
substitution position on the pyrimidoindole ring of the tetrazole moiety (Figure 1).
These newly prepared analogs were evaluated and several analogs of this compound
were identified to be effective in promoting UCB HSC ex vivo expansion.
2
. Results and discussion
2
.1. Chemistry
As a continuous effort to advancement our UCB stem cell program, we used
UM171 as the model structure and made a variety of substitutions on its 4 position as
well as on its 6/7 position substituent switching. Specifically, the core structure of
pyrimidoindole ring was unchanged and the benzyl substituent at the 2 position was
maintained due to its likely positive contribution toward the increased HSC ex vivo
expansion activity [51]. The substitution groups at the 4 position were selected based
on diversification, common pharmacophores and availability. The tetrazole moiety
was altered from the 7 to 6 position of the pyrimidoindole scaffold. Therefore two
series of analogs were generated, series A (compounds 1-11) and series B (compounds
1
2-37), respectively.
Analogs 1-11 were synthesized starting from 3-nitrobenzonitrile in 9 steps
according to a modified procedure for the preparation of UM171 (Scheme 1) [53].
The cyano group was converted to a tetrazole moiety by reacting with sodium azide to
give B001 in 90% yield, which was methylated with methyl iodide to afford B002 in
7
2% isolated yield. It should be noted that the methylation of B001 yielded two
products, 1-methyl-5-(3-nitrophenyl)-2H-tetrazole (10~15%) and the desired
-methyl-5-(3-nitrophenyl)-2H-tetrazole (B002).The 1-methylated by-product was
2
removed by recrystallization (1-3 times). The nitro group of B002 was partially
reduced by nickel powder/hydrazine to hydroxylamine intermediate that was then
5

ACCEPTED MANUSCRIPT
treated with acetyl chloride to give the acetylated product B004 in 84% combined
yield. Intermediate B004 was treated with malononitrile in chloroform in the presence
of triethylamine to give B005 in 58% yield via a [3,3]-sigmatropic rearrangement [54].
Acylation of B005 with phenylacetyl chloride gave B006 in 80% yield, which was
then cyclized in the presence of potassium t-butoxide to yield B007 in around 75%
yield. Chlorination of B007 with POCl at the 4 position afforded B008 in 92% yield,
3
which could then be substituted by various amino derivatives to give analogs 1-11.
Analogs Q01-Q08 were prepared using similar methods as described for the
preparation of B001-B008, respectively with 4-nitrobenzonitrile as the starting
material; while analogs 12-37 were prepared according to similar procedures as
described for the preparation of analogs 1-11 (Scheme 2).
2
.2. Pharmacology
2
.2.1. Primary screening and the discovery of analog 11 as the most potent
+
-
agent in this series for promoting UCB-derived CD34 CD38 cell ex vivo
expansion
Studies indicated that 0.5-5% human UCB cells express CD34 cells and LTC-ICs
+
-
(
long term cell initiating cells) are highly enriched in CD34 CD38 cells [1].
+
-
Therefore, CD34 CD38 are commonly used as immunophenotypic markers to assess
the HSC subpopulations. In this study, the synthesized analogs were measured for
+
-
their activities to promote human UCB CD34 CD38 cell ex vivo expansion. A higher
+
-
expression of CD34 CD38 phenotype indicates a more abundant HSC capacity after
+
-
compound treatment; whereas a decrease in CD34 CD38 expression (or a positive
expression of CD38) reflects a lower number of HSCs and increased differentiation.
+
-
The primary screening was performed to evaluate the increase of UCB CD34 CD38
cells for each compound under conditions optimized for HSC expansion (HSC
specific serum replacement combined with thrombopoietin, stem cell factor, flt3
+
ligand) [55]. Primary human UCB-derived CD34 cells from mobilized donors were
evaluated for CD34 and CD38 expression by flow cytometer following 7 days of
6

ACCEPTED MANUSCRIPT
incubation (Figure 2). The screening setup and criteria were illustrated in Figure 2A.
It should be noted that the control group contained cytokines (stimulating the
proliferation of HSCs, but mostly leading to differentiation) and had several folds of
expansion in terms of total cells. The main purpose of a good HSC ex vivo expansion
agent is not only to further expand the HSCs on or above the cytokines' control, but
+
-
also to maintain a high percentage of true HSCs that are defined by the CD34 CD38
markers in this assay. During the primary screening, 6 compounds, labeled 4, 6, 9,
11,26 and 35, showed good ex vivo expansion activities as demonstrated by their
+
-
higher expression of CD34 CD38 phenotype which implicated more HSCs capacity
(Figure 2B), while the other 31 compounds did not show sufficient activity (Figure
+
-
2
B,2C, Table 1). Data in the 4th column of Table 1 under "CD34 CD38 (%)" indicate
the percentage of HSCs in the media after 7 days of incubation. The culture media
+
-
treated with analogs 4, 6, 9, 11, 26 and 35 contained 30-70% of CD34 CD38 cells,
which were significantly higher than 19.1% for the control. In comparison, the 7-day
culture media of SR1 and UM171 were reported to maintain ~61% [50] and ~67%
+
[
51], respectively, of CD34 HSCs in separate biological assays. It also should be
pointed out that UM171 was in a fed-batch culture that was similar to a 3-D culture
systemwhich was reported to be applied on human UCB cells and yielded
a rapid (12-day) 11-fold increase of HSCs with self-renewing, multilineage
repopulating ability [56]. Thus a fed-batch culture is much more efficient than a
medium change or 2-D culture method as in the case for this study. Data in column 5
+
-
of Table 1 show the proportion/folds of expansion of CD34 CD38 cells normalized
with control. Analogs 4, 6, 9, 11, 26 and 35 displayed 1.59~3.65 folds of increase in
+
-
the number of CD34 CD38 cells. The activities of analogs 4 and 11 in this category
are comparable or superior to those of most currently available small molecules in
terms of HSC ex vivo expansion [9,35]. Overall, series A (1-11) analogs (with the
tetrazole substituted at the 7 position of the pyrimidoindole ring) are more potent than
series B (12-37) analogs in proliferating and maintaining UCB HSCs. However, a
more in-depth structure-activity relationship (SAR) cannot be easily concluded due to
a limited number of active analogs, lack of more diversified structures, or different
7

ACCEPTED MANUSCRIPT
mechanisms of action among them. This lack of SAR phenomenon seems to be a
common feature for most currently known HSC ex vivo expansion agents. It should be
noted that some compounds displayed low/good EC values (Table 1 column 6), but
50
+
-
the magnitudes of CD34 CD38 expression promoted by them were significantly
smaller than those of the 6 active analogs or even lower than that of the control group
probably due to their low efficiency, cytotoxicity, induced differentiation, or inhibition
of vehicle cytokines' induced expansion (Figure 2C). Analogs 4 and 11 demonstrated
the highest activities among the six and their EC values were assessed to be 473.4
5
0
nM and 30.0 nM, respectively (Figure 2D,2E, Table 1). Overall, analog 11 offered the
+
-
best promise in promoting CD34 CD38 cell ex vivo expansion and thus is merited for
further investigation.
+
2
.2.2. Analog 11 promotes UCB-derived CD34 cell and LT-HSC ex vivo
expansion
Compound 11 was further evaluated in a range of expanded concentrations for its
+
-
+
-
UCB CD34 CD38 cell ex vivo expansion activity. The absolute CD34 CD38 cell
count was determined (normalized with control) after 7 days of culture (Figure 3).
The results indicated that compound 11 was 4 to 5 times more effective than the
+
-
control in promoting CD34 CD38 cell expansion, with an optimal test concentration
around 165 nM (Figure 3A). In order to further evaluate the effect of 11 on
UCB-derived HSC expansion ex vivo, we assessed the phenotypic markers of
subpopulations and their absolute numbers at 165 nM concentration. Markers and
representative flow cytometric gating strategy and frequencies of each subsets were
shown (Figure 3B). Arrows from left to right displayed the layer-by-layer
+
+
-
subpopulations from HSCs (CD34 , CD34 CD38 ) to more primitive LT-HSCs
+
-
-
+
+
(
CD34 CD38 CD45RA CD90 CD49f ). The subpopulations' proportions (% of total)
of representative samples were presented in corresponding gating graphs (Figure 3B).
Quantifications of flow cytometric assay of proportion of each subpopulation and
absolute count (normalized with day 0) are presented in Figure 3C and Figure 3D,
respectively. Cells with analog 11 treatment retained high levels of CD34 expression
8

ACCEPTED MANUSCRIPT
(52.20±2.01%); whereas cells cultured with cytokines alone had significantly lower
+
-
CD34 expression (25.91±0.01%) (Figure 3C). Similarly, CD34 CD38 cells in the 11
treated medium also retained higher proportion (44.43±1.83%) than in the control
(21.64±0.01%) (Figure 3C). The results of absolute cell count assay also indicated
that analog 11 supported a 5-fold increase in expansion of total cells, a 5-fold increase
+
+
-
in CD34 cells and a 4.4-fold increase in CD34 CD38 cell subpopulations compared
to the data in day 0 (Figure 3D), which are comparable or superior to those of
reported small molecule HSC ex vivo expansion agents [9,35]. Analog 11 had a 2.4,
+
+
-
4
.9 and 4.4 folds of increase in total cells, CD34 cells, and CD34 CD38 cells,
respectively, compared to control (Figure 3D). More interestingly, LT-HSCs
+
-
-
+
+
(
CD34 CD38 CD45RA CD90 CD49f ), which have been more rigorously defined as
the most primitive HSC population with the highest repopulating capacity and can be
reversibly brought into cell cycle through extrinsic cues [57,58], were not affected in
terms of proportion, but were significantly increased in terms of absolute numbers, by
analog 11（Figure 3D). Similar phenomenon was also reported for UM171 [51,56].
One probable interpretation is that expansion medium supplemented with cytokines
stimulated the repopulation and differentiation of LT-HSCs [14], resulting in
remarkably high expansion of progenitor cell subsets; whereas 11 treatment was likely
to promote LT-HSC self-renewal instead of differentiation. Thus, the LT-HSCs in 11
treated group greatly outnumbered those in the control group and avoided exhaustion.
2
.2.3. Functional study and cytotoxicity assessment of analog 11
The physiologic function of HSCs is multi-lineage differentiation in response to
stressful events for hematopoietic system restoration. Therefore, the hematopoietic
colony-forming unit (CFU) assay was performed to assess the potential of analog 11
co-cultured HSCs to form multiple lineages. Cells treated with 11 were collected and
counted, readjusted to a density of 1,000 cells per 35 mm well, and then seeded in
semi‐solid MethoCult H4434 medium for 14 days of incubation. Types of colonies
and their amounts were classified and scored by morphology; burst-forming
unit-erythroid (BFU-E), colony-forming unit-granulocyte (CFU-G), colony-forming
9

ACCEPTED MANUSCRIPT
unit-macrophage (CFU-M), colony-forming unit-granulocyte/macrophage (CFU-GM)
and colony-forming unit-Mix (CFU-Mix) numbers were assessed (Figure 4A). Cells
pre-treated with analog 11 could notably increase the number of colony-forming cells
and commendably maintain the potential of multi-lineage differentiation (Figure 4A).
Pretreated with 11 at a concentration of 165 nM, cells produced around 254 total
CFUs, a 1.4-fold increase compared to control (Figure 4A，p＜0.001). Additionally,
UCB cells pretreated with 11 have increased granulocyte differentiation as
demonstrated by the increase of CFU-G and CFU-GM, which might be beneficial for
immune system reconstitution and infection prevention after hematopoietic stem cell
transplantation [59]. It should be noted that the level of BFU-E in 11 treated cells is
significantly lower than that in the control, though the mechanism is not clear. This
result along with the phenotypic assessment by flow cytometry suggest that analog 11
could significantly promote HSC ex vivo expansion and its expanded stem cells retain
adequate multi-lineage differentiation capacity.
Cell viability study was conducted to assess the effect of analog 11 on human
mononuclear THP-1cell line using cell counting CCK-8 (Figure 4B). The results
indicated that analog 11, at up to 1600 nM concentration, did not show noticeable
cytotoxicity against THP-1 cells, neither did it show significant cytotoxicity against
+
human primary CD34 cells at test concentration of 165 nM (Figure 3B FSC/SSC
gating). Thus, analog 11 is safe and may potentially be useful in clinical application
for HSC ex vivo expansion.
3
. Conclusions
Despite the tremendous efforts and progress made in the last decades, the current
HSC ex vivo expansion agents can only promote the hematopoietic progenitor cell
engraftment. The newly discovered expansion agent UM171 offers the most promise
by promoting LT-HSC expansion and inhibiting differentiation. We synthesized a total
of 37 pyrimidoindole derivatives and identified 6 of them to possess good activities in
+
-
promoting UCB CD34 CD38 cell ex vivo expansion through a series of biological
1
0

ACCEPTED MANUSCRIPT
assessments. Analog 11 was found to be the most overall promising compound in both
+
+
-
potency and efficiency in promoting UCB CD34 and CD34 CD38 cell expansion
and maintaining expanded cells' stemness. In addition, analog 11 seems to repopulate
+
-
-
+
+
the CD34 CD38 CD45RA CD90 CD49f LT-HSCs and maintain cells' stemness by
inhibiting their differentiation, rescuing HSCs from exhaustion, senescence and
apoptosis. Finally, UCB HSCs expanded by analog 11 retained colony-forming unit
property and adequate multi-lineage differentiation capacity. Overall, the results from
this initial study indicated that analog 11 was as potent as those reported small
molecules in HSC ex vivo expansion activity. Combined with its excellent
pharmacological activity and its non-cytotoxicity at high concentration, analog 11 is
one of the promising ex vivo expansion agents and may be warranted for additional
investigation for clinical potential.
4
. Experimental Section
4
.1. Chemistry
All reagents or solvents purchased commercially were used directly without
further purification. Reactions were monitored by analytical thin-layer
chromatography (TLC) on silica gel F254 glass plates and visualized under UV light
(254 and 365 nm) and temperatures were recorded using regular thermometers
without correction. Flash column chromatography was performed on silica gel
(200–300 mesh). Melting points were measured using capillary method without
1
correction. H NMR spectra were recorded with a Bruker Avance III 400 and 500
MHz NMR spectrometers at room temperature. Chemical shifts were recorded as
parts per million (ppm) downfield to tetramethylsilane (TMS). The following
abbreviations are used for multiplicity of NMR signals: s) singlet, d) doublet, t) triplet,
q) quartet, m) multiplet, dd) doublet of doublets, td) triplet of doublets; dt) doublet of
1
3
triplets, dq) doublet of quartets, b) broad, C NMR spectra were recorded with
Bruker Avance III 400 and 500 MHz NMR spectrometers (100 MHz).
1
1

ACCEPTED MANUSCRIPT
4
.1.1. 5-(3-Nitrophenyl)-2H-tetrazole (B001)
B001 was prepared according to a modified procedure by Demko and Sharpless
60]. Briefly, to a 1 L flask was added meta-nitrobenzonitrile (30.00 g, 0.20 mol),
[
sodium azide (14.50 g, 0.22 mol), zinc bromide (45.70 g, 0.20 mol), and water (500
o
mL). The mixture was heated to 100 C and stirred at that temperature for 24 h,
cooled to rt, poured into ice-cold water (500 mL), and added with 3 N HCl to pH = 5
(~100 mL). The mixture was extracted with ethyl acetate (3 x 200 mL), dried, and
concentrated to one third of the volume and put in refrigerator overnight to give a
o
1
white solid (34.8 g, yield 90%). mp 63.5-64.5 C; H NMR (DMSO-d , ppm) δ
6
8
.85-8.86 (t, 1H), 8.48-8.51 (dt, 1H), 8.43-8.46 (dq, J=8.3, 2.3, 0.9Hz, 1H), 7.91-7.95
1
3
(
t, J=8.0Hz, 1H); C NMR (DMSO, ppm) δ 155.4, 148.7, 133.5, 131.7, 126.6, 126.0,
1
21.9.
4
.1.2. 2-Methyl-5-(3-nitrophenyl)-2H-tetrazole (B002)
B002 was prepared according to similar procedures by Ali et al [53] and by Wang
et al [61]. Briefly, to a solution of B001 (33.00 g, 0.17 mol) in DMF (150 mL) was
added with anhydrous Na CO (28.63 g, 0.21 mol), methyl iodide (29.41 g, 0.21 mol).
2
3
o
The mixture was stirred at 90 C for 1 h, cooled to rt, and poured into ice-water (1L).
The solid was collected by filtration and recrystallized from ethyl acetate/petroleum
o
ether to give a white solid (26.0 g, yield 72%). mp 99.8-100.2 C (ref [53] mp
o
1
1
00-101 C); H NMR (DMSO-d , ppm) δ 8.66-8.67 (t, 1H), 8.40-8.42 (dt, J=7.8Hz,
6
1
3
1
H), 8.33-8.35 (dt, 1H), 7.81-7.85 (t, J=8.0Hz,1H), 4.46 (s, 3H); C NMR (DMSO,
1
ppm) δ 162.8, 148.6, 132.7, 131.5, 128.7, 125.4, 120.9; ref [53] H NMR (400 MHz,
DMSO-d ) δ 8.77 8.69 (m, 1H), 8.46 (d, J = 7.8 Hz, 1H), 8.41 8.34 (m, 1H), 7.87 (t,
6
−
−
J = 8.0 Hz, 1H), 4.47 (s, 3H). Thus, B002 is confirmed to be the correct 2'-methylated
isomer.
4
.1.3. N-Hydroxy-N-(3-(2-methyl-2H-tetrazol-5-yl)phenyl)acetamide (B004)
B004 was prepared according to a procedure by Kruger and Meier [62]. Briefly,
to a solution of B002 (30.0 g, 0.15 mol) in THF (350 mL) was added nickel powder
1
2

ACCEPTED MANUSCRIPT
o
(
10.0 g, 0.17 mol). The mixture was cooled to 0 C and added portion-wise with
o
hydrazine (7.30 g, 0.14 mol) and stirred at 0 C for 30 min, then at rt overnight,
o
o
cooled to 0 C, carefully added with NaHCO (54.00 g, 0.64 mol), stirred at 0 C for
3
3
2
0 min, and slowly added with acetyl chloride (13.77 g, 0.17 mol), and stirred at rt for
h. The mixture was filtered and the filtrate was concentrated to afford a solid that
was recrystallized from ethyl acetate to give a white solid (28.0g, yield 84%). mp
o
1
1
15.8-117.1 C; H NMR (DMSO-d , ppm) δ 10.83 (s, 1H), 8.43 (s, 1H), 7.82-7.84 (d,
6
1
3
J=5.3Hz, 2H), 7.54-7.58 (t, J=7.9Hz, 1H), 4.45 (s, 3H), 2.28 (s, 3H); C NMR
DMSO, ppm) δ 170.8, 164.4, 142.8, 129.9, 127.6, 122.5, 121.9, 117.7, 23.1.
(
4
.1.4. 2-Amino-6-(2-methyl-2H-tetrazol-5-yl)-1H-indole-3-carboxamide (B005)
B005 was prepared according to a procedure by Tomioka et al [54]. Briefly, a
solution of B004 (26.0 g, 0.11 mol) and malononitrile (7.4 g, 0.11 mol) in chloroform
o
(
500 mL) was stirred at 0 C for 30 min and then added triethylamine (11.28 g, 0.11
o
mol) dropwise, stirred at 0 C for an additional 30 min and at rt for 1h. The solid was
collected by filtration and washed with chloroform and then transferred into a flask,
added with methanol (200 mL) and triethylamine (11.28 g, 0.11 mol) and refluxed for
1
0h. After cooled to rt, the solid was collected by filtration and recrystallized from
o
1
methanol to afford a white solid (16 g, yield 58%). mp 207.6-209.3 C; H NMR
(
DMSO-d , ppm) δ 10.80 (s, 1H), 7.82 (s, 1H), 7.64-7.68 (q, J=8.2Hz, 2H), 7.03 (s,
6
1
3
2
1
2
H), 6.60 (s, 2H), 4.39 (s, 3H); C NMR (DMSO, ppm) δ 168.8, 166.0, 154.5, 133.0,
+
28.1, 118.9, 117.3, 117.1, 108.1, 87.3, 39.9; LC/MS-m/z: 258.1 [M+1] (exact mass:
57.1).
4
.1.5. 6-(2-Methyl-2H-tetrazol-5-yl)-2-(2-phenylacetamido)-1H-indole-3-
carboxamide (B006)
A solution of B005 (14.6 g, 56.3 mmol) and triethylamine (17.1 g, 0.17 mol) in
o
DMF (200 mL) was stirred at 0 C and added phenylacetyl chloride (17.4 g, 0.11 mol)
dropwise and then stirred at rt overnight. The mixture was then poured into ice cold
water (500 mL) and the solid was collected by filtration and recrystallized from ethyl
1
3

ACCEPTED MANUSCRIPT
o
1
acetate to afford a white solid (17.0 g, yield 80%). mp 288.6-289.9 C; H NMR
(
DMSO-d , ppm) δ 12.23 (s, 1H), 11.76 (s, 1H), 8.29 (s, 1H), 7.96-8.00 (m, J=8.4Hz,
6
1
3
1
H), 7.79-7.81 (m, J=8.2Hz, 1H), 7.26-7.40 (m, 7H), 4.41 (s, 3H), 3.91 (s, 2H); C
NMR (DMSO, ppm) δ 170.3, 168.4, 165.6, 143.1, 134.8, 133.0, 130.1 (2C), 129.2
2C), 127.6, 124.7, 119.9, 119.8, 119.2, 111.1, 92.3, 43.9.
(
4
4
.1.6. 2-Benzyl-7-(2-methyl-2H-tetrazol-5-yl)-3H-pyrimido[4,5-b]indol-
(9H)-one (B007)
B007 was prepared according to a procedure by Yadav et al [63]. Briefly, a
solution of B006 (16.3 g, 43.4 mmol) and potassium t-butoxide (29.2 g, 0.26 mol) in
isopropanol (500 mL) was refluxed for 12h and then concentrated to one third of the
volume and poured into ice-water (300 mL), added 6N HCl to adjust to pH = 6. The
solid was collected by filtration and recrystallized from methanol 3 times to give a
o
1
white solid product (11.6 g, yield 74.8%). mp 300.5-302.4 C; H NMR (DMSO-d ,
6
ppm) δ 12.48 (b, 1H), 12.40 (b, 1H), 8.07-8.20 (m, 3H), 7.91-7.97 (m, J=12.7Hz, 1H),
13
7
1
1
.26-7.41 (m, 4H), 4.43 (s, 3H), 4.04 (s, 2H); C NMR (DMSO, ppm) δ 165.3, 160.2,
59.4, 155.7, 137.0, 136.0, 129.6 (2C), 129.0 (2C), 127.3, 124.2, 122.7, 121.4, 119.8,
09.8, 98.6, 40.1.
4
.1.7. 2-Benzyl-4-chloro-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]
indole (B008)
A solution of B007 (6.3 g, 16.7 mmol) and phorphorous oxychoride (100 mL)
was refluxed for 12h and then concentrated to around one fifth of the volume and
poured into ice-water (200 mL), adjusted to pH=8 with 10% NaOH aqueous solution.
The solid was collected by filtration and recrystallized from ethyl acetate/petroleum
o
1
ether to give a light yellow solid (6.1 g, yield 92%). mp 281.8-283.1 C; H NMR
(
(
(
DMSO-d , ppm) δ 12.90 (s, 1H), 8.32-8.35 (d, J=8.3Hz, 1H), 8.19 (s, 1H), 8.04-8.07
6
1
3
dd, J=8.3Hz, 1H), 7.22-7.38 (m, J=7.7Hz, 5H), 4.46 (s, 3H), 4.30 (s, 2H); C NMR
DMSO, ppm) δ 166.4, 164.6, 157.8, 152.2, 139.1, 138.7, 129.7 (2C), 128.9 (2C),
1
26.9, 126.5, 123.3, 120.0, 119.7, 110.1, 109.1, 45.3.
1
4

ACCEPTED MANUSCRIPT
4
9
.1.8. 2-Benzyl-N-(4-chloro-3-fluorophenyl)-7-(2-methyl-2H-tetrazol-5-yl)-
H-pyrimido[4,5-b]indol-4-amine (1)
To a flask was added B008 (0.4 g, 1.07 mmol), DMSO (10 mL), K CO (0.3 g,
2
3
2
.14 mmol), 3-chloro-4-fluoroaniline (0.31 g, 2.14 mmol). The mixture was heated
o
and stirred at 90 C for 8h, cooled to rt, poured into ice-water (50 mL). The solid was
collected by filtration and recrystallized from methanol 4 times to afford a white solid
o
1
(
0.23 g, yield 45%). mp 289-291 C; H NMR (DMSO-d , ppm) δ 12.34 (s, 1H), 9.01
6
(d, 1H), 8.56-8.58 (d, J=8.2Hz, 1H), 8.15 (s, 1H), 8.10-8.11 (d, J=5.4Hz, 1H),
7
.96-7.97 (d, J=8.2Hz, 1H), 7.75-7.77 (m, 1H), 7.32-7.39 (m, J=6.2Hz, 5H), 7.22-7.24
1
3
(
t, J=7.0Hz, 1H), 4.45 (s, 3H), 4.13 (s, 2H); C NMR (DMSO, ppm) δ 166.3, 165.2,
1
1
58.1, 154.6, 152.5, 139.5, 137.5, 129.7 (2C), 128.8 (2C), 126.7, 123.8, 123.8, 123.1,
22.5, 120.9, 119.1, 118.6, 116.7, 116.6, 109.3, 95.6, 46.1, 40.3; LC/MS-m/z: 485.1
+
[
M+1] (exact mass: 484.1); Analysis (%) found: C61.78, H3.69, N22.97 (Calcd for
C H ClFN : C61.92, H3.74, N23.11).
25
18
8
4
.1.9. N-(2-(1H-Indol-3-yl)ethyl)-2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-
H-pyrimido[4,5-b]indol-4-amine (2)
9
Analog 2 was prepared according to the procedure as described for the
preparation of analog 1 by reacting B008 with tryptamine and obtained in 74% yield.
o
1
mp 288-290 C; H NMR (DMSO-d , ppm) δ 12.07 (s, 1H), 10.86 (s, 1H), 8.42 (m,
6
J=8.3Hz, 1H), 8.11 (m, 1H), 7.92 (m, J=8.2Hz, 1H), 7.78 (m, J=7.8Hz, 1H), 7.02-7.84
1
3
(
(
(
m, 10H), 4.45 (s, 3H), 4.13 (s, 2H), 3.88-3.93 (m, 2H), 3.08-3.12 (m, 2H); C NMR
DMSO, ppm) δ 166.7, 165.4, 157.6, 157.2, 139.9, 136.8, 136.8, 129.5 (2C), 128.6
2C), 127.9, 126.5, 123.1, 122.8, 121.9, 121.4, 119.1, 118.7, 118.5, 112.6, 111.9,
+
1
09.1, 94.0, 46.4, 41.8, 40.1, 26.0; LC/MS-m/z: 500.4 [M+1] (exact mass: 499.2);
Analysis (%) found: C69.70, H4.94, N25.07 (Calcd for C H N : C69.72, H5.04,
2
9
25
9
N25.23).
4
.1.10. 4-(2-((2-Benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-
1
5

ACCEPTED MANUSCRIPT
4
-yl)amino)ethyl)phenol (3)
Analog 3 was prepared according to the procedure as described for the
preparation of analog 1 by reacting B008 with p-hydroxyphenethylamine and
o
1
obtained in 62% yield. mp 252-254 C; H NMR (DMSO-d , ppm) δ 12.03 (s, 1H),
6
9
7
6
2
1
1
.20 (s, 1H), 8.38-8.40 (d, J=8.3Hz, 1H), 8.10 (s, 1H), 7.90-7.92 (d, J=8.2Hz, 1H),
.39-7.42 (m, 2H), 7.28-7.32 (m, 2H), 7.20-7.22 (m, 1H), 7.03-7.06 (d, J=8.4Hz, 2H),
.69-6.71 (d, J=8.4Hz, 2H), 4.44 (s, 3H), 4.09 (s, 2H), 3.72-3.77 (m, 2H), 2.81-2.85 (t,
1
3
H); C NMR (DMSO, ppm) δ 166.7, 165.4, 157.5, 157.1, 156.1, 139.9, 136.8, 130.2,
30.0 (2C), 129.6 (2C), 128.6 (2C), 126.5, 122.8, 121.9, 121.7, 118.5, 115.6 (2C),
+
09.1, 94.0, 46.3, 42.9, 40.1, 35.1; LC/MS-m/z: 477.1 [M+1] (exact mass: 476.2);
Analysis (%) found: C67.88, H5.01, N23.34 (Calcd for C H N O: C68.05, H5.08,
2
7
24
8
N23.51).
4
4
.1.11. 4-((2-Benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-
-yl)amino)-1,4-trans-cyclohexanol (4)
Analog 4 was prepared according to the procedure as described for the
preparation of analog 1 by reacting B008 with trans-4-hydroxycyclohexylamine and
o
1
obtained in 63% yield. mp 285-287 C; H NMR (DMSO-d , ppm) δ 12.02 (s, 1H),
6
8
.43-8.45 (d, J=8.2Hz, 1H), 8.10 (s, 1H), 7.89-7.91 (dd, J=8.2Hz, 1H), 7.40-7.41 (d,
J=7.2Hz, 2H), 7.30 (m, J=7.5Hz, 2H), 7.21 (m, J=7.3Hz, 1H), 6.73-6.75 (m, J=8.0Hz,
1
1
1
1
4
H), 4.65-4.66 (d, J=4.4Hz, 1H), 4.44 (s, 3H), 4.22-4.29 (m, 1H), 4.07 (s, 2H),
1
3
.92-1.94 (m, 4H), 1.55-1.64 (m, 2H), 1.28-1.37 (m, 2H); C NMR (DMSO, ppm) δ
66.5, 165.4, 157.6, 156.7, 139.8, 136.8, 129.7 (2C), 128.5 (2C), 126.5, 122.8, 122.2,
21.8, 118.4, 109.0, 94.0, 69.1, 49.4, 46.1, 40.1, 35.0 (2C), 30.6 (2C); LC/MS-m/z:
+
55.2 [M+1] (exact mass: 454.2); Analysis (%) found: C65.97, H5.61, N24.54
(
Calcd for C H N O: C66.06, H5.77, N24.65).
25 26 8
4
.1.12. 2-Benzyl-N-(4-chloro-2-methylphenyl)-7-(2-methyl-2H-tetrazol-5-yl)-
H-pyrimido[4,5-b]indol-4-amine (5)
9
Analog 5 was prepared according to the procedure as described for the
1
6

ACCEPTED MANUSCRIPT
preparation of analog 1 by reacting B008 with 2-methyl-4-chloroaniline and obtained
o
1
in 52% yield. mp 294-295 C; H NMR (DMSO-d , ppm) δ 12.23 (s, 1H), 8.79 (s,
6
1
H), 8.32-8.34 (d, J=7.8Hz, 1H), 8.13 (s, 1H), 7.90-7.91 (d, J=8.0Hz, 1H), 7.41-7.43
(m, 2H), 7.26-7.29 (m, 5H), 7.19-7.20 (m, 1H), 4.44 (s, 3H), 3.98 (s, 2H), 2.15 (s, 3H);
1
3
C NMR (DMSO, ppm) δ 166.3, 165.2, 158.1, 155.9, 139.5, 137.3, 137.2, 130.2,
1
9
29.8, 129.6 (2C), 128.9, 128.5 (2C), 126.5, 126.2, 123.5, 122.7, 121.5, 118.7, 109.2,
+
4.9, 45.9, 18.4; LC/MS-m/z: 480.9 [M+1] (exact mass: 480.2); Analysis (%) found:
C64.78, H4.19, N22.97 (Calcd for C H ClN : C64.93, H4.40, N23.30).
2
6
21
8
4
.1.13. 2-Benzyl-N-(3,4-dimethoxyphenethyl)-7-(2-methyl-2H-tetrazol-5-yl)-
H-pyrimido[4,5-b]indol-4-amine (6)
9
Analog 6 was prepared according to the procedure as described for the
preparation of analog 1 by reacting B008 with 3,4-dimethoxyphenethylamine and
o
1
obtained in 73% yield; mp 252-253 C; H NMR (DMSO-d , ppm) δ 12.03 (s, 1H),
6
8
.37-8.40 (d, J=8.2Hz, 1H), 8.09 (s, 1H), 7.89-7.92 (d, J=8.2Hz, 1H), 7.38-7.42 (m,
J=7.5Hz, 3H), 7.27-7.31 (m, J=7.5Hz, 2H), 7.19 (m, J=7.3Hz, 1H), 6.84-6.88 (m,
J=8.2Hz, 2H), 6.77-6.78 (m, J=8.1Hz, 1H), 4.44 (s, 3H), 4.08 (s, 2H), 3.77-3.82 (m,
1
3
2
1
1
4
H), 3.69-3.71 (6H, 2OMe), 2.88-2.91 (t, 2H); C NMR (DMSO, ppm) δ 166.6,
65.3, 157.5, 157.1, 149.1, 147.7, 139.9, 136.8, 132.7, 129.6 (2C), 128.6(2C), 126.5,
22.8, 121.8, 121.7, 121.0, 118.5, 113.0, 112.4, 109.1, 94.0, 56.0, 55.7, 46.3, 42.7,
+
0.1, 35.4; LC/MS-m/z: 521.1 [M+1] (exact mass: 520.2); Analysis (%) found:
C66.90, H5.35, N21.54 (Calcd for C H N O : C66.91, H5.42, N21.52).
2
9
28
8
2
4
.1.14. 2-Benzyl-N-(4-methoxyphenethyl)-7-(2-methyl-2H-tetrazol-5-yl)-9H-
pyrimido[4,5-b]indol-4-amine (A007)
Analog 7 was prepared according to the procedure as described for the
preparation of analog 1 by reacting B008 with 4-methoxyphenethylamine and
o
1
obtained in 66% yield. mp 262-263 C; H NMR (DMSO-d , ppm) δ 12.03 (s, 1H),
6
8
.38-8.40 (d, J=8.3Hz, 1H), 8.09 (s, 1H), 7.90-7.92 (d, J=8.2Hz, 1H), 7.40-7.42 (m,
J=7.1Hz, 3H), 7.28-7.32 (m, J=7.5Hz, 2H), 7.15-7.22 (m, J=7.3Hz, 3H), 6.86-6.88 (m,
17

ACCEPTED MANUSCRIPT
J=8.6Hz, 2H), 4.44 (s, 3H), 4.09 (s, 2H), 3.74-3.79 (m, 2H), 3.72 (3H, OMe),
1
3
2
1
.86-2.90 (t, 2H); C NMR (DMSO, ppm) δ 166.6, 165.4, 158.1, 157.5, 157.1, 139.9,
36.8, 132.1, 130.1 (2C), 129.6 (2C), 128.6 (2C), 126.5, 122.8, 121.6, 121.8, 118.5,
+
114.3 (2C), 109.1, 94.0, 55.5, 46.3, 42.8, 40.1, 35.1; LC/MS-m/z: 491.1 [M+1]
(exact mass: 490.2); Analysis (%) found: C68.47, H5.18, N22.69 (Calcd for
C H N O: C68.55, H5.34, N22.84).
28
26
8
4
.1.15. 2-Benzyl-N-(3-ethynylphenyl)-7-(2-methyl-2H-tetrazol-5-yl)-9H-
pyrimido[4,5-b]indol-4-amine (8)
Analog 8 was prepared according to the procedure as described for the
preparation of analog 1 by reacting B008 with 3-ethynylaniline and obtained in 59%
o
1
yield. mp 264-266 C; H NMR (DMSO-d , ppm) δ 12.33 (s, 1H), 8.98 (s, 1H),
6
8
7
4
1
1
4
.54-8.56 (d, J=8.3Hz, 1H), 8.15 (s, 1H), 8.00 (s, 1H), 7.94-7.97 (d, J=8.3Hz, 1H),
.82-7.84 (d, J=8.1Hz, 1H), 7.32-7.41 (m, J=7.4Hz, 5H), 7.18-7.24 (m, J=7.3Hz, 2H),
13
.45 (s, 3H), 4.24 (s, 1H), 4.12 (s, 2H); C NMR (DMSO, ppm) δ 166.3, 165.2, 158.1,
54.8, 140.4, 139.5, 137.5, 129.7 (2C), 129.1, 128.8 (2C), 126.6, 126.5, 125.3, 123.8,
23.2, 122.9, 122.1, 121.0, 118.6, 109.3, 95.7, 84.1, 80.8, 46.1, 40.1; LC/MS-m/z:
+
57.2 [M+1] (exact mass: 456.2); Analysis (%) found: C70.89, H4.24, N24.47
(
Calcd for C H N : C71.04, H4.42, N24.55).
27 20 8
4
.1.16. 2-Benzyl-7-(2-methyl-2H-tetrazol-5-yl)-N-(tetrahydro-2H-pyran-4-yl)-
H-pyrimido[4,5-b]indol-4-amine (9)
9
Analog 9 was prepared according to the procedure as described for the
preparation of analog 1 by reacting B008 with 4-aminotetrahydro-2H-pyrane and
o
1
obtained in 59% yield. mp 280-281 C; H NMR (DMSO-d , ppm) δ 12.04 (s, 1H),
6
8
.45-8.47 (d, J=8.2Hz, 1H), 8.08 (s, 1H), 7.89-7.91 (dd, J=8.2Hz, 1H), 7.37-7.39 (d,
J=7.7Hz, 2H), 7.27-7.30 (t, J=7.5Hz, 2H), 7.18-7.21 (t, J=7.3Hz, 1H), 6.88-6.89 (d,
J=7.8Hz, 1H), 4.46-4.49 (m, 1H), 4.44 (s, 3H), 4.06 (s, 2H), 3.93-3.95 (m, 2H),
1
3
3
1
.41-3.46 (m, 2H), 1.75-1.88 (m, 4H); C NMR (DMSO, ppm) δ 166.4, 165.3, 157.6,
56.5, 139.8, 136.8, 129.7 (2C), 128.5 (2C), 126.4, 122.9, 122.3, 121.8, 118.4, 109.1,
1
8

ACCEPTED MANUSCRIPT
+
9
4.1, 67.0 (2C), 47.4, 46.1, 40.1, 32.9 (2C); LC/MS-m/z: 441.2 [M+1] (exact mass:
4
40.2); Analysis (%) found: C65.28, H5.43, N25.31 (Calcd for C H N O: C65.44,
24
24
8
H5.49, N25.44).
4
.1.17. 2-Benzyl-N,N-dimethyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido
[4,5-b]indol-4-amine (10)
Analog 10 was prepared according to the procedure as described for the
preparation of analog 1 by reacting B008 with dimethylamine hydrochloride
o
1
triethylamine and obtained in 78% yield. mp 245-247 C; H NMR (DMSO-d , ppm)
6
δ 12.21 (s, 1H), 8.09 (s, 1H), 8.03-8.05 (d, J=8.4Hz, 1H), 7.90-7.92 (d, J=8.4Hz, 1H),
7
4
1
9
.38-7.39 (d, J=7.1Hz, 2H), 7.27-7.31 (t, J=7.5Hz, 2H), 7.17-7.21 (t, J=7.3Hz, 1H),
13
.44 (s, 3H), 4.08 (s, 2H), 3.30 (s, 6H); C NMR (DMSO, ppm) δ 165.2, 165.1, 160.3,
59.0, 139.6, 137.3, 129.6 (2C), 128.6 (2C), 126.5, 123.5, 123.0, 121.9, 118.8, 109.2,
+
5.4, 45.9, 40.1, 40.1; LC/MS-m/z: 385.2 [M+1] (exact mass: 384.2); Analysis (%)
found: C65.45, H5.18, N29.13 (Calcd for C H N : C65.61, H5.24, N29.15).
2
1
20
8
4
.1.18. 2-Benzyl-N-(2-(6,7-dimethoxy-1.2.3,4-tetrahydroisoquinolino)ethyl)-
-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-amine (11)
7
2
-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolino)ethylamine
according to a modified procedure by Wu at al [64]. Briefly, a solution of
-bromoethylamine hydrobromide (10.0 g, 48.8 mmol) and triethylamine (7.4 g, 73.2
mmol) in ethanol (50 mL) was cooled in ice and added with BOC anhydride (12.8 g,
8.6 mmol) under stirring. The mixture was stirred at rt overnight, concentrated in
was
prepared
2
5
vacuo, extracted with ethyl acetate, washed with brine, and dried over anhydrous
sodium sulfate. Removal of the solvent afforded a light yellow oil (10 g, ~91% yield).
A solution of BOC-2-bromoethylamine (2.3 g, 10.4 mmol) in dichloromethane (25
mL) was added with 6,7-dimethyl-1,2,3,4-tetrahydroisoquinoline hydrochloride (2.0 g,
8
.8 mmol) and triethylamine (2.6 g, 26.1 mmol), and stirred at rt overnight, extracted
with dichloromethane, washed with brine and dried over anhydrous sodium sulfate.
After workup, the crude was purified by a silica gel column to afford
1
9

ACCEPTED MANUSCRIPT
N-(tert-butyloxycarboamidoethyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
1
(
C002) as a light yellow oil (2.6 g). H NMR (CDCl , ppm) δ 6.61 (s, 1H), 6.53 (s,
3
1H), 5.15 (b, 1H), 3.84 (s, 3H), 3.85 (s, 3H), 3.56 (s, 2H), 3.32-3.33 (m, J=5.4Hz, 2H),
2.81-2.84 (t, J=5.7Hz, 2H), 2.71-2.74 (t, J=5.8Hz, 2H), 2.62-2.65 (t, J=5.9Hz, 2H),
1.44 (s, 9H). The oily intermediate was then dissolved in dichloromethane (10 mL),
added with trifluoroacetic acid (1 mL) and stirred at rt for 4h. The solvent was
removed in vacuo and the residue was added with water (10 mL) and 10% aqueous
NaOH to adjust the pH=10. The product was extracted with dichloromethane, washed
with brine and dried over anhydrous sodium sulfate. After workup, a light yellow oil
was obtained (~1.6 g, ~95% yield) and used directly in the next step without further
purification.
Analog 11 was prepared according to the procedure as described for the
preparation of analog 1 by reacting B008 with 2-(6,7-dimethoxy-1,2,3,4-
o
1
tetrahydroisoquinolino)ethylamine and obtained in 51% yield. mp 241-243 C; H
NMR (DMSO-d , ppm) δ 12.03 (s, 1H), 8.35-8.37 (d, J=8.2Hz, 1H), 8.08 (s, 1H),
6
7
7
3
.88-7.90 (d, J=8.2Hz, 1H), 7.39-7.41 (d, J=7.2Hz, 2H), 7.27-7.31 (d, J=7.4Hz, 3H),
.18-7.22 (t, J=7.3Hz, 1H), 6.63 (s, 1H), 6.66 (s, 1H), 4.43 (s, 3H), 4.08 (s, 2H),
1
3
.80-3.85 (m, 2H), 3.69-3.70 (6H, 2OMe), 3.58 (s, 2H), 2.71-2.76 (m, 6H); C NMR
(DMSO, ppm) δ 166.6, 165.3, 157.5, 157.2, 147.6, 147.3, 139.9, 136.8, 129.6 (2C),
1
5
28.6 (2C), 127.1, 126.5, 126.4, 122.8, 121.8, 121.6, 118.5, 112.3, 110.4, 109.1, 94.0,
+
7.3, 56.0, 55.9, 55.7, 51.1, 46.3, 40.1, 38.3, 28.7; LC/MS-m/z: 576.4 [M+1] (exact
mass: 575.3); Analysis (%) found: C66.71, H5.62, N21.79 (Calcd for C H N O :
32
33
9
2
C66.77, H5.78, N21.90).
4
.1.19. 5-(4-Nitrophenyl)-2H-tetrazole (Q01)
To a flask was added 4-nitrobenzonitrile (10.0 g, 67.52 mmol), sodium azide (4.8
g, 74.27 mmol), zinc bromide (15.2 g, 67.52 mmol), and water (150 mL). The mixture
o
was stirred and heated to 100 C overnight, cooled to rt and poured into ice-water,
extracted with ethyl acetate, washed with brine, and dried over sodium sulfate. The
solvent was removed and the solid was recrystallized from ethanol to give Q01 as a
2
0

ACCEPTED MANUSCRIPT
o
1
white solid (11.6 g, yield 90%). mp 205-207 C; H NMR (DMSO-d , ppm) δ
6
13
8
1
.41-8.43 (d, 2H), 8.27-8.29 (d, 2H); C NMR (DMSO, ppm) δ 155.9, 149.2, 131.1,
28.7 (2C), 125.1 (2C).
4
,1,20. 2-Methyl-5-(4-nitrophenyl)-2H-tetrazole (Q02)
A solution of Q01 (10.0 g, 52.32 mmol) in DMF (50 mL) was added with
anhydrous K CO (8.6 g, 62.78 mmol), MeI (9.0 g, 62.78 mmol) and was heated and
2
3
o
stirred at 90 C for 1 hr. After cooled to rt, the reaction mixture was pourted into
ice-water, filtered to give a solid that was further purified from crystallization in ethyl
acetate/petroleum ether to afford Q02 as a white solid (8.6 g, yield 80%). mp 165-166
o
1
13
C; H NMR (CDCl , ppm) δ 8.32-8.38 (m, 4H), 4.47 (s, 3H); C NMR (DMSO,
3
ppm) δ 162.9, 148.9, 133.2, 128.0 (2C), 125.0 (2C).
4
.1.21. N-Hydroxy-N-(4-(2-methyl-2H-tetrazol-5-yl)phenyl)acetamide (Q04)
A solution of Q02 (5 g, 24.37 mmol) in THF (100 mL) was added with nickel
o
powder (1.67 g, 28.40 mmol), cooled to 0 C, and then dropwise added with 85%
o
hydrazine hydrate (3 g, 52.46 mmol). The reaction was then stirred at 0 C for 30 min
o
and at rt overnight. It was then cooled to 0 C and added with NaHCO (8.5 g, 0.1 mol)
3
o
and stirred at 0 C for 30 min, added with acetyl chloride (1.9 g, 24.2 mmol), stirred at
rt for 2 hr, filtered off the solid and washed with THF. The filtrate was concentrated in
vacuo to give a solid that was recrystallized from ethyl acetate/petroleum ether to give
o
1
Q04 as a light yellow solid (5.1 g, yield 84%). mp 141-142 C; H NMR (DMSO-d ,
6
ppm) δ 10.82 (s, 1H, OH), 8.04-8.06 (d, 2H), 7.86-7.88 (d, 2H), 4.42 (s, 3H, N-Me),
1
3
2
.27 (s, 3H, acetyl); C NMR (DMSO, ppm) δ 170.9, 164.2, 143.7, 127.0, 123.0,
1
20.2, 23.2.
4
.1.22. 2-Amino-5-(2-methyl-2H-tetrazol-5-yl)-1H-indole-3-carboxamide (Q05)
A solution of Q04 (5.1 g, 21.87 mmol) in chloroform (100 mL) was added with
o
malononitrile (1.44 g, 21.87 mmol), triethylamine (2.2 g, 21.87 mmol) at 0 C and
stirred at rt for 30 min. The solid was filtered, washed with chlorochorm, dried, and
2
1

ACCEPTED MANUSCRIPT
then was placed in a 100 mL flask and added with triethylamine (2.2 g 21.87 mmol)
and methanol (60 mL), heated to reflux for 6 hr. After cooled to rt, the solid was
o
1
filtered, dried to afford Q05 (4.4 g, yield 78%). mp 209.6-211.4 C; H NMR
(
DMSO-d , ppm) δ 10.85 (s, 1H, indole-1H), 8.16 (s, 1H), 7.59-7.61 (dd, 1H),
6
1
3
7
.25-7.27 (d, 1H), 6.89 (s, 2H), 6.58 (s, 2H), 4.41 (s, 3H, N-Me); C NMR (DMSO,
ppm) δ 169.1, 166.3, 153.8, 134.3, 126.7, 119.4, 117.8, 115.3, 114.8, 87.2, 39.9.
4
.1.23. 5-(2-Methyl-2H-tetrazol-5-yl)-2-(2-phenylacetamido)-1H-indole-3-
carboxamide (Q06)
A solution of Q05 (4.3 g, 16.59 mmol) in dioxane (200 mL) was added with
triethylamine (5.0 g, 49.77 mmol), phenylacetyl chloride (5.13g, 33.18 mmol), and
stirred at rt overnight. The mixture was concentrated to about one third of the volume
and poured into ice-water. The solid was filtered and dried and recrystallized from
o
1
ethyl acetate to afford Q06 as a white solid (5.3 g, yield 82%). mp 293.5-294.8 C; H
NMR (DMSO-d , ppm) δ 12.19 (s, 1H), 11.57 (s, 1H), 8.45 (s, 1H), 7.77-7.78 (d, 1H),
6
13
7
.67-7.69 (d, 1H), 7.28-7.40 (m, 7H), 4.42 (s, 3H, N-Me), 3.91 (s, 2H); C NMR
(DMSO, ppm) δ 170.3, 168.5, 165.9, 142.2, 134.9, 134.1, 130.0 (2C) 129.1 (2C),
1
27.6, 123.4, 120.5, 117.4, 113.4, 92.9, 43.8.
4
.1.24. 2-Benzyl-6-(2-methyl-2H-tetrazol-5-yl)-3,9-dihydro-4H-pyrimido[4,5-b]
indol-4-one (Q07)
A solution of Q06 (5.0 g, 13.31 mmol) in isopropanol (100 mL) was added with
6
.25 N NaOH (15.7 mL, 97.83 mmol) and heated to reflux for 6 hr, cooled to rt, added
water (300 mL), and adjusted pH=6 with 6 N HCl. The solid was filtered, washed
with water, dried and recrystallized from methanol to give Q07 as a white solid (3.5 g,
o
1
yield 73.6%). mp 281.3-282.9 C; H NMR (DMSO-d , ppm) δ 12.40-12.50 (d, 2H,
6
3
4
,9-NHs), 8.68 (b, 1H), 8.01 (b, 1H), 7.57 (b, 1H). 7.26-7.39 (m, 5H), 4.43 (s, 3H),
+
.04 (s, 2H); LC/MS-m/z: 358.3 [M+1] (exact mass: 357.1).
4
.1.25. 2-Benzyl-4-chloro-6-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]
2
2

ACCEPTED MANUSCRIPT
indole (Q08)
A solution of Q07 (1.5 g, 4.2 mmol) in phosphorus oxychloride (15 mL) was
refluxed overnight, cooled to rt, and poured into ice-water. The solid was filtered,
washed with water and dried, recrystallized from ethyl acetate to give Q08 as a white
o
1
solid (1.4 g, yield 89%). mp 269.3-271.6 C; H NMR (DMSO-d , ppm) δ 12.83 (s,
6
1
4
1
4
H, 9-NH), 8.74 (s, 1H), 8.16-8.18 (dd, 1H), 7.63-7.65 (d, 1H), 7.21-7.34 (m, 5H),
1
3
.42 (s, 3H), 4.26 (s, 2H); C NMR (DMSO, ppm) δ 166.4, 164.8, 157.8, 152.1,
40.1, 138.7, 129.6 (2C), 128.8 (2C), 126.9, 126.5, 120.8, 120.2, 118.6, 113.4, 109.3,
5.3, 40.1.
4
.1.26. N1-(2-Benzyl-6-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-
yl)-N2,N2-bis(pyridin-2-ylmethyl)ethane-1,2-diamine (12)
To a flask was added N-BOC-1,2-ethylenediamine (3.0 g, 18.72 mmol), water (20
mL), NaOH (3.0 g, 74.88 mmol), 2-chloromethylpyridine HCl (6.4 g, 39.31 mmol).
o
The mixture was stirred at 70 C for 4 hr, cooled to rt, extracted with ethyl acetate,
washed with brine, dried over sodium sulfate. Solvent was removed and the crude was
purified by a silica gel column to give intermediate Q09, tert-butyl
1
(
2-(bis(pyridin-2-ylmethyl)amino)ethyl)carbamate, as an oil (5.1 g, yield 80%). H
NMR (CDCl , ppm) δ 8.45-8.47 (dt, 2H), 7.53-7.57(td, 2H), 7.31-7.35 (dd, 2H),
3
7
1
.05-7.08 (td, 2H), 5.88 (b, 1H), 3.79 (s, 4H), 3.15-3.17 (t, 2H), 2.61-2.64 (t, 2H),
.37 (s, 9H). Intermediate 9 (5.0 g, 14.6 mmol) was dissolved in dichloromethane (10
mL) and added with 6 N HCl (5 mL) and stirred at rt for 2 hr. The organic phase was
separated and the aqueous phase was adjusted to pH=10 with 4 N NaOH and
extracted with dichloromethane 3 times. The organic phase was combined and washed
with brine and dried, concentrated to give intermediate Q10 as an oil (3.2 g, yield
1
9
7
2
0.4%). H NMR (DMSO-d , ppm) δ 8.33-8.35 (dd, 2H), 7.45-7.49 (td, 2H),
6
.27-7.30 (dd, 2H), 6.95-6.98 (td, 2H), 3.66 (s, 4H), 2.60-2.63 (t, 2H), 2.47-2.50 (t,
H), 2.40 (b, 2H).
To a flask was added Q08 (0.4 g, 1.07 mmol), DMF (5 mL), anhydrous K CO
2
3
(
0.3 g, 2.14 mmol), and intermediate Q10 (0.5 g, 2.14 mmol). The mixture was stirred
2
3

ACCEPTED MANUSCRIPT
o
at 90 C for 4 hr, cooled to rt, and poured into ice-water. The solid was filtered ,
washed with water, and dried, recrystallized from methanol to give product 12 (0.35 g,
o
1
yield 56%). mp 195.1-196.9 C; H NMR (DMSO-d , ppm) δ 12.08 (s, 1H), 8.97 (s,
6
1
4
1
1
6
H), 8.43-8.44 (d, 2H), 8.05-8.07 (dd, 1H), 7.47-7.53 (m, 6H), 7.15-7.24 (m, 7H),
13
.42 (s, 3H), 3.81-3.90 (m, 8H), 2.80-2.84 (t, 2H); C NMR (DMSO, ppm) δ 166.3，
65.8, 160.0 (2C), 157.4, 157.1, 149.2 (2C), 139.7, 138.0, 136.8 (2C), 129.5 (2C),
28.5 (2C), 126.4, 123.2, 122.8 (2C), 122.4 (2C), 120.6, 119.7, 119.5, 112.0, 94.1,
+
0.2 (2C), 53.6, 46.1, 38.4; LC/MS-m/z: 582.4 [M+1] (exact mass: 581.3); Anal (%)
found: C67.96, H5.22, N26.28 (Calcd for C H N : C68.14, H5.37, N26.49).
3
3
31 11
4
.1.27. 2-Benzyl-N-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolino)ethyl)-6-
(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-amine (13)
Analog 13 was prepared according to the procedure of 12 by reacting
intermediate
Q08
with
6,7-dimethoxy-2-(N-aminoethyl)-1,2,3,4-tetrahydro-
o
1
isoquinoline and obtained as a white solid in 64.4% yield. mp 243.3-244.7 C; H
NMR (DMSO-d , ppm) δ 12.06 (s, 1H), 8.89 (s, 1H)，8.01-8.03 (d, 1H），7.53（d,
6
1
H），7.47 (t, 1H), 7.39-7.41 (d, 2H), 7.27-7.31 (t, 2H)，7.20-7.22 (t, 1H)，6.62-6.65
(d, 2H)，4.44 (s, 3H)，4.07 (s, 2H), 3.82-3.84 (m, 2H), 3.69-3.70 (d, 6H), 3.59 (s, 2H),
1
3
2
.74 (b, 6H); C NMR (DMSO, ppm): δ 166.4，165.8, 157.3, 157.2, 147.6, 147.3,
1
39.9, 138.0, 129.6 (2C), 128.6 (2C), 127.1, 126.5, 126.4, 123.2, 120.5, 119.2, 119.4,
112.2, 112.0, 110.4, 94.1, 57.3, 55.9, 55.9, 55.7, 51.3, 46.2, 40.1, 38.4, 28.7;
+
LC/MS-m/z: 576.3 [M+1] (exact mass: 575.3); Anal (%) found: C 66.73, H 5.67, N
2
2.02 (Calcd for C H N O : 66.77, H 5.78, N 21.90).
32 33 9 2
4
.1.28. (1-(2-Benzyl-6-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-
yl)piperidin-3-yl)methanol (14)
Analog 14 was prepared according to the procedure of 12 by reacting
intermediate Q08 with 3-hydroxymethylpiperidine and obtained as a solid in 62%
o
1
yield. mp 223.8-224.6 C; H NMR (DMSO-d , ppm) δ 12.28 (s, 1H), 8.43 (s, 1H),
6
8
.06-8.08 (d, 1H), 7.58-7.60 (d, 1H), 7.38-7.40 (d, 2H), 7.27-7.31 (t, 2H), 7.18-7.21 (t,
2
4

ACCEPTED MANUSCRIPT
1
H), 4.52-4.55 (t, 1H), 4.43 (s, 3H), 4.35-4.38 (d, 1H), 4.21-4.24 (d, 1H), 4.12 (s, 2H),
.42-3.46 (m, 2H), 3.06-3.08 (t, 1H), 2.88-2.94 (t, 1H), 1.87-1.90 (m, 3H), 1.74-1.77
3
1
3
(
m, 1H), 1.30-1.38 (m, 1H); C NMR (DMSO, ppm): δ 165.5, 165.5, 160.5, 159.0,
1
6
4
6
39.6, 138.6, 129.6 (2C), 128.6 (2C), 126.6, 123.4, 120.4, 120.3, 119.6, 112.5, 96.9,
+
4.2, 51.4, 48.9, 45.9, 40.1, 39.3, 27.5, 25.2; LC/MS-m/z: 455.3 [M+1] (exact mass:
54.2); Anal (%) found: C 63.34, H 5.76, N 23.63 (Calcd for C H N O + H O: C
2
5
26
8
2
3.54, H 5.97, N 23.71).
4
.1.29. N-(2-(1H-Indol-3-yl)ethyl)-2-benzyl-6-(2-methyl-2H-tetrazol-5-yl)-9H-
pyrimido[4,5-b]indol-4-amine (15)
Analog 15 was prepared according to the procedure of 12 by reacting
intermediate Q08 with 3-(2-aminoethyl)indole and obtained as a solid in 70.5% yield.
o
1
mp 286.5-288.7 C; H NMR (DMSO-d , ppm) δ 12.08 (s, 1H), 10.85 (s, 1H), 8.99 (s,
6
1
3
1
H), 8.04 (b, 1H), 7.73-7.75 (d, 2H), ), 7.02-7.28 (m, 9H), 4.46 (s, 3H), 4.14 (s, 2H),
1
3
.90 (b, 2H), 3.11 (b, 2H); C NMR (DMSO, ppm): δ 166.4, 165.9, 157.4, 157.1,
40.0, 138.0, 136.8, 129.5 (2C), 128.6 (2C), 128.0, 126.5 (2C), 123.0 (2C), 120.6,
119.6, 119.1, 118.7, 112.7, 111.9, 94.2, 46.4, 41.9, 40.1, 26.0; LC/MS-m/z: 500.3
+
[
M+1] (exact mass: 499.2); Anal (%) found: C 67.21, H 5.19, N 24.15 (Calcd for
C H N + H O: C 67.30, H 5.26, N 24.36).
29
25
9
2
4
.1.30. (1-(2-Benzyl-6-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-
yl)piperidin-4-yl)methanol (16)
Analog 16 was prepared according to the procedure of 12 by reacting
intermediate Q08 with 4-hydroxymethylpiperidine and obtained as a solid in 63%
o
1
yield. mp 219.7-220.3 C; H NMR (DMSO-d , ppm) δ 12.29 (s, 1H), 8.41 (s, 1H),
6
8
1
1
1
.05-8.07 (t, 1H), 7.58-7.60 (d, 1H), 7.37-7.39 (d, 1H), 7.27-7.31 (t, 1H), 7.17-7.21 (t,
H), 4.60-4.63 (t, 1H), 4.45 (s, 3H), 4.32-4.34 (d, 2H), 4.11 (s, 2H), 3.06-3.12 (t, 2H),
13
.87-1.90 (d, 2H), 1.76 (b, 1H), 1.36-1.41 (q, 2H); C NMR (DMSO, ppm): δ 165.5,
65.5, 160.5, 159.0, 139.6, 138.6, 129.6 (2C), 128.6 (2C), 126.6, 123.5, 120.3, 120.3,
1
19.7, 112.4, 96.8, 66.2, 48.0 (2C), 45.9, 40.1, 39.0, 29.2 (2C); LC/MS-m/z: 455.3
2
5

ACCEPTED MANUSCRIPT
+
[
M+1] (exact mass: 454.2); Anal (%) found: C 65.88, H 5.59, N 24.61 (Calcd for
C H N O: C 66.06, H 5.77, N 24.65).
25
26
8
4
.1.31. 4-((2-benzyl-6-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-
yl)amino)cyclohexan-1-ol (17)
Analog 17 was prepared according to the procedure of 12 by reacting
intermediate Q08 with trans-4-aminocyclohexan-1-ol and obtained as a solid in 67%
o
1
yield. mp 289.6-290.3 C; H NMR (DMSO-d , ppm) δ 12.03 (s, 1H), 8.93 (s, 1H),
6
8
1
1
1
9
4
5
.01-8.03 (d, 1H), 7.52-7.54 (d, 1H), 7.20-7.40 (m, 5H), 7.03-7.05 (d, 1H), 4.62 (s,
H), 4.45 (s, 3H), 4.26 (b, 1H), 4.05 (s, 2H), 3.47 (b, 1H), 1.91-1.93 (m, 4H),
1
3
.60-1.63 (m, 2H), 1.30-1.33 (m, 2H); C NMR (DMSO, ppm): δ 166.2, 165.9, 157.5,
56.8, 139.9, 138.0, 129.7 (2C), 128.5 (2C), 126.4, 123.2, 120.5, 120.0, 119.4, 111.8,
+
4.2, 69.3, 49.5, 46.1, 35.1 (2C), 30.5 (2C); LC/MS-m/z: 455.3 [M+1] (exact mass:
54.2); Anal (%) found: C 65.91, H 5.63, N 24.66 (Calcd for C H N O: C 66.06, H
2
5
26
8
.77, N 24.65).
4
.1.32. 2-Benzyl-N-(4-methoxyphenethyl)-6-(2-methyl-2H-tetrazol-5-yl)-9H-
pyrimido[4,5-b]indol-4-amine (18)
Analog 18 was prepared according to the procedure of 12 by reacting
intermediate Q08 with 4-methoxyphenethylamine and obtained as a solid in 56%
o
1
yield. mp 273.7-275.1 C; H NMR (DMSO-d , ppm) δ 12.06 (s, 1H), 8.95 (s, 1H),
6
8
2
3
1
.03-8.05 (d, 1H), 7.67 (t, 1H), 7.55-7.57 (d, 1H), 7.42-7.44 (d, 2H), 7.28-7.32 (m,
H), 7.16-7.20 (m, 3H), 6.87-6.89 (d, 2H), 4.45 (s, 3H), 4.10 (s, 2H), 3.75-3.77 (t, 2H),
1
3
.73 (s, 3H), 2.86-2.90 (t, 2H); C NMR (DMSO, ppm): δ 166.4, 165.8, 158.1, 157.4,
57.0, 140.0, 138.0, 132.2, 130.1 (2C), 129.2 (2C), 128.6 (2C), 126.5, 123.2, 120.6,
+
1
19.6, 114.3, 111.9, 94.2, 55.5, 46.3, 42.9, 35.1; LC/MS-m/z: 491.3 [M+1] (exact
mass: 490.2); Anal (%) found: C 65.88, H 5.31, N 21.82 (Calcd for C H N O +
2
8
26
8
H O: C 66.13, H 5.55, N 22.03).
2
4
.1.33. 2-Benzyl-N-(3,4-dimethoxyphenethyl)-6-(2-methyl-2H-tetrazol-5-yl)-9H-
2
6

ACCEPTED MANUSCRIPT
pyrimido[4,5-b]indol-4-amine (19)
Analog 19 was prepared according to the procedure of 12 by reacting
intermediate Q08 with 3,4-dimethoxyphenethylamine and obtained as a solid in 66%
o
1
yield. mp 273.6-274.5 C; H NMR (DMSO-d , ppm) δ 12.06 (s, 1H), 8.95 (s, 1H),
6
8
3
1
1
4
6
.03-8.05 (d, 1H), 7.57-7.63 (m, 2H), 7.20-7.43 (m, 5H), 6.78-5.87 (m, 3H), 4.45 (s,
1
3
H), 4.10 (s, 2H), 3.79 (b, 2H), 3.71 (s, 6H), 2.90 (b, 2H); C NMR (DMSO, ppm): δ
66.4, 165.8, 157.4, 157.0, 149.1, 147.7, 140.0, 138.0, 132.8, 129.7 (2C), 128.6 (2C),
26.5, 123.2, 121.0, 120.6, 119.6, 119.6, 112.9, 112.4, 111.9, 94.2, 56.0, 55.7, 46.3,
+
2.8, 40.1, 35.5; LC/MS-m/z: 521.1 [M+1] (exact mass: 520.2); Anal (%) found: C
6.76, H 5.28, N 21.63 (Calcd for C H N O : C 66.91, H 5.42, N 21.52).
2
9
28
8
2
4
4
.1.34. N-(2-((2-Benzyl-6-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-
-yl)amino)ethyl)quinoline-2-carboxamide (20)
To a flask was added methyl quinoline-2-carboxylate (0.4 g, 2.14 mmol),
methanol (10 mL), and ethylene-1,2-diamine (2.0 g, 33.3 mmol). The mixture was
stirred at rt for 4 hr, concentrated to dryness to give an oil (0.42 g, crude yield 91%).
The obtained oil was dissolved in DMF (6 mL), added with intermediate Q08 (0.4 g,
o
1
.07 mmol) and K CO (0.3 g, 2.14 mmol) and stirred at 90 C for 4 hr, cooled to rt,
2
3
poured into ice-water, filtered, washed with water, dried, and recrystallized from ethyl
o
1
acetate to give 20 as a white solid (0.32 g, yield 54%). mp 285.4-287.3 C; H NMR
(
DMSO-d , ppm) δ 12.09 (s, 1H), 9.17 (b, 1H), 9.02 (s, 1H), 8.54-8.56 (d, 1H),
6
8
.20-8.22 (d, 1H), 8.03-8.06 (m, 3H), 7.69-7.83 (m, 3H), 7.53-7.55 (d, 1H), 7.38-7.40
(d, 2H), 7.24-7.27 (t, 2H), 7.17-7.18 (t, 1H). 4.43 (s, 3H), 4.08 (s, 2H), 3.93-3.94 (b,
1
3
2
1
1
5
4
H), 3.74-3.75 (b, 2H); C NMR (DMSO, ppm): δ 166.3, 165.2, 165.1, 157.4, 157.3,
50.7, 146.4, 139.6, 138.3, 138.0, 130.9, 129.6 (2C), 129.6, 129.3, 128.6 (2C), 126.5,
+
23.3, 120.5, 119.7, 119.2, 111.9, 94.2, 46.2; LC/MS-m/z: 555.4 [M+1] (exact mass:
54.2); Anal (%) found: C 66.98, H 4.56, N 25.09 (Calcd for C H N O: C 67.13, H
3
1
26 10
.73, N 25.26).
4
.1.35. 4-(2-((2-Benzyl-6-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-
27

ACCEPTED MANUSCRIPT
4
-yl)amino)ethyl)phenol (21)
Analog 21 was prepared according to the procedure of 12 by reacting
intermediate Q08 with 4-hydroxyphenethylamine and obtained as a solid in 52% yield.
o
1
mp 294.6-296.1 C; H NMR (DMSO-d , ppm) δ 12.06 (s, 1H), 9.20 (s, 1H), 8.95 (s,
6
1
H), 8.03-8.05 (d, 1H), 7.63 (t, 1H), 7.54-7.56 (d, 1H), 7.18-7.42 (m, 5H), 7.05-7.08
(d, 2H), 6.71-6.74 (d, 2H), 4.45 (s, 3H), 4.10 (s, 2H), 3.72-3.77 (m, 2H), 3.82-3.86 (t,
1
3
2
H); C NMR (DMSO, ppm): δ 166.4, 165.8, 157.4, 157.0, 156.1, 140.0, 138.0,
1
30.4, 130.0 (2C), 129.6 (2C), 128.6 (2C), 126.5, 123.2, 120.6, 119.6, 115.7 (2C),
+
1
11.9, 94.1, 46.3, 43.1 35.2; LC/MS-m/z: 477.1 [M+1] (exact mass: 476.2); Anal (%)
found: C 67.92, H 4.87, N 23.40 (Calcd for C H N O: C 68.05, H 5.08, N 23.51).
2
7
24
8
4
.1.36. tert-Butyl(2-((2-benzyl-6-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]
indol-4-yl)amino)ethyl)carbamate (22)
Analog 22 was prepared according to the procedure of 12 by reacting
intermediate Q08 with N-BOC-ethylene-1,2-diamine and obtained as a solid in 54%
o
1
yield. mp 266.0-267.9 C; H NMR (DMSO-d , ppm) δ 12.07 (s, 1H), 8.95 (s, 1H),
6
8
.02-8.04 (dd, 1H), 7.53-7.55 (t, 1H), 7.41-7.43 (d, 2H), 7.27-7.31 (t, 2H), 7.17-7.21
(t, 1H), 7.08-7.10 )t, 1H), 4.41 (s, 3H), 4.05 (s, 2H), 3.66-3.67 (m, 2H), 3.28-3.31 (m,
1
3
2
1
4
H), 1.37 (s, 9H); C NMR (DMSO, ppm): δ 166.3, 165.8, 157.3, 157.1, 156.5, 139.8,
38.0, 129.7 (2C), 128.6 (2C), 126.5, 123.3, 120.5, 119.7, 119.6, 111.9, 94.2, 78.2,
+
6.2, 41.3, 40.1, 39.7, 28.7; LC/MS-m/z: 500.3 [M+1] (exact mass: 499.2); Anal (%)
found: C 62.29, H 5.64, N 25.07 (Calcd for C H N O : C 62.51, H 5.85, N 25.23).
2
6
29
9
2
4
.1.37. 2-Benzyl-N-(3-chloro-4-fluorophenyl)-6-(2-methyl-2H-tetrazol-5-yl)-9H-
pyrimido[4,5-b]indol-4-amine (23)
Analog 23 was prepared according to the procedure of 12 by reacting
intermediate Q08 with 3-chloro-4-fluoroaniline and obtained as a solid in 69% yield.
o
1
mp 289.0-290.2 C; H NMR (DMSO-d , ppm) δ 12.38 (s, 1H), 9.37 (b, 1H), 9.02 (s,
6
1
H), 8.10-8.12 (d, 1H), 8.01-8.03 (t, 1H), 7.57 (b, 1H), 7.59-7.61 (d, 1H), 7.31-7.39
1
3
(
m, 5H), 7.20-7.24 (m, 1H), 4.45 (s, 3H), 4.11 (s, 2H); C NMR (DMSO, ppm): δ
2
8