Design, synthesis and biological evaluation of novel condensed pyrrolo[1,2- c]pyrimidines featuring morpholine moiety as PI3Kα inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.05.036

Abstract Four series of condensed pyrrolo[1,2-c]pyrimidines 6a-d, 8a-d, 10a,b and 12a-e designed as PI3Kα inhibitors were synthesized and evaluated for inhibitory activity and selectivity toward different PI3K isoforms. The tested compounds displayed PI3K

Full text of DOI:10.1016/j.ejmech.2015.05.036

Accepted Manuscript
Design, Synthesis and Biological Evaluation of Novel Condensed
Pyrrolo[1,2-c]Pyrimidines Featuring Morpholine Moiety as PI3Kα Inhibitors
Marwa A. Ibrahim, Sahar M. Abou-Seri, Associate Professor of Pharmaceutical
Chemistry, Mona M. Hanna, Mohamed M. Abdalla, Nehad A. El Sayed
PII:
S0223-5234(15)30063-5
10.1016/j.ejmech.2015.05.036
EJMECH 7915
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 October 2014
Revised Date: 30 March 2015
Accepted Date: 22 May 2015
Please cite this article as: M.A. Ibrahim, S.M. Abou-Seri, M.M. Hanna, M.M. Abdalla, N.A. El Sayed,
Design, Synthesis and Biological Evaluation of Novel Condensed Pyrrolo[1,2-c]Pyrimidines Featuring
Morpholine Moiety as PI3Kα Inhibitors, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.05.036.
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ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Novel Condensed Pyrrolo[1,2-c]Pyrimidines
Featuring Morpholine Moiety as PI3Kα Inhibitors
Marwa A. Ibrahima, Sahar M. Abou-Seria,*, Mona M. Hannaa, Mohamed M. Abdallab, Nehad A.
El Sayeda
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini
Street, Cairo, P.O. Box 11562, Egypt.
bResearch Unit, Saco Pharm. Co., 6th October City, Egypt
Graphical abstract
Novel series of morpholino-pyrimidopyrrolopyrimidines 6a-d, 8a-d and 10a,b, and morpholino-
pyridopyrrolopyrimidine-2-carbonitriles 12a-e were designed and synthesized as PI3K
inhibitors. Compounds 8a-d and 12a-e proved to be potent and selective PI3Kα inhibitors.

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Novel Condensed Pyrrolo[1,2-c]Pyrimidines
Featuring Morpholine Moiety as PI3Kα Inhibitors
Marwa A. Ibrahima, Sahar M. Abou-Seria,*, Mona M. Hannaa, Mohamed M. Abdallab, Nehad A.
El Sayeda
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini
Street, Cairo, P.O. Box 11562, Egypt.
bResearch Unit, Saco Pharm. Co., 6th October City, Egypt
* Corresponding author: Sahar Mahmoud Abou-Seri;
Associate Professor of Pharmaceutical Chemistry, Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Cairo University, Kasr El-Aini street, Cairo, P.O. Box, 11562, Egypt.
Tel.: +2-02-33144107, +2-1227059742; fax: +2-02-2362 8426.
E-mail address: saharshaarawy_69@yahoo.com
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Abstract
Four series of condensed pyrrolo[1,2-c]pyrimidines 6a-d, 8a-d, 10a,b and 12a-e designed as
PI3Kα inhibitors were synthesized and evaluated for inhibitory activity and selectivity toward
different PI3K isoforms. The tested compounds displayed PI3Kα kinase inhibitory activity at
either low micromolar or nanomolar level. In particular, the morpholino-
pyrimidopyrrolopyrimidinones 8a-d and morpholino-pyridopyrrolopyrimidine-2-carbonitriles
12a-e proved to be highly potent and selective PI3Kα inhibitors (IC50 =0.1-7.7nM). Moreover,
the target compounds exhibited considerable cytotoxic activity against cervical cancer cell line
HeLa that over-expresses p110α (0.21-1.99 µM). Molecular modeling simulation revealed that,
the designed compounds docked well into p110α active site and their complexes are stabilized by
a key H-bonding with the backbone amide of Val851 as well as other favorable hydrophobic and
H-bond interactions with different amino acids within the enzyme active site.
Keywords: PI3K inhibitor; morpholino derivatives; pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine;
pyrido[3,2-e]pyrrolo[1,2-c]pyrimidine-2-carbonitrile.
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1. Introduction
Phosphatidylinositol-3-kinases (PI3Ks) are a family of intracellular lipid kinases which has
been discovered since the last two decades. They are classified into three classes I, II and III
based on their structure and substrate specificity. Class I PI3K is divided into two subclasses,
class IA (p110α, β and δ) and class IB (p110γ) based on their mode of activation [1,2]. The PI3K
signaling pathway regulates the cellular responses and plays a critical role in maintaining the
balance between cellular survival and apoptosis. Active PI3K phosphorylates
phosphatidylinositol 4,5-bisphosphate into phosphatidylinositol 3,4,5-trisphosphate which in turn
propagates intracellular signaling, activating downstream signaling proteins that promote cell
growth and survival [1,3,4]. Therefore, aberrant activation of PI3K signaling can have a
predominant role in uncontrolled cell proliferation and may enhance migration which favors
malignant tumor formation [1]. The importance of this pathway in cancer is highlighted by the
frequent mutation of PIK3CA gene that codes for p110α in many cancers and mutational
inactivation of PTEN (the tumor suppressor phosphatase which antagonizes PI3K action) in
more than 50% of different types of cancer [5,6]. Thus, targeting PI3K with small molecule
inhibitors provides an attractive opportunity for cancer therapy. Multiple efforts are under way in
academia or pharmaceutical community to develop clinically relevant inhibitors of PI3K
signaling pathway, especially inhibitors of the p110α isoform which has been linked to cancer
survival and resistance to therapy [3,5,7].
Surveying the literatures revealed that, a large array of morpholino derivatives were reported
as p110α inhibitors (Fig. 1). In 1994, LY294002 I was developed as PI3K inhibitor based on the
natural flavone, quercetin [8]. The crystal structure of LY294002 I in complex with PI3Kγ
revealed that the morpholine oxygen is engaged in a key hydrogen bond interaction with the
catalytic site [4]. This finding provided a starting point for designing PI3K inhibitors, where a
morpholine rest was used to gain affinity towards the enzyme [8-10]. In this context, a large
number of inhibitors have been designed with diverse scaffolding to find novel hits. Different
morpholino derivatives with monocyclic central core were cited. For instance, the 5-cyano-6-
morpholino-pyrimidine WJD008 II selectively inhibited p110α isoform and displayed superior
antiproliferative activity against a panel of cancer cells [7]. Moreover, the dimorpholino-
pyrimidine derivative buparlisib (BKM-120) III, a pan-class I PI3K inhibitor, is currently under
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evaluation in patients with advanced solid tumor [11,12]. Likewise, novel bicyclic heterocycles
bearing morpholine moiety have been emerged as PI3Kα inhibitors including the imidazo[1,2-
a]pyrazine derivative IV [13] and the orally bioavailable thienopyrimidine GDC-0941 V which
entered phase I clinical trials in cancer patients [14]. Although, a considerable number of p110α
inhibitors with monocyclic and bicyclic cores were synthesized [7,9,12-14], to date, only a few
inhibitors with central tricyclic scafold were reported. For example, the oxazino[4,3-e]purine VI
was designed to target the over-expressed p110α in glioblastoma [15]. Additionally,
pyridofuropyrimidine PI-103 VII has excellent potency for p110α and p110β isoforms at
nanomolar level [16].
2. Rational Design
A throughout analysis of large array of morpholine-based p110α inhibitors reported in the
literature revealed that the main pharmacophore describing p110α inhibitors’ interactions with
their binding site could be represented by: 1- A hydrogen-bond acceptor group that is centered on
the morpholine oxygen atom which is implicated in a key hydrogen bond interaction with
Val851 amide backbone within p110α active site. 2-Two hydrophobic regions were also defined,
these regions were represented by a central heteroaromatic ring and an aromatic ring or lipophilic
side chain –located at meta position to the morpholine moiety- bearing H-bond donor or acceptor
group which is involved in H-bond interaction with Tyr836, Asp810 and/or Lys802 (Fig. 2)
[8,17,18].
Recent research findings in our labs disclosed the new pyrimidopyrrolopyrimidine
derivative, 1-((4-chlorobenzylidene)amino)-3-thioxo-pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidin-6-
one VIII as potent cytotoxic agent [19]. Since the pyrimidopyrrolopyrimidine core in VIII could
provide the appropriate scaffolding to place both the morpholine oxygen and the hydroxyl
phenyl moiety of VII near the catalytic region for optimum binding, we initiated a research work
directed at the design and synthesis of new series of PI3Kα inhibitiors through replacing the
central tricyclic heteroaromatic core in VII by pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine ring
system in 6a-d, hoping to obtain a new series of PI3Kα inhibitors with potent antitumor activity
(Fig. 3). The adopted molecular manipulations involved varying the substituent responsible for
H-bond interaction on the meta aromatic ring of our targeted compounds. Although, it was
reported that 3-OH phenyl group afforded potent inhibitors, these inhibitors suffered from
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extensive metabolism [8]. In the present study, we relied mainly on designing the methoxy
derivatives which are supposed to avoid extensive metabolism, but still able to form H-bond with
the specified amino acid residues. The pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine core was also
appended by ethyl or phenyl substituents, in addition to a thioxo group to address different
hydrophobic interactions with the binding site amino acids.
In a second approach the chromone nucleus in the potent PI3K inhibitor LY294002 I was
replaced by pyrimidopyrrolopyrimidinone in 8a-d, as the carbonyl oxygen may have the
potential to be involved in H-bond interaction with the enzyme active site. Also, the morpholine
moiety was grafted through one carbon spacer to impart some sort of flexibility and allow proper
binding with the active site. Moreover, new trends towards designing potent and selective PI3K
inhibitors were based on either introducing a second morpholine or morpholinomethyl moiety to
the central heterocyclic core in BKM-120 III and IV [11,13] or attaching another morpholine
rest to the meta position of the phenyl ring in oxazino[4,3-e]purine derivative VI [15].
Consequently, the 1,3-dimorpholino congeners 10a,b were synthesized to explore the influence
of such structure modification on the inhibitory effect of pyrimidopyrrolopyrimidinones 8c-d
(Fig.3).
Finally, bioisosteric replacement of the pyrimidine nitrogen of 6a-d with a pendant nitrile
led to the design of compounds 12a-e (Fig.3).
Since, the design of the target morpholino-pyrimidopyrrolopyrimidines 6a-d, 8a-d and
10a,b, and morpholino-pyridopyrrolopyrimidine-2-carbonitriles 12a-e was based on the
hypothesis that such compounds could mimic the interactions of known p110α inhibitors with
the enzyme active site, therefore, molecular docking approach was used to support the design
hypothesis prior synthesis. As the designed compounds may exist as racemic mixtures or
mixtures of diastereomers in case of 8b, 8d and 10b, thus docking of all the possible isomers of
the new inhibitors into the crystal structure of p110α kinase domain in complex PI-103 VII
[PDB ID: 4L23] [20] was performed using Molecular Operating Environment MOE version
2009.10 [21]. Affinity of the compounds to the target enzyme was judged by comparing the
binding free energy (docking score) and the binding mode of the target compounds with that of
the co-crystallized ligand PI-103. Analysis of docking results (Table1 and supplementary
material) revealed that all isomers of the designed compounds fit into the active site of p110α
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and most of them displayed comparable docking scores and binding modes similar to that of PI-
103 (Fig. 4 and 5). Moreover, the morpholine oxygen of all the isomers showed the potential to
be engaged in hydrogen bond interaction with Val851. Such hydrogen bond is considered crucial
for PI3Kα inhibitory activity [2,10,20] and novel compounds reported to be stabilized by this
central interaction become deeply embedded into the binding cleft of PI3K, suggesting their
potential activity as p110α inhibitors.
Regarding the potential effect of compounds’ chirality on binding interactions and p110α
inhibitory activity, the modeling results revealed no significant difference between 10aR and
10aS enantiomers of compounds 6a-d and 12a-e. Both enantiomers of these compounds
displayed the same binding modes with no significant difference in docking scores and H-bonds
length, therefore they are expected to be equally active (Fig.4, and supplementary material).
However, docking results of the morpholinomethyl derivatives 8a-d and 10a,b showed
preferential binding of isomers having an absolute R configuration at C10a regardless of the
absolute configuration at the methylene spacer (C1’). For example, the carbonyl oxygen of
compound 8d with (10aR, 1’R) and (10aR, 1’S) absolute configuration participated in an
additional H-bond interaction with Tyr836 while the (10aS, 1’S) and (10aS, 1’R) diastereomers
did not (see supplementary material).
3. Results and discussion
3.1. Chemistry
Schemes 1-3 illustrate the synthetic pathways adopted for the preparation of the targeted
compounds 6a-d, 8a-d, 10a,b and 12a-e. Reaction of 2-dicyanomethylidinopyrrolidine 1 with
ethyl or phenyl isothiocyanates followed by sodium borohydride mediated reduction afforded the
enaminonitrile derivatives 3a,b as reported [19,22-24]. Acylation of the intermediates 3a,b with
3- or 4-anisoyl chloride followed by in situ intramolecular cyclization gave the tricyclic
pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidinones 4a-d. Chlorination of the latter, using excess POCl3
was achieved only upon using drops of dimethylformamide (DMF) to yield the chloro-
intermediates 5a-d. These intermediates 5a-d were reacted directly with morpholine, in dry DMF
and in the presence of anhydrous potassium carbonate as a base to furnish the desired 3-aryl-1-
morpholino-pyrimidopyrrolopyrimidines 6a-d. (Scheme 1)
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Chloroacylation of the bicyclic o-aminonitriles 3a,b proceeded via stirring with chloroacetyl
chloride or chloropropionyl chloride overnight in dry benzene, then intramolecular cyclization
was achieved by reflux with ethanolic hydrochloric acid to produce the 3-(chloro-alkyl)
pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidinones 7a-d. Condensation of the chloro-alkyl derivatives
7a-d with morpholine in refluxing absolute ethanol furnished the targeted 3-morpholinoalkyl-
pyrimidopyrrolopyrimidinones 8a-d. Alternatively, the 1,3-dimorpholino-congeners 10a,b were
synthesized through the dichloro-intermediates 9a,b obtained by chlorination of 3-(chloro-
alkyl)pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidinones 7a-d with POCl3, followed by nucleophilic
substitution of chlorine atoms with morpholine using the same synthetic procedure adopted for
the preparation of compounds 8a-d. (Scheme 2)
Finally, the o-aminonitrile key intermediate 3a was refluxed with appropriate aromatic
aldehydes and malononitrile in absolute ethanol to afford the corresponding 1-amino-
pyridopyrrolopyrimidine-2-carbonitrile derivatives 11a-e. The desired morpholino-pyrido[3,2-
e]pyrrolo[1,2-c]pyrimidines 12a,c were synthesized by coupling the amino derivatives 11a,c
with two equivalents of 2-chloroethanol in refluxing mixture of dry DMF/dry dioxane using
anhydrous potassium carbonate as an acid binder and dehydrating agent (Method A). On the
other hand, reaction of the amino intermediates 11b,d,e with 2-chloroethanol failed to produce
the morpholino-derivatives 12b,d,e. However, the targeted compounds 12b,d,e were obtained
upon heating the 1-amino-pyridopyrrolopyrimidines 11b,d,e with the more reactive bis(2-
bromoethyl)ether and anhydrous potassium carbonate in a mixture of dry DMF/dry dioxane
(Method B). (Scheme 3)
3.2. Biological evaluation
PI3K p110α inhibitory activity of the targeted morpholino-pyrimidopyrrolopyrimidines 6a-
d, 8a-d, 10a,b and morpholino-pyridopyrrolopyrimidine-2-carbonitriles 12a-e was assayed in
vitro using Scintillation Proximity Assay (SPA) [16,25]. Furthermore, to examine selectivity for
p110α, the inhibitory activity of the designed compounds was evaluated against other PI3K
isoforms namely p110β (class IA), p110γ (class IB), p110δ (class IA) and PI3K C2β (class II) and
the data are presented in Table 2.
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3.2.1. PI3K p110α inhibitory activity
As shown in Table 2, all the tested compounds displayed p110α kinase inhibitory activity
at either low micromolar or nanomolar level. Interestingly, the morpholino-
pyrimidopyrrolopyrimidines 6a-d, 8a-d and 10a,b displayed a wide range of p110α inhibitory
activity with IC50 ranging from 0.1 nM to 5.49 ꢀM. While, the 3-aryl-1-morpholino-
pyrimidopyrrolopyrimidines 6a-d exhibited moderate inhibitory activity (IC50 = 0.234 - 0.695
ꢀM), the 3-morpholinoalkyl-pyrimidopyrrolopyrimidinones 8a-d had superior potency (IC50
=
0.1 - 0.8 nM) and the dimorpholino-derivatives 10a,b were the least potent (IC50 = 2.34 - 5.49
ꢀM). On the other hand, 3-aryl-1-morpholino-pyridopyrrolopyrimidine-2-carbonitriles 12a-e
were able to inhibit the enzyme activity at single digit nanomolar concentration (IC50 = 1.2 - 7.7
nM).
Analysis of the data in Table 2 revealed that, the substitution pattern on the 3-aryl-1-
morpholino-pyrimidopyrrolopyrimidines 6a-d affected the inhibitory potency, the 5-ethyl
derivatives 6a,b (IC50 = 0.234 and 0.387 ꢀM, respectively) exhibited almost 2 fold increase in
p110α inhibitory potency than the corresponding phenyl derivatives 6c,d (IC50 = 0.695 and 0.678
ꢀM, respectively). Such decrease in potency may be explained by unfavorable steric effect of the
more bulky phenyl group at position-5 of the pyrimidopyrrolopyrimidine core. Conversly,
varying substituent position at the 3-aryl moiety has slight effect on the inhibitory potency of
compounds 6a-d. The highest activity in this series was observed with the 3-OCH3 analogue 6a
(IC50= 0.234 ꢀM); it is note worthy that this result is consistent with docking studies as
compound 6a was perfectly overlayed with the co-crystallized ligand (Fig. 4), and was able to
form two hydrogen bonds with the active site of PI3K similar to the co-crystallized ligand with
comparable docking score (-14.42 and -13.53, respectively) Table 1.
Regarding, the most potent series, 3-morpholinoalkyl-pyrimidopyrrolopyrimidinones 8a-d
which showed exceptional enzyme inhibitory potency, the incorporation of a methyl group to the
methylene spacer between the morpholine rest and the pyrimidopyrrolopyrimidinone core in
8b,d (IC50 = 0.3 and 0.1 nM, respectively) resulted in about 3-5 fold increase in potency
compared to compounds 8a,c with unbranched methylene spacer (IC50 = 0.8 and 0.5 nM,
respectively). The molecular simulation studies suggested that, the steric repulsion between the
amidic NH and the methyl group in compound 8b,d forced the carbonyl oxygen to attain a
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proper orientation for optimum binding with Tyr836 in the catalytic site (H-bond length = 1.70
and 1.76 Å, respectively), Figure 5A, Table 1. Moreover, the 5-phenyl derivatives 8c,d (IC50 =
0.5 and 0.1 nM, respectively) showed 1.6-3 fold increase in potency than their 5-ethyl
counterparts 8a,b (IC50 = 0.8 and 0.3 nM, respectively); inspection of the top docking poses of
p110α-8c,d complexes elucidated that the phenyl ring is stabilized by hydrophobic interactions
with the side chains of Met772 and Ser919 (Fig. 5A). Structurally, Met772 exists in the flexible
loop of p110α and is reported to stabilize the complex of pyridofuropyrimidine inhibitors with
PI3K p110α through hydrophobic interactions [20].
Surprisingly, introduction of a second morpholine moiety to position-1 of the 3-
morpholinoalkyl-pyrimidopyrrolopyrimidinones 8c-d (IC50 = 0.5 and 0.1 nM, respectively) to
yield the 1,3-dimorpholino congeners 10a,b resulted in a dramatic decrease in the inhibitory
potency (IC50 = 2.34 and 5.49 ꢀM, respectively). This outcome suggested that introducing a
second morpholine moiety at position-1 may produce a spectacular steric clash at the bottom of
ATP binding site [20]. Moreover, the incorporation of an additional methyl group to the
methylene linker in 10b led to 2 fold reduction in potency.
With respect to the targeted 3-aryl-1-morpholino-pyridopyrrolopyrimidine-2-carbonitriles
12a-e, it was envisaged that replacing the pyrimidopyrrolopyrimidine scaffold in 6 by the
pyridopyrrolopyrimidine-2-carbonitrile one in 12 could be beneficial for kinase inhibitory
activity. Results in Table 2 revealed that such structural modification augmented the inhibitory
potency of the 3-aryl-1-morpholino-pyrimidopyrrolopyrimidines 6a,b (IC50 = 0.234 and 0.387
ꢀM, respectively) by 60 - 300 fold (c.f. 3-aryl-1-morpholino-pyridopyrrolopyrimidine-2-
carbonitrile 12b,c IC50 = 3.8 and 1.2 nM, respectively).
Exploring the effect of different substituents on the 3-phenyl ring of the pyridopyrrolo
pyrimidine-2-carbonitrile scaffold, revealed that the 3-OH analogue 12a (IC50 = 7.7 nM) had the
least activity in this set. Replacing the 3-OH group in 12a by the more lipophilic OCH3 group
afforded the 3-OCH3 analogue 12b (IC50 = 3.8 nM) with 2 fold enhanced potency. Shifting the
OCH3 substituent in 12b to para position afforded the most potent compound 12c (IC50 = 1.2
nM) indicating that substitution at position-4 is favourable for activity. The previous finding was
confirmed by the fact that the incorporation of 4-OCH3 group to the derivatives 12a,b to obtain
12d,e (IC50 = 3.8 and 1.9 nM, respectively) raised the inhibitory activity by 2 fold. The top
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ranked and populated predicted binding modes of compounds 12a-e disclosed that the cyano
group in this series is stabilized by hydrophobic interactions with Ile848 and Ile 932 and
participated in dipole-dipole interaction with Tyr836 (the cyano nitrogenis is directed toward the
the carbon atom bearing OH of Tyr836 side chain, C≡Nδ-….δ+C
̶ OH distance = 3.17 Å) , Figure
5B. Dipole-dipole interactions are considered key contributers to the overall strength of drug-
protein interactions.[26] Moreover, docking models of the most potent derivatives in this series
12c-e (IC50 = 1.2, 3.8 and 1.9 nM, respectively) revealed that the 4-OCH3 oxygen accepts
hydrogen bond from Lys802 (Table 1), this hydrogen bond interaction was reported to enhance
the p110α inhibitory potency of some pyridofuropyrimidine derivatives and could justify the
high activity of the 4-OCH3 phenyl substituted compounds 12c-e [20].
3.2.2. PI3K isoforms selectivity
Development of PI3K-targeted remedies faced two paradigms either pan inhibition of all
PI3K isoforms or isoform selective inhibition. Pan inhibition sounds good based on the theory
that simultaneous blocked of all PI3K isoforms in cells will disregulate PI3K signaling pathway
and hinder the ability of tumor cells to grow, survive or proliferate, but it will lead to undesirable
toxicities. Accordingly, isoform selective PI3K inhibition is considered as an alternative
approach to modulate the PI3K cell signaling pathway [10]. Inhibition of PI3K p110α activity
may offer a specific way to treat multiple types of human cancers, since mutations in PIK3CA
gene have been frequently observed in a variety of cancers including colon, breast, ovarian, brain
and gastric cancers [27]. As shown in Table 2, the targeted 3-morpholinoalkyl-
pyrimidopyrrolopyrimidines 8a-d and morpholino-pyridopyrrolopyrimidine-2-carbonitriles 12a-
e can be considered potent and selective p110α inhibitors. The selectivity of these compounds for
p110α (class IA) over p110β (class IA) was not very high; p110β is also considered as a potential
target for cancer and the concomitant inhibition of both isoforms could potentiate the anticancer
activity of these compounds [10,16]. Conversely, these compounds displayed excellent
selectivity profile for p110α over p110γ, p110δ and PI3K C2β isoforms.
On the other hand, most of the 3-aryl-1-morpholino-pyrimidopyrrolopyrimidines 6a-d and the
dimorpholino derivatives 10a,b displayed good selectivity for p110α over p110β and p110γ but
were fairly selective toward p110δ and PI3K C2β.
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3.2.3. In vitro cytotoxic activity
The anticancer activity of the targeted compounds 6a-d, 8a-d, 10a,b, and 12a-e were
evaluated in vitro against two cancer cell lines reported to over-express PI3K p110α (cervical
cancer, HeLa) or contain mutated PIK3CA (ovarian cancer, SKOV3) [28,7]. Cytotoxicity was
assessed using the Sulfo-Rhodamine –B-stain (SRB) assay method described by Skehan et al and
the obtained IC50 values are listed in Table 3. The results showed that all the tested compounds
displayed cytotoxic activity against HeLa cancer cell line at low micromolar or sub-micromolar
concentration with IC50 values ranging from 0.21- 1.99 µM. Meanwhile, SKOV-3 cancer cells
were less sensitive to the designed compounds (IC50 = 1.34- 78.45 µM).
4. Conclusion
The present study reports the design and synthesis of novel series of morpholino-
pyrimidopyrrolopyrimidines 6a-d, 8a-d and 10a,b, and morpholino-pyridopyrrolopyrimidine-2-
carbonitriles 12a-e as PI3K inhibitors. Molecular docking studies were performed to support the
implemented design strategies. The designed compounds fit into the active site of PI3K
α with
comparable docking scores and similar binding modes to the co-crystallized ligand. The target
compounds were evaluated for their PI3Kα inhibitory activity as well as their selectivity against
PI3Kβ, PI3Kγ, PI3Kδ and PI3K C2β isoforms. The results revealed that the 3-morpholinoalkyl-
pyrimidopyrrolopyrimidinones 8a-d and the 3-aryl-1-morpholino-pyridopyrrolopyrimidine-2-
carbonitriles 12a-e proved to be potent and selective PI3Kα inhibitors at low nanomolar levels.
These results were in consistency with the analysis of the docking results. Moreover, the new
compounds displayed considerable cytotoxic effect against HeLa cancer cell line at low
micromolar or sub-micromolar level.
5. Experimental
5.1. Chemistry
Melting points were determined on Electrothermal Stuart SMP3 digital melting point
apparatus and are uncorrected. The infrared (IR) spectra were recorded using a Shimadzu FT-IR
1650 (Perkin Elmer) spectrophotometer as potassium bromide discs at Faculty of Pharmacy,
Cairo University. The proton nuclear resonance (1H NMR) spectra were recorded on Bruker
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spectrometer at 400 MHz NMR, at Micro-analytical Unit, Faculty of Pharmacy, Cairo University
or Joel spectrometer at 300 MHz NMR, at Faculty of Science, Cairo University, using
13
tetramethylsilane (TMS) as the internal standard. The C-NMR spectra were run using Bruker
spectrometer at 100 MHz NMR or on Joel spectrometer at 75 MHz NMR. The mass spectra were
performed on Finnigan SSQ 7000 Gas chromatograph-Mass spectrophotometer at the
Microanalytical Centre, Faculty of Science, Cairo University. The reactions were monitored by
TLC (silica gel, aluminum sheets with fluorescence indicator F254, Merck) using
chloroform/methanol (9.5:0.5 v/v) as eluent. The purity of the newly synthesized compounds
was assessed by TLC. Elemental microanalyses were performed at the Microanalytical Center,
Faculty of Science, Cairo University or at The Regional Center for Mycology and
Biotechnology, Al-Azhar University.
Compounds 1 [22,30], 2a [23], 2b [19], 3a [23], 3b [19] and 11c [23] were prepared according to
the reported procedures.
5.1.1. General procedure for the preparation of 5-Ethyl/phenyl-3-(3- or 4-methoxyphenyl)- 6-
thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidin-1(2H)-ones 4a-d.
To a suspension of 3a,b (10 mmol) in dry benzene (20 ml), 3 or 4-anisoyl chloride (11 mmol)
was added dropwise and the reaction mixture was refluxed. The obtained solid was filtered while
hot, washed with benzene then chloroform, dried and crystallized from methanol.
5.1.1.1.
3-(3-Methoxyphenyl)-5-ethyl-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-
e]pyrrolo[1,2-c] pyrimidin-1(2H)-one (4a). Compound 4a was prepared from 3a and 3-anisoyl
chloride by refluxing for 4 h. Yellow crystals, yield 64%; m.p. 294-296 ºC. IR (KBr, cm-1): 3149
1
(NH), 1668 (C=O). H NMR (DMSO-d6-300MHz, δ, ppm): 1.29 (t, 3H, J= 6.9 Hz), 1.87-1.90
(m, 3H), 2.75-2.83 (m, 1H), 3.60-3.67 (m, 1H), 3.80-3.90 (m, 4H), 4.43-4.54 (m, 3H), 7.14 (d,
1H, J= 8.4 Hz), 7.42 (t, 1H, J= 8.1 Hz), 7.70-7.75 (m, 2H), 12.70 (br. s, 1H, D2O exchangeable).
MS m/z (%): 356 (M+, 66), 327 (100). Anal. Calcd. for C18H20N4O2S (356.44): C, 60.65; H,
5.66; N, 15.72. Found: C, 60.68; H, 5.69; N, 15.80.
5.1.1.2.
3-(4-Methoxyphenyl)-5-ethyl-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-
e]pyrrolo[1,2-c] pyrimidin-1(2H)-one (4b). Compound 4b was prepared from 3a and 4-anisoyl
chloride by refluxing for 4 h. Yellow crystals, yield 68%; m.p. 350 ºC (decomp.). IR (KBr, cm-1):
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1
3178 (NH), 1662 (C=O). H NMR (DMSO-d6-300MHz, δ, ppm): 1.28 (t, 3H, J= 6.9 Hz), 1.81-
1.94 (m, 3H), 2.81-2.84 (m, 1H), 3.66-3.7 (m, 1H), 3.79-3.84 (m, 4H), 4.42-4.55 (m, 3H), 7.07
(d, 2H, J= 9.0 Hz), 8.11 (d, 2H, J= 9.0 Hz), 12.5 (br. s, 1H, D2O exchangeable). MS m/z (%):
356 (M+, 10), 135 (100). Anal. Calcd. for C18H20N4O2S (356.44): C, 60.65; H, 5.66; N, 15.72.
Found: C, 60.72; H, 5.69; N, 15.90.
5.1.1.3.
3-(3-Methoxyphenyl)-5-phenyl-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-
e]pyrrolo[1,2-c] pyrimidin-1(2H)-one (4c). Compound 4c was prepared from 3b and 3-anisoyl
chloride by refluxing for 10 h. Pale yellow solid, yield 70%; m.p. 280-282 ºC. IR (KBr, cm-1):
1
3400 (NH), 1664 (C=O). H NMR (DMSO-d6-300MHz, δ, ppm): 1.90-2.00 (m, 3H), 2.85-2.95
(m, 1H), 3.65-3.75 (m, 4H), 3.85-3.95 (m, 1H), 4.71-4.75 (m, 1H), 7.02 (d, 1H, J= 8.4 Hz ),
7.23-7.54 (m, 8H). 13C NMR (DMSO-d6-100 MHz , δ, ppm): 21.89, 34.00, 52.36, 55.63, 56.37,
98.42, 112.47, 119.10, 120.09, 127.89, 128.67, 130.22, 131.30, 141.08, 159.65, 176.23 (C=S).
MS m/z (%): 404 (M+, 21), 135 (100). Anal. Calcd. for C22H20N4O2S (404.48): C, 65.33; H,
4.98; N, 13.85. Found: C, 65.39; H, 5.02; N, 14.01.
5.1.1.4.
3-(4-Methoxyphenyl)-5-phenyl-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-
e]pyrrolo[1,2-c] pyrimidin-1(2H)-one (4d). Compound 4d was prepared from 3b and 4-anisoyl
chloride by refluxing for 10 h. Pale yellow solid, yield 73%; m.p. 242 ºC (decomp.). IR (KBr,
cm-1): 3155 (NH), 1643 (C=O). 1H NMR (DMSO-d6-300MHz, δ, ppm): 1.95-2.10 (m, 3H), 2.85-
2.95 (m, 1H), 3.60-3.75 (m, 1H), 3.76 (s, 3H), 3.80-3.95 (m, 1H), 4.65-4.75 (m, 1H), 6.90 (d,
2H, J= 8.7 Hz), 7.18-7.28 (m, 2H), 7.35-7.45 (m, 3H), 7.69 (d, 2H, J= 9.0 Hz), 12.55 (br. s, 1H,
D2O exchangeable). MS m/z (%): 404 (M+, 100). Anal. Calcd. for C22H20N4O2S (404.48): C,
65.33; H, 4.98; N, 13.85. Found: C, 65.41; H, 5.03; N, 13.98.
5.1.2. General procedure for the preparation of 1-chloro-3-(3- or 4-methoxyphenyl)-5-ethyl or
phenyl-8,9,10,10a-tetrahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-6(5H)-thiones 5a-d.
A suspension of 4a-d (1.5 mmol) in phosphorus oxychloride (5ml) in the presence of few drops
of DMF was gently heated till the dissolution, then refluxed for 8-10 h. Excess phosphorus
oxychloride was removed under reduced pressure; the reaction mixture was cooled to room
temperature and poured dropwise with stirring onto crushed ice, then neutralized with ammonia
solution. The formed solid was filtered, washed several times with water and dried. The product
was employed directly in the next step without purification.
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5.1.3. General procedure for the preparation of 5-ethyl/phenyl-3-(3- or 4-methoxyphenyl)-1-
morpholino-8,9,10,10a-tetrahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-6(5H)-thiones 6a-d.
A suspension of 5a-d (1.5 mmol), anhydrous potassium carbonate (0.28 g, 2 mmol) and
morpholine (0.13 ml, 1.5 mmol) in dry DMF (5ml) was heated at 70ºC for 14 h. The reaction
mixture was allowed to cool to room temperature, poured onto ice/water mixture (30 ml) and
neutralized with acetic acid. The separated solid was filtered, washed with water then ethanol,
dried and crystallized from DMF.
5.1.3.1.
5-Ethyl-3-(3-methoxyphenyl)-1-morpholino-8,9,10,10a-tetrahydropyrimido[5,4-
e]pyrrolo[1,2-c]pyrimidine-6(5H)-thione (6a). Greenish powder, yield 62%; m.p. 178-180 ºC. IR
(KBr, cm-1): 1645 (C=N). 1H NMR (DMSO-d6-400MHz, δ, ppm): 1.17 (t, 3H, J= 6.8 Hz), 1.84-
1.91 (m, 3H), 2.66-2.69 (m, 1H), 3.24-3.29 (m, 4H), 3.64-3.69 (m, 2H), 3.75-3.85 (br. s, 5H),
3.93-4.00 (m, 2H), 4.34-4.38 (m, 1H), 4.45-4.52 (m, 2H), 7.14 (d, 1H, J= 8.0 Hz), 7.43-7.48
(m, 1H), 7.70-7.74 (m, 2H). MS m/z (%): 426 (M++1, 100). Anal. Calcd. for C22H27N5O2S
(425.55): C, 62.09; H, 6.40; N, 16.46. Found: C, 62.14; H, 6.47; N, 16.59.
5.1.3.2.
5-Ethyl-3-(4-methoxyphenyl)-1-morpholino-8,9,10,10a-tetrahydropyrimido[5,4-
e]pyrrolo[1,2-c]pyrimidine-6(5H)-thione (6b). Pale brown solid, yield 59%; m.p. 283-285 ºC. IR
(KBr, cm-1): 1676 (C=N). 1H NMR (DMSO-d6-300MHz, δ, ppm): 1.16 (t, 3H, J= 6.3Hz), 1.82-
1.88 (m, 3H), 2.60-2.68 (m, 1H), 3.26-3.29 (m, 4H (overlapped)), 3.63-3.67 (m, 2H), 3.83 (s,
3H), 3.95-4.01 (m, 4H), 4.30-4.35 (m, 2H), 4.40-4.45 (m, 1H), 7.06 (d, 2H, J= 8.7 Hz), 8.11 (d,
2H, J= 8.7 Hz). MS m/z (%): 426 (M++1, 0.4), 127 (100). Anal. Calcd. for C22H27N5O2S
(425.55): C, 62.09; H, 6.40; N, 16.46. Found: C, 62.14; H, 6.43; N, 16.58.
5.1.3.3.
3-(3-Methoxyphenyl)-1-morpholino-5-phenyl-8,9,10,10a-tetrahydropyrimido[5,4-
e]pyrrolo[1,2-c]pyrimidine-6(5H)-thione (6c). Pale brown solid, yield 64%; m.p. 312-315 ºC. IR
(KBr, cm-1): 1637 (C=N). 1H NMR (DMSO-d6-300MHz, δ, ppm): 1.64-1.90 (m, 3H), 2.71-2.80
(m, 1H), 3.26-3.32 (m, 2H), 3.61-3.68 (m, 2H, (overlapped)), 3.70-3.95 (m, 9H), 4.47-4.52 (m,
1H), 7.1 (d, 1H, J= 8.1 Hz ), 7.21-7.44 (m, 8H). Anal. Calcd. for C26H27N5O2S (473.59): C,
65.94; H, 5.75; N, 14.79. Found: C, 66.20; H, 5.55; N, 14.61.
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5.1.3.4.
3-(4-Methoxyphenyl)-1-morpholino-5-phenyl-8,9,10,10a-tetrahydropyrimido[5,4-
e]pyrrolo[1,2-c]pyrimidine-6(5H)-thione (6d). Pale brown solid, yield 66%; m.p. 287-289 ºC. IR
1
(KBr, cm-1): 1647 (C=N). H NMR (DMSO-d6-300MHz, δ, ppm): 1.6-1.89 (m, 3H), 2.71-2.74
(m, 1H), 3.25-3.35 (m, 2H), 3.57-3.78 (m, 7H), 3.78-4.05 (m, 4H), 4.45-4.47 (m, 1H), 6.85 (d,
2H, J= 8.7 Hz), 7.17 (d, 2H, J= 8.7 Hz), 7.33-7.54 (m, 3H), 7.62 (d, 2H, J= 9.0 Hz). MS m/z
(%): 474 (M++1, 38), 57 (100). Anal. Calcd. for C26H27N5O2S (473.59): C, 65.94; H, 5.75; N,
14.79. Found: C, 65.93; H, 5.78; N, 14.85.
5.1.4.General procedure for the preparation of 3-(1’-chloroethyl)/(chloromethyl)-5-ethyl/ 5-
phenyl-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidin-1(2H)-ones
7a-d.
To an ice cooled suspension of 3a,b (1mmol) in dry benzene (15ml), chloroacetyl chloride or
chloropropionyl chloride (1.1 mmol) was added drop wise and the reaction mixture was stirred
over night at room temperature. The solvent was removed by distillation under vacuum, the
residue was azeotroped twice with benzene (2 x 5 ml) and the solvent was removed by
distillation. The obtained residue was refluxed with saturated solution of ethanolic hydrochloric
acid (10 ml) for 3-6 h, the reaction mixture was left to cool, and the separated crystals were
filtered, washed with aqueous ethanol, dried and recrystallized from absolute ethanol.
5.1.4.1.
3-(Chloromethyl)-5-ethyl-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-
e]pyrrolo[1,2-c] pyrimidin-1(2H)-one (7a). Shiny white crystals, yield 63%; m.p. 243-245 ºC. IR
(KBr, cm-1): 3400 (NH), 1643 (C=O). 1H NMR (DMSO-d6-300MHz, δ, ppm): 1.19 (t, 3H, J= 6.9
Hz), 1.82-1.89 (m, 3H), 2.77-2.80 (m, 1H), 3.65-3.67 (m, 1H), 3.79-3.83 (m, 1H), 4.34-4.44 (m,
3H), 4.47 (s, 2H), 12.8 (br. s, 1H, D2O exchangeable). Anal. Calcd. for C12H15ClN4OS (298.79):
C, 48.24; H, 5.06; N, 18.75. Found: C, 48.39; H, 4.87; N, 18.51.
5.1.4.2. 3-(1’-Chloroethyl)-5-ethyl-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-
e]pyrrolo[1,2-c]pyrimidin-1(2H)-one (7b). The compound was obtained in 49 % yield as a mixture of
diastereoisomers. The diastereomeric ratio, as determined by 1H NMR analysis, was found to be 50/50.
White crystals, m.p. 343-345 ºC. IR (KBr, cm-1): 3433 (NH), 1647 (C=O). 1H NMR (CDCl3-
300MHz, δ, ppm): 1.34 (t, 3H, J= 6.9 Hz), 1.89 (d, 3H, J= 6.9 Hz), 1.97-2.05 (m, 3H), 2.98-3.00
(m, 1H), 3.87-3.90 (m, 1H), 3.98-4.05 (m, 1H), 4.43-4.58 (m, 3H), 4.86 (q, 1H, J= 6.9 Hz). MS
15

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m/z (%): 312 (M+, 54), 314 (M++2, 21), 283 (100). Anal. Calcd. for C13H17ClN4OS (312.82): C,
49.91; H, 5.48; N, 17.91. Found: C, 49.74; H, 5.26; N, 17.79.
5.1.4.3.
3-(Chloromethyl)-5-phenyl-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-
e]pyrrolo[1,2-c]pyrimidin-1(2H)-one (7c). Pale yellow crystals, yield 45%; m.p. 322 ºC
1
(decomp.). IR (KBr, cm-1): 3427 (NH), 1651 (C=O). H NMR (DMSO-d6-300MHz, δ, ppm):
1.90-1.96 (m, 3H), 2.87-2.95 (m, 1H), 3.67-3.75 (m, 1H), 3.82-3.95 (m, 1H), 4.18 (s, 2H), 4.60-
4.66 (m, 1H), 7.15 (d, 2H, J= 9.0 Hz), 7.32-7.42 (m, 3H), 12.80 (br. s, 1H, D2O exchangeable).
MS m/z (%): 346 (M+, 100), 348 (M++2, 39). Anal. Calcd. for C16H15ClN4OS (346.83): C, 55.09;
H, 4.36; N, 16.15. Found: C, 55.09; H, 4.16; N, 15.90.
5.1.4.4. 3-(1’-Chloroethyl)-5-phenyl-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-
e]pyrrolo[1,2-c] pyrimidin-1(2H)-one (7d). The compound was obtained in 51 % yield as a mixture
of diastereoisomers. The diastereomeric ratio, as determined by 1H NMR analysis, was found to be 50/50.
Shiny white crystals; m.p. 290-292 ºC. IR (KBr, cm-1): 3433 (NH), 1653 (C=O). 1H NMR
(DMSO-d6-300MHz, δ, ppm): 1.27 and 1.33 (2d, 3H, J= 6.6 Hz, J= 6.6 Hz ), 1.90-2.00 (m, 3H),
2.80-2.95 (m, 1H), 3.65-3.75 (m, 1H, H-8), 3.81-3.97 (m, 1H), 4.58-4.60 (m, 1H), 4.69-4.74 (m,
1H), 7.11-7.13 (m, 2H), 7.32-7.40 (m, 3H), 12.73 (br. s, 1H, D2O exchangeable). MS m/z (%):
360 (M+,100), 362 (M++2, 39). Anal. Calcd. for C16H15ClN4OS (360.86): C, 56.58; H, 4.75; N,
15.53. Found: C, 56.37; H, 5.00; N, 15.23.
5.1.5. General procedure for the preparation of 5-ethyl/phenyl-3-(morpholinomethyl)/(1’-
morpholinoethyl)-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidin-
1(2H)-ones 8a-d.
A mixture of 7a-d (10 mmol) and morpholine (1.7 ml, 20 mmol) in absolute ethanol (10 ml) was
refluxed for 6 h. The solvent was removed by distillation under vacuum; the residue was
dissolved in dilute HCl and filtered. The filtrate was rendered alkaline with ammonia solution
and extracted with chloroform (3 x 10 ml). The combined chloroformic extract was washed with
water, dried over anhydrous sodium sulphate and evaporated to dryness. The residue was
crystalized from ethanol.
4.1.5.1.
5-Ethyl-3-(morpholinomethyl)-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-
e]pyrrolo[1,2-c] pyrimidin-1(2H)-one (8a). White crystals, yield 98%; m.p. 220-222 ºC. IR
16

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(KBr, cm-1): 3433 (NH), 1649 (C=O). 1H NMR (CDCl3-300MHz, δ, ppm): 1.29 (t, 3H, J= 6.9
Hz), 1.85-2.04 (m, 3H), 2.59 (t, 4H, J= 4.5 Hz), 2.93-3.00 (m, 1H), 3.49 (s, 2H), 3.75-3.87 (m,
5H), 3.93-4.01 (m, 1H), 4.37-4.60 (m, 3H). 13C NMR (CDCl3-100 MHz , δ, ppm): 14.05, 21.77,
33.04, 42.71, 52.46, 53.61, 55.62, 60.52, 66.63, 98.50, 152.90, 157.41, 160.01 (C=O), 176.16
(C=S). MS m/z (%): 349 (M+, 62), 64 (100). Anal. Calcd. for C16H23N5O2S (349.45): C, 54.99;
H, 6.63; N, 20.04. Found: C, 55.01; H, 6.62; N, 20.21.
5.1.5.2. 5-Ethyl-3-(1’-morpholinoethyl)-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-
e]pyrrolo[1,2-c] pyrimidin-1(2H)-one (8b). The compound was obtained in 90 % yield as a mixture
of diastereoisomers. The diastereomeric ratio, as determined by 1H NMR analysis, was found to be 50/50.
White crystals; m.p. 189-191 ºC. IR (KBr, cm-1): 3433 (NH), 1647 (C=O). 1H NMR (CDCl3-
300MHz, δ, ppm): 1.29 (t, 3H, J= 6.9 Hz), 1.39 (d, 3H, J= 6.9 Hz), 1.86-2.00 (m, 3H), 2.56-2.60
(m, 4H), 2.95-3.05 (m, 1H), 3.50-3.60 (m, 1H), 3.78-3.84 (m, 5H), 3.95-4.05 (m, 1H), 4.42-4.58
(m, 3H). 13C NMR (CDCl3-100 MHz , δ, ppm): 12.31, 14.01, 21.79, 32.95, 42.85, 52.46, 55.64,
62.56, 66.73, 98.36, 152.82, 152.88, 160.10 (C=O), 176.18 (C=S). MS m/z (%): 363 (M+, 6), 249
(100). Anal. Calcd. for C17H25N5O2S (349.45): C, 56.17; H, 6.93; N, 19.27. Found: C, 56.23; H,
6.98; N, 19.35.
5.1.5.3
5-Phenyl-3-(morpholinomethyl)-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-
e]pyrrolo[1,2-c]pyrimidin-1(2H)-one (8c). Buff crystals, yield 82%; m.p. 248-250 ºC. IR (KBr,
cm-1): 3400 (NH), 1651 (C=O). 1H NMR (CDCl3-300MHz, δ, ppm): 1.97-2.12 (m, 3H), 2.48 (t,
4H, J= 5.7 Hz), 3.07-3.10 (m, 1H), 3.26 (s, 2H), 3.72 (t, 4H, J= 6.0 Hz), 3.83-3.91(m, 1H), 4.01-
4.07 (m, 1H), 4.62-4.70 (m, 1H), 7.19-7.21 (m, 2H), 7.39-7.46 (m, 3H), 10.15 (br. s, 1H, D2O
13
exchangeable). C NMR (CDCl3-100 MHz , δ, ppm): 22.12, 33.23, 52.53, 53.34, 56.48, 65.62,
99.18, 128.31, 128.90, 130.80, 140.53, 153.23, 160.36 (C=O), 176.83 (C=S). MS m/z (%): 397
(M+, 52), 235 (100). Anal. Calcd. for C20H23N5O2S (397.49): C, 60.43; H, 5.83; N, 17.6. Found:
C, 60.24; H, 6.04; N, 17.91.
5.1.5.4. 5-Phenyl-3-(1’-morpholinoethyl)-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-
e]pyrrolo[1,2-c] pyrimidin-1(2H)-one (8d). The compound was obtained in 86 % yield as a mixture
of diastereoisomers. The diastereomeric ratio, as determined by 1H NMR analysis, was found to be 50/50.
17

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White crystals; m.p. 242-243 ºC. IR (KBr, cm-1): 3400 (NH), 1649 (C=O). 1H NMR (CDCl3-
300MHz, δ, ppm): 1.00 and 1.06 (2d, 3H, J= 6.9 Hz, J= 6.6 Hz), 2.01-2.10 (m, 3H), 2.43-2.50
(m, 4H), 3.07-3.15 (m, 1H), 3.30-3.45 (m, 1H), 3.71-3.74 (m, 4H), 3.80-3.91 (m, 1H), 4.00-4.15
(m, 1H), 4.59-4.63 (m, 1H), 7.18-7.22 (m, 2H), 7.37-7.44 (m, 3H). 13C NMR (CDCl3-100 MHz ,
δ, ppm): 11.63, 21.97, 33.04, 49.70, 52.30, 56.31, 62.01, 66.69, 127.78, 128.27, 130.40, 140.20,
160.00 (C=O), 170.00 (C=S). MS m/z (%): 411 (M+, 4), 326 (100). Anal. Calcd. for
C21H25N5O2S (411.52): C, 60.29; H, 6.12; N, 17.02. Found: C, 60.98; H, 6.36; N, 16.84.
5.1.6. General procedure for the preparation of 1-chloro-3-(1’-chloroethyl)/(chloromethyl)-5-
phenyl-8,9,10,10a-tetrahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-6(5H)-thiones 9a,b.
A suspension of 7c,d (1.5 mmol) and phosphorous oxychloride (3 ml) in dry chloroform (10 ml)
was refluxed for 6-10 h, then cooled to room temperature and poured portion wise on crushed ice
with stirring. The reaction mixture was rendered alkaline with ammonia solution and extracted
with chloroform (3 x 10 ml). The combined chloroformic extract was washed with water, dried
over anhydrous sodium sulphate and evaporated to dryness under vacuum. The residue was
employed directly in the next reaction without purification.
5.1.7. General procedure for the preparation of 1-morpholino-3-(morpholinomethyl)/(1’-
morpholinoethyl)-5-phenyl-8,9,10,10a-hexahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-
6(5H)-thiones 10a,b.
The obtained residue 9a,b (1.5 mmol) was dissolved in absolute ethanol (10ml) and refluxed
with morpholine (20 mmol, 1.7 ml) for 10-14 h. The reaction mixture was left to cool, poured on
crushed ice and the pH was adjusted to 7 with acetic acid. The separated solid was filtered,
washed several times with water to get rid of excess morpholine. The product was purified by
preparative TLC using chloroform/methanol mixture (9:1) as the mobile phase.
5.1.7.1.
1-Morpholino-3-(morpholinomethyl)-5-phenyl-8,9,10,10a-hexahydropyrimido[5,4-
e]pyrrolo[1,2 -c]pyrimidine-6(5H)-thione (10a). Brown solid, yield 38%; m.p. 186-188 ºC. IR
(KBr, cm-1): 1659 (C=N). 1H NMR (CDCl3-300MHz, δ, ppm): 1.90-2.10 (m, 3H), 2.95-3.00 (m,
1H), 3.08-3.14 (m, 4H), 3.40-3.50 (m, 1H), 3.60-3.64 (m, 4H), 3.80-3.90 (m, 1H), 3.96-4.11 (m,
8H), 4.31 (s, 2H), 4.70-4.75 (m, 1H), 7.16 (d, 2H, J=8.1 Hz), 7.37-7.44 (m, 3H). MS m/z (%):
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466 (M+, 46), 77 (100). Anal. Calcd. for C24H30N6O2S (466.60): C, 61.78; H, 6.48; N, 18.01.
Found: C, 61.71; H, 6.64; N, 18.09.
5.1.7.2.
1-Morpholino-3-(1’-morpholinoethyl)-5-phenyl-8,9,10,10a-hexahydropyrimido[5,4-
e]pyrrolo[1,2 -c]pyrimidine-6(5H)-thione (10b). The compound was obtained in 40 % yield as a
mixture of diastereoisomers, after chormatography on silica gel. The diastereomeric ratio, as determined
1
by H NMR analysis, was found to be 60/40. Pale green solid; m.p. 273-275 ºC. IR (KBr, cm-1):
1664 (C=N). 1H NMR (CDCl3-400MHz, δ, ppm): 1.16 and 1.22 (2d, 3H, J= 6.9 Hz, J= 6.6 Hz),
2.01-2.14 (m, 3H), 2.35-2.43 (m, 4H), 2.80-2.90 (m, 1H), 3.14-3.18 (m, 2H), 3.38-3.42 (m, 2H),
3.53-3.62 (m, 4H), 3.74-3.80 (m, 3H, H-8), 3.88-3.96 (m, 3H, H-8), 4.10-4.20 (m, 1H), 4.70-4.73
13
(m, 1H), 7.22-7.28 (m, 2H), 7.37-7.47 (m, 3H). C NMR (CDCl3-100 MHz, δ, ppm): 12.21,
21.89, 32.96, 45.35, 48.86, 49.67, 49.76, 54.89, 62.05, 62.16, 66.95, 67.29, 97.69, 127.48,
128.31, 129.68, 136.75, 151.01, 155.09, 160.92 (C=S). MS m/z (%): 481 (M++1, 18), 77 (100).
Anal. Calcd. for C25H32N6O2S (480.63): C, 62.47; H, 6.71; N, 17.49. Found: C, 62.12; H, 7.08;
N, 17.39.
5.1.8. General procedure for the preparation of 1-amino-3-aryl-5-ethyl-6-thioxo-5,6,8,9,10,10a-
hexahydropyrido[3,2-e]pyrrolo[1,2-c]pyrimidine-2-carbonitriles 11a-e. A mixture of the
enaminonitrile 3a (1.11g, 5 mmol), malononitrile (0.33g, 5 mmol) and the appropriate aldehyde
(5 mmol) in absolute ethanol was refluxed for 5 h. The separated crystals were filtered, washed
with ethanol and recrystallized from DMF.
5.1.8.1.
1-Amino-3-(3-hydroxyphenyl)-5-ethyl-6-thioxo-5,6,8,9,10,10a-hexahydropyrido[3,2-
e]pyrrolo[1,2-c]pyrimidine-2-carbonitrile (11a). Yellow solid, yield 39%; m.p. 350 ºC
1
(decomp.). IR (KBr, cm-1): 3440-3217 (NH2), 3329 (OH), 2206 (C≡N), 1693 (C=N). H NMR
(DMSO-d6-300MHz, δ, ppm): 1.26 (t, 3H, J= 6.9Hz), 3.49-3.54 (m, 4H), 4.39-4.44 (m, 3H), 4.56
(q, 2H, J= 6.9Hz), 6.86 (d, 1H, J= 7.8Hz), 7.27 (t, 1H, J= 7.8Hz), 7.37-7.41 (m, 3H), 7.49 (s,
1H), 9.63 (br. s, 1H, D2O exchangeable). Anal. Calcd. for C19H19N5OS (365.45): C, 62.44; H,
5.24; N, 19.16. Found: C, 62.47; H, 5.23; N, 19.32.
5.1.8.2.
1-Amino-3-(3-methoxyphenyl)-5-ethyl-6-thioxo-5,6,8,9,10,10a-hexahydropyrido[3,2-
e]pyrrolo [1,2-c]pyrimidine-2-carbonitrile (11b). Pale yellow solid, yield 37%; m.p. 322-324 ºC.
IR (KBr, cm-1): 3429 and 3340 (NH2), 2198 (C≡N), 1689 (C=N). 1H NMR (DMSO-d6-300MHz,
19

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δ, ppm): 1.24 (t, 3H, J= 6.6Hz), 3.45-3.49 (m, 4H), 3.78 (s, 3H), 4.33-4.38 (m, 3H), 4.50-4.60
(m, 2H), 6.98 (d, 1H, J= 7.8Hz), 7.37 (t, 1H, J= 7.8Hz), 7.42-7.51 (m, 4H). MS m/z (%): 379
(M+ 4), 179 (100). Anal. Calcd. for C20H21N5OS (379.48): C, 63.30; H, 5.58; N, 18.46. Found:
,
C, 63.10; H, 5.37; N, 18.48.
5.1.8.3.
1-Amino-3-(4-methoxyphenyl)-5-ethyl-6-thioxo-5,6,8,9,10,10a-hexahydropyrido[3,2-
e]pyrrolo [1,2-c]pyrimidine-2-carbonitrile (11c) [23]. Yellow solid, yield 35%; m.p. 344-346 ºC
1
(as reported). IR (KBr, cm-1): 3435 and 3209 (NH2), 2204 (C≡N), 1691 (C=N). H NMR
(DMSO-d6-300MHz): δ 1.25 (t, 3H, J= 6.9Hz), 3.47-3.52 (m, 4H), 3.82 (s, 3H), 4.37-4.42 (m,
3H), 4.50 (q, 2H, J= 6.6Hz ), 7.00 (d, 2H, J=8.7Hz), 7.34 (s.br., 2H), 7.93 (d, 2H, J= 8.7Hz).
Anal. Calcd. for C20H21N5OS (379.48): C, 63.30; H, 5.58; N, 18.46. Found: C, 63.03; H, 5.67; N,
18.00.
5.1.8.4.
1-Amino-3-(3-hydroxy-4-methoxyphenyl)-5-ethyl-6-thioxo-5,6,8,9,10,10a-
hexahydropyrido[3,2-e]pyrrolo[1,2-c]pyrimidine-2-carbonitrile (11d). Pale yellow solid, yield
41%; m.p. 249-251 ºC. IR (KBr, cm-1): 3392-3217 (NH2), 3309 (OH), 2208 (C≡N), 1691 (C=N).
1H NMR (DMSO-d6-300MHz, δ, ppm): 1.27(t, 3H, J= 6.6Hz), 3.51-3.6 (m, 4H), 3.84 (s, 3H),
4.41-4.46 (m, 3H), 4.60 (q, 2H, J= 6.6Hz ), 7.03 (d, 1H, J= 8.7Hz), 7.35 (br. s, 2H, D2O
exchangeable), 7.44 (d, 1H, J= 8.4Hz), 7.62 (s, 1H), 9.28 (br. s, 1H, D2O exchangeable). Anal.
Calcd. for C20H21N5O2S (395.48): C, 60.74; H, 5.35; N, 17.71. Found: C, 60.61; H, 5.01; N,
17.80.
5.1.8.5. 1-Amino-3-(3,4-dimethoxyphenyl)-5-ethyl-6-thioxo-5,6,8,9,10,10a-hexahydropyrido[3,2-
e] pyrrolo[1,2-c]pyrimidine-2-carbonitrile (11e). Yellow solid, yield 35%; m.p. 297-299 ºC. IR
(KBr, cm-1): 3498 and 3211 (NH2), 2198 (C≡N), 1693 (C=N). 1H NMR (DMSO-d6-300MHz, δ,
ppm): 1.24 (t, 3H, J= 6.6Hz), 3.47-3.52 (m, 4H), 3.79 (s, 3H), 3.83 (s, 3H), 4.36-4.41 (m, 3H),
4.52 (q, 2H, J= 6.9Hz), 7.00 (d, 1H, J= 8.4Hz), 7.34 (br. s, 2H, D2O exchangeable), 7.52-7.57
(m, 2H). Anal. Calcd. for C21H23N5O2S (409.50): C, 61.59; H, 5.66; N, 17.10. Found: C, 61.68;
H, 5.71; N, 17.23.
5.1.9. General procedure for the preparation of 3-aryl-5-ethyl-1-morpholino-6-thioxo-
5,6,8,9,10,10a-hexahydropyrido[3,2-e]pyrrolo[1,2-c]pyrimidine-2-carbonitriles 12a-e.
20

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Method A: A mixture of 11a or 11c (0.4 g, 1 mmol), chloroethanol (0.18 ml, 2.5 mmol) and
anhydrous potassium carbonate (0.7 g, 5 mmol) in a mixture of dry DMF/dry dioxane (1:1) was
refluxed for 24 h-48 h (monitored by TLC), then filtered while hot. The filtrate was allowed to
cool and poured on water. The resulted solid was filtered, washed with water, dried and
crystallized from DMF.
Method B: A mixture of 11b, 11d or 11e (1 mmol), bis(2-bromoethyl)ether (0.13 ml, 1 mmol)
and anhydrous potassium carbonate (0.7 gm, 5 mmol) in a mixture of dry DMF/dioxane (1:1)
was heated at 70 ºC for 80-120 h (monitored by TLC). For compounds 12b and 12e, the resulted
solid was filtered while hot, washed with water, dried and crystallized from DMF. In case of
compound 12d, the reaction mixture was filtered while hot; the filtrate was allowed to cool,
poured on water and neutralized with acetic acid. The separated solid was filtered, washed with
water, dried and crystallized from DMF.
5.1.9.1.
3-(3-Hydroxyphenyl)-5-ethyl-1-morpholino-6-thioxo-5,6,8,9,10,10a-
hexahydropyrido[3,2-e] pyrrolo[1,2-c]pyrimidine-2-carbonitrile (12a). Yellow solid, yield 56%;
1
m.p. >350 ºC. IR (KBr, cm-1): 3304 (OH), 2200 (C≡N), 1691 (C=N). H NMR (DMSO-d6-
300MHz, δ, ppm): 1.22 (t, 3H, J= 6.6Hz), 3.40-3.55 (m, 4H), 3.65-3.80 (m, 8H), 4.35-4.45 (m,
3H), 4.54-4.60 (m, 2H), 6.86 (d, 1H, J= 7.5Hz), 7.29 (t, 1H, J= 7.8Hz), 7.36 (d, 1H, J= 7.5Hz),
7.43 (s, 1H). MS m/z (%): 435 (M+, 20), 69 (100). Anal. Calcd. for C23H25N5O2S (435.54): C,
63.43; H, 5.79; N, 16.08. Found: C, 63.45; H, 5.83; N, 16.31.
5.1.9.2.
3-(3-Methoxyphenyl)-5-ethyl-1-morpholino-6-thioxo-5,6,8,9,10,10a-
hexahydropyrido[3,2-e] pyrrolo[1,2-c]pyrimidine-2-carbonitrile (12b). Yellow solid, yield 66%;
1
m.p. 358 ºC (decomp.). IR (KBr, cm-1): 2204 (C≡N), 1697 (C=N). H NMR (DMSO-d6-
300MHz, δ, ppm): 1.15-1.25 (m, 3H), 3.10-3.40 (m, 2H), 3.50-3.65 (m, 6H), 3.70-3.90 (m, 7H),
4.30-4.45 (m, 3H), 4.50-4.65 (m, 2H), 7.04 (br. s, 1H), 7.39 (br. s, 1H), 7.52 (br. s, 2H). MS m/z
(%): 449 (M+, 21), 69 (100). Anal. Calcd. for C24H27N5O2S (449.57): C, 64.12; H, 6.05; N,
15.58. Found: C, 64.16; H, 6.06; N, 15.63.
5.1.9.3.
3-(4-Methoxyphenyl)-5-ethyl-1-morpholino-6-thioxo-5,6,8,9,10,10a-
hexahydropyrido[3,2-e] pyrrolo[1,2-c]pyrimidine-2-carbonitrile (12c). Yellow solid, yield 65%;
m.p. 315-317 ºC. IR (KBr, cm-1): 2202 (C≡N), 1680 (C=N). 1H NMR (DMSO-d6-300MHz, δ,
ppm): 1.20-1.25 (m, 3H), 3.11-3.25 (m, 4H), 3.50-3.60 (m, 4H), 3.83 (s, 3H), 4.15-4.25 (m, 4H),
21

ACCEPTED MANUSCRIPT
4.40-4.47 (m, 2H), 4.50-4.65 (m, 3H), 7.05 (d, 2H, J= 8.1Hz), 7.49 (d, 2H, J= 8.1Hz). MS m/z
(%): 449 (M+, 0.16), 310 (100). Anal. Calcd. for C24H27N5O2S (449.57): C, 64.12; H, 6.05; N,
15.58. Found: C, 64.23; H, 6.08; N, 15.71.
5.1.9.4.
3-(3-Hydroxy-4-methoxyphenyl)-5-ethyl-1-morpholino-6-thioxo-5,6,8,9,10,10a-
hexahydropyrido [3,2-e]pyrrolo[1,2-c]pyrimidine-2-carbonitrile (12d). Yellow solid, yield 58%;
m.p. 264-266 ºC. IR (KBr, cm-1): 3419 (OH), 2208 (C≡N), 1689 (C=N). 1H NMR (DMSO-d6-
300MHz, δ, ppm): 1.21 (t, 3H, J= 6.8Hz), 3.08 (br. s, 2H), 3.18 (br. s, 2H), 3.37-3.45 (m, 2H
(overlapped)), 3.53 (br. s, 1H), 3.62-3.65 (m, 1H), 3.82-3.86 (m, 7H), 4.08 (br. s, 2H), 4.26-4.31
(m, 1H), 4.44-4.52 (m, 1H), 6.99 (d, 1H, J= 8.6Hz), 7.45 (s, 1H), 7.54 (d, 1H, J= 8.2Hz), 8.62
(br. s, 1H). Anal. Calcd. for C24H27N5O3S (465.57): C, 61.92; H, 5.85; N, 15.04. Found: C,
62.08; H, 5.91; N, 15.21.
5.1.9.5.
3-(3,4-Dimethoxyphenyl)-5-ethyl-1-morpholino-6-thioxo-5,6,8,9,10,10a-
hexahydropyrido[3,2-e] pyrrolo[1,2-c]pyrimidine-2-carbonitrile (12e). Yellow solid, yield 42%;
1
m.p. 346-348 ºC. IR (KBr, cm-1): 2222 (C≡N), 1691 (C=N). H NMR (DMSO-d6-300MHz, δ,
ppm): 1.27 (br. s, 3H), 3.13 (br. s, 2H), 3.21 (br. s, 2H), 3.57-3.65 (m, 4H), 3.70-3.85 (m, 7H),
4.35 (br. s, 1H), 4.40-4.50 (br. s, 2H), 4.60-4.70 (m, 2H), 7.05-7.15 (m, 1H), 7.60-7.70 (m, 2H).
MS m/z (%): 479 (M+, 1), 81 (100). Anal. Calcd. for C25H29N5O3S (479.59): C, 62.61; H, 6.09;
N, 14.60. Found: C, 62.79; H, 6.13; N, 14.84.
5.2. Scintillation proximity assay (SPA) for p110α, p110β, p110γ, p110δ and PI3K C2β
GST-tagged bovine p110α, GST-tagged human p110β, His-tagged p110γ, GST-tagged
p110δ and Glu-tagged PI3K C2β were expressed in a Sf9/Baculovirus system and purified as
fusion proteins. The test compounds were dissolved in DMSO, further diluted to obtain an
optimal range of concentrations for treatments to a final volume of 0.5 µL (10-fold serial
dilution were made to provide a total of nine drug concentrations plus control, concentration
range 10-6 102 µM). Each enzyme was mixed in 25 µL of buffer solution (p110α, β, γ assay: 20
̶
mM Tris–HCl (pH 7.4), 160 mM NaCl, 2 mM dithiothreitol, 30 mM MgCl2, 0.4 mM EDTA, and
0.4 mM EGTA; PI3K C2β assay: 20 mM Tris–HCl (pH 7.4), 160 mM NaCl, 2 mM
dithiothreitol, 5 mM MgCl2, 15 mM CaCl2, and 0.4 mM EDTA). Then, 25 µL of 5 mM Tris–HCl
supplemented with 1 µg PI (Sigma), 0.125 µCi [γ -33P]ATP (Amersham Pharmacia), and 2 ꢀM
22

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non-radiolabeled ATP (Sigma) were added to the mixture to initiate the reaction. After allowing
the reaction to proceed at room temperature for 120 min, 0.2 mg of wheat germ agglutinin-
coated SPA beads (Amersham) in 150 µL PBS was added, and the mixture was left to stand for 5
min and then centrifuged at 300g for 2 min. The radioactivity was measured using TopCount
(Packard). IC50 values are given as means of at least two separate determinations with typical
variations of less than ±20%. The reported IC50 values of LY294002 I and PI-103 VII are cited
for comparison [16,25].
5.3. In vitro cytotoxic activity
Cytotoxic activity was measured in vitro using the Sulfo-Rhodamine –B-stain (SRB) assay
method described by Skehan et al [32]. Human cancer cell lines were supplemented with 10%
heat inactivated FBS, 50 units/mL of penicillin and 50 g/mL of streptomycin and maintained at
37°C in a humidified atmosphere containing 5% CO2. The cells were maintained as “monolayer
culture” by serial subculturing. Exponentially growing cells were collected using 0.25% Trypsin-
EDTA and seeded in 96-well plates at 1000-2000 cells/well in DMEM supplemented medium.
After 24 h, cells seeded in 96-well plates were treated with various concentrations of the tested
compounds for 72 h. The medium was removed, and the cells adhered to the plate were fixed
with 10% trichloroacetic acid and stained for 10 min at room temperature with 0.4% SRB
dissolved in 1% acetic acid. The plates were air dried for 24 h. After three washings using 1%
acetum, sulfo-rhodamine-B was dissolved in 100 µl of buffer containing 10 mM Tris-base, and
the OD value was measured at 520 nm with a multiwell spectrophotometer (VersaMax). The
inhibitory rate on cell proliferation was calculated by using the formula (OD520 nm control cells -
OD520 nm treated cells)/OD520 nm control cells x 100%). The average IC50 values were calculated
from at least three independent tests.
5.4. Molecular docking
All molecular modeling calculations and docking studies were carried out using Molecular
Operating Environment (MOE) software version 2009.10 [21]. The target compounds were drawn
on MOE. The structures were subjected to energy minimization using Hamiltonian-Force Field-
MMFF94x and the force field partial charges were calculated for each molecule. Stochastic
23

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conformational analysis was run for each molecule using default settings and the four most stable
conformers for each compound were retained.
The X-ray crystal structure of PI3Kα in complex with PI-103 was downloaded from
http://www.rscb.org/pdb (PDB ID: 4L23) [20]. The protein-ligand complex obtained from the
protein data bank was prepared for docking as follows: 1-The enzyme was 3D protonated, where
hydrogen atoms were added at their standard geometry, the partial charges were computed and the
system was optimized. 2-Deletion of chain B of the protein together with co-crystallized water
molecules was performed. 3-The binding pocket has been defined, isolated and then the back bone
was hidden.
Flexible ligand-rigid receptor docking of the most stable conformers was done with MOE-
DOCK using Alpha triangle placement method and London dG as a scoring function. The obtained
poses were subjected to force field refinement using the same scoring function. Ten of the most
stable docking models for each ligand were retained with the best scored conformation. In order to
validate the docking procedure, PI-103 was docked into the active site of 4L23. The docking results
showed a near perfect alignment with the original ligand as obtained from the X-ray crystallography
pdb file. The re-docked ligand showed rmsd of 0.6058 with docking score of -13.5347 Kcal/mol and
displayed the same binding interactions.
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26

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Figure captions
Figure 1: Structure of reported morpholine-based PI3K inhibitors I-VII.
Figure 2: Simple diagram describing the pharmacophore for PI3K p110α inhibitors.
Figure 3: Schematic diagram describing the design of the targeted compounds 6a-d, 8a-d, 10a,b
and 12a-e.
Figure 4: Superimposition of the cocrystallized ligand PI-103 (blue) and compound 6a [S
enantiomer (A) and R enantiomer (B)] (yellow) docked in the active site of p110α
enzyme. Hydrogen atoms and some residues were omitted for clarity.
.
Figure 5: The best scored docking poses of the 3-morpholinoalkyl-pyrimidopyrrolopyrimidinone
derivative 8d [R,R diastereomer] (A) and the 3-aryl-1-morpholino-
pyridopyrrolopyrimidine-2-carbonitrile derivatives 12c [R enantiomer] (B) within
p110α active site. Hydrogen atoms and some residues were omitted for clarity.
Scheme 1. Reagents and solvents: i: RNCS, (C2H5)3N, CH2Cl2; ii: NaBH4, EtOH; iii: 3- or 4-
OCH3-PhCOCl, benzene; iv: POCl3/drops of DMF; v: morpholine, DMF, K2CO3.
Scheme 2. Reagents and solvents: i. ClCH2COCl or ClCH(CH3)COCl, benzene; ii. Ethanolic
HCl; iii. Morpholine, EtOH; iv. POCl3/CHCl3.
Scheme 3. Reagents and solvents: i. (di)substituted benzaldehyde, CH2(CN)2, EtOH; ii.
ClCH2CH2OH, K2CO3, dry DMF/ dry dioxane, (11a or c); iii. BrCH2CH2OCH2CH2Br,
K2CO3, dry DMF/ dry dioxane, (11b,d or e)
27

ACCEPTED MANUSCRIPT
Table 1
Docking scores (Kcal/mol) and bond interactions of the targeted morpholinyl-
pyrimidopyrrolopyrimidine derivatives 6a-d, 8a-d, 10a,b, the targeted morpholinyl-
pyridopyrrolopyrimidine-2-carbonitrile derivatives 12a-e and PI-103 with amino acids of PI3Kα
active site.
Cpd.
6a
R1
R2
Docking
score
No. of
H-bonds
Distance
(Aº)
Amino acid
involved
Molecular
structure
O of morpholine
O of 3-OCH3
C2H5 3-OCH3
C2H5 4-OCH3
C6H5 3-OCH3
C6H5 4-OCH3
-14.42
-12.05
-13.73
-12.48
-11.85
-13.32
-11.69
-13.46
-11.59
-11.19
2
2
2
2
2
2
2
2
2
1
2.08
1.80
Val851
Tyr836
2.10
1.88
Val851
Lys802
O of morpholine
O of 4-OCH3
6b
6c
2.19
1.83
Val851
Lys802
O of morpholine
O of 3-OCH3
2.20
2.30
Val851
Lys802
O of morpholine
O of 4-OCH3
6d
8a
C2H5
C2H5
C6H5
C6H5
C6H5
C6H5
H
CH3
H
1.74
2.17
Val851
Tyr836
O of morpholine
O of C=O
1.75
1.70
Val851
Tyr836
O of morpholine
O of C=O
8b
1.89
2.10
Val851
Tyr836
O of morpholine
O of C=O
8c
CH3
H
1.73
1.76
Val851
Tyr836
O of morpholine
O of C=O
8d
1.75
2.70
Val851
Gln859
O of morpholine
O of morpholine
10a
10b
CH3
1.84
Val851
O of morpholine