Design, synthesis, biological evaluation and molecular modeling study of new thieno[2,3-d]pyrimidines with anti-proliferative activity on pancreatic cancer cell lines

Article abstract of DOI:10.1016/j.bioorg.2019.103472

Pancreatic cancer is one of the most challenging diseases with seven months only as median survival time due to its poor prognosis. Several enzymes are blamed for the progress of pancreatic cancer especially, platelet-derived growth factor receptors (PDGFRs), this in turn makes them promising targets for its treatment. In this study, twenty eight new compounds based on thieno[2,3-d]pyrimidine scaffold were synthesized as anti-pancreatic cancer agents mimicking the benzofuro[3,2-d]pyrimidine derivative, amuvatinib. Various linkers including amides, esters, ketones, urea and thiourea derivatives were utilized to study their effect on the anti-proliferative activity of these compounds. Most of the tested compounds revealed good cytotoxic activities against pancreatic carcinoma cell line PANC-1. Compound 9d showed the highest cytotoxicity with an IC₅₀ value of 5.4 μM. Furthermore, 9d showed excellent platelet derived growth factor receptor (PDGFR-α) inhibitory activity, with IC₅₀ value 0.155 μM. Docking study was carried out into PDGFR-α active site which showed comparable binding mode to that of FDA approved PDGFR-α inhibitor, imatinib. 3D-Quantitative structure activity relationship (QSAR) model was built up with five-featured pharmacophore which could be implemented for emerging effective lead structures. These compounds could serve as a new chemotype for discovering new agents for pancreatic cancer therapy.

Full text of DOI:10.1016/j.bioorg.2019.103472

Journal Pre-proofs
Design, synthesis, biological evaluation and molecular modeling study of new
thieno[2,3-d]pyrimidines with anti-proliferative activity on pancreatic cancer
cell lines
Mohamed S.H. Salem, Yasmine M. Abdel Aziz, Mohamed S. Elgawish,
Mohamed M. Said, Khaled A.M. Abouzid
PII:
S0045-2068(19)30611-X
DOI:
https://doi.org/10.1016/j.bioorg.2019.103472
YBIOO 103472
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
19 April 2019
22 October 2019
23 November 2019
Please cite this article as: M.S.H. Salem, Y.M. Abdel Aziz, M.S. Elgawish, M.M. Said, K.A.M. Abouzid, Design,
synthesis, biological evaluation and molecular modeling study of new thieno[2,3-d]pyrimidines with anti-
proliferative activity on pancreatic cancer cell lines, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/
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Design, synthesis, biological evaluation and molecular
modeling study of new thieno[2,3-d]pyrimidines with anti-
proliferative activity on pancreatic cancer cell lines
Mohamed S.H. Salema,, Yasmine M. Abdel Aziz a,*, Mohamed S. Elgawishb,
Mohamed M. Saida and Khaled A.M. Abouzid c,d**
aPharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Suez Canal
University, Ismailia 41522, Egypt.
bMedicinal Chemistry Department, Faculty of Pharmacy, Suez Canal University,
Ismailia 41522, Egypt.
cPharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University,
Abassia, Cairo 11566, Egypt.
dDepartment of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of
Sadat City, Menoufia, 32897, Egypt
Corresponding authors
** Corresponding authors: Khaled.abouzid@pharma.asu.edu.eg; Tel.: +20-
1222165624
* Yasmine M. Abdel Aziz, mohamad_yasmine@yahoo.com, +20 1277718225
Abstract
Pancreatic cancer is one of the most challenging diseases with seven months
only as median survival time due to its poor prognosis. Several enzymes are blamed
for the progress of pancreatic cancer especially, platelet-derived growth factor
receptors (PDGFRs), this in turn makes them promising targets for its treatment. In
this study, twenty eight new compounds based on thieno[2,3-d]pyrimidine scaffold
were synthesized as anti-pancreatic cancer agents mimicking the benzofuro[3,2-
d]pyrimidine derivative, amuvatinib. Various linkers including amides, esters,
ketones, urea and thiourea derivatives were utilized to study their effect on the anti-
proliferative activity of these compounds. Most of the tested compounds revealed
good cytotoxic activities against pancreatic carcinoma cell line PANC-1. Compound
9d showed the highest cytotoxicity with an IC50 value of 5.4 µM. Furthermore, 9d
showed excellent platelet derived growth factor receptor (PDGFR-α) inhibitory
activity, with IC50 value 0.155 µM. Docking study was carried out into PDGFR-α
active site which showed comparable binding mode to that of FDA approved PDGFR-
α inhibitor, imatinib. 3D- Quantitative structure activity relationship (QSAR) model

was built up with five-featured pharmacophore which could be implemented for
emerging effective lead structures. These compounds could serve as a new chemotype
for discovering new agents for pancreatic cancer therapy.
Keywords
Thieno[2,3-d]pyrimidine; PDGFR-α; Kinase inhibitor; Cytotoxic effect; PANC-1;
Pancreatic cancer; 3D-QSAR; ADME.
1- Introduction
Pancreatic cancer is one of the leading causes of death prompting more than
400,000 deaths annually [1]. This is attributed to the low five-year survival rate
(8.2%) in comparison with other common cancer types [2]. The treatment of
pancreatic cancer faces great challenges; only about 10% of patients presenting with
resectable disease suitable for potentially curative surgery [3]. For many years, 5-
fluorouracil (5-FU) (Fig. 1) was considered as the optimum chemotherapeutic agent
for pancreatic cancer. In 1997, gemcitabine (Fig. 1) has become the drug of choice for
pancreatic cancer cases, showing a modest survival benefit over 5-FU and better
improvement of disease related symptoms [4]. However, therapeutic outcome has not
upgraded significantly over the past years upon using those conventional treatments
due to their limited effect and substantial toxicity. Consequently, a new trend of
anticancer agents that target pancreatic cancer at a molecular level is needed to treat
this deadly disease [5].
Several proteins were found to be overexpressed in pancreatic cancer cases.
Among these proteins are platelet derived growth factor receptors (PDGFRs) and in a
lower percentage of cases stem cell factor receptors (SCFRs) or c-KIT [6-8]. Both are
cell surface tyrosine kinase receptors with a unique structural relationship suggesting
a common evolutionary origin. It is reasonable that the c-KIT ligands have structural
features in common with PDGFR ligands [9]. Multi-targeted receptor tyrosine kinase
inhibitors that target both c-KIT and PDGFR-α are expected to have a great efficacy
in the treatment of pancreatic cancer [10].
2- Rationale and design
In this study, we planned to design and synthesize series of novel potential
therapeutics for pancreatic cancer in which tyrosine kinase activity, especially

PDGFR-α and c-KIT is highly expressed [6-8]. Amuvatinib (Fig. 2), which reveals a
powerful cytotoxicity against pancreatic cancer cell line PANC-1, was selected as a
hit compound due to its more selectivity on PDGFR-α and c-KIT suggesting less side
effects and more potency [10, 11]. Three regions were modified in amuvatinib (Fig.
2) with the aid of well-documented structure-activity relationship (SAR), and the
synthetic convenience of amuvatinib [12].
In order to get novel analogues and clarify the structure-activity relationships,
we introduced the following modifications for amuvatinib. Benzofuro[3,2-
d]pyrimidine of amuvatinib was replaced with alicyclic thieno[2,3-d]pyrimidine core
structures in our target compounds. Thieno[2,3-d]pyrimidine scaffold achieves a
promising potency towards tyrosine kinases via occupying the hinge binding region
instead of adenine ring of ATP [13]. Piperazine was used as linker mimicking that of
amuvatinib. Amides, esters, ketones, urea and thiourea derivatives were all used in the
hydrogen bond binding region to reveal the effect of changing the heteroatom, the
significance of presence of secondary nitrogen and the optimal number of atoms
between linker and aromatic moiety. Different bulky aromatic moieties were also
introduced occasionally comprising electron withdrawing groups to study their effect
on the activity. The ability of synthesized compounds to inhibit the growth of human
pancreatic carcinoma (PANC-1) cell line was assessed. Moreover, the most active
compound was further evaluated for its inhibitory activity towards PDGFR-α. 3D-
QSAR model was developed using biological activity results to relate the molecular
structure to its activity by a mathematical model.
3- Results and discussion
3.1.
Chemistry
Target compounds were synthesized as sketched in (Schemes 1-3). The
starting materials 1a-h were prepared via the reaction of chloroacetyl chloride with a
variety of substituted anilines [14-19]. Similarly, the precursors 2-4 were prepared by
the reaction of chloroacetyl chloride with naphthols, or diphenyl amine in
dichloromethane in presence of triethylamine or in the presence of pyridine in case of
phenol (Scheme 1). On the other hand, 2-aminothiophene-3-carbonitrile derivatives
5a,b were obtained via Gewald’s reaction, which involves a multi-component
condensation between cyclopentanone or cyclohexanone, malononitrile and elemental

sulfur in 70% ethanol in presence of morpholine [20]. The precursors 5a,b were
cyclized through reflux in formic acid and sulfuric acid mixture to give compounds
6a,b [21]. Refluxing 6a,b in phosphorus oxychloride yielded the chlorinated
derivatives 7a,b [22], which were reacted with anhydrous piperazine in isopropanol
affording the key intermediates 8a,b [23] (Scheme 2). The final step in the synthesis
of compounds 9-12 and compounds 14-18, involved nucleophilic substitution of
chloro of intermediates 1-4 or bromo of phenacyl bromide with intermediates 8a,b.
Moreover, the synthesis of compounds 13a-d and 19a-d involved the nucleophilic
addition of intermediates 8a,b to isocyanate and isothiocyanate derivatives (Schemes
3a,b). The structures of novel compounds were characterized via 1H NMR, 13C NMR,
FT-IR, EI-MS and elemental analyses. In addition to HPLC analyses were performed
to determine their purity (≥95%).
3.2.
Biological evaluation
3.2.1. In vitro anticancer activity against PANC-1 cell line
The potential cytotoxicity of newly synthesized compounds was evaluated
against PANC-1 cell line via SulfoRhodamine-B (SRB) assay. As shown in (Table 1),
compound 9d showed the best inhibitory activity with IC50 value 5.4 μM (Table 2a)
which is comparable to the reported data of amuvatinib with IC50 value 3 μM [10].
Compounds 13b, 13c, 14c, 19b and 19c also displayed a low activity at two digits
micromolar concentration, with IC50 values of 13.49, 15.78, 15.39, 14.41 and 17.82
μM respectively followed by compounds 13a, 13d, 14d, 19a and 19d with IC50 values
of 33.76, 31.57, 31.91, 33.23 and 29.56 μM respectively. The remaining compounds,
especially 14e (Table 2b), showed the least cytotoxic activity with higher IC50 values.
3.2.2. In vitro PDGFR-α assay
In order to prove the success of fragment-based drug design approach, we extended
the biological enzyme assay inhibitory activity to PDGFR-α. The IC50 value of
compound 9d was evaluated compared to reference PDGFR-α inhibitor imatinib (Fig.
3). Compound 9d showed excellent PDGFR-α inhibitory activity, at the sub
micromolar level with IC50 value 0.155 µM, which was three fold minor imatinib with
IC50 value 0.049 µM (Fig. 4).

3.3.
Molecular modeling
3.3.1. 3D-QSAR
In this study, pharmacophore model was developed using pharmacophore
modeling task of Schrodinger (10.1). Twenty-eight synthesized compounds having in
most cases a promising activity against PANC-1 cancer cell line, which overexpresses
PDGFR-α and c-KIT enzymes [6, 8], were used for developing a 3D-QSAR
pharmacophore model. Five sites or variants were chosen, to obtain the optimal
pattern of features which are shared by the most active compounds. Phase provides a
fixed group of six pharmacophore features: H-bond donor (D), H-bond acceptor (A),
hydrophobic group (H), aromatic ring (R), negatively charged group (N) and
positively charged group (P) (Supplementary: Table 1). Five pharmacophore models
were generated with different combination of variants (AAHHR, AAHRR, AARRR,
AHRRR and HHRRR). Among these five models, AAHHR exhibited the supreme
alignment with the most active compounds and showed the best survival scoring
function, consequently it was selected to generate 3D-QSAR model [24].
3.3.1.1.
Developing a pharmacophore model
Ten predictive pharmacophore models (hypothesis) of the variant AAHHR
were created through the alignment of diverse conformations of the denoted training
set ligands just before binding them with the created models. All created
pharmacophore models contained a minimum of five chemical features. The model
which lacks essential features for ligand binding is not presupposed to be affordable
for distinguishing between active and inactive ligands. The model exhibited the
highest survival score was chosen for creating an atom-based alignment of the newly
synthesized compounds (Supplementary: Table 2). Model AAHHR.36 was chosen
due to its ability to discriminate between active and inactive ligands in terms of atom-
based alignment. The unique arrangement of chemical sites or features accompanied
by the optimal distances between them is shown in pharmacophore model
(AAHHR.36) (Fig. 5).
The two hydrogen bond acceptors in 21 training set are mapped to N1-
pyrimidine and N3-pyrimidine. While the two hydrophobic groups are mapped to
cyclopentyl or cyclohexyl and piperazine. Whereas aromatic ring feature is mapped to
thiophene ring. A dataset of 21 compounds (training set) were used to generate an

atom-based 3D-QSAR hypothesis (Supplementary: Fig. 1a). The alignment obtained
from the best model AAHHR.36 was employed to create a 3D-QSAR model. The
alignment of the training set compounds to the developed pharmacophore model is
presented in (Supplementary: Fig. 1b). The seven compounds (test set) also showed
the same feature map of training set (Supplementary: Fig. 1c). Moreover, the reported
amuvatinib with potent anti-pancreatic cancer activity exhibited the same feature map;
N1-pyrimidine and N3-pyrimidine showed hydrogen bond acceptors nature. Whereas
piperazine and phenyl ring showed the same feature map of cyclohexyl and
piperazine. While furan showed the same feature of thiophene in synthesized
compounds (Supplementary: Fig. 1d) [10].
3.3.1.2.
3D-QSAR contour map analysis
The generated 3D-QSAR pharmacophore model accompanied by positive and
negative effects of hydrophobic/nonpolar, H-bond donor, electron withdrawing, and
other features mapped on the most active ligand were depicted in (Supplementary:
Fig. 2). Blue areas suggested promising features encouraging enzyme ligand
interactions, whereas red areas suggested unfavorable features discouraging
interactions.
The H-bond donor feature can be revealed by comparing the activity of
compounds 9b, 9d, and 9f that do not differ structurally from each other except in the
substitution on the phenyl ring. Compounds 9b and 9f having alkyl and bulky
substitutions respectively, on phenyl ring in position 3 which is unfavorable in terms
of activity. The generated pharmacophore model suggests that m-substitution on
phenyl ring affords unfavorable configuration inside the receptor which directs the
amide linker, responsible for H-bond formation, away from blue area containing side
chains of amino acids that act as H-bond acceptors, and near to red cubes showed
undesirable interactions with this amide linker. Alternatively, compound 9d with the
highest activity has a blue area around N4-piperazine and amide linker, as para
substitution on phenyl ring affords favorable configuration (Fig. 6). This explanation
is obviously shown in compounds containing NHCS and NHCO linkers (compounds
14a-f and 19a-d) compared to COOR (compounds 15 and 16).
Ortho and meta substitutions of phenyl group are unfavorable for activity,
whereas unsubstituted and p-substituted phenyl rings are favorable for activity. This is

validated by the proposed compounds containing unsubstituted or p-substituted
phenyl ring (9d, 14c and 19a-c) showed the most promising activity. p-Chloro (9d
and 14c) and p-fluoro (19c), o- and p-dichloro (9e and 14d) and unsubstituted phenyl
ring (13a,b and 19a,b) containing compounds achieved the highest activity in
comparison with m-methyl (9b and 14a), m-bromo (9f and 14e), o-methyl (9a) and o-
methoxy (9c) containing compounds. Although the replacement of the phenyl ring by
aliphatic chain (ethyl) could kept hopeful activity, the presence of these phenyl rings
improve the activity, because of their (
) stacking interactions with the side
휋 ― 휋
chains of some amino acids. This is validated by the fact that compounds (13b and
19b), which have phenyl rings, display higher activity when compared to compounds
(13d and 19d) having aliphatic chains. The increment of ring size to naphthyl group
in compounds (10a, b and 15) should enhance the hydrophobic interaction and thus it
increases the activity, however, in our study, naphthyl group has lower activity than
phenyl. This is may be because bulkier groups at this position will occupy red cube or
clash with hydrophobic residue in receptor site. Additionally, the distance between
piperazine ring and phenyl ring is important for activity. Four-bond distance is
optimum for anticancer activity because this distance directs the phenyl group inside
the hydrophobic pocket of enzyme. In case of three-bond distance the phenyl group
sets near red cube which is unfavorable for inhibitory activity. This can be shown in
some compounds containing four-bond distance between these two moieties like 9d,
14c, 14d, 14f, and amuvatinib. However, other compounds show low activity
although they contain four not three-bond distance, as there are many factors affecting
their biological activity, including the substitution on phenyl ring (9b, 9f, 14a, and
14e), type of hydrogen bonding region (11, and 16), and the ring size in naphthyl
derivatives (10, and 15). On the other hand, compound 18 contains only three-bond
distance between phenyl and piperazine rings, the thing which is suggested to be the
reason of its lower activity as suggested by our developed 3D-QSAR model.
Definitely, compounds 13a-d, and 19a-d show good activity although their three-
bond distance, but the urea and thiourea hydrogen bonding regions affect positively
on their activity. Briefly, the distance between these two moieties can have some
effect on anti-proliferative activity, but we do believe that this effect is not that much
critical in comparison with other factors.
3.3.1.3.
Pharmacophore validation

Generated 3D-QSAR models are not valid for predicting the new compounds
activities and retrieving the active ones from database unless they are statistically
significant. The (AAHHR.36) model was selected based on its validation. A 3D-
QSAR study was carried on the seven synthesized compounds (test set) to explore the
outcome of three-dimensional arrangement of chemical features on the PANC-1
cytotoxicity (Supplementary: Table 3).
The stability of pharmacophore model is revealed by the huge value of F
(165.9) and high degree of confidence, which is reinforced by the minor value of
variance ratio P (1.249e). Furthermore, the data used to create the regression model
are the most suitable for the QSAR analysis, the thing which is distinct from minor
values of standard deviation (0.277) of the regression and RMSE (0.543) as well as
high value of r2 (squared correlation coefficient value) (0.954). The model was also
validated by external validated correlation coefficient (q2 = 0.641). The statistical
parameter q2 is more trustworthy and robust than r2, as it is attained via external
validation through separating the data into training and test groups. Extra validation
for the created model (AAHHR.36) was accomplished through mapping of one
compound with reported cytotoxicity against PANC-1 cell line (amuvatinib) [10].
Moreover, comparing its fit value, which was detected as 1.89 (Supplementary: Table
3), with fit values of the tested compounds and comparing its predicted with its
experimental activity using ligand pharmacophore mapping protocol [24].
The predicted anti-cancer activities of these compounds and the dataset via the
generated model were enormously close to those experimentally measured
(Supplementary: Table 3). This fact highlights the value of this generated model as a
tool for the prediction of cytotoxicity of newly synthesized compounds [25].
3.3.2. Molecular docking of compound 9d in the active site of PDGFR-α
Up to date, there are only three available crystal structures of PDGFR-α on
protein data bank (PDB). Two of them are present with no inhibitory ligands (PDB
ID: 5K5X), and (PDB ID: 6A32) and the other in complex with imatinib analogue
(PDB ID: 5GRN). In order to provide a potential rationalization for the detected in
vitro PDGFR-α activity, docking studies were performed for the protein tyrosine
kinase, PDGFR-α (PDB ID: 5GRN) containing imatinib structural analogue. The

synthesized compounds were docked into the ATP binding pocket as thieno[2,3-
d]pyrimidines scaffold mimicking the adenine ring of ATP and represents hinge-
binding motif. The pose of synthesized thieno[2,3-d]pyrimidine derivatives (9d and
14e) and imatinib are identical, the thing which means that the biological activity
might be the same (Supplementary: Fig. 3a). In detail, the docking results showed that
our inhibitors form hydrogen bonds, hydrophobic contact and hydrophobic
hydrophobic interactions. Two nitrogens of piperazine and amide of the most active
compound 9d acted as hydrogen bond donor with key amino acids in active pocket
Asp836 with distance of 3.66 Å and 1.81 Å respectively (Supplementary: Fig. 3b). A
halogen bond is also formed between Cl and Phe856. A non-covalent -cation
휋
interaction is obviously shown in most of thieno[2,3-d]pyrimidines series between
protonated nitrogen of piperazine and imidazole of Hie816 . The presence of aromatic
ring besides hydrogen donor/acceptor is general criteria of protein kinase inhibitors.
Aromatic moiety showed favorable van der Waals interactions with Ile843, Tyr849,
Phe856, and Leu857 as shown in compound 9d (Supplementary: Fig. 3b). In the same
manner, imatinib showed similar interactions in the active domain like our studied
compounds. However, imatinib showed an extra hydrogen bond with key amino acid
Cys677 (Supplementary: Fig. 3c). The competitive binding of the designed
compounds to ATP binding domain made our compound belonging to type I kinase
inhibitors.
Extra precision glide docking of compound 9d with active domain of PDGFR-
α showed a reasonable docking score of -8.224 kJ/mol and a glide E-model value of -
82.42 kcal mol-1, compared to imatinib that gave a docking score of -13.405 kJ/mol
and a glide E-model value of - 152.50 kcal mol-1.
3.3.3. in silico ADME predictive study
To predict the drug-likeness of the proposed compounds as a potent anticancer
agent, their ADME (Absorption, Distribution, Metabolism and Excretion) properties
were investigated using QikProp program v4.3 of the Schrodinger. This function
provides an assessment of the physicochemical properties and the bioavailability of
the designed compounds based on their chemical structures. Many pharmacokinetic
parameters were also investigated as presented in Table 3 such as polar surface area
(PSA), QPPCaco (predicted apparent Caco-2 cell permeability in nm s-1), QPPMDCK

(predicted apparent MDCK cell permeability in nm s-1). Moreover, QPlogS (predicted
aqueous solubility), QPlogKhsa (prediction of binding to human serum albumin), and
percent human oral absorption (predicted human oral absorption on 0-100% scale).
One of the most important properties required in drugs is the oral activity. It is
well known that an orally active compound should not have more than two violations
of the Lipinski rule, the thing which is achieved in our designed compounds
confirming their drug-likeness properties. The oral bioavailability of the drug
molecules could be controlled by the value of rotatable bonds (0–15) and polar
surface area (PSA) (7–200Å). The designed compounds showed a reasonable PSA
compared to amuvatinib and imatinib suggesting their oral activity.
Another important property is the intestinal absorption or permeation. Caco-2
cell permeability (QPPCaco) is the good model to anticipate the intestinal permeation
by passive diffusion transport. Thiourea derivatives 19b, 19c and 19d showed higher
permeability than urea derivative 19a. We attributed this to the fact that the oxygen
atom is more basic and more hydrophilic than sulfur atom. Generally, the proposed
compounds exhibited good intestinal permeability, even higher than imatinib, which
can be enough for efficacy. However, the intestinal permeability of amuvatinib, still
higher than our designed compounds suggesting that this intestinal permeability can
be improved after further modifications. The lower permeability of imatinib can be
attributed to the large number of hydrogen bond acceptors, compared to our design
compounds. Most of the test compounds showed a good BBB permeability
(QPPMDCK>25) as well, compared to Amuvitinib.
The long duration of action is another important property of the approved
drugs as decreasing the dose frequency is highly desirable, especially to cancer
patients. This feature is related to the binding ability of a drug to Plasma albumin
which is expected to be high in the designed compounds. On another hand QPlogS
and QPlogPo/w are descriptors of aqueous solubility and octanol/water partition
coefficient; the test compounds were found to be in the permissible range (Table 3).
Briefly, our designed compounds were considered promising lead molecules for
designing potent and selective PGEFRα inhibitors with excellent membrane
permeability and oral bioavailability.
4- Conclusion

Based on the scaffold of amuvatinib with the tricyclic benzofuro[3,2-
d]pyrimidine core, novel thieno[2,3-d]pyrimidine series were designed. Biological
studies have led to the identification of novel small molecules that displayed a
significant inhibitory activity against pancreatic carcinoma with IC50 values in the low
micromolar-range. 3D-QSAR model was constructed with five-featured pharmacophore
(AAHHR): two hydrogen bond acceptors (A) equivalent to N1-pyrimidine and N3-
pyrimidine, two hydrophobic groups (H) equivalent to cyclopentyl/cyclohexyl and
piperazine and aromatic ring feature (R) equivalent to thiophene ring. The model was
developed to relate structural features of the newly synthesized compounds to their
activity. Moreover, the model exhibited good correlation (r2), cross validation (q2)
coefficients and excellent agreement between experiment and predictive activity. A
major observation is that amuvatinib and all thieno[2,3-d]pyrimidine series have the
same orientation and occupy the same position. Consequently, this study highlights
various essential moieties that play a crucial role in activity and provides an enhanced
explanation of the structure activity relationship. Also, many of these newly
synthesized compounds could be hit molecules for discovering new anti-pancreatic
cancer agents.
5- Experimental section
5.1.
Chemistry
Reagents for synthesis were purchased from Sigma-Aldrich, Merck and Alfa Aesar
and used without any extra purification. Completion of reactions was monitored using
thin-layer chromatography (TLC) benefitting from different solvent systems as
mobile phases and silica gel Merck 60 (F254) as stationary phase. The spots were
checked under UV light illumination (254 and 366 nm). The melting points of
compounds were determined by means of Stuart melting point apparatus SMP10 and
are uncorrected. Fourier-Transform Infrared (FT-IR) spectra were recorded using KBr
discs on a Vector 22 Infrared spectrophotometer (υmax in cm-1), with ratio (1drug: 3
KBr). 1H NMR and 13C NMR spectra were run at Bruker 400 MHz
spectrophotometer at Center for Drug Discovery and Development Research, Ain
Shams University. Chemical shift values are presented as parts per million (ppm)
using tetramethyl silane (TMS) as an internal standard. Both electron impact mass
spectra (EI-MS) and elemental analyses were measured by Thermo Scientific ISQLT

mass spectrometer and Thermo Scientific Flash 2000 elemental analyzer respectively,
at the Regional Center for Mycology and Biotechnology, Al-Azhar University. The
synthetic procedures for the target compounds are illustrated in (Schemes 1- 3).
5.1.1. General procedure for the preparation of compounds 9a-f and 14a-f
The target compounds 9a-f and 14a-f were prepared by nucleophilic
substitution of chloro of 2-chloro-N-substitutedphenyl acetamide derivatives 1a-h
with 4-(1-piperazinyl) thieno[2,3-d]pyrimidine derivatives 8a,b . Briefly, sodium
bicarbonate (2.36 mmol, 0.2 g) and sodium iodide (2.36 mmol, 0.51 g) were added to
a solution of intermediates 8a,b (2.36 mmol) and intermediates 1a-h (2.36 mmol) in
DMF (30 mL). The reaction mixture was stirred for 36 h at room temperature, then
added to water (50 mL) affording a precipitate which was filtered under vacuum and
left to dry. The resulting solid was recrystallized from absolute ethanol to give the
titled compounds.
5.1.1.1.
N-(2-Methylphenyl)-2-[4-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-
d]pyrimidin-4-yl)piperazin-1-yl] acetamide 9a. Yellowish white crystals, 87% yield;
m.p. 192-193 ⁰C; FT-IR (KBr, cm-1): υ 3416 (NH), 3020 (CH aromatic), 2922 (CH
1
aliphatic), 1688 (-NH-C=O), 1586 (C=C), 1541 (C=N); H NMR (400 MHz, CDCl3)
δ 9.34 (s, 1H, NH D2O exchangeable), 8.51 (s, 1H, pyrimidine H), 8.08 (d, J=7.8 Hz,
1H, ArH), 7.15-7.25 (m, 2H, ArH), 7.06 (d, J=8.2 Hz, 1H, ArH), 3.70 (m, 4H,
piperazine), 3.39 (s, 2H, -CH2CO-), 3.04 (m, 4H, -CH2-CH2-CH2-), 2.95 (m, 4H,
piperazine), 2.48 (m, 2H, -CH2-CH2-CH2-), 2.32 (s, 3H, ArCH3); MS: (Mwt.: 407.5):
m/z, 407.3 [M+, (10.88%)], 269 (100%); Anal. Calcd for C22H25N5OS: C, 64.84; H,
6.18; N, 17.18; Found: C, 64.78; H, 6.14; N, 17.1.
5.1.1.2.
N-(3-Methylphenyl)-2-[4-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-
d]pyrimidin-4-yl)piperazin-1-yl] acetamide 9b. White crystals, 90% yield; m.p. 65-66
⁰C; FT-IR (KBr, cm-1): υ 3416 (NH), 3021 (CH aromatic), 2922 (CH aliphatic), 1685
1
(-NH-C=O), 1595 (C=C), 1545 (C=N); H NMR (400 MHz, CDCl3) δ 9.39 (s, 1H,
NH D2O exchangeable), 8.51 (s, 1H, pyrimidine H), 7.41-7.45 (m, 2H, ArH), 7.22 (t,
J=7.95 Hz, 1H, ArH), 6.94 (d, J=7.34 Hz, 1H, ArH), 3.73 (m, 4H, piperazine), 3.42
(s, 2H, -CH2CO-), 3.03 (m, 4H, -CH2-CH2-CH2-), 2.98 (m, 4H, piperazine), 2.45

(quint, J=6.85 Hz, 2H, -CH2-CH2-CH2-), 2.34 (s, 3H, ArCH3); MS: (Mwt.: 407.5):
m/z, 407.2 [M+, (8.60%)], 376.15 (100%); Anal. Calcd for C22H25N5OS: C, 64.84; H,
6.18; N, 17.18; Found: C, 64.77; H, 6.0; N, 17.15.
5.1.1.3.
N-(2-Methoxyphenyl)-2-[4-(6,7-dihydro-5H-
cyclopenta[4,5]thieno[2,3-d]pyrimidin-4-yl)piperazin-1-yl] acetamide 9c. White
crystals, 92% yield; m.p. 182-183 ⁰C; FT-IR (KBr, cm-1): υ 3420 (NH), 3016 (CH
aromatic), 2924 (CH aliphatic), 1677 (-NH-C=O), 1600 (C=C), 1551 (C=N); 1H NMR
(400 MHz, CDCl3) δ 9.69 (s, 1H, NH D2O exchangeable), 8.51 (s, 1H, pyrimidine H),
8.32 (d, J=7.09 Hz, 1H, ArH), 7.08 (t, J =7.34 Hz, 1H, ArH), 6.97 (t, J=7.46 Hz, 1H,
ArH), 6.90 (d, J=7.82 Hz, 1H, ArH), 3.90 (s, 3H, ArOCH3-), 3.75 (m, 4H,
piperazine), 3.42 (s, 2H, -CH2CO-), 3.03 (m, 4H, -CH2-CH2-CH2-), 2.97 (m, 4H,
piperazine), 2.45 (quint, J=6.85 Hz, 2H, -CH2-CH2-CH2-); MS: (Mwt.: 423.5): m/z,
423.14 [M+, (100%)]; Anal. Calcd for C22H25N5O2S: C, 62.39; H, 5.95; N, 16.54;
Found: C, 62.28; H, 5.88; N, 16.38.
5.1.1.4.
N-(4-Chlorophenyl)-2-[4-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-
d]pyrimidin-4-yl)piperazin-1-yl] acetamide 9d. Yellow crystals, 95% yield; m.p. 76-
77 ⁰C; FT-IR (KBr, cm-1): υ 3415 (NH), 3026 (CH aromatic), 2923 (CH aliphatic),
1
1685 (-NH-C=O), 1618 (C=C), 1548 (C=N); H NMR (400 MHz, CDCl3) δ 9.12 (s,
1H, NH D2O exchangeable), 8.48 (s, 1H, pyrimidine H), 7.51 (d, J=6.85 Hz, 2H,
ArH), 7.28 (d, J=6.85 Hz, 2H, ArH), 3.62 (m, 4H, piperazine), 3.21 (s, 2H, -CH2CO-
), 3.01 (m, 4H, -CH2-CH2-CH2-), 2.79 (m, 4H, piperazine), 2.44 (m, 2H, -CH2-CH2-
CH2-); 13C NMR (101 MHz, CDCl3) δ 173.81, 167.59, 160.51, 151.51, 140.62,
136.05, 135.42, 129.43, 129.11, 120.69, 117.66, 61.89, 53.29, 49.39, 31.52, 29.88,
28.21; MS: (Mwt.: 427.95): m/z, 427.8 [M+, (100%)]; Anal. Calcd for C21H22ClN5OS:
C, 58.94; H, 5.18; N, 16.36; Found: C, 58.60; H, 5.00; N, 16.25.
5.1.1.5.
N-(2,4-Dichlorophenyl)-2-[4-(6,7-dihydro-5H-
cyclopenta[4,5]thieno[2,3-d]pyrimidin-4-yl)piperazin-1-yl] acetamide 9e. Yellow
crystals, 93% yield; m.p. 156-157 ⁰C; FT-IR (KBr, cm-1): υ 3415 (NH), 3023 (CH
aromatic), 2924 (CH aliphatic), 1695 (-NH-C=O), 1590 (C=C), 1544 (C=N); 1H NMR
(400 MHz, CDCl3) δ 9.97 (s, 1H, NH D2O exchangeable), 8.52 (s, 1H, pyrimidine H),

8.31 (d, J3=8.56 Hz, 1H, ArH), 7.41 (d, J4=2.2 Hz, 1H, ArH), 7.25 (dd, J3=8.56, Hz
J4=2.2 Hz, 1H, ArH), 3.48-3.95 (m, 6H, piperazine and -CH2CO-), 2.99-3.11 (m, 8H,
-CH2-CH2-CH2- and piperazine), 2.48 (quint, J=7.10 Hz, 2H, -CH2-CH2-CH2-); MS:
(Mwt.: 462.4): m/z, 461.2 [M+, (100%)]; Anal. Calcd for C21H21Cl2N5OS: C, 54.55;
H, 4.58; N, 15.15; Found: C, 54.30; H, 4.56; N, 15.14.
5.1.1.6.
N-(3-Bromophenyl)-2-[4-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-
d]pyrimidin-4-yl)piperazin-1-yl] acetamide 9f. Yellow crystals, 90% yield; m.p. 68-
70 ⁰C; FT-IR (KBr, cm-1): υ 3415 (NH), 3019 (CH aromatic), 2924 (CH aliphatic),
1
1685 (-NH-C=O), 1593 (C=C), 1545 (C=N); H NMR (400 MHz, CDCl3) δ 9.52 (s,
1H, NH D2O exchangeable), 8.54 (s, 1H, pyrimidine H), 7.88 (s, 1H, ArH), 7.57 (d,
J=7.09 Hz, 1H, ArH), 7.30 (d, J=7.31 Hz, 1H, ArH), 7.22 (t, J=7.23 Hz, 1H, ArH),
3.76 (m, 4H, piperazine) 3.46 (s, 2H, -CH2CO-), 3.07 (m, 4H, -CH2-CH2-CH2-), 3.00
(m, 4H, piperazine), 2.50 (m, 2H, -CH2-CH2-CH2-); MS: (Mwt.: 472.4): m/z, 472.5
[M+, (8.69%)], 389.1 (100%); Anal. Calcd for C21H22BrN5OS: C, 53.39; H, 4.69; N,
14.83; Found: C, 52.99; H, 4.75; N, 14.76.
5.1.1.7.
N-(3-Methylphenyl)-2-[4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4-yl)piperazin-1-yl] acetamide 14a. White crystals, 88% yield; m.p. 70-
72 ⁰C; FT-IR (KBr, cm-1): υ 3416 (NH), 3021 (CH aromatic), 2927 (CH aliphatic),
1
1686 (-NH-C=O), 1594 (C=C), 1556 (C=N); H NMR (400 MHz, CDCl3) δ 9.07 (s,
1H, NH D2O exchangeable), 8.57 (s, 1H, pyrimidine H), 7.37-7.43 (m, 2H, ArH),
7.24 (t, J=8.07 Hz, 1H, ArH), 6.95 (d, J=7.58 Hz, 1H, ArH), 3.50 (m, 4H,
piperazine), 3.24 (s, 2H, -CH2CO-), 2.89-2.97 (m, 4H, -CH2-CH2-CH2-CH2-), 2.84
(m, 4H, piperazine), 2.36 (s, 3H, ArCH3), 1.83-1.97 (m, 4H, -CH2-CH2-CH2-CH2-);
MS: (Mwt.: 421.5): m/z, 421.5 [M+, (4.74%)], 69.0 (100%); Anal. Calcd for
C23H27N5OS: C, 65.53; H, 6.46; N, 16.61; Found: C, 65.18; H, 6.59; N, 16.88.
5.1.1.8.
N-(2,6-Dimethylphenyl)-2-[4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4-yl)piperazin-1-yl] acetamide 14b. Orange crystals, 95% yield; m.p.
165-167 ⁰C; FT-IR (KBr, cm-1): υ 3419 (NH), 3020 (CH aromatic), 2940 (CH
1
aliphatic), 1681 (-NH-C=O), 1595 (C=C), 1560 (C=N); H NMR (400 MHz, CDCl3)
δ 8.65 (s, 1H, NH D2O exchangeable), 8.56 (s, 1H, pyrimidine H), 7.08-7.13 (m, 3H,

ArH), 3.46-3.51 (m, 4H, piperazine), 3.31 (s, 2H, -CH2CO-), 2.87-2.98 (m, 8H, -CH2-
CH2-CH2-CH2- and piperazine), 2.25 (s, 6H, CH3-Ar-CH3), 1.83-1.97 (m, 4H, -CH2-
CH2-CH2-CH2-); 13C NMR (101 MHz, CDCl3) δ 168.72, 161.88, 151.53, 136.21,
135.15, 133.62, 128.21, 127.31, 127.14, 121.69, 110.31, 61.69, 53.82, 50.81, 26.78,
25.89, 23.21, 18.62; MS: (Mwt.: 435.5): m/z, 435.5 [M+, (13.79%)], 421 (100%);
Anal. Calcd for C24H29N5OS: C, 66.18; H, 6.71; N, 16.08; Found: C, 66.39; H, 6.87;
N, 15.84.
5.1.1.9.
N-(4-Chlorophenyl)-2-[4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4-yl)piperazin-1-yl] acetamide 14c. Orange crystals, 96% yield; m.p. 80-
81 ⁰C; FT-IR (KBr, cm-1): υ 3420 (NH), 3026 (CH aromatic), 2929 (CH aliphatic),
1
1689 (-NH-C=O), 1596 (C=C), 1556 (C=N); H NMR (400 MHz, CDCl3) δ 9.15 (s,
1H, NH D2O exchangeable), 8.56 (s, 1H, pyrimidine H), 7.54 (d, J=8.56 Hz, 2H,
ArH), 7.30 (d, J=8.56 Hz, 2H, ArH), 3.48 (m, 4H, piperazine), 3.24 (s, 2H, -CH2CO-
), 2.89-2.95 (m, 4H, -CH2-CH2-CH2-CH2-), 2.81-2.86 (m, 4H, piperazine), 1.82-1.97
(m, 4H, -CH2-CH2-CH2-CH2-); MS: (Mwt.: 441.98): m/z, 441.2 [M+, (26.99%)], 45.0
(100%); Anal. Calcd for C22H24ClN5OS: C, 59.78; H, 5.47; N, 15.85; Found: C,
60.03; H, 5.31; N, 15.97.
5.1.1.10.
N-(2,4-Dichlorophenyl)-2-[4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4-yl)piperazin-1-yl] acetamide 14d. Yellow crystals, 98% yield; m.p.
166-168 ⁰C; FT-IR (KBr, cm-1): υ 3414 (NH), 3020 (CH aromatic), 2930 (CH
1
aliphatic), 1637 (-NH-C=O), 1590 (C=C), 1556 (C=N); H NMR (400 MHz, CDCl3)
δ 9.95 (s, 1H, NH D2O exchangeable), 8.57 (s, 1H, pyrimidine H), 8.46 (d, J3=8.8 Hz,
1H, ArH), 7.41 (d, J4=2.2 Hz, 1H, ArH), 7.28 (dd, J3=8.56, Hz J4=2.2 Hz, 1H, ArH),
3.50 (m, 4H, piperazine), 3.29 (s, 2H, -CH2CO-), 2.90-2.96 (m, 4H, -CH2-CH2-CH2-
CH2-), 2.85-2.88 (m, 4H, piperazine), 1.82-1.98 (m, 4H, -CH2-CH2-CH2-CH2-); MS:
(Mwt.: 476.4): m/z, 476.5 [M+, (17.9%)], 465.12 (100%); Anal. Calcd for
C22H23Cl2N5OS: C, 55.46; H, 4.87; N, 14.70; Found: C, 55.72; H, 4.99; N, 14.54.
5.1.1.11.
N-(3-Bromophenyl)-2-[4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4-yl)piperazin-1-yl] acetamide 14e. Yellow crystals, 95% yield; m.p. 99-
101 ⁰C; FT-IR (KBr, cm-1): υ 3442 (NH), 3018 (CH aromatic), 2920 (CH aliphatic),

1
1678 (-NH-C=O), 1596 (C=C), 1556 (C=N); H NMR (400 MHz, CDCl3) δ 9.17 (s,
1H, NH D2O exchangeable), 8.57 (s, 1H, pyrimidine H), 7.81 (s, 1H, ArH), 7.52 (d,
J=7.83 Hz, 1H, ArH), 7.20-7.25 (m, 2H, ArH), 3.49 (m, 4H, piperazine), 3.25 (s, 2H,
-CH2CO-), 2.89-2.93 (m, 4H, -CH2-CH2-CH2-CH2-), 2.83 (m, 4H, piperazine), 1.81-
1.97 (m, 4H, -CH2-CH2-CH2-CH2-); MS: (Mwt.: 486.43): m/z, 486.5 [M+, (17.2%)],
353.2 (100%); Anal. Calcd for C22H24BrN5OS: C, 54.32; H, 4.97; N, 14.40; Found: C,
54.65; H, 4.88; N, 14.65.
5.1.1.12.
N-(4-Bromophenyl)-2-[4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4-yl)piperazin-1-yl] acetamide 14f. Yellow crystals, 93% yield; m.p. 79-
80 ⁰C; FT-IR (KBr, cm-1): υ 3420 (NH), 3023 (CH aromatic), 2931 (CH aliphatic),
1
1686 (-NH-C=O), 1590 (C=C), 1530 (C=N); H NMR (400 MHz, CDCl3) δ 9.15 (s,
1H, NH D2O exchangeable), 8.56 (s, 1H, pyrimidine H), 7.40-7.53 (m, 4H, ArH),
3.48 (m, 4H, piperazine), 3.23 (s, 2H, -CH2CO-), 2.89-2.92 (m, 4H, -CH2-CH2-CH2-
CH2-), 2.83 (m, 4H, piperazine), 1.84-1.94 (m, 4H, -CH2-CH2-CH2-CH2-); MS:
(Mwt.: 486.43): m/z, 486.5 [M+, (14.3%)], 53.0 (100%); Anal. Calcd for
C22H24BrN5OS: C, 54.32; H, 4.97; N, 14.40; Found: C, 54.60; H, 4.84; N, 14.72.
5.1.2. General procedure for the preparation of compounds 10-12 and 15-17
The objective compounds 10-12 and 15-17 were set up by nucleophilic
substitution of chloro of naphthyl chloroacetate derivatives 2a,b, phenyl chloroacetate
3, or 2-chloro-N,N-diphenyl acetamide 4 with 4-(1-piperazinyl) thieno[2,3-
d]pyrimidine derivatives 8a,b . In brief, sodium bicarbonate (2.36 mmol, 0.2 g) and
sodium iodide (3.27 mmol, 0.7 g) were added to a solution of intermediates 8a,b (2.36
mmol) and intermediates 2-4 (2.36 mmol) in DMF (30 mL). The reaction mixture was
stirred for 18 h at room temperature, then added to water (50 mL) affording a
precipitate which was filtered under vacuum and left to dry. The resulting solid was
recrystallized from ethyl acetate/n-hexane (3:1) to give the titled compounds.
5.1.2.1.
Naphthalen-1-yl-2-[4-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-
d]pyrimidin-4-yl)-piperazin-1-yl] acetate 10a. White crystals, 55% yield; m.p. 88-89
⁰C; FT-IR (KBr, cm-1): υ 3023 (CH aromatic), 2927 (CH aliphatic), 1768 (-O-C=O),
1601 (C=C), 1561 (C=N); 1H NMR (400 MHz, CDCl3) δ 8.49 (s, 1H, pyrimidine H),

7.87 (m, 2H, ArH), 7.76 (m, 1H, ArH), 7.53 (m, 2H, ArH), 7.47 (m, 1H, ArH), 7.29
(m, 1H, ArH), 3.91 (s, 2H, -CH2COO-), 3.82 (m, 4H, piperazine), 3.03-3.11 (m, 8H, -
CH2-CH2-CH2- and piperazine), 2.45 (m, 2H, -CH2-CH2-CH2-); MS: (Mwt.: 444.5):
m/z, 444.5 [M+, (8.1%)], 392.12 (100%); Anal. Calcd for C25H24N4O2S: C, 67.54; H,
5.44; N, 12.60; Found: C, 67.50; H, 5.33; N, 12.36.
5.1.2.2.
Naphthalen-2-yl-2-[4-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-
d]pyrimidin-4-yl)-piperazin-1-yl] acetate 10b. White crystals, 70% yield; m.p. 101-
103 ⁰C; FT-IR (KBr, cm-1): υ 3021 (CH aromatic), 2925 (CH aliphatic), 1768 (-O-
1
C=O), 1601 (C=C), 1562 (C=N); H NMR (400 MHz, CDCl3) δ 8.50 (s, 1H,
pyrimidine H), 7.85 (m, 3H, ArH), 7.60 (s, 1H, ArH), 7.50 (m, 2H, ArH), 7.28 (m,
1H, ArH), 3.62-3.84 (m, 6H, -CH2COO- and piperazine), 2.95-3.10 (m, 8H, -CH2-
CH2-CH2- and piperazine), 2.46 (m, 2H, -CH2-CH2-CH2-); MS: (Mwt.: 444.5): m/z,
444.5 [M+, (34.42%)], 368.45 (100%); Anal. Calcd for C25H24N4O2S: C, 67.54; H,
5.44; N, 12.60; Found: C, 67.23; H, 5.23; N, 12.5.
5.1.2.3.
Phenyl-2-[4-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-
4-yl)-piperazin-1-yl] acetate 11. Yellow crystals, 68% yield; m.p. 86-88 ⁰C; FT-IR
(KBr, cm-1): υ 3019 (CH aromatic), 2926 (CH aliphatic), 1776 (-O-C=O), 1592
1
(C=C), 1548 (C=N); H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H, pyrimidine H), 7.39
(t, J=7.58 Hz, 2H, ArH), 7.24 (t, J=7.24 Hz, 1H, ArH), 7.11 (d, J=7.83 Hz, 2H,
ArH), 3.73 (m, 4H, piperazine), 3.65 (s, 2H, -CH2COO-), 3.02 (m, 4H, -CH2-CH2-
CH2-), 2.96 (m, 4H, piperazine), 2.40 (quint, J=6.85 Hz, 2H, -CH2-CH2-CH2-); MS:
(Mwt.: 394.5): m/z, 394.5 [M+, (4.45%)], 269 (100%); Anal. Calcd for C21H22N4O2S:
C, 63.94; H, 5.62; N, 14.20; Found: C, 63.75; H, 5.55; N, 14.05.
5.1.2.4.
N,N-Diphenyl-2-[4-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-
d]pyrimidin-4-yl)piperazin-1-yl] acetamide 12. Yellow crystals, 98% yield; m.p. 169-
171 ⁰C; FT-IR (KBr, cm-1): υ 3026 (CH aromatic), 2922 (CH aliphatic), 1678 (-N-
1
C=O), 1618 (C=C), 1546 (C=N); H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H,
pyrimidine H), 7.29-7.49 (m, 10H, ArH), 3.96 (m, 4H, piperazine), 3.64 (s, 2H, -
CH2CO-), 3.36 (m, 4H, piperazine), 3.01 (m, 4H, -CH2-CH2-CH2-), 2.45 (quint,
J=6.85 Hz, 2H, -CH2-CH2-CH2-); 13C NMR (101 MHz, CDCl3) δ 163.61, 159.17,

151.64, 141.23, 135.22, 130.45, 128.84, 127.61, 125.81, 124.04, 116.62, 58.19, 51.37,
46.81, 31.52, 29.82, 28.21; MS: (Mwt.: 469.6): m/z, 469.5 [M+, (9.55%)], 348.87
(100%); Anal. Calcd for C27H27N5OS: C, 69.06; H, 5.80; N, 14.91; Found: C, 69.09;
H, 5.70; N, 14.87.
5.1.2.5.
Naphthalen-2-yl-2-[4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4-yl)-piperazin-1-yl] acetate 15. White crystals, 60% yield; m.p. 123-125
⁰C; FT-IR (KBr, cm-1): υ 3016 (CH aromatic), 2926 (CH aliphatic), 1768 (-O-C=O),
1600 (C=C), 1561 (C=N); 1H NMR (400 MHz, CDCl3) δ 8.55 (s, 1H, pyrimidine H),
7.79-7.89 (m, 3H, ArH), 7.59 (s, 1H, ArH), 7.50 (m, 2H, ArH), 7.24 (m, 1H, ArH),
3.66 (s, 2H, -CH2COO-), 3.53-3.59 (m, 4H, piperazine), 2.87-2.95 (m, 8H, -CH2-CH2-
CH2-CH2- and piperazine), 1.80-1.99 (m, 4H, -CH2-CH2-CH2-CH2-); MS: (Mwt.:
458.6): m/z, 458.5 [M+, (13.3%)], 43.11 (100%); Anal. Calcd for C26H26N4O2S: C,
68.10; H, 5.71; N, 12.22; Found: C, 67.94; H, 5.84; N, 12.29.
5.1.2.6.
Phenyl-2-[4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)-
piperazin-1-yl] acetate 16. Yellow crystals, 65% yield; m.p. 113-115 ⁰C; FT-IR (KBr,
cm-1): υ 3020 (CH aromatic), 2937 (CH aliphatic), 1760 (-O-C=O), 1591 (C=C), 1558
(C=N); 1H NMR (400 MHz, CDCl3) δ 8.53 (s, 1H, pyrimidine H), 7.40 (t, J=7.58 Hz,
2H, ArH), 7.25 (t, J=7.58 Hz, 1H, ArH), 7.11 (d, J=7.82 Hz, 2H, ArH), 3.60 (s, 2H, -
CH2COO-), 3.52 (m, 4H, piperazine), 2.88-2.95 (m, 8H, -CH2-CH2-CH2-CH2- and
piperazine), 1.80-1.97 (m, 4H, -CH2-CH2-CH2-CH2-); 13C NMR (101 MHz, CDCl3) δ
161.88, 160.76, 151.91, 150.16, 136.83, 135.24, 129.23, 127.20, 126.11, 121.56,
115.93, 57.98, 52.06, 49.97, 26.53, 25.68, 22.65; MS: (Mwt.: 408.5): m/z, 408.5 [M+,
(14.29%)], 62.28 (100%); Anal. Calcd for C22H24N4O2S: C, 64.68; H, 5.92; N, 13.71;
Found: C, 64.83; H, 5.78; N, 13.98.
5.1.2.7.
N,N-Diphenyl-2-[4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4-yl)piperazin-1-yl] acetamide 17. Yellow crystals, 95% yield; m.p. 156-
157 ⁰C; FT-IR (KBr, cm-1): υ 3023 (CH aromatic), 2935 (CH aliphatic), 1680 (-N-
1
C=O), 1594 (C=C), 1556 (C=N); H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H,
pyrimidine H), 7.29-7.43 (m, 10H, ArH), 3.42 (m, 4H, piperazine), 3.21 (s, 2H, -
CH2CO-), 2.88 (m, 4H, piperazine), 2.73 (m, 4H, -CH2-CH2-CH2-CH2-), 1.77-1.95

(m, 4H, -CH2-CH2-CH2-CH2-); MS: (Mwt.: 483.6): m/z, 483.6 [M+, (1.66%)], 45.01
(100%); Anal. Calcd for C28H29N5OS: C, 69.54; H, 6.04; N, 14.48; Found: C, 69.81;
H, 6.27; N, 14.71.
5.1.3. General procedure for the preparation of compounds 13a-d and 19a-d
The aimed compounds 13a-d and 19a-d were synthesized by nucleophilic
addition of 4-(1-piperazinyl) thieno[2,3-d]pyrimidine derivatives 8a,b to isocyanate
and isothiocyanate derivatives [26]. In short, mixtures of intermediates 8a,b (3 mmol)
and aryl isocyanate, aryl isothiocyanate or ethyl isothiocyanate (4.5 mmol) were
refluxed in absolute ethanol (20 mL) for 3 h. The precipitated solid was cooled,
filtered, washed with cold water and dried under vacuum. Finally, the solid was
recrystallized from ethanol/acetone (3:1) to provide the titled compounds.
5.1.3.1.
N-Phenyl-4-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-
4-yl) piperazine-1-carboxamide 13a. Buff solid, 90% yield; m.p. 212-213 ⁰C; FT-IR
(KBr, cm-1): υ 3416 (NH), 3020 (CH aromatic), 2896 (CH aliphatic), 1633 (-N-C=O-
1
NH-), 1593 (C=C), 1554 (C=N); H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H,
pyrimidine H), 7.45 (d, J=8.07 Hz, 2H, ArH), 7.27 (t, J=7.83 Hz, 2H, ArH), 7.14 (s,
1H, NH D2O exchangeable), 7.02 (t, J=7.34 Hz, 1H, ArH), 3.78 (m, 4H, piperazine),
3.75 (m, 4H, piperazine), 3.01-3.08 (m, 4H, -CH2-CH2-CH2-), 2.49 (quint, J=7.09 Hz,
2H, -CH2-CH2-CH2-); 13C NMR (101 MHz, CDCl3) δ 161.81, 155.11, 148.71, 142.12,
138.86, 135.78, 128.99, 128.92, 123.61, 120.32, 116.49, 49.21, 44.02, 32.31, 29.87,
28.56; MS: (Mwt.: 379.5): m/z, 379.5 [M+, (7.56%)], 307.15 (100%); Anal. Calcd for
C20H21N5OS: C, 63.30; H, 5.58; N, 18.46; Found: C, 63.30; H, 5.48; N, 18.35.
5.1.3.2.
N-Phenyl-4-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-
4-yl) piperazine-1-carbothioamide 13b. Yellow solid, 97% yield; m.p. 142-144 ⁰C;
FT-IR (KBr, cm-1): υ 3416 (NH), 3023 (CH aromatic), 2926 (CH aliphatic), 1594
(C=C), 1551 (C=N), 1265 (-N-C=S-NH-); 1H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H,
pyrimidine H), 7.76 (s, 1H, NH D2O exchangeable), 7.32 (t, J=7.82 Hz, 2H, ArH),
7.20 (d, J=7.58 Hz, 2H, ArH), 7.14 (t, J=7.34 Hz, 1H, ArH), 4.02 (m, 4H,
piperazine), 3.72 (m, 4H, piperazine), 3.02 (m, 4H, -CH2-CH2-CH2-), 2.46 (quint,
J=7.15 Hz, 2H, -CH2-CH2-CH2-); MS: (Mwt.: 395.5): m/z, 395.3 [M+, (4.73%)],

269.2 (100%); Anal. Calcd for C20H21N5S2: C, 60.73; H, 5.35; N, 17.71; Found: C,
60.72; H, 5.32; N, 17.65.
5.1.3.3.
N-(4-Fluorophenyl)-4-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-
d]pyrimidin-4-yl) piperazine-1-carbothioamide 13c. Yellow solid, 92% yield; m.p.
188-190 ⁰C; FT-IR (KBr, cm-1): υ 3416 (NH), 3021 (CH aromatic), 2919 (CH
1
aliphatic), 1617 (C=C), 1546 (C=N), 1274 (-N-C=S-NH-); H NMR (400 MHz,
CDCl3) δ 8.43 (s, 1H, pyrimidine H), 7.79 (s, 1H, NH D2O exchangeable), 7.23 (dd,
J3=8.44, Hz J4=4.77 Hz, 2H, ArH), 7.01 (t, J=8.44 Hz, 2H, ArH), 4.09 (m, 4H,
piperazine), 3.79 (m, 4H, piperazine), 3.00-3.07 (m, 4H, -CH2-CH2-CH2-), 2.47
(quint, J=7.09 Hz, 2H, -CH2-CH2-CH2-); MS: (Mwt.: 413.5): m/z, 413.2 [M+,
(14.9%)], 269.2 (100%); Anal. Calcd for C20H20FN5S2: C, 58.09; H, 4.87; N, 16.94;
Found: C, 58.00; H, 4.55; N, 17.00.
5.1.3.4.
N-Ethyl-4-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-4-
yl) piperazine-1-carbothioamide 13d. White solid, 87% yield; m.p. 78-80 ⁰C; FT-IR
(KBr, cm-1): υ 3415 (NH), 2925 (CH aliphatic), 1544 (C=N), 1289 (-N-C=S-NH-); 1H
NMR (400 MHz, CDCl3) δ 8.47 (s, 1H, pyrimidine H), 5.93 (s, 1H, NH D2O
exchangeable), 4.08 (m, 4H, piperazine), 3.88 (m, 4H, piperazine), 3.73 (m, 2H, -NH-
CH2-CH3), 3.02-3.09 (m, 4H, -CH2-CH2-CH2-), 2.49 (quint, J=6.79 Hz, 2H, -CH2-
CH2-CH2-), 1.27 (t, J=7.34 Hz, 3H, -NH-CH2-CH3); MS: (Mwt.: 347.5): m/z, 347.2
[M+, (4.3%)], 43.0 (100%); Anal. Calcd for C16H21N5S2: C, 55.30; H, 6.09; N, 20.15;
Found: C, 55.20; H, 6.02; N, 19.89.
5.1.3.5.
N-Phenyl-4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)
piperazine-1-carboxamide 19a. Buff solid, 78% yield; m.p. 217-219 ⁰C; FT-IR (KBr,
cm-1): υ 3419 (NH), 3020 (CH aromatic), 2941 (CH aliphatic), 1633 (-N-C=O-NH-),
1594 (C=C), 1560 (C=N); 1H NMR (400 MHz, CDCl3) δ 8.56 (s, 1H, pyrimidine H),
7.38 (d, J=7.58 Hz, 2H, ArH), 7.31 (t, J=7.70 Hz, 2H, ArH), 7.07 (t, J=7.09 Hz, 1H,
ArH), 6.49 (s, 1H, NH D2O exchangeable), 3.69 (m, 4H, piperazine), 3.48 (m, 4H,
piperazine), 2.88-2.99 (m, 4H, -CH2-CH2-CH2-CH2-), 1.82-1.99 (m, 4H, -CH2-CH2-
CH2-CH2-); MS: (Mwt.: 393.5): m/z, 393.2 [M+, (11.39%)], 268 (100%); Anal. Calcd
for C21H23N5OS: C, 64.10; H, 5.89; N, 17.80; Found: C, 64.37; H, 5.75; N, 17.67.

5.1.3.6.
N-Phenyl-4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)
piperazine-1-carbothioamide 19b. Yellow solid, 95% yield; m.p. 140-142 ⁰C; FT-IR
(KBr, cm-1): υ 3416 (NH), 3021 (CH aromatic), 2926 (CH aliphatic), 1597 (C=C),
1
1556 (C=N); 1269 (-N-C=S-NH-), H NMR (400 MHz, CDCl3) δ 8.53 (s, 1H,
pyrimidine H), 7.43 (s, 1H, NH D2O exchangeable), 7.36 (t, J=7.83 Hz, 2H, ArH),
7.14-7.19 (m, 3H, ArH), 3.99 (m, 4H, piperazine), 3.51 (m, 4H, piperazine), 2.88-2.93
(m, 4H, -CH2-CH2-CH2-CH2-), 1.79-1.97 (m, 4H, -CH2-CH2-CH2-CH2-); MS: (Mwt.:
409.6): m/z, 409.2 [M+, (18.45%)], 362.9 (100%); Anal. Calcd for C21H23N5S2: C,
61.58; H, 5.66; N, 17.10; Found: C, 61.80; H, 5.61; N, 17.36.
5.1.3.7.
N-(4-Fluorophenyl)-4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4-yl) piperazine-1-carbothioamide 19c. Buff solid, 90% yield; m.p. 184
⁰C; FT-IR (KBr, cm-1): υ 3416 (NH), 3019 (CH aromatic), 2909 (CH aliphatic), 1612
(C=C), 1556 (C=N); 1268 (-N-C=S-NH-), 1H NMR (400 MHz, CDCl3) δ 8.52 (s, 1H,
pyrimidine H), 7.45 (s, 1H, NH D2O exchangeable), 7.18 (dd, J3=8.68, Hz J4=4.77
Hz, 2H, ArH), 7.04 (t, J=8.56 Hz, 2H, ArH), 3.99-4.06 (m, 4H, piperazine), 3.48-3.56
(m, 4H, piperazine), 2.87-2.94 (m, 4H, -CH2-CH2-CH2-CH2-), 1.79-1.98 (m, 4H, -
CH2-CH2-CH2-CH2-); 13C NMR (101 MHz, CDCl3) δ 183.98, 168.59, 161.51, 159.18,
151.36, 136.05, 135.89, 126.81, 126.19, 126.10, 121.47, 116.05, 115.82, 49.92, 48.58,
26.73, 25.84, 22.94, 22.77; MS: (Mwt.: 427.56): m/z, 427.13 [M+, (22.63%)], 356.13
(100%); Anal. Calcd for C21H22FN5S2: C, 58.99; H, 5.19; N, 16.38; Found: C, 58.71;
H, 5.42; N, 16.25.
5.1.3.8.
N-Ethyl-4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)
piperazine-1-carbothioamide 19d. Off white solid, 80% yield; m.p. 177-179 ⁰C; FT-
IR (KBr, cm-1): υ 3420 (NH), 2930 (CH aliphatic), 1558 (C=N), 1294 (-N-C=S-NH-);
1H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H, pyrimidine H), 5.55 (s, 1H, NH D2O
exchangeable), 4.00 (m, 4H, piperazine), 3.75 (m, 2H, -NH-CH2-CH3), 3.51 (m, 4H,
piperazine), 2.89-2.94 (m, 4H, -CH2-CH2-CH2-CH2-), 1.79-1.96 (m, 4H, -CH2-CH2-
CH2-CH2-), 1.28 (t, J=7.34 Hz, 3H, -NH-CH2-CH3); MS: (Mwt.: 361.5): m/z, 361.2
[M+, (20.3%)], 289 (100%); Anal. Calcd for C17H23N5S2: C, 56.48; H, 6.41; N, 19.37;
Found: C, 56.66; H, 6.49; N, 19.51.

5.1.4. Synthesis
of
1-Phenyl-2-[4-(5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4-yl)piperazin-1-yl] ethenone 18
Compound 18 was provided by nucleophilic substitution of bromo of phenacyl
bromide with 4-(piperazin-1-yl)-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidine
8b . Potassium carbonate (6.66 mmol, 0.92 g) was added to a solution of intermediate
8b (6.66 mmol, 1.83 g) and phenacyl bromide (6.66 mmol, 1.33 g) in DMF (30 mL).
The reaction mixture was stirred for 36 h at room temperature, then added to water
(150 mL) to afford a precipitate which was filtered under vacuum and allowed to dry.
The solid residue was recrystallized from ethyl acetate to impart 18. Yellow solid,
60% yield; m.p. 143-145 ⁰C; FT-IR (KBr, cm-1): υ 3028 (CH aromatic), 2921 (CH
1
aliphatic), 1694 (Ar-C=O), 1619 (C=C), 1551 (C=N); H NMR (400 MHz, CDCl3) δ
8.53 (s, 1H, pyrimidine H), 8.03 (d, J=7.34 Hz, 2H, ArH), 7.58 (t, J=7.34 Hz, 1H,
ArH), 7.48 (t, J=7.34 Hz, 2H, ArH), 3.91 (s, 2H, -CH2-CO), 3.52 (m, 4H, piperazine),
2.87-2.94 (m, 4H, -CH2-CH2-CH2-CH2-), 2.81 (m, 4H, piperazine), 1.79-1.97 (m, 4H,
-CH2-CH2-CH2-CH2-); MS: (Mwt.: 392.5): m/z, 392.2 [M+, (8.12%)], 286.26 (100%);
Anal. Calcd for C22H24N4OS: C, 67.32; H, 6.16; N, 14.27; Found: C, 67.59; H, 6.29;
N, 14.45.
5.2.
Biological evaluation
5.2.1. Measurement of cytotoxicity against PANC-1 cell line
The ability of newly synthesized compounds to inhibit the growth of human
pancreatic carcinoma PANC-1 cell line (Merck KGaA, Darmstadt, Germany #
87092802), was assessed using the method of Skehan and compared with imatinib
(Merck KGaA) [27]. Exponentially expanding cells were gathered and seeded into 96-
well microtiter plates at the proper concentrations (1000-2000 cells/ 0.33 cm2 well).
Afterward, plates were incubated at 37 ⁰C in a moistened atmosphere for 24 h.
Subsequently, cells were incubated with ascending concentrations of the tested
synthesized compounds dissolved in DMSO (0.01, 0.1, 1, 10 & 100 μM) for 72 h.
Following incubation, 10% trichloroacetic acid (150 μL/well) was used to fix the cells
at 4 ⁰C for 1 h followed by washing with water 3 times at least. Later, 0.4%
SulfoRhodamine-B (SRB) dissolved in 1% acetic acid (70 μL/well) was used to stain

cells at room temperature. Wells were rinsed with extra 1% acetic acid to eliminate
unbound dye. Plates were then air dried for 24 h and kept away from UV radiation.
Tris base 10 mM, (pH=7.4) (150 μL/well) was added to solubilize the dye on a shaker
at 1600 rpm for 5 min. ELISA micro plate reader (ChroMate-4300, Florida-US) was
used to measure the optical density at 564 nm and IC50 values were determined using
sigmoidal concentration response curve fitting models (Sigma plot software).
5.2.2. Measurement of inhibitory activity of compound 9d against PDGFR-α
Compound 9d was evaluated against PDGFR-α using HTScanTM PDGFR-α
Kinase Assay Kit according to Cell Signaling Technology's instructions. 10 µL of
ATP (10 mM) and 1.25 mL of substrate peptide (6 µM) were mixed to prepare 2X
ATP/substrate cocktail ([substrate]= 3µM, [ATP]= 40 µM). Afterward, DTT/Kinase
buffer was prepared and sent to enzyme tube creating 4X reaction cocktail
([enzyme]= 0.8 Units/µL). 12.5 µL/well of prediluted compound 9d (dissolved in
DMSO), 12.5 µL of the 4X reaction cocktail, and 2X ATP/substrate cocktail were
incubated together at room temperature for 30 minutes. The reaction was stopped,
diluted with D2O and moved to a 96-well Streptavidin-coated plate to be incubated at
room temperature for 1 h with primary antibody and Phospho-Tyrosine Monoclonal
Antibody (P-Tyr-100) #9411, 1:1000 in PBS/T. Similarly, europium labeled anti-
mouse IgG 1:500 in PBS/T was incubated with the reaction mixture at room
temperature for 30 minutes. At last, Time-Resolved Plate Reader was used to detect
615 nm Fluorescence emission.
5.3.
Molecular Modelling
Molecular modelling was performed by Schrodinger computational software
using Maestro 10.1 at Faculty of Pharmacy, Suez Canal University (Complete
experiment are available in Supplementary information).
Acknowledgments
The authors are very grateful for the holding company for biological products
and vaccines VACSERA (Cell Culture Unit) for performing the in vitro cytotoxic
activity against PANC-1 cell line and PDGFR-α inhibition assay. The authors have
declared no conflict of interests.

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Tables and Figures
Table 1: In vitro cytotoxic activity of the synthesized compounds on pancreatic
cancer cell line PANC-1.
Cpd.
IC50 (µM) Cpd. IC50 (µM)
Amuvatinib
3.00 [10]
46.31
13d
14a
14b
14c
14d
14e
14f
15
31.57
71.64
>100.00
15.39
31.91
>100
Imatinib
9a
65.59
9b
>100.00
60.21
9c
9d
5.40
9e
44.50
35.98
70.11
52.12
39.49
74.39
33.23
14.41
17.82
29.56
9f
>100.00
>100.00
95.15
10a
10b
11
16
17
88.97
18
12
74.74
19a
19b
19c
19d
13a
13b
13c
33.76
13.49
15.78

Table 2a: Assay and growth curve of highest active compound 9d.
Conc.
0
Mean
1.62
1.17
1.13
1.06
1.02
1.00
SD
Viability
100.00
72.04
9d
120
100
80
60
40
20
0
1.59
1.17
1.11
1.09
1.02
1.00
1.65
1.62
1.17
1.18
1.04
1.02
0.99
0.03
0.00
0.04
0.02
0.01
0.02
Cpd. 9d
IC50 = 5.4
6.25
12.5
25
1.17
1.12
1.04
1.01
1.03
70.04
65.29
50
62.65
0
25
50
75
100
Conc. µg/well
100
61.93
Table 2b: Assay and growth curve of lowest active compound 14e.
Conc.
14e
Mean
SD
Viability%
120
100
80
60
40
20
0
Cpd. 14e
IC50 = 154.6
0
1.91
1.60
1.51
1.55
1.39
0.48
1.88
1.70
1.51
1.38
1.35
0.77
1.81
1.53
1.50
1.40
1.41
0.60
1.87
1.61
1.51
1.44
1.38
0.61
0.05
0.08
0.00
0.09
0.03
0.13
100.00
86.29
80.75
77.35
73.96
32.87
6.25
12.5
25
50
0
25
50
75
100
100
Conc. µg/well

Table 3: Results of ADME study for the synthesized compounds in comparison with amuvatinib and imatinib.
% Human
Comp.
No.
QPP
Mol-MWa DonorHBb AcceptHBc QPlogPo/wd #rotore PSAf QPlogSg QPlogHERGh QPPCacoi
QPlogKhsak Oral
MDCKj
Absorption l
Amuvatinib 447.511
1
2
1
1
1
1
1
1
7
4.736
3.595
5.045
5.349
5.688
5.897
4.67
2
7
3
3
3
3
3
3
72.397 -6.331 -6.298
87.981 -4.665 -9.075
42.898 -6.275 -6.978
41.484 -6.697 -7.076
22.918 -6.525 -7.083
23.303 -6.896 -6.941
24.258 -5.469 -5.867
41.497 -5.873 -7.085
3773.205 4115.661 0.634
76.297 37.517 0.53
100
Imatinib
9d
493.61
10.5
5.5
5.5
5
81.69
96.302
100
425.975
440.002
421.617
439.608
373.573
405.557
888.752 2151.993 0.928
888.566 2058.698 1.063
1944.678 4143.099 1.117
1954.348 7498.889 1.152
1921.707 4010.873 0.789
891.907 837.201 0.943
14c
19b
100
19c
5
100
19d
5
100
19a
5.5
4.844
100
Acceptable Ranges:
a
b
f
c
g
k
d
<500amu;
0-15;
<5;
7-200;
<25 poor, >500 great;
<10;
< 0.5;
-1.5 – 1.5;
<5;
< - 5;
e
h
i
j
l
<25 poor, >500 great;
>80% is high, <25% is poor.