Discovery and optimization of pyrrolo[1,2-a]pyrazinones leads to novel and selective inhibitors of PIM kinases

Article abstract of DOI:10.1016/j.bmc.2013.09.054

A novel series of PIM inhibitors was derived from a combined effort in natural product-inspired library generation and screening. The novel pyrrolo[1,2-a]pyrazinones initial hits are inhibitors of PIM isoforms with IC₅₀ values in the low microm

Full text of DOI:10.1016/j.bmc.2013.09.054

Accepted Manuscript
Discovery and Optimization of Pyrrolo[1,2-a]Pyrazinones Leads to Novel and
Selective Inhibitors of PIM Kinases
Francesco Casuscelli, Elena Ardini, Nilla Avanzi, Elena Casale, Giovanni Cervi,
Matteo D’Anello, Daniele Donati, Daniela Faiardi, Ronald D. Ferguson,
Gianpaolo Fogliatto, Arturo Galvani, Aurelio Marsiglio, Danilo G. Mirizzi,
Marisa Montemartini, Christian Orrenius, Gianluca Papeo, Claudia Piutti,
Barbara Salom, Eduard R. Felder
PII:
S0968-0896(13)00834-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.09.054
BMC 11138
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 July 2013
13 September 2013
20 September 2013
Please cite this article as: Casuscelli, F., Ardini, E., Avanzi, N., Casale, E., Cervi, G., D’Anello, M., Donati, D.,
Faiardi, D., Ferguson, R.D., Fogliatto, G., Galvani, A., Marsiglio, A., Mirizzi, D.G., Montemartini, M., Orrenius,
C., Papeo, G., Piutti, C., Salom, B., Felder, E.R., Discovery and Optimization of Pyrrolo[1,2-a]Pyrazinones Leads
to Novel and Selective Inhibitors of PIM Kinases, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/
10.1016/j.bmc.2013.09.054
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Discovery and Optimization of Pyrrolo[1,2-
a]Pyrazinones Leads to Novel and Selective
Inhibitors of PIM Kinases
Leave this area blank for abstract info.
Francesco Casuscelli, Elena Ardini, Nilla Avanzi, Elena Casale, Giovanni Cervi, Matteo D’Anello, Daniele
Donati, Daniela Faiardi, Ronald D. Ferguson, Gianpaolo Fogliatto, Arturo Galvani, Aurelio Marsiglio, Danilo
G. Mirizzi, Marisa Montemartini, Christian Orrenius, Gianluca Papeo, Claudia Piutti, Barbara Salom and
Eduard R. Felder
O
H
O
N
H
O
N
H
N
N
O
N
N
Br
S
H
N
CO 2H
Cl
NH
Br
O
N
N
15a
1
PIM1 IC50 = 3nM
PIM2 IC50 = 73nM
PIM3 IC50 = 12nM
PIM1 IC50 = 1.441 µM
Longamide B

Bioorganic & Medicinal Chemistry
journal hom epage: www.elsevier.com
Discovery and Optimization of Pyrrolo[1,2-a]Pyrazinones Leads to Novel and
Selective Inhibitors of PIM Kinases
Francesco Casuscellia,∗ Elena Ardinia, Nilla Avanzia, Elena Casalea, Giovanni Cervia, Matteo D’Anelloa,
Daniele Donatia, Daniela Faiardia, Ronald D. Fergusona†, Gianpaolo Fogliattoa, Arturo Galvania, Aurelio
Marsiglioa, Danilo G. Mirizzia, Marisa Montemartinia, Christian Orreniusa, Gianluca Papeoa, Claudia
Piuttia, Barbara Saloma, Eduard R. Feldera
aOncology, Nerviano Medical Sciences, viale Pasteur 10, 20014, Nerviano (MI) I
† Current address: Merck & Co., Inc., 126 E. Lincoln Ave., Rahway, NJ 07065
ARTICLE INFO
ABSTRACT
Article history:
Received
A novel series of PIM inhibitors was derived from a combined effort in natural product-inspired
library generation and screening. The novel pyrrolo[1,2-a]pyrazinones initial hits are inhibitors
of PIM isoforms with IC50 values in the low micromolar range. The application of a rational
optimization strategy, guided by the determination of the crystal structure of the complex in the
kinase domain of PIM1 with compound 1, led to the discovery of compound 15a, which is a
potent PIM kinases inhibitor exhibiting excellent selectivity against a large panel of kinases,
representative of each family. The synthesis, structure–activity relationship studies, and
pharmacokinetic data of compounds from this inhibitor class are presented herein. Furthermore,
the cellular activities including inhibition of cell growth and modulation of downstream targets
are also described.
Received in revised form
Accepted
Available online
Keywords:
PIM kinases
Natural Product Scaffold
PyrroloPyrazinone
Kinase selectivity
Anti-cancer agents
2013 Elsevier Ltd. All rights reserved.
1. Introduction
The gene family of PIM (Proviral Integration site of mouse Moloney leukemia virus) consists of three members PIM1, PIM2 and
PIM3, encoding for serine/threonine-specific kinases belonging to the group of CAMK protein kinases.1 PIM kinases are highly
homologous to each other and have partially overlapping functions and expression patterns. Unlike most other protein kinases, PIM
kinases are constitutively active enzymes, whose activity is largely regulated at the level of expression and stability. In hematopoietic
cells, expression of PIM kinases is stimulated by numerous growth factors and cytokines such as interleukins.2 The Pim-1 gene has been
———
∗ Corresponding author. Tel.: +39 0331-581326; fax: +39 0331-581347; e-mail: francesco.casuscelli@nervianoms.com

found to function as a proto-oncogene. When overexpressed it induces lymphomas in transgenic mice even though by itself, Pim1 is not
a strong oncogene. Only when expressed with other oncogenic proteins, particularly those involved in proliferation and cell survival
such as c-Myc, it exerts a potent synergistic irreversible transforming effect.3,4 PIM kinases have been found to phosphorylate a number
of proteins involved in proliferation, cell cycle progression and apoptosis resistance. Notably, PIMs phosphorylate and inactivate the
pro-apoptotic protein BAD. PIMs were found to be involved in c-Myc transcription, both through phosphorylation of c-Myc, which
increases c-Myc stability, as well as phosphorylation of promoter histones in c-Myc target genes.5 Subsequent to the first publication of
PIM1 in complex with an inhibitor,6 a significant amount of structural information has been reported,7 as well as a number of
inhibitors.2b,8 The structures revealed a novel hydrogen bonding pattern in the hinge region, where the presence of a proline residue
prevents the formation of sequential hydrogen donor-acceptor interactions with hinge binders, typical for other kinases. After a first
series of inhibitors culminating with the clinical trial of the multikinase inhibitor SGI-1776,9 a second, more selective generation
emerged. Generally, the trend reported in the literature for PIM inhibitors reveals that PIM3 inhibition goes along with PIM1 and
selectivity is observed over PIM2.10,11 The increasing number of patents and publications dealing with PIM kinases indicates an intense
interest to develop potent and selective inhibitors of PIM kinases.2,5 The major challenge in this field is to increase the cellular potency
by inhibiting not only PIM1 but more importantly PIM2. Therefore, the new generation of PIM inhibitors must aim at a high potency on
PIM2 to ensure an irreversible cellular fate.11 Moreover, the numerous interactions and signaling pathways in which PIM1 is involved
open many possibilities for combination approaches with tyrosine kinase inhibitors,12 PI3K/AKT pathway inhibitors13 or direct pro-
apoptotic stimulators14 to further enhance responses and to limit resistance and toxicity.9 As part of our search for novel anticancer
drugs, one of our goals is the design of pan-PIM inhibitors with the ability to suppress the enzymatic activity of all three PIM kinases,
while maintaining good selectivity towards all other kinases. Ultimately, we aim to identify inhibitors with adequate in vivo properties
to achieve a desirable pharmacodynamic response resulting in tumor growth inhibition. In this paper we disclose the synthesis and
biological evaluation of a series of pyrrolo[1,2-a]pyrazinone derivatives leading to the discovery of single digit nanomolar PIM
inhibitors. Heterocyclic compounds containing the pyrrolo[1,2-a]pyrazinone moiety occur in a number of natural products of marine
sponge origin, generally known as pyrrole alkaloids.15 This intriguing chemical architecture attracted the attention of many researchers
engaged in total synthesis of natural products,16 however to date, its application as a scaffold in medicinal chemistry programs is
underappreciated. On the other hand, natural product-inspired compounds provide a rich source of novel scaffolds for drug discovery
possessing interesting properties such as asymmetric and sp3-rich molecular templates, which might be advantageous in exploring
demanding biological targets. 17,18
Table 1. Properties of compound 1
Cmpd
1
PIM1 IC50a µM
1.441
Others Kinasesb µM
>10.00
HLMc
81
PAMPAd
48
cLogPe
5.2
LEf
LLE g
0.65
Fsp3h
0.44
Sol. µg/mli
68
0.34
a IC50 value is shown as the mean of duplicate experiments. b tested on a panel of 56 kinases17 [ATP] = 2Km, Bcr-Abl, ACK1, AKT1, ALK, Aur1, Aur2, BUB1,
BRK, CDC7/DBF4, CDK2/CYCA, CHK1, CK2, EEF2K, EGFR1, ERK2, EphA2, FAK, FGFR1, FLT3, GSK3β, Haspin, IGFR1, IKK2, IR, JAK1, JAK2, JAK3,
KIT, LCK, LYN, MAPKAPK2, MELK, c-MET, MNK2, MPS1, MST4, NEK6, NIM1, P38α, PAK4, PDGFR, PDK1, PERK, PKAα, PKCβ, PLK1, RET, ROS1,
SULU1, Syk, TRKA, TLK2, TYK2, VEGFR2, VGFR3, ZAP70. cIntrinsic Clearance Human Liver Microsomes (ml/min/kg). d Parallel artificial membrane
permeability assay, apparent permeability Papp (x10-6cm/s). eCalculated logarithm of lipophilicity using ACD/Labs 12.0. fLigand Efficiency LE = -
g
1.4log(IC50)/HAC; HAC = heavy atom count. Lipophilic Ligand Efficiency LLE = -Log(IC50)-cLogP.22 hFsp3=(number of sp3 hybridized carbons/total carbon
count). i Thermodynamic Solubility pH 7.
2. Design and synthesis
2.1. Hit Identification
Compound libraries based on the pyrrolo[1,2-a]pyrazinone scaffold, a molecular skeleton embedded in alkaloids such as Longamide
B,19 were prepared, aimed at the development of a biologically active chemotype within a chemical space suitable for drug discovery20
and compatible with binding the active site of kinases. As a first step, to efficiently screen for attractive starting points in kinase
inhibition programs a diversified expansion was prepared in a combinatorial fashion. Promising PIM kinase inhibition was observed

within an array of sixty compounds prepared performing Suzuki, Heck and Sonogashira cross-coupling reactions (for R1 diversification)
and amides coupling with aliphatic amines (for R2 modification).20 The identification of initial hits was achieved by profiling the library
in a panel of 56 kinases for a selectivity screening campaign, which afforded the PIM1 kinase inhibitor pyrrolo[1,2-a]pyrazinone 1 with
an IC50 value of 1.441 μM (Fig. 1).
O
O
1
H
H
O
2
Br
N
8
N
3
7
NH
N
N
N
R1
4
Br
5
6
S
H
N
H
N
CO2H
O
R2
O
(-)-Longamide B
PIM1 IC50 = >10.0 µM
1
Library scaffold
PIM1 IC50 = 1.441 µM
Figure 1. Scaffold and library design
Although compound 1 demonstrated good inhibition of the PIM1 enzyme and excellent selectivity with an ATP competitive
mechanism, it suffered from suboptimal drug-like properties including high lipophilicity and low metabolic stability that resulted in
modest cell-based activity, (Table 1). However, 1 showed both a sufficient molecular complexity (as measured by Fsp3)21 and a
stereocenter, features frequently encountered in promising compounds for drug discovery.
To further increase our understanding of the inhibitor series binding mode, and to guide its optimization, a crystal structure of the
complex of the kinase domain of PIM1 with compound 1 was determined to 1.9 Å resolution (Fig. 2A).
A
B
Figure 2. A. The 1.9 Å resolution crystal structure of compound 1 bound to PIM1 (PDB code 4bzn).23
Diagrams showing the 2Fo-Fc electron density map associated with inhibitor 1 contoured at the 1σ level. The inhibitor is shown with yellow carbon atoms and
the protein is shown with green carbon atoms. B Transparent surface representation of the structure with a close-up of the ATP binding site as seen from the
solvent. All visualizations were captured with PyMol.24

Compound 1 occupies the ATP pocket without forming the canonical pattern of multiple hydrogen bonds with the unique kinase
hinge region sequence, due to the presence of Pro123 that prevents such interaction.7c The sole interaction with the hinge is between the
backbone carbonyl of Glu121 and one aromatic hydrogen of the thiophene ring.25 As already observed for several PIM1 inhibitors, the
apparent features in the binding of 1 are the polar interactions that the compound establishes with the conserved residues Lys67 and
Asp186.7d In fact, the carbonyl oxygen of the lactam ring of 1 is hydrogen bonded with Lys67 and makes a water-mediated interaction
with Glu89, while the amide nitrogen forms a hydrogen bond with Asp186. In addition, the glycine-rich loop Phe49 adopts the ‘turned
in’, inactive6c, 7 conformation that forms favorable hydrophobic contacts with the inhibitor. The nitrogen of the amide tethered to
position 4 establishes a water-mediated hydrogen bond with the sugar pocket Asp128 side chain and the backbone carbonyl of Glu171.
Further, hydrophobic interactions occur between the neopentyl moiety and the hydrophobic patch made up by the residues of Leu44,
Val126, and Leu174. Interestingly, preliminary SAR data indicated that the absence of the neopentyl hydrophobic group or its
substitution with smaller alkyl groups caused significant loss of activity,26 thus revealing the importance of lipophilic substituents in this
part of the site (the hydrophobic channel). This requirement, besides the lack of polar hinge-region interactions, underlines the key role
of hydrophobic contributions in PIM1 ligand binding. It is worth noting that, although the crystallization experiment was performed
with a racemic mixture of compound 1, the electron density relative to the inhibitor is well defined and reveals that only one of the two
enantiomers, presumably the most active, is bound. From the structure, it was assumed that the preferred absolute stereochemistry was
(S).
These initial structural data revealed how diverse compounds can satisfy the unusual binding requirements of PIM1. Combined with
the modular nature of the pyrrolo[1,2-a]pyrazinone scaffold synthesis, this data provided the foundation for the optimization of this
class towards inhibitory effects on PIM targets.
2.2. Chemistry
Although our targeted scaffold is reminiscent of Longamide B in its core constitution, the absence of an activating bromine atom on
the pyrrole ring called for the application of a different preparation process than our first published scheme in the natural product
synthesis.16b A more recently developed approach27 seemed more suitable to cope with the negative effects of the missing bromine on
the intramolecular aza-Michael reaction yield. Consequently, the preparation of our pyrrolo[1,2-a]pyrazinone scaffold follows the steps
outlined in Scheme 1.
The treatment of bromo crotonate 2a with diformyl imide sodium salt afforded 2b in high yields; further deprotection of the imide to
obtain the trifluoroacetyl ammonium salt 2c was accomplished in the presence of a mixture of TFA and absolute EtOH, under reflux.
Successive coupling reaction of commercially available pyrrole 3 with the aminocrotonate 2c in the presence of DIPEA delivered amide
4, which smoothly cyclized in the presence of a catalytic amount of DBU, leading to the pyrrolo[1,2-a]pyrazinone skeleton 5 through a
regioselective intramolecular aza-Michael reaction. Hydrolysis of the ester moiety gave the acid 6. Compounds 5 and 6 were used
respectively as starting materials for the solution and the solid-phase synthesis approach of scaffold expansion.
Scheme 1. Synthesis of pyrrolo[1,2-a]pyrazinone scaffold
O
-
CF3CO2
O
O
O
a
I
b
+
NH3
N
Br
O
O
O
O
2a
2b
2c
I
I
O
I
O
O
N
N
N
c
d
e
O
H
NH
NH
NH
N
H
CCl3
O
OH
O
O
3
5
6
4
O
O
Scheme 1. Reagents and conditions (a) Na+(OHC)2N-, NaI, CH3CN, reflux, 18 h, 96%; (b) TFA, EtOH, 90°C, 18 h; (c) amine 2c, DIPEA, CH3CN, rt, 18 h,
71%; (d) DBU(cat), CH3CN, 1h, 96%; (e) LiOH, THF, MeOH, H2O, rt, 18h 70%.

In order to explore the pyrrolo[1,2-a]pyrazinone core as novel and unusual kinase inhibitor scaffold, two diversity points were
elaborated: the carbon 7 of the pyrrole ring (R1), and the amido-group (R2) as shown in Figure 1. To follow this expansion strategy, an
initial focused library of compounds was selected from a carefully designed virtual library and was synthesized using solid-phase
chemistry.
The general route for the decoration of pyrrolo[1,2-a]pyrazinone is shown in scheme 2. The synthesis started with the reductive
amination of polymer-supported aldehyde 7 with the selected amines. Subsequently, these resulting secondary amines 8 were coupled
with the 7-iodo-pyrrole carboxylic acid 6 in the presence of HBTU as activating agent to yield the corresponding amides 9. A Suzuki
coupling between the iodo-pyrrole and the boronic esters or acids gave the 7-aryl pyrrolo[1,2-a]pyrazinone derivatives, which were
finally cleaved from the resin under acid conditions to afford an array of compounds including derivatives 1 and 10.
Scheme 2. Solid-Phase synthesis of pyrrolo[1,2-a]pyrazinone derivatives
R1
I
Pol
O
O
O
O
Pol
Pol
O
O
a
b
N
c, d
O
N
NH
NH
HN
R2
O
O
O
O
N
HN
R2
R2
7
8
9
1 R1 = thiophen-3-yl;
R2 = neopentyl
10a-h
Scheme 2. Reagents and conditions Pol is for butyryl aminomethyl resin (copolystyrene-1%DVB) (a) R2NH2, NaBH(AcO)3, AcOH, THF, rt, 20 h; (b) 6, HBTU,
DIPEA, DMF, rt, 24 h; (c) PdCl2(dppf)*CH2Cl2, R1-B(OH)2, Cs2CO3, DME, 80°C, 6 h; (d) TFA:CH2Cl2, 1:1, 1h 2 cycles.
Alternatively, a solution phase approach was also performed. Differently to the solid-phase approach, the cross-coupling reaction
was initiated as reported in Scheme 3. Thus, Suzuki reaction on ester 5 delivered compounds 11, which were quantitatively converted to
the corresponding acids 12 in the presence of LiOH in a solution of THF/MeOH/water at room temperature. Finally, coupling with
selected amines afforded the final racemic amides 13.
Scheme 3. Synthesis of pyrrolo[1,2-a]pyrazinone derivatives
R1
R1
I
R1
O
O
O
O
a
b
c
N
N
N
N
NH
NH
NH
NH
NH
R2
O
O
5
O
O
OH
O
O
11a R1= Ph-m-CF3
12a R1= Ph-m-CF3
13a-l table 3;
14a-i table 4;
15a-d table 5;
16a-c table 5;
11b R1= Ph-m-F
12b R1= Ph-m-F
11c R1= Ph-3,4-F
12c R1= Ph-3,4-F
11d R1= 4-(2-F-Pyridyl)
12d R1= 4-(2-F-Pyridyl)
12e R1= Ph-m-Cl
11e R1= 3-(1-boc-pyrazole)
11f R1= Ph-m-Cl
12f R1= Ph-5-Cl-2-F
11g R1= Ph-5-Cl-2-F
Scheme 3. Reagents and conditions (a) PdCl2(dppf)*CH2Cl2, R1-B(OH)2, Cs2CO3, DME, 80°C, 6 h; (b) LiOH, THF, MeOH, H2O, rt, 4h, (c) R2NH2, HBTU,
DIPEA, dioxane, 60°C, 6 h.
Advanced exploration of the two diversity points on the pyrrolo[1,2-a]pyrazinone scaffold was planned with different aromatic
moieties (R1) and with chiral amines (R2) synthesized for the purpose. The strategy of performing first the Suzuki step and then the
coupling with the (S) chiral amines leading to 14 as a pair of diastereoisomers (4R,S) and (4S,S). This approach largely focused upon an
amide-diversity investigation. The resulting diastereoisomers 14, 15 or 16 were separated by standard chromatography techniques.
Suitable optically active amines 20 were prepared in three steps from the corresponding chiral N-Boc amino alcohols in a
stereoconservative fashion, as shown in Scheme 4.
Scheme 4. Synthesis of chiral amines

R3
R3
R3
R3
O
O
O
a
b
c
R4
R4
O
N
O
N
O
N
H2N
2.HCl
20
H
H
H
OH
O
S
O
O
17
18
19
20a R3 = Cyclohexyl, R4= N-Me-piperazine
20b R3= t-But, R4= N-Me-piperazine
20c R3= Me, R4= N-Me-piperazine
20d R3 = Cyclohexyl, R4= 4-dimethylamino
piperidine
Scheme 4. Reagents and conditions (a) MsCl, TEA, CH2Cl2, -10°C- 0°C, 2 h; (b) amines (R4), THF, 70°C, 8 h; (c) HCl 4M in dioxane , rt, 24 h
3. Results and Discussion
In a kinase specificity screening of a natural-product-inspired compound library the pyrrolo[1,2-a]pyrazinone compound 1 was
identified as a PIM1 inhibitor suitable as a starting point for lead identification. Compound 1 does not form the classical pair or triplet
of hydrogen bonds with the kinase hinge region. It may therefore be considered as an ATP competitive, non ATP mimetic inhibitor. The
high selectivity observed within this chemical class across the kinase panel is therefore not too surprising.
Analysis of the crystal structure of PIM1 bound to compound 1, as previously observed, indicated that hydrophobic interactions play
an essential role in the binding of this enzyme. Given the fact that induced fit or stereoelectronic effects of large hydrophobic groups are
not trivial to predict, we decided to probe the PIM1 active site with a series of reasonably sized substituents in both R1 and R2 (see
compounds 10 Scheme 2). Initial modification of the hinge-binding region, while maintaining the neopentyl amide substituent of
compound 1, allowed us to explore the SAR of different aryl and heteroaryl R1 substituents on the pyrrole ring 10 (Table 2).
In reviewing the PIM1 biochemical binding data for this series, a certain preference for phenyl groups possessing hydrophobic meta-
substituents appeared, and compound 10f resulted the most interesting. Moving the bulky substituent from meta to the para-position
killed the activity of 10h. Noteworthy, compound 10b was poorly active on PIM1 (5.471 µM), and showed also a modest inhibition of
GSK3β (4.608 µM), indicating that the presence of a typical hinge binder such as the pyridine ring allowed interaction with additional
kinases.
Table 2. Neopentylamides (racemic) (IC50, µM)a
O
R1
NH
N
H
N
O
Cmpd
1
R1
PIM1
1.441
2.544
5.471
2.548
1.716
1.937
0.521
1.275
>10
thiophen-3-yl
10a
10b
10c
10d
10e
10f
10g
10h
phenyl
pyridin-4-yl
4-fluorophenyl
2-fluorophenyl
3-fluorophenyl
3-trifluoromethylphenyl
3-trifluoromethoxyphenyl
4-trifluoromethoxyphenyl
aThe ATP concentrations used in the assays were 2·Km. IC50 values are an average of two or more determinations.

Following these preliminary results, we further explored the series by modifying both the amide substituents R2 (in which a wide
diversity was allowed), and R1. In Table 3 the biochemical activity for the most potent members of this series is reported, along with the
antiproliferative activity against a PIM1-dependent cell line (Ba/F3/PIM1) and selected ADME properties. On the basis of the data
reported in Table 3, it appears that the combined overall bulk of R1 and R2 negatively affects the activity of the compounds due to
intramolecular steric hindrance in the bioactive conformation (13a-c vs 13d-f). For this reason the preferred substituents on the R1 aryl
group were halogens. Gratifyingly, cyclohexylmethyl amine derivatives 13e and 13f exhibited a 7-fold higher potency compared to
compound 1. Compounds 13g and 13h were designed to potentially achieve additional interactions in the binding site. The presence of
pyrimidine or pyrazole heterocycles close to the hinge region, could engage in a hydrogen bonding interaction with the carbonyl of
Glu121. This expectation seemed substantiated with 13g, which showed increased affinity for PIM1. Conversely the binding effect of
the pyrazole ring did not stand out significantly in terms of potency. The NH of the pyrazole ring imparted promiscuous recognition of
the ATP-site. Hence, compound 13h affected PIM kinases together with CDK2(0.060µM), CDC7(0.284µM), and GSK3(0.386µM).
Compounds 13i and 13j bearing a furanyl-methyl group in R2, resulted in a significantly improved biochemical potency compared to
10f. Excessive bulk within R1 may lead to a dramatic drop in activity, as exemplified when comparing inactive 13l with 13k (methyl vs.
fluoro respectively).
Table 3. R1, R2 diversification on racemic scaffold (IC50, µM)a
O
R1
NH
N
H
N
O
R2
Cmpd
10f
R1
3-trifluoromethylphenyl
“
R2
neopentyl
PIM1
0.521
0.614
0.838
0.375
0.124
0.073
0.094
0.033
1.098
0.125
0.041
0.132
>5
PIM2
Ba/F3/PIM1b
Caco2c
HLMd
13a
13b
13c
13d
13e
13f
3-fluorobenzyl
3.62
3.13
>5
“
tetrahydro-2H-pyran-4-yl
“
cyclohexylmethyl
2-fluorophenyl
3-fluorophenyl
3,4-difluorophenyl
2-chloropyrimidin-4-yl
1H-pyrazol-4-yl
3,4-difluorophenyl
3-chlorophenyl
5-chloro-2-fluorophenyl
“
3.12
2.46
4.47
1.315
0.675
2.222
0.980
2.406
>5
1.786
1.107
1.273
>5
11.9
11.24
11.8
0.9
400
48.5
400
197
“
“
13g
13h
13i
“
3-fluorobenzyl
furan-2-ylmethyl
3.644
2.427
13j
“
“
“
33.1
97.4
13k
13l
5-chloro-2-methyphenyl
aAs in Table 2. bData given are mean values derived from at least three replicates. cApparent permeability Papp (x10-6cm/s) is measured from the apical side to the
basolateral side. dStability in human liver microsomes (mL/min/kg)
Compounds with activity below 0.2 µM on PIM1, were progressed to the cell-based assay, demonstrating a promising confirmation
of activity in most cases. However, cellular physical and metabolic barriers are an unavoidable impediment to strict trending between
biochemical and proliferative inhibition performance. The high susceptibility to human liver metabolism remained an additional

concern, as some of these compounds tested in the human liver microsomes assay revealed a moderate (13e) to high (13d, 13f-g and
13j) intrinsic clearance.
A closer inspection of the 3D structure (Fig. 2B) of compound 1 in PIM1 revealed that the R2 substituent occupies the hydrophobic
space close to the solvent accessible region while the amide nitrogen interacts through a water molecule with the polar residues Asp128
and Glu171. Hence, we reasoned that improvement of PIM kinase potency could be obtained by introducing a branched R2 substituent
bearing two arms: one hydrophobic and one hydrophilic. Likely, the flexible side-chain ending with a hydrophilic group would be
projected towards the ribose binding pocket, an electronegative region of the protein formed by residues Asp128, Asp131, Glu171,
Asn172 and the carbonyl backbone of Glu171 (Fig. 2B). This would entail a range of interactions, directly or water-mediated, with
polar side chains. Additionally, the lipophilic group would maintain its position in the hydrophobic channel.
Furthermore, as previously observed from the crystallographic data, a chiral centre at C4 of the pyrazinone ring could be beneficial.
To assess the potential of this approach, chiral amino alcohols and amines with both hydrophobic and hydrophilic characteristics were
selected and prepared. The use of such chiral building blocks generated a mixture of diastereoisomers, which were resolved by standard
LC techniques thus avoiding the need of expensive and costly chiral separation tools. By applying the previously described synthetic
route (Scheme 3), pairs of diastereoisomers 14 were synthesized from intermediate 12, and easily resolved by chromatography. The
results of our efforts to introduce a solvent-exposed polar group via flexible linkers to the Cα position of R2 are shown in Table 4.
Remarkably, this modification with branched chains (Table 4) allowed a significant and effective step towards nanomolar PIM2
potencies and as a result, even more potent PIM1 compounds. Compound 14a for instance reached an IC50 value of 0.097 µM against
PIM2 and 0.013 µM against PIM1. Compared with its diastereoisomer 14b, a clear influence of the core piperazinone ring
stereochemistry was also observed. Thus the crystal structure of 14a bound to PIM1 was resolved in order to confirm the binding mode
and the absolute configuration of the asymmetric centre.

Table 4. Investigation of chiral amphiphilic amides (IC50, µM)a
O
O
R1
R1
NH
NH
N
N
H
H
B
A
N
N
R2
R2
O
O
Cmpd
14a
R1
R2
PIM1
0.013
PIM2
0.097
Ba/F3/PIM1a
>10
Caco2a
0.9
HLMa
6.5
*
B
2-fluoropyridin-4-yl
NH2
14b
14c
A
A
2-fluoropyridin-4-yl
3-fluorophenyl
“
“
l
0.150
0.010
0.502
0.184
>10
0.6
1.1
8.3
*
14d
14e
A
A
“
“
0.016
0.087
0.252
0.516
1.533
14.6
OH
*
*
3.723
3.050
2.7
1.3
55
OH
OH
14f
A
“
0.141
0.684
87.8
*
14g
14h
A
A
“
“
0.012
0.233
0.275
0.956
>10
0.8
10.1
6.7
NH2
*
*
N
14i
A
“
0.009
0.150
0.490
1.0
N
N
aAs in Table 3
Figure 3. PIM1 binding site showing the 2Fo-Fc electron density associated with 14a.23 contoured at 1.0 σ. The Inhibitor is shown with yellow carbon atoms
and protein is shown with green carbon atoms.
The binding mode of molecule 14a reflects the mode of compound 1 (Fig. 3). The lactam moiety establishes the same polar
interactions seen for 1. The pyridine ring occupies the “hinge binding” region with the fluorine atom pointing toward the hydrophobic
pocket while the phenyl group is well placed in the hydrophobic channel. As expected, the structural data also confirmed that the
increased affinity for PIM1 observed with compound 14a is the result of the water-mediated interaction between the positively charged
nitrogen atom of the amino methyl residue with the Asp128 and Asp131 side chains.

Comparison of PIM1 inhibitory activity of diastereoisomer 14a (S,S; scaffold B, 0.013 µM) and 14b (R,S; scaffold A, 0.150 µM)
revealed a potency difference of an order of magnitude. Activity on PIM2 showed similar trends with a 5-10 fold preference for the
(S,S) configuration. These stereochemical assignments were based upon X-ray data (Fig. 3). Notably, the 3-D structure also suggested
that the amine moiety R2 with (S)-configuration allowed the key interactions with polar side chains. These structural observations
together with our experience with previous scaffolds, outlined the stringent requirement in obtaining tight binding against both PIM1
and PIM2. Therefore, the synthesis of compounds with the R-configured amines was not considered.
Although the derivative with the amino methyl side chain performed slightly better in the biochemical assay than the corresponding
hydroxy methyl, poor antiproliferative activity was observed, likely a consequence of a poor permeability (14c vs 14e). Additionally,
14c shows a similar PIM1 potency as its cyclohexyl analog 14g. On the contrary, the cyclic analogue 14h, displayed a drop in affinity
of about 10 fold. We were also pleased with the tendency to increased HLM metabolic stability as reported in Table 4.
Despite significant improvements, compounds 14a, 14c and 14g were still devoid of significant cellular activity. This might be
ascribed to the poor permeability shown by positively charged compounds at physiological pH. Actually, the compounds that possess
high biochemical activity are found to be quite soluble (≥ 200 μM) at either pH 3 or 7, but have low membrane permeability.
On the other hand, the amine can strongly contribute to binding affinity. Thus we tried to attenuate this polarity and thereby the
energy of desolvation by preparing molecules where the amine was part of a six membered ring such as the methylpiperazine. Synthesis
of compound 14i represents a step in the right direction, as it exhibited an increased biochemical activity and submicromolar cellular
activity.
We therefore envisioned compounds possessing the chiral cyclohexyl, methyl-amino moieties in the R2 position as well as an
expanded array of substituted aromatic R1-groups that would provide additional SAR regarding the balance between PIM1-PIM2
biochemical potency versus cellular activity.
The results clearly show areas in which further optimization efforts were needed. Permeability remained a major concern, and,
despite having gained significant affinity for PIM1, the activity against PIM2 remained relatively weak. Since the R2 moiety had
provided such a boost to biochemical activity, we first maintained this group constant and directed our optimization efforts toward the
R1 aryl moiety. Different R1 groups with the best meta-substituent, were also synthesized in the attempt to address the membrane
permeability issues.
We were pleased to find that replacing fluorine with chlorine (15a) gave a 3-fold enhancement of affinity for PIM1 and 2-fold for
PIM2, and cellular activity was also improved. Unfortunately additional analogues (15b-d) with the best R1 groups identified so far,
were slightly less potent and a flat SAR was observed. Since the overall profile of pyrrolo[1,2-a]pyrazinone derivatives looked
promising, we extended our exploration in order to improve the PIM2 binding affinity. To this end, an attempt was made to find
differences in the binding mode of 15a between PIM1 and PIM2 through structural analyses based on docking runs. Results of docking
studies carried out on 15a in PIM1 match those previously observed with 1 and 14a. Comparing the structure for compound 15a docked
to PIM1 with the crystal structure of PIM228 no clear conformational differences could be detected, and consequently the lower affinity
for PIM2 should be unrelated to the contact interactions and might instead result from a change in protein dynamics, a mechanism
suggested by others.29 Although fitting well into the ATP pocket, it was possible to observe that 15a lacks some interactions.. The
lactam moiety of 15a reproduced the bidentate network of H-bonds observed for 1 and 14a with Lys67 and Glu89 and both the
cyclohexyl and the phenyl group are accommodated in the hydrophobic channel defined by Leu44, Arg122, Val126 (Ala122 for PIM2),
and Leu174. The N-methylpiperazine group extended into the solvent-accessible region beyond the polar side chains of Asp128,
Asp131 and Glu171, an indication that it may assume the role of a handle interacting favourably with the water network. New
derivatives were thus designed in order to explore the hydrophobic shell formed between the residue Leu44,
Val126/Ala122(PIM1/PIM2) and Leu174. En route to this aim, further analogues were designed containing either the branched amine
with an additional heterocyclic moiety or alkyl and cycloalkyl groups. Table 5 shows compounds obtained from this investigation.

Table 5. R1 variations combined with chiral R2 residues bearing cyclic tertiary amines (IC50, µM)a
O
R1
NH
N
H
N
O
R2
Cmpd
14i
R1
R2
PIM1
0.009
PIM2
0.150
Ba/F3/PIM1a
0.490
Caco2a
1.0
HLMa
6.7
*
3-fluorophenyl
N
N
15a
15b
3-chlorophenyl
“
“
0.003
0.026
0.073
0.250
0.125
0.156
8.4
5.9
3,4-difluorophenyl
5-chloro-2-
15c
“
“
0.002
0.132
0.147
fluorophenyl
15d
16a
2-fluoropyridin-4-yl
3-chlorophenyl
0.013
0.001
0.095
0.036
2.480
0.480
0.33
1.01
*
5.0
N
N
*
16b
16c
“
“
0.005
0.53
0.262
>5.0
N
N
*
N
N
aAs in Table 3
As depicted in Table 5, the replacement of the piperazine ring with 4-dimethylamino piperidine ring 16a, although enhancing the
affinity to both isoforms, was detrimental to cell activity. The increased basicity of the 4-dimethylaminopiperidine ring (dimethylamino
nitrogen calculated pKa = 9.3830) compared to the methylpiperazine ring (piperazine N-Me nitrogen calculated pKa = 7.6030) could
explain the drop in cellular activity through a decrease in cellular permeability, as confirmed by Caco-2 assay data. Replacing the
cyclohexyl substituent with smaller groups such as t-butyl 16b or methyl 16c resulted in a decrease of affinity. Therefore, compound
15a was selected for further profiling, as it represented the best combination of biochemical and cellular potency within this set of
analogues. Compound 15a was selected for additional characterization and initially was screened against a panel of 176 kinases to
evaluate its selectivity. Overall, remarkable selectivity over the covered part of the kinome was observed, where no protein kinases were
inhibited >30% by 15a at 0.1 μM concentration. We subsequently determined the IC50 values on an in-house kinase selectivity panel
composed of 56 kinases (Table 6).17
Table 6. Kinase Selectivitya
cmpd
PIM1
0.003
PIM2
0.073
PIM3
0.012
Others Kinasesb
>10.00
15a
aIC50 (µM) values reported were the averages of at least two independent dose-response curves. The ATP concentrations used in these assays were 2Km. b Kinase
selectivity panel as reported in Table 1.
Inhibitor 15a exhibits low nanomolar activity against PIM1, PIM2 and PIM3 and shows a high degree of selectivity for PIM kinases
(Table 6). Multiple PIM inhibition is in line with the high sequence similarity between the PIM proteins and the literature data, which
2,5,10
reports widespread parallelism of PIM1 and PIM3 inhibitory activity, and generally weaker PIM2 affinities.
Based on previous
reports of PIM inhibition evolutions,25, 31 we assembled a panel of cell lines in order to profile our lead compound’s selectivity vs. the
PIM-dependent cell lines.

Table 7. Anti-proliferative activities of inhibitor 15a in a cell lines panel.
Cell lines
Ba/F3/PIM1
MV4-11
IC50 µM at 72h
0.125
1.529
1.929
3.313
3.704
5.928
6.658
7.134
>10
LP-1
LNCaP
CAPAN-1
NCI-H929
RPMI 8226
KMS-11
Ba/F3
15a impaired the survival of murine Ba/F3 cells made IL-3 independent by overexpression of PIM1, at a sub-micromolar
concentration. It also impaired survival of human leukemic cell lines MV4-11 at a micromolar concentration, which is consistent with
its biochemical data. Additionally, the modest activity towards multiple myeloma cell lines might be related to the low PIM2 inhibition,
since it has been well demonstrated that PIM2 is involved in the survival of multiple myeloma cells.32 Pleasantly, 15a exhibited
interesting activity against LNCaP and CAPAN-1 cell lines, in agreement with the literature reports about the sensitivity of prostate and
pancreatic cancer cell lines towards PIM inhibition.33 To demonstrate that the cytotoxicity of 15a was dependent on the inhibition of
PIM kinases, the status of PIM biomarkers was investigated. 15a was incubated with murine Ba/F3/PIM1 cells, which were
subsequently lysed for analysis of their phosphorylation status by SDS-PAGE followed by Western blotting. As shown in Figure 4, 15a
efficiently and dose-dependently promoted c-Myc degradation5 and inhibited the phosphorylation of both BAD S112 and 4EBP1
T37/T46.
c
0.1 0.5 1
5
10 µM
c-myc
pBAD
Ser-112
4EBP1
pT37/T46
GAPDH
Figure 4. Western blot showing the effect of compound 15a on the components of the PIM pathway in murine Ba/F3/PIM1 cells. GAPDH staining was used
as a loading control.
These observations suggest that 15a has potential anticancer activity thanks to its activity against PIM kinases, while the lack of a
stringent correlation between biochemical inhibition and cellular antiproliferative activity may be due to limiting physico-chemical
properties.
As reported in Table 8, compound 15a possessed high water solubility at physiological pH and low-moderate Caco-2 apparent
permeability (8 x10‐6cm/s) with low efflux. Plasma protein binding was high in all species, including human. No inhibition of the subset
of the tested cytochromes was observed. Liver microsomes metabolic stability was moderate and also hepatocytes clearance in different
species was moderate. We further profiled the inhibitor on a hERG ion channel assay (indicative of potential myocardial toxicity due to
QT prolongation34) where it showed negligible activity.

Table 8. ADME profile of compound 15a.
Sol a µg/mL
Caco2b
Efflux
PPB HRMc
HLMd
12.6
Hep CL HRMDe
5.9/21/63/11
CYPf µM
>10
hERGI at10µM
16%
85
8.1
2.1
97.0/96.4/98.0
a Thermodynamic Solubility at pH 7. bApparent permeability apical to basolateral Caco-2 permeability. cPlasma protein binding human/rat/mice. d Intrinsic
Clearance in human liver microsomes (mL/min/kg). eHalf-life in the presence of in vitro preparations of hepatocytes clearance human/rat/mouse/dog
(mL/min/kg). fInhibition of cytochromes CYP2D6, CYP2C9, CYP3A4 (IC50).
Table 9. PK parameters in mouse for compound 15a i.v. and oral dosing, 8.2 mg kg-1 to female BALB/c mice in 10% DMSO
0.1% Tween-20 in saline solution
CL
Vdss
T1/2 (h)
AUC
Fos (%)
0.65
(mL/min/kg)
(L/kg)
(μM.h)
IVa
27.99
11.32
13.58
1.25
8.75
0.05
OSa
aThree animals were used for each dosing group. All values correspond to the average mean of the group
The overall satisfactory in vitro properties of 15a prompted us to an in vivo pharmacokinetic (PK) evaluation in mouse. 15a was
widely distributed (Vss 11.32 L) as a reflection of its overtly basic nature, but cleared slowly from the general circulation with a plasma
clearance lower than the mouse hepatic blood flow. The compound has a plasma half-life higher than 10 hours. Despite the low
clearance, the plasma exposure value following per os dose indicates the almost complete lack of oral availability (Table 9).
The poor oral availability of 15a was in line with the low-moderate Caco-2 apparent permeability, likely resulted from a combination
of high lipophilicity (cLogP = 6.67), ionizable state and moderate polar surface area (PSA = 69). These results suggested that further
optimization work is required to obtain a balanced physico-chemical properties profile in order to achieve both an appropriate level of
permeability and a favorable PK profile.
4. Conclusion
In summary, our research efforts in the synthesis of a natural product-inspired chemotype for targeted inhibition of ATP processing
enzymes produced the pyrrolo[1,2-a]pyrazinone series of PIM inhibitors. The kinome specificity and the modular nature of its
synthesis, make the pyrrolo[1,2-a]pyrazinone an attractive chemical class. Its bioactivity potential, together with the structural
arrangement of the modification site and the drug-likeness make this scaffold a valuable starting point for medicinal chemistry
investigations. SAR developed around the bicyclic core and amide moieties combined with structure-based design led to the discovery
of a new generation of PIM inhibitors, of which compound 15a is the most promising member, exhibiting low nanomolar activity
against PIM kinases and an excellent selectivity against a large panel of kinases. We established that 15a was functionally active in cells
and that it inhibits PIM signal transduction. Modulation by 15a of the targets downstream of PIM kinases, 4EBP1, pBAD, and c-Myc,
was observed in Ba/F3/PIM1 cells. Despite important biochemical activity improvement a gap remains between enzyme and cellular
potencies. With the available X-ray structures of the inhibitors in complex with the PIM1 protein, structural refinements of this series
are accessible. The strong effects of physicochemical properties on bioavailability and PK have been assessed in a preliminary study,
which lays the ground for further optimization work towards therapeutically useful PIM inhibitors.
5. Experimental
5.1. Chemistry
All solvents and reagents, unless otherwise stated, were commercially available, of the best grade and were used without further
purification. All experiments dealing with moisture-sensitive compounds were carried out under dry nitrogen or argon atmosphere.
Thin-layer chromatography was performed on Merck Silica Gel 60 F254 pre-coated plates. Column chromatography was conducted

1
under medium pressure on silica (Merck Silica Gel 40–63 μm). H-NMR spectra were recorded on a Varian INOVA 400 spectrometer
1
1
(operating at 400.5 MHz for H) and equipped with a 5 mm H{13C,15N} z axis PFG indirect detection probe. Chemical shifts were
referenced with respect to the residual solvents signals.35 Data are reported as follows: chemical shift (δ), multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, br s = broad signal, td = triplet of doublet, dd = doublet of doublets, ddd = doublet of doublets of
doublets, m = multiplet), coupling constants (Hz), and number of protons. Electrospray (ESI) mass spectra were obtained on a Finnigan
LCQ ion trap. HPLC–UV–MS analyses were carried out combining the ion trap MS instrument with HPLC system SSP4000 (Thermo
Separation Products) equipped with an auto-sampler LC Pal (CTC Analytics) and UV6000LP diode array detector (UV detection 215–
400 nm). Instrument control, data acquisition and processing were performed by using Xcalibur 1.2 software (Finnigan). HPLC
chromatography was run at room temperature, and 1 mL/min flow rate, using a Waters X Terra RP 18 column (4.6 x 50 mm; 3.5 μm).
Mobile phase A was ammonium acetate 5 mM buffer (pH 5.5 with acetic acid)/acetonitrile 90:10, and mobile phase B was ammonium
acetate 5 mM buffer (pH 5.5 with acetic acid)/acetonitrile 10:90; the gradient was from 0% to 100% B in 7 min then hold 100% B for 2
min before re-equilibration. Mass data given as m/z ratio. ESI(+) high resolution mass spectra (HRMS) were obtained on a Waters Q-
Tof Ultima directly connected with a micro HPLC 1100 Agilent as previously described.36 Optical rotation measurements were
performed on a Perkin Elmer 241 polarimeter and reported as follows: [α]DT (c = g/100 mL, solvent). Yields are unoptimized.
5.1.1. Ethyl (2E)-4-(diformylamino)but-2-enoate (2b)
A solution of ethyl-4-bromo-crotonate 2a (1 g, 5.18 mmol), diformylimide sodium salt (0.59 g, 6.22 mmol) and sodium iodide (0.78
g, 5.18 mmol) in dry acetonitrile (25 mL) was stirred under reflux overnight. The solvent was evaporated under vacuum and the residue
partitioned between dichloromethane and water (1:1, 40 mL). The aqueous phase was back-extracted with dichloromethane (3 x 15 mL)
and the combined organic layers were dried over Na2SO4. The solvent was evaporated under vacuum and the residue purified by flash
chromatography (hexane-EtOAc 6:4) to obtain the title compound as a light-brown solid (0.92 g, 96% yield). In alternative, Et2O was
added to the deep brown oil residue to obtain the product as a crystalline solid.
1H NMR (400 MHz, DMSO-d6) δ 9.00 (s, 2H), 6.77 (dt, J=15.8, 4.5 Hz, 1H), 5.87 (dt, J=15.8, 2.0 Hz, 1H), 4.25 (dd, J= 4.5, 2Hz
2H), 4.11 (q, J=7.1 Hz, 2H), 1.20 (t, J=7.1 Hz, 3H); LCMS : m/z 208 [M+Na]+ at r.t. 3.32 min.; HRMS (ESI) calcd for C8H11NNaO4 [M
+ Na ]+ 208.0580 found 208.0581
5.1.2. Ethyl (2E)-4-aminobut-2-enoate trifluoroacetate(2c)
A solution of ethyl (2E)-4-(diformylamino)but-2-enoate 2b (0.89 mg, 4.8 mmol) in a mixture trifluoroacetic acid-ethanol (absolute)
(2:1, 10 mL) was stirred under reflux overnight. The reaction was followed by LC-MS and stopped when complete conversion was
achieved. The solvent was evaporated under vacuum to obtain compound ethyl (2E)-4-aminobut-2-enoate trifluoroacetate as brown oil
(undetermined yield), which was used without further purification in the next step. LCMS : m/z 130 [M+H]+ at r.t. 1.20 min.
5.1.3. Ethyl (2E)-4-{[(4-iodo-1H-pyrrol-2-yl)carbonyl]amino}but-2-enoate (4)
Ethyl (2E)-4-aminobut-2-enoate trifluoroacetate 2c (0.97 g, 4 mmol) was added to a solution of 2,2,2-trichloro-1-(4-iodo-1H-pyrrol-
2-yl)ethanone 3 (0.68 g, 2 mmol) and diisopropylethylamine (DIPEA) (2.7 mL, 16 mmol) in dichloromethane (20 mL) and the reaction
mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum and the residue purified by flash
chromatography (SiO2, hexane-EtOAc 1:1) to obtain compound 4 as an off-white solid (0.48 g, 68% yield).1H NMR (400 MHz,
DMSO-d6) δ 11.82 (br. s., 1H), 8.39 (t, J=5.8 Hz, 1H), 7.01 (dd, J=2.9, 1.5 Hz, 1H), 6.96 (dd, J=2.5, 1.5 Hz, 1H), 6.90 (dt, J=15.7, 4.7
Hz, 1H), 5.87 (dt, J=15.7, 1.8 Hz, 1H), 4.12 (q, J=7.1 Hz, 2H), 3.99 - 4.06 (m, 2H), 1.21 (t, J=7.1 Hz, 3H); LCMS : m/z 349 [M+H]+ at
r.t. 4.73 min.; HRMS (ESI) calcd for C11H14IN2O3 [M + H ]+ 349.0044 found 349.0046
5.1.4. Ethyl (7-iodo-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl)acetate (5)
Diaza(1,3)bicyclo[5.4.0]undecane (DBU) (0.04 mL, 0.3 mmol) was added to a solution of ethyl (2E)-4-{[(4-iodo-1H-pyrrol-2-
yl)carbonyl]amino}but-2-enoate 4 (0.45 g, 1.3 mmol) in acetonitrile (8 mL) and the reaction mixture was stirred at room temperature
for 30 min. The solvent was evaporated under vacuum and the residue purified by flash chromatography (SiO2, hexane-EtOAc 1:1) to
obtain the title compound as an off-white solid (0.35 g, 79% yield).1H NMR (400 MHz, DMSO-d6) δ 7.72 (br. s., 1H), 7.13 (d, J=1.7

Hz, 1H), 6.72 (d, J=1.7 Hz, 1H), 4.61 - 4.69 (m, 1H), 4.05 - 4.15 (m, 2H), 3.66 (ddd, J=13.1, 4.1, 1.8 Hz, 1H), 3.31 - 3.38 (m, 1H), 2.82
(d, J=7.1 Hz, 2H), 1.19 (t, J=7.1 Hz, 3H); LCMS : m/z 349 [M+H]+ at r.t. 4.15 min.; HRMS (ESI) calcd for C11H14IN2O3 [M + H ]+
349.0044 found 349.0037
5.1.5. (7-iodo-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl)acetic acid (6)
In a 25 mL single-neck round-bottom flask, lithium hydroxide monohydrate (63 mg, 1.5 mmol, 2 eq) was added to a solution of 5
(260 mg, 0.75 mmol, 1 eq) in THF-H2O (1:1, 8 mL) and the reaction mixture was stirred at room temperature overnight. The organic
phase was washed with CH2Cl2 (2 x 10 mL). The aqueous phase was acidified with HCl (1 M) until pH=1 and extracted with EtOAc (3
x 10 mL). The combined organic layers were dried over Na2SO4 and the solvent was evaporated under vacuum to obtain the title
compound as an off-white solid (229 mg, 95% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.55 (s, 1H), 7.71 (d, J=2.8 Hz, 1H), 7.13 (d,
J=1.6 Hz, 1H), 6.71 (d, J=1.6 Hz, 1H), 4.55 - 4.65 (m, 1H), 3.59 - 3.68 (m, 1H), 3.27 - 3.41 (m, 1H), 2.75 (d, J=6.8 Hz, 2H); LCMS :
m/z 320 [M+H]+ at r.t. 3.06 min.; HRMS (ESI) calcd for C9H10IN2O3 [M + H ]+ 320.9731 found 320.9741
5.1.6. General synthetic procedure for compounds 1, 10a-10h
4-(4-formyl-3-methoxyphenoxy)butyryl aminomethyl resin (copolystyrene-1%DVB) 0.98 mmol/g 7 (2.5 g, 2.45 mmol, 1 eq.) was
suspended in 20 ml of dry THF and amines (9.8 mmol, 4 eq.) was added. The resulting suspension was shaken at room temperature for
2 h. Then acetic acid (0.147 g, 2.45 mmol, 1 eq.) and NaBH(AcO)3 (1.557 g, 7.35 mmol, 3 eq.) were added and the final suspension was
shaken for 16 h at room temperature. The resin was rinsed with THF (twice) MeOH (twice) DCM (twice), MeOH (twice), DMF
(twice) and DCM (three times) then dried under nitrogen flux. The resin 8 was used in the next step.A solution of compound 6 (1.563 g,
4.9 mmol, 2 eq), DIPEA (1.266 g, 9.8 mmol, 4 eq) and HBTU (1.857 g, 4.9 mmol, 2 eq) in 20 ml of dry DMF was stirred for 30 min
and then was added to resin 8 (2.5 g, 2.45 mmol, 1 eq). The final suspension was shaken for 24h at room temperature. The resin was
rinsed with a cycle of DMF, MeOH, DCM (3 times), DCM (3 times) and 1,4-dioxane (once) and dried under nitrogen flux. The resin 9
was then used in the next step. A mixture of resin 9 (0.100 g, 0.098 mmol, 1 eq), PdCl2(dppf).CH2Cl2 (0.032 g, 0.039 mmol, 0.4 eq),
boronic acid (0.49 mmol, 5 eq) and cesium carbonate (0.127 g, 0.392 mmol, 4 eq in 0.5 mL of H2O) in 1 mL of DME was stirred at 80
°C overnight, under an argon atmosphere. The resin was rinsed with a cycle of DMF, MeOH, DCM (3 times), DCM (3 times) and 1,4-
dioxane (once) and dried under nitrogen flux. The resin (0.100 g, 0.098 mmol) was suspended in a solution of TFA-DCM (1:1, 1 mL)
and shaken for 2 h at room temperature. The solution was collected and the resin was rinsed with DCM (collected as well), and a second
cycle was performed. The final washing was performed with MeOH. All the collected organic layers were dried under reduced pressure
and the title compounds were purified by preparative HPLC.
5.1.7. N-(2,2-dimethylpropyl)-2-[1-oxo-7-(thiophen-3-yl)-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-
yl]acetamide (1)
Yield: 24%; 8.3 mg; as a white amorphous solid 1H NMR (400 MHz, DMSO-d6) δ 7.88 (t, J=6.2 Hz, 1H), 7.64 (d, J=2.8 Hz, 1H),
7.50 (dd, J=4.9, 2.9 Hz, 1H), 7.49 (dd, J=2.9, 1.5 Hz, 1H), 7.30 (dd, J=4.9, 1.5 Hz, 1H), 7.28 (d, J=1.7 Hz, 1H), 6.97 (d, J=1.7 Hz, 1H),
4.63 (tt, J=7.1, 3.8 Hz, 1H), 3.67 (ddd, J=13.0, 4.2, 1.0 Hz, 1H), 2.92 (dd, J=13.1, 6.3 Hz, 1H), 2.86 (dd, J=13.1, 6.1 Hz, 1H), 2.75 (dd,
J=14.9, 7.0 Hz, 1H), 2.64 (dd, J=14.9, 7.0 Hz, 1H), 0.79 (s, 9H); LCMS : m/z 346 [M+H]+ at r.t. 5.41 min.; HRMS (ESI) calcd for
C18H24N3O2S [M + H ]+ 346.1584 found 346.1595.
5.1.8. N-(2,2-dimethylpropyl)-2-(1-oxo-7-phenyl-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-
yl)acetamide (10a)
1
Yield: 34%; 28 mg; as a white amorphous solid. H NMR (400 MHz, DMSO-d6) δ 7.88 (t, J=6.6 Hz, 1H), 7.67 (d, J=2.8 Hz, 1H),
7.53 (dd, J=8.2, 1.2 Hz, 2H), 7.37 (d, J=1.8 Hz, 1H), 7.32 (dd, J=8.2, 7.4 Hz, 2H), 7.15 (dd, J= 7.4, 1.2 Hz, 1H), 7.02 (d, J=1.8 Hz, 1H),
4.66 (tt, J=7.1, 3.8 Hz, 1H), 3.68 (ddd, J=13.0, 4.2, 1.5 Hz, 1H), 2.92 (dd, J=13.2, 6.6 Hz, 1H), 2.86 (dd, J=13.2, 6.6 Hz, 1H), 2.78 (dd,
J=15.0, 6.9 Hz, 1H), 2.66 (dd, J=15.0, 7.1 Hz, 1H), 0.79 (s, 9H); LCMS : m/z 340 [M+H]+ at r.t. 5.49 min.; HRMS (ESI) calcd for
C20H26N3O2 [M + H ]+ 340.2020 found 340.2026.

5.1.9. N-(2,2-dimethylpropyl)-2-[1-oxo-7-(pyridin-4-yl)-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-
yl]acetamide (10b)
1
Yield: 13%; 9.0 mg; as a white solid. H NMR (400 MHz, DMSO-d6) δ 8.67 (d, J=6.8 Hz, 2H), 8.09 (d, J=6.8 Hz, 2H), 7.97 (d,
J=1.7 Hz, 1H), 7.93 (d, J=3.3 Hz, 1H), 7.89 (t, J=6.2 Hz, 1H), 7.49 (d, J=1.7 Hz, 1H), 4.77 (tt, J=7.2, 3.6 Hz, 1H), 3.73 (ddd, J=13.1,
4.4, 1.2 Hz, 1H), 2.95 (dd, J=13.1, 6.2 Hz, 1H), 2.77 - 2.86 (m, 2H), 2.70 (dd, J=15.1, 6.6 Hz, 1H), 0.75 (s, 9H). LCMS : m/z 341
[M+H]+ at r.t. 4.07 min. HRMS (ESI) calcd for C19H25N4O2 [M + H ]+ 341.1972 found 341.1987.
5.1.10. N-(2,2-dimethylpropyl)-2-[7-(4-fluorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-
yl]acetamide (10c)
Yield: 22%; 7.9 mg; as a off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.85 (t, J=6.2 Hz, 1H), 7.65 (d, J=2.9 Hz, 1H), 7.53 (dd,
J=8.8, 5.4 Hz, 2H), 7.32 (d, J=1.8 Hz, 1H), 7.12 (dd, J=8.8, 5.4 Hz, 2H), 6.98 (d, J=1.8 Hz, 1H), 4.62 (tt, J=7.2, 3.8 Hz, 1H), 3.65 (ddd,
J=12.9, 4.2, 1.1 Hz, 1H), 2.89 (dd, J=13.1, 6.2 Hz, 1H), 2.82 (dd, J=13.1, 6.2 Hz, 1H), 2.74 (dd, J=14.9, 7.2 Hz, 1H), 2.62 (dd, J=14.9,
7.2 Hz, 1H), 0.76 (s, 9H); LCMS : m/z 358 [M+H]+ at r.t. 5.57 min.; HRMS (ESI) calcd for C20H25FN3O2 [M + H ]+ 358.1926 found
358.1935.
5.1.11. N-(2,2-dimethylpropyl)-2-[7-(3-fluorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-
yl]acetamide (10d)
1
Yield: 45%; 16.3 mg as off-white solid. H NMR (400 MHz, DMSO-d6) δ 7.89 (t, J=6.2 Hz, 1H), 7.70 (d, J=2.4 Hz, 1H), 7.45 (d,
J=1.7 Hz, 1H), 7.33 - 7.41 (m, 2H), 7.09 (d, J=1.7 Hz, 1H), 6.96 (td, J=8.6, 2.3 Hz, 1H), 4.66 (tt, J=7.2, 3.7 Hz, 1H), 3.68 (ddd, J=12.9,
4.1, 1.2 Hz, 1H), 2.93 (dd, J=13.1, 6.2 Hz, 1H), 2.85 (dd, J=13.1, 6.2 Hz, 1H), 2.78 (dd, J=14.9, 7.2 Hz, 1H), 2.66 (dd, J=14.9, 7.1 Hz,
1H), 0.79 (s, 9H); LCMS : m/z 358 [M+H]+ at r.t. 5.64 min.; HRMS (ESI) calcd for C20H25FN3O2 [M + H ]+ 358.1926 found 358.1939.
5.1.12. N-(2,2-dimethylpropyl)-2-[7-(2-fluorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-
yl]acetamide (10e)
Yield: 40%;14.1 mg as white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.87 (t, J=6.3 Hz, 1H), 7.72 (d, J=3.2 Hz, 1H), 7.63 - 7.70 (m,
1H), 7.37 (t, J=2.1 Hz, 1H), 7.14 - 7.24 (m, 3H), 7.08 (t, J=1.4 Hz, 1H), 4.70 (tt, J=7.0, 3.7 Hz, 1H), 3.71 (ddd, J=13.1, 4.4, 1.1 Hz,
1H), 2.91 (dd, J=13.1, 6.3 Hz, 1H), 2.84 (dd, J=13.1, 6.3 Hz, 1H), 2.78 (dd, J=15.0, 7.0 Hz, 1H), 2.64 (dd, J=15.0, 7.0 Hz, 1H), 0.77 (s,
9H); LCMS : m/z 358 [M+H]+ at r.t. 5.61 min.; HRMS (ESI) calcd for C20H25FN3O2 [M + H ]+ 358.1926 found 358.1934.
5.1.13. N-(2,2-dimethylpropyl)-2-{1-oxo-7-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrrolo[1,2-
a]pyrazin-4-yl}acetamide (10f)
Yield: 26%;10.3 mg as white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.89 (t, J=6.2 Hz, 1H), 7.81 - 7.87 (m, 2H), 7.73 (d, J=2.6 Hz,
1H), 7.55 (d, J=1.8 Hz, 1H), 7.54 (t, J=7.5 Hz, 1H), 7.49 (d, J=7.5 Hz, 1H), 7.16 (d, J=1.8 Hz, 1H), 4.67 (tt, J=7.2, 3.8 Hz, 1H), 3.69
(ddd, J=13.0, 4.3, 1.3 Hz, 1H), 2.96 (dd, J=13.1, 6.2 Hz, 1H), 2.75 - 2.87 (m, 2H), 2.67 (dd, J=14.9, 7.2 Hz, 1H), 0.78 (s, 9H); LCMS :
m/z 408 [M+H]+ at r.t. 6.20 min.; HRMS (ESI) calcd for C21H25F3N3O2 [M + H ]+ 408.1894 found 408.1901.
5.1.14. N-(2,2-dimethylpropyl)-2-{1-oxo-7-[3-(trifluoromethoxy)phenyl]-1,2,3,4-tetrahydropyrrolo[1,2-
a]pyrazin-4-yl}acetamide (10g)
1
Yield: 18%;7.5 mg as off-white solid. H NMR (400 MHz, DMSO-d6) δ 7.88 (t, J=6.3 Hz, 1H), 7.72 (d, J=2.8 Hz, 1H), 7.58 (ddd,
J=7.8, 1.2, 0.9 Hz, 1H), 7.49 (d, J=1.8 Hz, 2H), 7.44 (t, J=8.0 Hz, 1H), 7.13 (dd, J=8.0, 1.2 Hz, 1H), 7.11 (d, J=1.8 Hz, 1H), 4.66 (tt,
J=7.1, 3.8 Hz, 1H), 3.69 (ddd, J=10.9, 3.8, 2.1 Hz, 1H), 2.94 (dd, J=13.1, 6.3 Hz, 1H), 2.83 (dd, J=13.1, 6.3 Hz, 1H), 2.79 (dd, J=14.9,
7.1 Hz, 1H), 2.66 (dd, J=14.9, 7.1 Hz, 1H), 0.78 (s, 9H); LCMS : m/z 424 [M+H]+ at r.t. 6.29 min.; HRMS (ESI) calcd for
C21H25F3N3O3 [M + H ]+ 424.1843 found 424.1856.
5.1.15. N-(2,2-dimethylpropyl)-2-{1-oxo-7-[4-(trifluoromethoxy)phenyl]-1,2,3,4-tetrahydropyrrolo[1,2-
a]pyrazin-4-yl}acetamide (10h)

Yield: 23%; 9.4 mg as a grey solid. 1H NMR (400 MHz, DMSO-d6) δ 7.88 (t, J=6.1 Hz, 1H), 7.70 (d, J=3.7 Hz, 1H), 7.65 (d, J=8.8
Hz, 2H), 7.42 (d, J=1.8 Hz, 1H), 7.30 (d, J=8.8 Hz, 2H), 7.06 (d, J=1.8 Hz, 1H), 4.67 (tt, J=7.3, 3.7 Hz, 1H), 3.69 (ddd, J=13.0, 4.3, 1.3
Hz, 1H), 2.92 (dd, J=13.1, 6.1 Hz, 1H), 2.85 (dd, J=13.1, 6.1 Hz, 1H), 2.78 (dd, J=14.9, 7.3 Hz, 1H), 2.66 (dd, J=14.9, 7.3 Hz, 1H),
0.78 (s, 9H); LCMS : m/z 424 [M+H]+ at r.t. 6.31 min.; HRMS (ESI) calcd for C21H25F3N3O3 [M + H ]+ 424.1843 found 424.1852.
5.1.16. General procedure for preparation of compounds 11a-g
In a Schlenk tube filled with argon, a mixture of 5 (1.0 g, 2.87 mmol, 1 eq), PdCl2(dppf).CH2Cl2 (0.082 g, 0.1 mmol, 0.035 eq) and
boronic acid (3.73 mmol, 1.3 eq) in 1,4 dioxane (10 mL, ) was degassed before addition of cesium carbonate (1.871 g, 5.74 mmol, 2 eq
in 2 mL of H2O) under argon atmosphere and the reaction was stirred at 80 °C for 4h. The mixture was filtered through SiO2 (washed
with EtOAc) and the solvent evaporated under vacuum. The residue was purified by flash chromatography (EtOAc-Hex).
5.1.17. Ethyl 7-(3-trifluoromethylphenyl)-1-oxo-1, 2,3,4-tetrahydropyrrolo[1,2-a]pyrazine-4-
carboxylate 11a
Yield: 73%; 0.770 g as a grey solid. 1H NMR (400 MHz, DMSO-d6) δ 7.86 - 7.91 (m, 2H), 7.73 (br. s., 1H), 7.67 (d, J=1.8 Hz, 1H),
7.55 - 7.58 (m, 1H), 7.56 (t, J=7.6 Hz, 1H), 7.50 (d, J=9.1 Hz, 1H), 7.18 (d, J=1.8 Hz, 1H), 4.61 - 4.70 (m, 1H), 4.11 (dq, J= 7.1 Hz,
2H), 3.70 (d, J=13.2, 3.6, 2.0 Hz, 1H), 3.39 (d, J=13.2, 5.0, 4.0 Hz, 1H), 2.94 (dd, J=15.2, 6.2 Hz, 1H), 2.90 (dd, J=15.2, 7.5 Hz, 1H),
1.18 (t, J=7.1 Hz, 3H); LCMS: m/z 367 [M+H]+ at r.t. 6.1 min.; HRMS (ESI) calcd for C18H18F3N2O3 [M + H ]+ 367.1264 found
367.1267.
5.1.18. Ethyl [7-(3-fluorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetate 11b
Yield 55%; as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 7.70 (d, J=2.2 Hz, 1H), 7.55 (d, J=1.8 Hz, 1H), 7.29 - 7.44 (m,
3H), 7.10 (d, J=1.8 Hz, 1H), 6.92 - 7.02 (m, 1H), 4.58 - 4.72 (m, 1H), 4.14 (q, J=7.1 Hz, 2H), 3.71 (ddd, J=13.1, 4.1, 1.7 Hz, 1H), 3.37
(ddd, J=13.1, 4.4, 4.1 Hz, 1H), 2.79 - 2.94 (m, 2H), 1.18 (t, J=7.1 Hz, 3H). LCMS: m/z 317 [M+H]+ at r.t. 5.47 min.; HRMS (ESI) calcd
for C17H18FN2O3 [M + H ]+ 317.1296 found 317.1294.
5.1.19. Ethyl [7-(3,4-difluorophenyl)-1-oxo-1, 2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetate 11c
Yield 86%; as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.71 (d, J=0.9 Hz, 1H), 7.65 (ddd, J=12.5, 7.9, 2.0 Hz, 1H), 7.52
(d, J=1.8 Hz, 1H), 7.30 - 7.44 (m, 2H), 7.10 (d, J=1.8 Hz, 1H), 4.64 (tt, J=6.7, 4.3 Hz, 1H), 4.10 (m, 2H), 3.72 (ddd, J=13.0, 4.1, 1.6
Hz, 1H), 3.39 (dt, J=13.0, 4.1, 3.7 Hz, 1H), 2.90 (dd, J=15.8, 7.1 Hz, 1H), 2.86 (dd, J=15.8, 7.1 Hz, 1H), 1.18 (t, J=7.1 Hz, 3H). LCMS:
m/z 335 [M+H]+ at r.t. 5.62 min.; HRMS (ESI) calcd for C17H17F2N2O3 [M + H ]+ 335.1202 found 335.1196.
5.1.20. Ethyl [7-(2-fluoropyridin-4-yl)-1-oxo-1, 2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetate 11d
Yield 40%; as a light brown amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.12 (d, J=5.4 Hz, 1H), 7.81 (d, J=1.8 Hz, 1H), 7.80
(br. s., 1H), 7.55 (dd, J= 7.5, 5.2 Hz, 1H), 7.37 (s, 1H), 7.30 (d, J=1.8 Hz, 1H), 4.61 - 4.74 (m, 1H), 4.10 (q, J=7.1 Hz, 2H), 3.72 (ddd,
J=13.0, 4.1, 1.6 Hz, 1H), 3.39 (dt, J=13.0, 4.1, 3.7 Hz, 1H), 2.93 (dd, J=16.7, 6.6 Hz, 1H), 2.89 (dd, J=16.7, 6.6 Hz, 1H), 1.17 (t, J=7.1
Hz, 3H). LCMS: m/z 318 [M+H]+ at r.t. 4.52 min.; HRMS (ESI) calcd for C16H17FN3O3 [M + H ]+ 318.1249 found 318.1245.
5.1.21. Tert-butyl 4-[4-(2-ethoxy-2-oxoethyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-7-yl]-1H-
pyrazole-1-carboxylate 11e
Yield 20%; as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.43 (d, J=0.7 Hz, 1H), 8.06 (d, J=0.7 Hz, 1H), 7.66 (d, J=2.0
Hz, 1H), 7.40 (d, J=1.7 Hz, 1H), 7.02 (d, J=1.7 Hz, 1H), 4.55 - 4.70 (m, 1H), 3.99 - 4.18 (m, 2H), 3.70 (ddd, J=13.1, 4.2, 1.6 Hz, 1H),
3.33-3.38 (m, 1H), 2.85 (d, J=7.4 Hz, 2H), 1.59 (s, 9H), 1.17 (t, J=7.1 Hz, 3H). LCMS: m/z 389 [M+H]+ at r.t. 5.17 min.; HRMS (ESI)
calcd for C19H25N4O5 [M + H ]+ 389.182 found 389.1812.
5.1.22. Ethyl [7-(3-chlorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetate 11f
Yield 60%; as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 7.71 (d, J=1.2 Hz, 1H), 7.64 (t, J=1.8 Hz, 1H), 7.58 (d, J=1.8
Hz, 1H), 7.54 (ddd, J=7.9 1.4, 1.0 Hz, 1H), 7.35 (t, J=7.9 Hz, 1H), 7.20 (ddd, J=7.9, 2.1, 1.0 Hz, 1H), 7.10 (d, J=1.8 Hz, 1H), 4.60 -
4.69 (m, 1H), 4.11 (q, J=7.1 Hz, 2H), 3.71 (ddd, J=13.1, 4.2, 1.9 Hz, 1H), 3.37 (ddd, J=13.1, 8.8, 4.2, Hz, 1H), 2.92(dd, J=15.6, 6.4 Hz,

1H), ), 2.89 (dd, J=16.0, 7.2 Hz, 1H), 1.18 (t, J=7.1 Hz, 3H). LCMS: m/z 333 [M+H]+ at r.t. 5.97 min.; HRMS (ESI) calcd for
C17H18ClN2O3 [M + H ]+ 333.1001 found 333.1005.
5.1.23. Ethyl [7-(5-chloro-2-fluorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetate
11g
Yield 59%; as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 7.78 (dd, J=6.8, 2.3 Hz, 1H), 7.75 (d, J=2.8 Hz, 1H), 7.53 (t,
J=2.0 Hz, 1H), 7.21 - 7.33 (m, 2H), 7.13 - 7.18 (m, 1H), 4.61 - 4.83 (m, 1H), 4.10 (q, J=7.3 Hz, 1H), 3.72 (ddd, J=13.7, 4.7, 1.7 Hz,
1H), 3.37 (ddd, J=13.7, 4.7, 4.0 Hz, 1H), 2.91(dd, J=16.0, 6.7 Hz, 1H), ), 2.87 (dd, J=16.0, 6.7 Hz, 1H), 1.17 (t, J=7.1 Hz, 3H). LCMS:
m/z 351 [M+H]+ at r.t. 5.21 min.; HRMS (ESI) calcd for C17H17ClFN2O3 [M + H ]+ 351.0906 found 351.0912.
5.1.24. General procedure for preparation of compounds 12a-f
Lithium hydroxide (0.240 g, 10 mmol, 10 eq) was added to a solution of ethyl pyrrolo[1,2-a]pyrazine-4-carboxylate derivatives 11
(1.0 mmol, 1 eq) in 10 mL of THF/MeOH/H2O (8:1:1) and the reaction mixture was stirred at room temperature for 16h. The organic
phase was evaporated. The aqueous phase was acidified with HCl 1 N until pH<1 and extracted with EtOAc (4 times). The combined
organic layers were dried over Na2SO4 and the solvent was evaporated under vacuum to obtain the product.
5.1.25. 1-oxo-7-[3-(trifluoromethyl)phenyl]-1,2,3, 4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl-acetic acid
12a
1
Yield 97% 0.330, as a off-white solid. H NMR (400 MHz, DMSO-d6) δ 11.70 - 13.48 (m, 1H), 7.81 - 7.96 (m, 2H), 7.72 (br. s.,
1H), 7.69 (d, J=1.8 Hz, 1H), 7.56 (t., J=7.8 Hz, 1H), 7.50 (d, J=7.8 Hz, 1H), 7.17 (d, J=1.8 Hz, 1H), 4.55 - 4.70 (m, 1H), 3.71 (ddd,
J=12.9, 4.0, 2.0 Hz, 1H), 3.39 (dd, J=12.9, 5.6, 4.0 Hz, 1H), 2.87 (dd, J=16.1, 5.8 Hz, 1H), 2.80 (dd, J=16.1, 7.8 Hz, 1H),; LCMS: m/z
339 [M+H]+ at r.t. 4.61 min.; HRMS (ESI) calcd for C16H14F3N2O3 [M + H ]+ 339.0951 found 339.0952.
5.1.26. [7-(3-fluorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetic acid 12b
Yield 90%; as a off-white solid. LCMS : m/z 289 [M+H]+ at r.t. 4.08 min.; HRMS (ESI) calcd for C15H14FN2O3 [M + H ]+ 289.2737
found 289.2730.
5.1.27. [7-(3,4-difluorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetic acid 12c
Yield 68%; off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.62 (br. s, 1H), 7.70 (br. s., 1H), 7.65 (ddd, J=12.4, 7.7, 1.8 Hz, 1H),
7.54 (d, J=1.8 Hz, 1H), 7.29 - 7.45 (m, 2H), 7.09 (d, J=1.8 Hz, 1H), 4.54 - 4.67 (m, 1H), 3.70 (ddd, J=13.6, 4.1, 1.9 Hz, 1H), 3.38 (ddd,
J=13.6, 5.0, 4.3 Hz, 1H), 2.83 (dd, J=16.0, 6.2 Hz, 1H), 2,79 (dd, J=16.0, 7.4 Hz, 1H). LCMS : m/z 307 [M+H]+ at r.t. 4.3 min.; HRMS
(ESI) calcd for C15H13F2N2O3 [M + H ]+ 307.0889 found 307.0899.
5.1.28. [7-(2-fluoro-4-pyridinyl)-1-oxo-1,2,3, 4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl] acetic acid 12d
Yield 50%; off-white solid. LCMS : m/z 290 [M+H]+ at r.t. 3.98 min.; HRMS (ESI) calcd for C14H13FN3O3 [M + H ]+ 290.2617
found 290.2620.
5.1.29. [7-(3-chlorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetic acid 12e
1
Yield 85%; 0.450, as a off-white solid. H NMR (400 MHz, DMSO-d6) δ 12.60 (br. s., 1H), 7.68 - 7.72 (m, 1H), 7.64 (t, J=1.8 Hz,
1H), 7.59 (d, J=1.8 Hz, 1H), 7.51 - 7.57 (m, 1H), 7.35 (t, J=7.9 Hz, 1H), 7.21 (ddd, J=7.9, 1.8, 1.0 Hz, 1H), 7.10 (d, J=1.8 Hz, 1H), 4.54
- 4.70 (m, 1H), 3.70 (ddd, J=13.2, 4.0, 2.5 Hz, 1H), 3.38 (ddd, J=13.2, 4.7, 4.0 Hz, 1H), 2.86 (dd, J=16.2, 6.0 Hz, 1H), 2.80 (dd, J=16.2,
7.6 Hz, 1H). LCMS : m/z 305 [M+H]+ at r.t. 4.11 min.; HRMS (ESI) calcd for C15H14ClN2O3 [M + H ]+ 305.0688 found 305.0691.
5.1.30. [7-(5-chloro-2-fluorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetic acid 12f
Yield 92%; off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.76 - 7.82 (m, 1H), 7.74 (m, 1H), 7.56 (s, 1H), 7.22 - 7.32 (m, 2H),
7.14 (s, 1H), 4.66 (m, 1H), 3.66 - 3.75 (dd, J= 12.4, 3.6 Hz, 1H), 3.394 (dd, J= 12.4, 3.6 Hz, 1H), 2.79 (d, J=6.7 Hz, 2H). LCMS : m/z
323 [M+H]+ at r.t. 4.3 min.; HRMS (ESI) calcd for C15H13FClN2O3 [M + H ]+ 323.0593 found 323.0591.
5.1.31. General procedure for preparation of compounds 13a-13l

A solution of pyrrolo[1,2-a]pyrazine-4-carboxylic acid derivatives 12 (0.27 mmol), N,N-diisopropylethylamine (DIPEA) (0.412 ml,
2.4 mmol) and 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, HBTU) (0.201 g, 0.54 mmol) in dry
dioxane (6 ml) was stirred for 15 min, then added the amines (0.54 mmol) and the final suspension was reacted at 80°C for 6 h. The
solvent was removed in vacuo generating a dark brown oil. The residue was purified by preparative HPLC.
5.1.32. N-(3-fluorobenzyl)-2-{1-oxo-7-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrrolo[1,2-
a]pyrazin-4-yl}acetamide13a
Yield 9%; 10.9 mg as a thick yellow oil. 1H NMR (400 MHz, DMSO-d6) δ 8.55 (t, J=6.3 Hz, 1H), 7.84 (m, 2H), 7.73 (d, J=3.3 Hz,
1H), 7.54-7.58 (m, 1H); 7.52 (d, J=1.8 Hz, 1H),7.48-7.52 (m, 2H), 7.17 (d, 1H), 7.13-7.16 (m, 1H), 6.93-7.01 (m, 3H), 4.71 (tt, J=7.3,
3.7 Hz, 1H), 4.38 (dd, J=15.3, 6.3 Hz, 1H), 4.19 (dd, J=15.3, 6.3 Hz, 1H), 3.71 (ddd, J=13.0, 4.2, 1.3 Hz, 1H), 3.36 - 3.42 (m, 1H), 2.82
(dd, J=15.5, 7.2 Hz, 1H), 2.70 (dd, J=15.5, 7.2 Hz, 1H), LCMS : m/z 446 [M+H]+ at r.t. 5.97 min.; HRMS (ESI) calcd for C23H20F4N3O2
[M + H ]+ 446.1486 found 446.1478
5.1.33. 2-{1-oxo-7-[3-(trifluoromethyl)phenyl]-1, 2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl}-N-
(tetrahydro-2H-pyran-4-yl)acetamide (13b)
1
Yield: 10%; 10.9 mg as a white amorphous solid. H NMR (400 MHz, DMSO-d6) δ 8.00 (d, J=7.6 Hz, 1H), 7.84(m, 2H), 7.71 (d,
J=3.1 Hz, 1H), 7.56 (t, J=7.7 Hz, 1H), 7.52 (d, J=1.8 Hz, 1H), 7.49 (d, J=7.7Hz, 1H), 7.17 (d, J=1.8 Hz, 1H), 4.65 (tt, J=7.1, 3.8 Hz,
1H), 3.65-3.80 (m, 4H), 3.32 (m, 3H), 2.70 (dd, J=15.2, 7.5 Hz, 1H), 2.57 (dd, J=15.2, 7.5 Hz, 1H), 1.60-1.68 (m, 2H), 1.17-1.39 (m,
2H) LCMS : m/z 422 [M+H]+ at r.t. 5.09 min.; HRMS (ESI) calcd for C21H23F3N3O3 [M + H ]+ 422.1686 found 422.1677.
5.1.34. N-(cyclohexylmethyl)-2-{1-oxo-7-[3-(trifluoromethyl)phenyl]-1,2, 3,4-tetrahydropyrrolo[1,2-
a]pyrazin-4-yl}acetamide (13c)
Yield: 10%; 11.3 mg as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 7.92 (t, J=5.9 Hz, 1H), 7.86 (d, J=7.6 Hz, 1H),
7.85 (s, 1H), 7.71 (d, J=3.4 Hz, 1H), 7.55 (t, J=7.6 Hz, 1H), 7.49 (d, J=1.8 Hz, 1H), 7.47-7.50 (m, 1H), 7.16 (d, J=1.8 Hz, 1H), 4.67 (tt,
J=7.2, 3.6 Hz, 1H), 3.69 (ddd, J= 12.7, 4.3, 1.1 Hz, 1H), 3.37 (m, 1H), 3.03 (dt, J=13.2, 5.9 Hz, 1H), 2.68 - 2.79 (m, 2H), 2.58 (dd,
J=14.6, 7.2 Hz, 1H); 1.42-1.54 (m, 5H), 1.21-1.29 (m, 1H), 0.91-1.01 (m, 3H), 0.69-0.79 (m, 2H), LCMS : m/z 434 [M+H]+ at r.t. 6.31
min.; HRMS (ESI) calcd for C23H27F3N3O2 [M + H ]+ 434.2050 found 434.2056.
5.1.35. N-(cyclohexylmethyl)-2-[7-(2-fluorophenyl)-1-oxo-1,2, 3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-
yl]acetamide (13d)
Yield: 15%; 15.8 mg as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 7.91 (t, J=6.2 Hz, 1H), 7.63 - 7.73 (m, 2H), 7.32
(t, J=1.4 Hz, 1H), 7.13 - 7.24 (m, 3H), 7.08 (t, J=1.4 Hz, 1H), 4.62 - 4.77 (m, 1H), 3.71 (dd, J=13.3, 4.9 Hz, 1H), 3.37 (m, 1H), 3.00 (dt,
J=13.2, 6.6 Hz, 1H), 2.65 - 2.79 (m, 2H), 2.53 - 2.60 (m, 1H); 1.45-1.54 (m, 5H), 1.21-1.30 (m, 1H), 0.94-1.05 (m, 3H), 0.69-0.81 (m,
2H), LCMS : m/z 384 [M+H]+ at r.t. 5.86 min.; HRMS (ESI) calcd for C22H27FN3O2 [M + H ]+ 384.2082 found 384.2085.
5.1.36. N-(cyclohexylmethyl)-2-[7-(3-fluorophenyl)-1-oxo-1,2, 3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-
yl]acetamide 13e)
Yield: 12%; 12.1 mg as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 7.93 (t, J=6.0 Hz, 1H), 7.69 (d, J=3.1 Hz, 1H),
7.28 - 7.42 (m, 4H), 7.09 (d, J=1.8 Hz, 1H), 6.88 - 7.01 (m, 1H), 4.58 - 4.74 (m, 1H), 3.69 (ddd, J=12.9, 4.3, 1.2 Hz, 1H), 3.36 (m, 1H),
3.01 (dt, J=13.2, 6.0 Hz, 1H), 2.65 - 2.80 (m, 2H), 2.56 (dd, J=14.2, 6.2 Hz, 1H 1H), 1.48-1.57 (m, 5H), 1.20 - 1.36 (m, 1H); 0.97-1.05
(m, 3H), 0.71-0.82 (m, 2H), LCMS : m/z 384 [M+H]+ at r.t. 5.86 min.; HRMS (ESI) calcd for C22H27FN3O2 [M + H ]+ 384.2082 found
384.2087.
5.1.37. N-(cyclohexylmethyl)-2-[7-(3,4-difluorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-
4-yl]acetamide (13f)
Yield: 11%; 11.7 mg as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 7.92 (t, J=5.8 Hz, 1H), 7.69 (d, J=3.2 Hz, 1H),
7.57 - 7.66 (m, 1H), 7.29 - 7.43 (m, 3H), 7.08 (d, J=1.8 Hz, 1H), 4.55 - 4.72 (m, 1H), 3.68 (ddd, J= 13.0, 4.0, 1.3 Hz, 1H), 3.35-3.39 (m,

1H), 3.01 (dt, J=13.1, 5.8 Hz, 1H), 2.60 (dd, J=15.0, 6.2 Hz, 1H 1H), 1.48-1.57 (m, 5H), 1.20 - 1.30 (m, 1H); 0.97-1.05 (m, 3H), 0.71-
0.82 (m, 2H), LCMS : m/z 402 [M+H]+ at r.t. 5.99 min.; HRMS (ESI) calcd for C22H26F2N3O2 [M + H ]+ 402.1988 found 402.1987.
5.1.38. N-(cyclohexylmethyl)-2-[7-(2-chloro-pyrimidin-4-yl)-1-oxo-1, 2,3,4-tetrahydropyrrolo[1,2-
a]pyrazin-4-yl]acetamide (13g)
Yield: 7%;7.5 mg as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J=5.4 Hz, 1H), 7.92 (t, J=5.9 Hz, 1H), 7.87
(d, J=3.7 Hz, 1H), 7.78 (d, J=5.4 Hz, 1H), 7.71 (d, J=1.7 Hz, 1H), 7.35 (d, J=1.7 Hz, 1H), 4.66 - 4.85 (m, 1H), 3.72 (dd, J=13.5, 4.1 Hz,
1H), 3.34 - 3.43 (m, 1H), 3.05 (dt, J=13.2, 6.7 Hz, 1H), 2.74 (dd, J=14.6, 8.7 Hz, 1H), 2.64 - 2.71 (m, 1H), 2.58 (dd, J=13.2, 6.7 Hz,
1H), 1.38-1.54 (m, 5H), 1.16-1.33 (m, 1H), 0.85-1.12 (m, 3H), 0.62-0.78 (m, 2H); LCMS : m/z 402 [M+H]+ at r.t. 5.06 min.; HRMS
(ESI) calcd for C20H25ClN5O2 [M + H ]+ 402.1692 found 402.1699.
5.1.39. N-(3-fluorobenzyl)-2-[1-oxo-7-(1H-pyrazol-4-yl)-1,2,3,4-tetrahydropyrrolo[1, 2-a]pyrazin-4-
yl]acetamide trifluoroacetate (13h)
Yield: 11%; 11.1 mg as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.53 (dd, J=6.3, 5.6 Hz, 1H), 7.74 (s, 2H), 7.59
(d, J=3.3 Hz, 1H), 7.18-7.25 (m, 1H), 6.98-7.04 (m, 3H), 7.10 (d, J=1.7 Hz, 1H), 6.83 (d, J=1.7 Hz, 1H), 4.64 (tt, J=7.0, 3.7 Hz, 1H),
4.36 (dd, J=15.0, 6.3 Hz, 1H ), 4.22 (dd, J=15.0, 5.6 Hz, 1H ), 3.68 (ddd, J=13.0, 4.2, 1.2 Hz, 1H), 2.74 (dd, J=15.0, 7.2 Hz, 1H), - 2.67
(dd, J=15.0, 7.2 Hz, 1H); LCMS : m/z 368 [M+H]+ at r.t. 3.87 min.; HRMS (ESI) calcd for C19H19FN5O2 [M + H ]+ 368.1518 found
368.1527.
5.1.40. 2-[7-(3,4-difluorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]-N-(furan-2-
ylmethyl)acetamide (13i)
1
Yield: 43%; 45 mg as a white amorphous solid. H NMR (400 MHz, DMSO-d6) δ 8.50 (t, J=5.6 Hz, 1H), 7.70 (d, J=2.7 Hz, 1H),
7.55 - 7.63 (d, J=4.4 Hz, 1H), 7.47 (dd, J=1.7, 0.7 Hz, 1H), 7.31 - 7.42 (m, 3H), 7.07 (d, J=1.7 Hz, 1H), 6.28 (dd, J=3.2, 1.7 Hz, 1H),
6.16 (dd, J=3.2, 0.7 Hz, 1H), 4.55 - 4.75 (m, 1H), 4.27 - 4.36 (dd, J= 15.7, 5.6 Hz, 1H), 4.15 - 4.26 (dd, J= 15.7, 5.6 Hz, 1H), 3.68 (ddd,
J=12.8, 4.2, 1.0 Hz, 1H), 3.29-3.31 (m, 1H), 2.74 (dd, J=14.9, 7.0 Hz, 1H); 2.63 (dd, J=14.9, 7.0 Hz, 1H); LCMS : m/z 386 [M+H]+ at
r.t. 10.28 min.; HRMS (ESI) calcd for C20H18F2N3O3 [M + H ]+ 386.1311 found 386.1312.
5.1.41. 2-[7-(3-chlorophenyl)-1-oxo-1,2,3, 4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]-N-(furan-2-
ylmethyl)acetamide (13j)
Yield: 38%;40 mg as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.52 (dd, J= 6.2, 5.1 Hz, 1H), 7.71 (d, J=2.8 Hz,
1H), 7.57 (b.s., 1H), 7.46 - 7.51 (m, 1H), 7.42 (s, 1H), 7.41 (s, 1H), 7.35 (t, J=7.9 Hz, 1H), 7.21 (dd, J=7.9, 1.6 Hz, 1H), 7.08 (d, J=1.8
Hz, 1H), 6.21 - 6.28 (m, 1H), 6.14 (d, J=3.2 Hz, 1H), 4.67 (ddd, J= 4.0, 3.8, 2.8 Hz, 1H), 4. 31 (dd, J=15.6, 6.2 Hz, 1H), 4.19 (dd,
J=15.6, 5.1 Hz, 1H), 3.68 (dd, J=13.2, 2.8 Hz, 1H), 3.35 (dt, J=13.2, 4.0 Hz, 1H), 2.74 (dd, J=13.2, 7.1 Hz, 1H), 2.64 (dd, J=13.2, 7.1
Hz, 1H); LCMS : m/z 384 [M+H]+ at r.t. 10. 7 min.; HRMS (ESI) calcd for C20H19ClN3O3 [M + H ]+ 384.1110 found 384.1114.
5.1.42. 2-[7-(5-chloro-2-fluorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]-N-(furan-2-
ylmethyl)acetamide (13k)
Yield: 52%; 26 mg as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.48 (t, J=5.6 Hz, 1H), 7.72 - 7.79 (m, 2H), 7.41 -
7.48 (m, 2H), 7.24 - 7.32 (m, 2H), 7.14 (t, J=1.6 Hz, 1H), 6.26 (dd, J=3.2, 1.8 Hz, 1H), 6.15 (dd, J=3.2, 0.9 Hz, 1H), 4.68 - 4.77 (m,
1H), 4.30 (dd, J=16.0, 5.8 Hz, 1H), 4.20 (dd, J=16.0, 5.4 Hz, 1H), 3.71 (dd, J=14.0, 1.2 Hz, 1H), 3.35 - 3.42 (m, 1H), 2.75 (dd, J=15.5,
7.3 Hz, 1H), 2.75 (dd, J=15.5, 6.5 Hz, 1H), LCMS : m/z 402 [M+H]+ at r.t. 5.47 min.; HRMS (ESI) calcd for C20H18ClFN3O3 [M + H]+
402.1015 found 402.1015.
5.1.43. 2-[7-(5-chloro-2-methylphenyl)-1-oxo-1,2,3, 4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]-N-(furan-2-
ylmethyl)acetamide (13l)
Yield: 62%; 31 mg as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.48 (t, J=5.6 Hz, 1H), 7.70 (br. s., 1H), 7.45 (dd,
J=1.8, 0.9 Hz, 1H), 7.33 (d, J=2.0 Hz, 1H), 7.25 (d, J=8.3 Hz, 1H), 7.10 - 7.22 (m, 2H), 6.86 (d, J=2.0 Hz, 1H), 6.28 (dd, J=3.2, 1.8 Hz,

1H), 6.15 (dd, J=3.2, 0.9 Hz, 1H), 4.65 - 4.75 (m, 1H), 4.17 - 4.35 (m, 2H), 3.71 (dd, J=12.8, 4.0 Hz, 1H), 3.34 - 3.42 (m, 1H), 2.77 (dd,
J=14.8, 7.2 Hz, 1H), 2.65 (dd, J=14.8, 6.4 Hz, 1H), 2.33 (s, 3H); LCMS : m/z 398 [M+H]+ at r.t. 5.65 min.; HRMS (ESI) calcd for
C21H21ClN3O3 [M + H]+ 398.1266 found 398.1266.
5.1.44. N-[(1S)-2-amino-1-phenylethyl]-2-[(4S)-7-(2-fluoro-4-pyridinyl)-1-oxo-1,2,3, 4-
tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetamide (14a) and N-[(1S)-2-amino-1-phenylethyl]-2-[(4R)-7-
(2-fluoro-4-pyridinyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetamide trifluoroacetate,
14b
A solution of [7-(2-fluoro-4-pyridinyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl] acetic acid 12d (65 mg, 0.22 mmol),
N,N-diisopropylethylamine (DIPEA) (0.348 ml, 2.02 mmol) and HBTU (102 mg, 0.27 mmol) in dry dioxane (5 ml) was stirred for 15
min, then was added to tert-butyl [(2S)-2-amino-2-phenylethyl]carbamate (63 mg, 0.27 mmol) and the final suspension was reacted for
2 h at 70°C. The solvent was removed in vacuo. The crude was then portioned between ethyl acetate and water, the organic layer dried
over Na2SO4 and the solvent removed in vacuo. Purification by flash chromatography on silica gel column (DCM/MeOH/NH4OH
95/5/0.1) gave as first eluted peak the compound tert-butyl [(2S)-2-({[(4R)-7-(2-fluoropyridin-4-yl)-1-oxo-1,2,3,4-
tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetyl}amino)-2-phenylethyl]carbamate (33 mg, 30% yield) as a pale yellow foam,and as
second eluted peak the compound tert-butyl [(2S)-2-({[(4S)-7-(2-fluoropyridin-4-yl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-
a]pyrazin-4-yl]acetyl}amino)-2-phenylethyl]carbamate as a off-white foam (35 mg, 32% yield). The products were stirred with 10
ml of a mixture of TFA/DCM (1:1) for 30 minutes at room temperature and the solvents removed in vacuo. N-[(1S)-2-amino-1-
phenylethyl]-2-[(4R)-7-(2-fluoro-4-pyridinyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetamide
trifluoroacetate,
14b, (30 mg, 90% yield) as a light yellow foam. 1H NMR (400 MHz, DMSO-d6) δ 8.61 (d, J=8.5 Hz, 1H), 8.11 (d, J=5.4 Hz, 1H), 7.87
(b.m. 3H), 7.82 (m, 1H), 7.68 (d, J=1.7 Hz, 1H), 7.31-7.40 (m, 6H), (d, J=1.7 Hz, 1H), 7.29 (d, J=1.7 Hz, 1H), 5.11 (td, J=8.5, 4.6 Hz,
1H), 4.63 - 4.72 (m, 1H), 3.62-3.67 (m, 1H), 3.33 (m, 1H), 3.00-3.15 (m, 2H), 2.78 (d, J=6.7 Hz, 7.452H). LCMS : m/z 408 [M+H]+ at
r.t. 3.69 min.;HRMS (ESI) calcd for C22H23FN5O2 [M + H]+ 408.1831 found 408.1846. N-[(1S)-2-amino-1-phenylethyl]-2-[(4S)-7-(2-
fluoro-4-pyridinyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetamide trifluoroacetate, 14a, Yield: 95%; 31 mg, as a
light yellow foam; [α]D22: +21.47 (c 0.61, MeOH). 1H NMR (400 MHz, DMSO-d6) δ 8.50 (d, J = 8.5 Hz, 1H), 8.09 (d, J = 5.4 Hz, 1H),
7.89 (br. s., 3H), 7.83 (d, J = 3.4 Hz, 1H), 7.44 (d, J = 1.7 Hz, 1H), 7.36-7.38 (m, 2H), 7.28 (d, J = 1.7 Hz, 1H), 7.19 (s, 1H), 7.10-7.16
(m, 5H), 5.08 - 5.17 (m, 1H), 4.61 - 4.75 (m, 1H), 3.73 (dd, J = 13.1, 4.0 Hz, 1H), 3.36 - 3.46 (m, 1H), 3.10 - 3.18 (m, 1H), 2.90 - 3.04
(m, 1H), 2.68 - 2.84 (m, 2H); LCMS : m/z 408 [M+H]+ at r.t. 3.74 min.; HRMS (ESI) calcd for C22H23FN5O2 [M + H]+ 408.1831 found
408.1836.
Compound 14c-h were prepared in a manner similar to that described for 14a
5.1.45. N-[(1S)-2-amino-1-phenylethyl]-2-[(4S)-7-(3-fluorophenyl)-1-oxo-1,2, 3,4-tetrahydropyrrolo[1,2-
a]pyrazin-4-yl]acetamide trifluoroacetate, 14c
Compound 14c was prepared from 12b according to the same procedure for compound 14a by using tert-butyl [(2S)-2-amino-2-
phenylethyl]carbamate in step c. Yield: 94%; 24.6 mg; as a pale yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J=8.4 Hz, 1H),
7.89 (br. s., 3H), 7.73 (br. s., 1H), 7.45 (t, J=1.6 Hz, 1H), 7.30 - 7.40 (m, 4H), 7.24 - 7.29 (m, 2H), 7.24 - 7.29 (m, 2H), 7.09 (d, J=1.6
Hz, 1H), 6.95 - 7.00 (m, 1H), 5.05 - 5.22 (m, J=3.5 Hz, 1H), 4.58 - 4.72 (ddd, J=6.9, 6.9, 3.3 Hz, 1H), 3.69-3.75 (m, 1H), 3.28-3.34 (m,
1H), 2.88-3.20 (b.m., 2H), 2.66-2.80 (m, 2H); LCMS : m/z 407 [M+H]+ at r.t. 4.52 min.; HRMS (ESI) calcd for C23H24FN4O2 [M + H]+
407.1878 found 407.1889.
5.1.46. N-[(1S)-1-cyclohexyl-2-hydroxyethyl]-2-[(4S)-7-(3-fluorophenyl)-1-oxo-1, 2,3,4-
tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetamide (14d)
Compound 14d was prepared from 12b according to the same procedure for compound 14a by using (2S)-2-amino-2-
1
cyclohexylethanol. Yield: 48%; as a off-white amorphous solid. H NMR (400 MHz, DMSO-d6) δ 7.68 (d, J=3.4 Hz, 1H), 7.63 (d,
J=9.2 Hz, 1H), 7.28 - 7.42 (m, 4H), 7.09 (d, J=1.8 Hz, 1H), 6.90 - 6.99 (m, 1H), 4.61 - 4.72 (m, 1H), 4.50 (t, J=5.4 Hz, 1H), 3.69 (ddd,

J=13.0, 3.4, 1.2 Hz, 1H), 3.60 (dq, J=9.2, 5.8 Hz, 1H), 2.79 (dd, J=14.8, 8.4 Hz, 1H), 2.58 (dd, J=14.8, 5.8 Hz, 1H), 1.56-1.32 (m, 6H),
0.7-0.95 (m, 5H); LCMS : m/z 414 [M+H]+ at r.t. 5.29 min.; HRMS (ESI) calcd for C23H29FN3O3 [M + H]+ 414.2188 found 414.2192.
5.1.47. 2-[(4S)-7-(3-fluorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]-N-[(1S)-2-
hydroxy-1-phenylethyl]acetamide (14e)
Compound 14e was prepared from 12b according to the same procedure for compound 14a by using (2S)-2-amino-2-phenylethanol.
Yield: 34%; as a off-white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.42 (d, J=8.3 Hz, 1H), 7.70 (br.s., 1H), 7.27 - 7.37 (m,
4H), 7.19-7.21 (m, 2H), 7.18-7.21 (m, 2H), 7.13-7.16 (m, 1H), 7.08 (d, J=1.3 Hz, 1H), 6.90 - 6.99 (m, 1H), 4.61 - 4.84-4.90 (m, 1H),
4.62-4.66 (m, 1H), 3.67 (ddd, J=13.1, 9.3, 1.2 Hz, 1H), 3.56 (dq, J=9.3, 5.8 Hz, 2H), 2.81 (dd, J=14.5, 7.2 Hz, 1H), 2.58 (dd, J=14.5,
6.7 Hz, 1H), 1.56-1.32 (m, 6H), 0.7-0.95 (m, 5H); LCMS : m/z 408 [M+H]+ at r.t. 4.97 min.; HRMS (ESI) calcd for C23H23FN3O3 [M +
H]+ 407.1718 found 407.1729.
5.1.48. 2-[(4S)-7-(3-fluorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]-N-[(2S)-1-
hydroxy-3-phenylpropan-2-yl]acetamide (14f)
Compound 14f was prepared from 12b according to the same procedure for compound 14a by using (2S)-2-amino-3-phenylpropan-
1
1-ol. Yield: 50%; as a off-white amorphous solid. H NMR (400 MHz, DMSO-d6) δ 7.93 (d, J=8.4 Hz, 1H), 7.65 (br.s., 1H), 7.45 (d
J=1.8 Hz, 1H), 7.31-7.39 (m, 4H), 7.17-7.21 (m, 2H), 7.11-7.13 (m, 2H), 7.08 (d J=1.8 Hz, 1H), 6.93 - 6.98 (m, 1H), 4.54 (ddd, J=7.1,
3.4, 3.4 Hz, 1H), 3.89 - 3.99 (m, 1H), 3.60 (dd, J=13.6, 3.2 Hz, 1H), 2.76 (dd, J=13.5, 5.7 Hz, 1H), 2.62 - 2.70 (m, 1H), 2.52 - 2.62 (m,
2H); LCMS : m/z 422 [M+H]+ at r.t. 4.65 min.; HRMS (ESI) calcd for C24H25FN3O3 [M + H]+ 422.1875 found 422.1878.
5.1.49. N-[(1S)-2-amino-1-cyclohexylethyl]-2-[(4S)-7-(3-fluorophenyl)-1-oxo-1,2,3, 4-
tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetamide hydrochloride (14g)
Compound 14g was prepared from 12b according to the same procedure for compound 14a by using tert-butyl [(2S)-2-amino-2-
1
cyclohexylethyl]carbamate. Yield: 10%; as a pale yellow foam. H NMR (400 MHz, DMSO-d6) δ 7.79 (d, J=9.3 Hz, 1H), 7.72 (d,
J=3.2 Hz, 1H), 7.32 - 7.40 (m, 3H), 7.31 (d, J=1.8 Hz, 1H), 7.10 (d, J=1.8 Hz, 1H), 6.85 - 7.00 (m, 1H), 4.69 (td, J=8.6, 3.7 Hz, 1H),
3.78 - 3.85 (m, 1H), 3.74 (dd, J=14.0, 4.1 Hz, 1H), 3.23-3.27 (m, 1H), 2.89 - 2.95 (m, 1H), 2.79 (dd, J=15.0, 9.0 Hz, 1H), 2.66-2.68 (m,
1H), 2.60-2.63 (m, 1H), 2.59-2.66 (m, 1H). LCMS : m/z 413 [M+H]+ at r.t. 9.27 min.; HRMS (ESI) calcd for C23H30FN4O2 [M + H]+
413.2348 found 413.2357.
5.1.50. (4S)-7-(3-fluorophenyl)-4-{2-[(2S)-4-methyl-2-phenylpiperazin-1-yl]-2-oxoethyl}-3,4-
dihydropyrrolo[1,2-a]pyrazin-1(2H)-one (14h)
Compound 14g was prepared from 12b according to the same procedure for compound 14a by using (3S)-1-methyl-3-
phenylpiperazine. Yield: 11%; as a pale yellow foam. LCMS : m/z 447 [M+H]+ at r.t. 6.98 min.; HRMS (ESI) calcd for C26H28FN4O2
[M + H]+ 447.2191 found 447.2198.
5.1.51. (1S)-1-cyclohexyl-2-(4-ethylpiperazin-1-yl)ethanamine trihydrochloride (20a)
Mesylchloride (180 μl, 2.32 mmol) was added dropwise to a solution of tert-butyl [(1S)-1-cyclohexyl-2-hydroxyethyl]carbamate
(17a) (4.0 g, 16.46 mmol) and TEA (6.8 mL, 49.38 mmol) in DCM (30 ml) at –0°C. The solution was stirred at 0°C under an
atmosphere of argon for 3h. The reaction mixture was poured into ice/water and the separated aqueous layer was re-extracted with
DCM (5 ml). The combined organic layers were washed with 0.5N HCl, water, saturated aqueous NaHCO3 sol and brine, dried
(MgSO4) and the solvent was removed in vacuo to afford (2S)-2-[(tert-butoxycarbonyl)amino]-2-cyclohexylethyl methanesulfonate (18)
as an orange oil (4.35 g) that was used in the next step without purification. 1H NMR (400 MHz, DMSO-d6) δ 6.87 (d, J=9.0 Hz, 1H),
4.20 (dd, J=10.2, 4.2 Hz, 1H), 4.08 (dd, J=10.3, 7.3 Hz, 1H), 3.46 - 3.55 (m, 1H), 3.15 (s, 3H), 1.38 (s, 9H). 2.16 g (6.73 mmol) of (2S)-
2[(tert-butoxycarbonyl)amino]-2-cyclohexylethyl methanesulfonate were dissolved in 30 ml of dry tetrahydrofuran and 7.491 ml (67.3
mmol) of methylpiperazine were added. The solution was refluxed under stirring for 6 h. The solution was then cooled and 80 ml of
diethylether were added to precipitate methylpiperazinium salts that were removed by filtration. The filtrate was evaporated to give a
crude that was purified by flash-chromatography (eluent: dichloromethane-methanol-ammonia 30% 97-3-0.3) to yield 1.315 g of a tert-

butyl [(1S)-1-cyclohexyl-2-(4-methylpiperazin-1-yl)ethyl]carbamate (19) (60 % yield). 1H NMR (400 MHz, DMSO-d6) δ 6.38 (d, J=9.4
Hz, 1H), 3.38 - 3.51 (m, J=10.1 Hz, 1H), 2.20 - 2.39 (m, 10H), 2.15 (s, 3H), 1.51 - 1.72 (m, 5H), 1.37 (s, 9H), 1.24 - 1.34 (m, J=8.2 Hz,
1H), 0.84 - 1.19 (m, 5H). The obtained compound 1.315 g (4.03 mmol) was dissolved in 10 ml of dichloromethane and 10 ml of HCl 4
M in dioxane were added drowise..The solution was stirred at room temperature for 3 h and then the solvent evaporated. The residue
was triturated with diethylether, giving 1.35 g of (1S)-1-cyclohexyl-2-(4-ethylpiperazin-1-yl)ethanamine dihydrochloride (20a) as
yellow hygroscopic solid (100% yield). 1H NMR (400 MHz, DMSO-D6) δ (ppm) 10.51 (br. s., 1H), 7.81 (br. s., 3H),2.97-3.15 (m, 5H),
2.85-2.94 (m, 1H), 2.54-2.68 (m, 3H), 2.74 (s, 3H), 2.35-2.46 (m, 2H), 1.43 - 1.80 (m, 6H), 0.99 - 1.29 (m, 5H) HRMS (ESI) calcd for
C13H28N3 [M + H]+ 226.2278 found 226.2283.
5.1.52. (2S)-3,3-dimethyl-1-(4-methylpiperazin-1-yl)butan-2-amine dihydrochloride (20b)
1
Yield: 95%; as a pale yellow amorphous hygroscopic solid. H NMR (400 MHz, DMSO-D6) δ (ppm) 10.28 (br. s., 1H), 7.64 (br. s.,
3H), 2.98-3.15 (m, 5H), 2.87-2.91 (m, 1H), 2.80-2.82 (m, 2H), 2.75 (s, 3H),2.52-2.67 (m, 3H), 0.96 (s, 9H)
5.1.53. (2S)-1-(4-methylpiperazin-1-yl)propan-2-amine trihydrochloride (20c)
1
Yield: 96%; as a pale yellow amorphous hygroscopic solid. H NMR (400 MHz, DMSO-D6) δ (ppm) 10.56 (b.s. 1H), 7.90 (br.s.,
2H), 2.88-3.11 (m, 5H), 2.74 (s, 3H), 2.59 - 2.69 (m, 2H), 2.52-2.56 (m, 4H), 1.17 (d, J=6.6 Hz, 3H); HRMS (ESI) calcd for C8H20N3
[M + H]+ 158.1652 found 158.1660.
5.1.54. 1-[(2S)-2-amino-2-cyclohexylethyl]-N,N-dimethylpiperidin-4-amine trihydrochloride(20d)
1
Yield: 90%; as a pale yellow amorphous hygroscopic solid. H NMR (400 MHz, DMSO-D6) δ (ppm) 10.73 (br. s., 1H), 8.45 (br. s.,
3H), 3.87-3.92 b.m. 2H), 3.55-3.73 (m, 3H), 2.92-3.19 (b.m. 4H), 2.74 (d, J=4.3 Hz, 6H)1.99-2.30 (b.m. 3H), 1.59-1.81 (m, 6H), 0.96-
1.25 (m, 5H). HRMS (ESI) calcd for C15H32N3 [M + H]+ 254.2591 found 254.2597.
5.1.55. N-[(1S)-1-cyclohexyl-2-(4-methylpiperazin-1-yl)ethyl]-2-[(4S)-7-(3-fluorophenyl)-1-oxo-1,2,3,4-
tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetamide trifluoroacetate (14i)
Compound 14i was prepared from 12b according to the same procedure for compound 14a by using compound 20a. Yield: 21%; as
1
light grey foam. H NMR (400 MHz, DMSO-d6) δ 7.75 (d, J=2.8 Hz, 1H), 7.73(d, J=8.6 Hz, 1H), 7.31 - 7.39 (m, 4H), 7.11 (d, J=1.8
Hz, 1H), 6.88 - 7.02 (m, 1H), 4.60 - 4.76 (m, 1H), 3.80 - 3.90 (m, 2H), 3. 73 (dd, J= 17.2, 5.7 Hz, 1H), 3.36 (dt, J=17.2, 4.4, 3.8 Hz,
1H), 2.78 - 2.84 (m, 1H), 2.76 (s, 3H), 2.59 (dd, J=14.7, 5.8 Hz, 2H), 2.52 (m, 4H), 1.35 - 1.55 (ddd, J=12.6, 12.6 Hz, 5H), 1.24 (br. s.,
1H), 0.89 - 1.04 (dd, J= 12.8, 11.4 Hz, 4H), 0.71 - 0.89 (m, 5H); LCMS : m/z 496 [M+H]+ at r.t. 4.95 min.; HRMS (ESI) calcd for
C28H39FN5O2 [M + H]+ 496.3083 found 496.3085.
5.1.56. 2-[(4S)-7-(3-chlorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]-N-[(1S)-1-
cyclohexyl-2-(4-methylpiperazin-1-yl)ethyl]acetamide (15a)
Compound 15a was prepared from 12e according to the same procedure for compound 14a by using compound 20a. Yield: 42%; as
light yellow foam. [α]D22: +27.60 (c 0.86, MeOH). 1H NMR (400 MHz, DMSO-d6) δ 7.72 (d, J = 2.9 Hz, 1H), 7.62 (d, J = 9.4 Hz, 1H),
7.58 (t, J = 1.8 Hz, 1H), 7.51 (ddd, J = 7.8, 1.8, 0.9 Hz, 1H), 7.39 (d, J = 1.8 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.18 (ddd, J = 7.8, 1.8,
0.9Hz, 1H), 7.09 (d, J = 1.8 Hz, 1H), 4.66 (ddd, J = 8.1, 5.6, 3.9 Hz, 1H), 3.75 - 3.89 (m, 1H), 3.70 (ddd, J = 13.0, 4.2, 1.5 Hz, 1H),
3.39 (m, 2H), 2.77 (dd, J = 14.5, 8.1 Hz, 1H), 2.56 (dd, J = 14.5, 5.6 Hz, 1H), 2.17-2.40 (m, 10H), 2.11 (s, 3H), 1.68 (m, 1H), 1.35-1. 51
(m, 4H), 1.20-1.28 (m, 1H), 0.75-1.10 (m, 5H); LCMS : m/z 512 [M+H]+ at r.t. 5.34 min.; HRMS (ESI) calcd for C28H39ClN5O2 [M +
H]+ 512.2787 found 512.2776.
5.1.57. N-[(1S)-1-cyclohexyl-2-(4-methylpiperazin-1-yl)ethyl]-2-[(4S)-7-(3,4-difluorophenyl)-1-oxo-
1,2,3, 4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetamide (15b)
Compound 15b was prepared from 12c according to the same procedure for compound 14a by using compound 20a. Yield: 49%; as
light grey foam. [α]D22: +41.78 (c 0.81, MeOH). H NMR (400 MHz, DMSO-d6) δ 7.73 (d, J = 3.1 Hz, 1H), 7.64 (d, J = 6.2 Hz, 1H),
1
7.61 (dd, J = 7.8, 1.7 Hz, 2H), 7.31 - 7.41 (m, 3H), 7.09 (d, J = 1.7 Hz, 1H), 4.65 (td, J = 6.3, 3.6 Hz, 1H), 3.76 - 3.86 (m, 1H), 3. 3.72

(dd, J = 12.9, 4.1 Hz, 1H), 3.40 (m, 2H), 2.77 (dd, J = 14.5, 8.3 Hz, 1H), 2.58 (dd, J = 14.5, 6.3 Hz, 1H), 2.28-2.45 (m, 11H), 1.36-
1.52 (m, 4H), 1.24 (m, 1H), 0.97 (m, 1H), 0.81 (m, 4H); LCMS : m/z 514 [M+H]+ at r.t. 5.09 min.; HRMS (ESI) calcd for
C28H38F2N5O2 [M + H]+ 514.2988 found 514.2989.
5.1.58. 2-[(4S)-7-(5-chloro-2-fluorophenyl)-1-oxo-1,2, 3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]-N-
[(1S)-1-cyclohexyl-2-(4-methylpiperazin-1-yl)ethyl]acetamide (15c)
Compound 15c was prepared from 12f according to the same procedure for compound 14a by using compound 20a. Yield: 35%; as
pale yellow foam. 1H NMR (400 MHz, DMSO-d6) δ 7.79 (d, J = 4.0 Hz, 1H), 7.72 - 7.77 (m, 1H), 7.64 (d, J = 9.7 Hz, 1H), 7.38 (s,
1H), 7.22 - 7.29 (m, 2H), 7.16 (s, 1H), 4.65 - 4.79 (m, 1H), 3.78-3.82 (m,1H), 3.75 (dd, J = 12.8, 4.1, Hz, 2H), 3.38 (m, 1H), 2.75 - 2.83
(m, J = 8.91 Hz, 1H), 2.78 (dd, J =14.8, 9.4 Hz, 1H), 2.74 (br. s., 4H), 2.55 (dd, J =14.8, 4.7 Hz, 1H), 2.15 - 2.40 (m, 4H), 1.10 - 1.56
(m, 8H), 0.64 - 1.01 (m, 6H); LCMS : m/z 530 [M+H]+ at r.t. 5.17 min.; HRMS (ESI) calcd for C28H38ClFN5O2 [M + H]+ 530.2693
found 530.2704.
5.1.59. N-[(1S)-1-cyclohexyl-2-(4-methylpiperazin-1-yl)ethyl]-2-[(4S)-7-(2-fluoropyridin-4-yl)-1-oxo-
1,2,3, 4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetamide (15d)
Compound 15d was prepared from 12d according to the same procedure for compound 14a by using compound 20a. Yield: 25%; as
a pale yellow solid: [α]D22: +52.42 (c 0.68, MeOH). H NMR (400 MHz, DMSO-d6) δ 9.53 (br. s., 1H), 8.10 (d, J = 5.3 Hz, 1H), 7.84
1
(d, J = 3.4 Hz, 1H), 7.67 (d, J = 9.0 Hz, 1H), 7.63 (s, 1H), 7.52 (d, J = 5.2 Hz, 1H), 7.34 (s, 1H), 7.30 (d, J = 1.7 Hz, 1H), 4.67 - 4.75
(m, 1H), 3.64 - 3.84 (m, 2H), 3.39 (m, 1H), 2.66-2.82 (m, 5H), 2.25-2.36 (m, 8H), 1.35-1.49 (m, 5 H), 1.24 (m, 1H), 0.99-0.87 ( m, 2H),
0.85-0.70 (m, 4H); LCMS : m/z 497 [M+H]+ at r.t. 4.24 min.; HRMS (ESI) calcd for C27H38FN6O2 [M + H]+ 497.3035 found 497.3020.
5.1.60. 2-[(4S)-7-(3-chlorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]-N-{(1S)-1-
cyclohexyl-2-[4-(dimethylamino)piperidin-1-yl]ethyl}acetamide (16a)
Compound 16a was prepared from 12e according to the same procedure for compound 14a by using compound 20d. Yield: 11%; as
an off-white solid. [α]D22: +53.60 (c 0.54, MeOH). 1H NMR (400 MHz, DMSO-d6) δ 7.72 (d, J = 2.8 Hz, 1H), 7.61 (d, J = 9.4 Hz, 1H),
7.58 (t, J = 1.9 Hz, 1H), 7.52 (dt, J = 7.9, 1.5, 0.9 Hz, 1H), 7.39 (d, J = 1.8 Hz, 1H), 7.33 (t, J = 7.9 Hz, 1H), 7.18 (ddd, J = 7.9, 0.92,
2.1, 0.9 Hz, 1H), 7.09 (d, J = 1.8 Hz, 1H), 4.56 - 4.77 (m, 1H), 3.74 - 3.89 (m, 1H), 3. 73 (dd, J = 12.8, 1.22 Hz, 1H), 3.37-3.41 (m,
1H), 2.85 (d, J = 10.8 Hz, 1H), 2.76 (dd, J = 14.5, 8.6 Hz, 1H), 2.55 (dd, J = 14.5, 5.6 Hz, 1H), 2.16 - 2.27 (m, 2H), 2.12 (s, 6H), 1.83 -
1.98 (m, 2H), 1.72 - 1.81 (m, 1H), 1.56 - 1.68 (m, 2H), 1.35 - 1.53 (m, 4H), 1.15 - 1.34 (m, 4H), 0.88 - 1.08 (m, 2H), 0.67 - 0.90 (m,
3H); LCMS : m/z 540 [M+H]+ at r.t. 6.02 min.; HRMS (ESI) calcd for C30H43ClN5O2 [M + H]+ 540.31 found 540.3110.
5.1.61. 2-[(4S)-7-(3-chlorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]-N-[(2S)-3,3-
dimethyl-1-(4-methylpiperazin-1-yl)butan-2-yl]acetamide hydrochloride (16b)
Compound 16b was prepared from 12e according to the same procedure for compound 14a by using compound 20b. Yield: 46%; as
an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.36 (br. s., 1H), 7.79 (br. s., 1H), 7.70 (d, J = 9.7 Hz, 1H), 7.59 (t, J = 1.7 Hz,
1H), 7.47 - 7.53 (m, 2H), 7.34 (t, J = 7.9 Hz, 1H), 7. 20 (dd, J = 7.9, 1.8 Hz, 1H), 7.11 (d, J = 1.7 Hz, 1H), 4.56 - 4.77 (m, 1H), 3.65 -
3.85 (m, 2H), 3.39 (bm, 2H), 3.04 (m, 1H), 2.81 (dd, J = 15.1, 6.7 Hz, 1H), 2.77 (s, 3H), 2.64-2.67 (m, 1H), 2.46-2.47 (m, 1H), 2.15-
2.36 (m, 8H), 0.79 (s, 9H); LCMS : m/z 486 [M+H]+ at r.t. 4.89 min.; HRMS (ESI) calcd for C26H37ClN5O2 [M + H]+ 486.2631 found
486.2624.
5.1.62. 2-[(4S)-7-(3-chlorophenyl)-1-oxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]-N-[(2S)-1-(4-
methylpiperazin-1-yl)propan-2-yl]acetamide hydrochloride (16c)
Compound 16c was prepared from 12e according to the same procedure for compound 14a by using compound 20c. Yield: 38%; as
light grey foam. 1H NMR (400 MHz, DMSO-d6) δ 9.53 (br. s., 1H), 7.88 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 2.7 Hz, 1H), 7.61 (t, J = 1.7
Hz, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.49 (s, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.20 (dd, J = 7.9, 1.1 Hz, 1H), 7.11 (d, J = 1.7 Hz, 1H), 4.57 -
4.68 (m, 1H), 3.87 - 4.00 (m, 1H), 3.70 (dd, J = 12.4, 3.6, Hz, 1H), 3.37-3.41 (m, 1H), 2.80 - 3.07 (m, 5H), 2.75 (br. s., 3H), 2.62 - 2.70

(m, 1H), 2.53 - 2.61 (m, 1H), 2.13 - 2.35 (m, 5H), 0.95 (d, J = 6.6 Hz, 3H); LCMS : m/z 444 [M+H]+ at r.t. 4.38 min.; HRMS (ESI)
calcd for C23H31ClN5O2 [M + H]+ 444.2161 found 444.2152.
5.2. Expression and Purification of PIM proteins
The GST-tagged versions of human PIM1, 2 and 3 were expressed and purified in bacteria following the published conditions.6, 28
Briefly, cells were harvested, lysed in buffer containing 100 mM TrisHCl pH 7.6, 150 mM NaCl, 20 mM DTT, 1 mM EDTA, 0.2%
CHAPS, and the soluble fraction was loaded onto a glutathione based affinity column and extensively washed. The GST tag was
removed by overnight incubation on column at 4°C with the site specific protease PreScission (GE), and the eluted proteins were then
subjected to a final polishing step with a SDX200 column in a final buffer containing 25 mM TRIS pH 7.6, 150 mM NaCl, 2 mM DTT.
5.3. Crystallographic methods
Crystals of the apo protein were obtained at 20 °C by the vapor diffusion method (sitting drops), mixing equal volumes of PIM1 (10
mg/ml in 25 mM TrisHCl pH 7.6; 150 mM NaCl; 2 mM DTT ) and the reservoir solution consisting of 20% PEG 3350K, 0.3 M NaCl,
0.1 M TrisHCl pH 7.6. The crystal complex of PIM-1 with either compound 1 and 14a was obtained by transferring crystals of the apo
protein in the reservoir solution containing 1 mM inhibitor and 5% DMSO (final concentration) and soaking them overnight..
Diffraction data were collected at the European Synchrotron Radiation Facility (Grenoble, France) on beamlines ID14-4 and ID23-2.
Indexing, integration and scaling were performed using DENZO and SCALEPACK.37 Model building and structure refinement were
iteratively carried out with COOT38 and Refmac in the CCP4 suite.39,40 The coordinates have been deposited in the Protein Data Bank
with code 4bzn, 4bzo for 1and 14a respectively, together with structure factors.
5.4. Molecular Modeling
All molecular docking and the generation of binding models were performed starting from reported crystallographic structures using
the QXP/FLO program. In docking, typical settings correspond to 2000 Monte Carlo conformational search steps and 10 final poses
saved. Polar hydrogen atoms were added and optimized with the available QXP/FLO tools.41
5.5. In Vitro Kinase Assays
The biochemical activity of compounds was determined by incubation with specific enzymes and substrates, followed by
quantization of the phosphorylated product.17 PIM1 showed a linear kinetic after a step of pre-activation by auto-phosphorylation in the
following conditions:1.7 µM PIM1 incubated for 1 h at 28° C in the presence of 125 μM ATP. Compounds were 3-fold serially diluted
from 10 to 0.0006 µM, then incubated for 60 min at room temperature in the presence of ATP/P33γ-ATP mix [= 2αKm], substrate
[≥3Km], and the specific enzyme in a final volume of 15µl of kinase buffer, using 384 well plates. The final concentration of DMSO
was 1%. PIM1(1nM), PIM2 (1.5nM), or PIM3 (1nM) enzymes were incubated with compounds in 50mM HEPES, pH 7.5 buffer
containing, 10 mM MgCl2, 1 mM DTT, 3 μM Na2VO3, 0.2 mg/mL BSA and 1.0% of DMSO at room temperature. The reaction was
initiated by addition of substrate AKTide (ARKRERTYSFGHHA) and ATP as required:
for PIM1 ATP = 200 μM; 33P-γ-ATP: 6 nM; AKTide substrate = 25 µM
for PIM2 ATP = 4 µM ; 33P-γ-ATP = 1 nM, AKTide substrate = 5 µM
for PIM3 ATP= 80µM, 33P-γ-ATP = 1 nM, AKTide substrate 30µM
After 60 min at room temperature, an amount of 60µl of Dowex resin (Supelco) in sodium formate buffer pH 3.00, was added to stop
the reaction and capture unreacted P33γ-ATP, separating it from the phosphorylated substrate in solution. After 60 min of rest, a
volume of 22 µl of supernatant was transferred to Optiplate 384well plates. After the addition of 50 µl of Microscint 40 (Packard), the
radioactivity was counted in the TopCount.
5.6. Cell proliferation assay

Ba/F3 cell line stable transfected with PIM1 was generated using the Flp-In System (Life technologies-Invitrogen).6 Briefly a Ba/F3
cell line containing an integrated FRT site was created according to the system instructions and the gene coding for full length PIM1
was cloned into the pEF5/FRT/V5-DEST plasmid. A co-transfection was performed with the previous vector and the pOG44 plasmid
on the new Ba/F3 cells which were first selected by their resistance to hygromycin B and then by their ability to grow in absence of IL-
3. Human leukemia cell line MV4-11, human multiple myeloma cell lines KMS-11, LP-1, NCI-H929 and RPMI 8226, human
carcinoma prostate cell lines LnCAP, and human adenocarcinoma pancreas cell line CAPAN-1, were obtained from ATCC and
maintained in RPMI 1640 plus 2mM Glutamine and 10% Fetal bovine serum. To evaluate the antiproliferative activity of the
compounds, cells were seeded into 96 or 384 wells plates at final concentration ranging from 10000-30000 cells/cm2 for adherent cells
and 100000/ml for non adherent, in appropiate medium plus 10% FCS. After 24hs cells were treated with serial dilutions of the
compounds dissolved in 0.1%DMSO and incubated at 37°C and 5 % CO2. After 72 hours the plates were processed using CellTiter-Glo
assay (Promega) following the manufacturer’s instruction. CellTiter-Glo was a homogenous method based on the quantification of the
ATP present, an indicator of metabolically active cells. ATP was quantified using a system based on luciferase and D-luciferin resulting
into light generation. The luminescent signal was proportional to the number of cells present in the culture. Briefly, 25 µl/well reagent
solution were added to each well and after 5 minutes, shacked microplates were red by Envision (PerkinElmer) luminometer. The
luminescent signal was proportional to the number of cells present in culture. Data obtained from compound versus vehicle treated cells
were compared using Assay Explorer (Symyx Technologies Inc.) software. IC50 was calculated using sigmoid interpolation curve.
5.7. Mechanism of action studies
The mechanism of action of the compound was investigated using Ba/F3/PIM 1 cells. Cells were treated for 24hs with the compound
or DMSO as control. Immunoblotting was done according to standard procedures and detection was performed with the correspondent
antibodies recognizing total cMyc, phospho-BAD S112 and phospho-4E-BP1(Thr 37-46) from Cell Signaling Technologies, and
GAPDH from Santa Cruz Biotechnology.
5.8. In Vitro ADME and PK.
High-throughput solubility, PAMPA, Caco-2 cell permeability, metabolic stability (human liver microsomes), intrinsic clearance in
hepatocytes and in vivo Pharmacokinetics data were acquired according to previously reported procedures.42
Acknowledgments
The authors are grateful to the Analytical group for the NMR and MS analysis. We also thank Franco Ciprandi, Viviana Desperati
and Eliana Lancerotto for HPLC compounds purification, and the group of Assay Development for the biochemical assay on kinases
panel.
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