Novel tetrahydropyrido[1,2-a]isoindolone derivatives (valmerins): Potent cyclin-dependent kinase/glycogen synthase kinase 3 inhibitors with antiproliferative activities and antitumor effects in human tumor xenografts

Article

pubs.acs.org/jmc

Novel Tetrahydropyrido[1,2‑a]isoindolone Derivatives (Valmerins):

Potent Cyclin-Dependent Kinase/Glycogen Synthase Kinase 3

Inhibitors with Antiproliferative Activities and Antitumor Eﬀects in

Human Tumor Xenografts

Rajaa Boulahjar,^†,‡Aziz Ouach,^†Chiurato Matteo,^†Stephane Bourg,^†Myriam Ravache,^⊥

̂

Remy le Guevel,^⊥Severine Marionneau,^#Thibauld Oullier,^#Olivier Lozach,^§Laurent Meijer,^∥,§

́

Christiane Guguen-Guillouzo,^⊥Saïd Lazar,^‡Mohamed Akssira,^‡Yves Troin, Gerald Guillaumet,^†

́

and Sylvain Routier*^,†

^†Institut de Chimie Organique et Analytique, Universite

Cedex 2, France

́

d’Orlea

́

ns, UMR CNRS 7311, rue de Chartres, BP 6759, 45067 Orlea

́

ns

^‡Laboratoire de Chimie, Bioorganique et Analytique, URAC 22 po

̂

le Repam, Universite Hassan II Mohammedia-Casablanca, BP 146,

́

28800 Mohammedia, Morocco

^§CNRS, “Protein Phosphorylation & Human Disease” group, USR3151, Station Biologique, B.P. 74, 29682 Roscoﬀ Cedex, France

^∥ManRos Therapeutics, Centre de Perharidy, 29680 Roscoﬀ, France

^⊥Plateforme ImPACcell-SFR BIOSIT UMS-CNRS3480 UMS-INSERM018, Universite

^#In vivo Platform, Cancer

opole Grand Ouest, 63 quai Magellan, 44000 Nantes, France

Clermont Universite, ENSCCF, Laboratoire de Chimie des Heterocycles et des Glucides, BP 10448, 63000 Clermont-Ferrand,

́

de Rennes 1, 35043 Rennes Cedex, France.

́

̂

́

France

S

* Supporting Information

ABSTRACT: The development of CDK and GSK3 inhibitors

has been regarded as a potential therapeutic approach, and a

substantial number of diverse structures have been reported to

inhibit CDKs and GSK-3β in recent years. Only a few

molecules have gone through or are currently undergoing

clinical trials as CDK and GSK inhibitors. In this paper, we

prepared valmerins, a new family containing the

tetrahydropyrido[1,2-a]isoindone core. The fused heterocycle was prepared with a straightforward synthesis that was

functionalized by a (het)arylurea. Twelve valmerins inhibited the CDK5 and GSK3 with an IC₅₀< 100 nM. A semiquantitative

kinase scoring was realized, and a cellular screening was done. At the end of our study, we investigated the in vivo potency of one

valmerin. Mice exhibited good tolerance to our lead, which proved its eﬃcacy and clearly blocked tumor growth. Valmerins

appear also as good candidates for further development as anticancer agents.

GSK3. This enzyme, which is encoded by two independent

genes, -α and -β, is interestingly involved in a broad range of bio-

logical processes such as stem-cell renewal, the cell-division cycle,

diﬀerentiation, apoptosis, circadian rhythm, transcription, and

insulin action, and its deregulation has been associated with many

diseases, such as metabolic disorders, CNS diseases, and cancer.^3,4

The development of CDK and GSK3 inhibitors has been

regarded as a potential therapeutic approach and a substantial

number of diverse structures have been reported to inhibit

CDKs and GSK-3β in recent years. Only a few molecules have

gone through or are currently undergoing clinical trials. Flavo-

piridol (Alvocidib) and its analogues P276-00, (R)-roscovitine

INTRODUCTION

■

Abnormal protein phosphorylation is frequently involved in the

development of various human pathogeneses. Among the 518

human genes encoding protein kinases, cyclin-dependent pro-

tein kinases (CDKs) and glycogen synthase kinase-3 (GSK3),

two serine/threonine kinases, have attracted considerable

attention because of their frequent deregulation in major

pathologies. CDKs are located in the nucleus and participate

mainly in processes of cell cycle control, transcription, and

post-transcriptional modiﬁcations but also in cell diﬀerentiation

and cancer cell death.^1,2Consequently, many pharmacological

inhibitors of CDKs have been found to display promising

antitumor and/or neuroprotective activities. Although a few

kinase inhibitors display a deﬁnite speciﬁcity proﬁle, many of

them are rather unselective and inhibit several kinases such as

Received: June 18, 2012

Published: October 19, 2012

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Figure 1. Some representative CDK and GSK3 inhibitors in clinical trials.

Figure 2.

(Seliciclib), R547, SNS-032, PD-0332991 (CDK4 and -6 selec-

tivity), AZD5438, AG-024322, and AT7519 are representative

examples (Figure 1).⁵⁻¹²Most of them target multiple CDKs

and are applied in the treatment of leukemia, melanoma

and solid tumors. Only nine GSK3β inhibitors are in develo-

pment, while the bis(indolylmaleimide) derivative named

enzastaurin (LY317615, phase II) is appropriate for brain

cancer.¹³

or from a β-hydroxylactam under intramolecular cyclization. In

our opinion, synthesis of the valmerins¹⁸(i.e., tetrahydropyrido-

[1,2-a]isoindolone(hetaryl)ureas) E will also be achieved using

as a key step the intramolecular cyclization of the β-hydro-

xylactam 4 which is readily accessible from the nitrophthalimide

1 (Scheme 1).

Scheme 1. Retrosynthetic Scheme

In the search for heterocyclic skeletons that could be used to

design new protein kinase inhibitors, we ﬁrst developed novel

indolocarbazole analogues and published original anticancer

series possessing strong cell eﬀects and exhibiting kinase inhibi-

tion.¹⁴Some of them have entered clinical trials. More recently,

we reported an original synthesis leading to het(aryl)indoli-

zinones A via an (acyl)iminium intermediate (Figure 2).¹⁵Thus,

we envisioned the use of our optimized synthetic pathway leading

to tetrahydropyrido[1,2-a]isoindolone B as the central core for

new CDK inhibitors. To establish our SAR studies, we will

start from the work of Honma et al. which described new

bis(het)aryl ureas C and D to optimize their selective CDK4

inhibitors.¹⁶

A complete SAR study was carried out using various urea

residues on E (alkyls, aryls, and heteroaryls) to aﬀord a very

interesting library exhibiting highly potent CDK/GSK3

inhibitors.¹⁹The best ﬁnal derivatives were next evaluated

against seven cancer cell lines, and, after selection we used our

leader for in vivo evaluations on xenograft mice. The results,

from synthesis to a full biological evaluation, are also described

in this paper.

Our chemical investigations indicated that the derivatives A are

readily available by condensation of a β-formylbenzoic acid with a

primary amine (i.e., 2-(2-methyl-1,3-dioxolan-2-yl)ethanamine)¹⁷

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a

Scheme 2

a

Key: (i) methyl vinyl ketone (1.2 equiv), Triton B (cat.), EtOAc, reﬂux, 12 h, 85%; (ii) ethylene glycol (2.0 equiv), PTSA·H₂O (cat.), toluene,

Dean−Stark, reﬂux, 12 h, 89%; (iii) NaBH₄(3.0 equiv), THF/MeOH (1/2), −20 °C, 15 min, 76% ; (iv) PTSA·H₂O (0.5 equiv), toluene, Dean−

Stark, reﬂux, 6 h, 73%; (v) HCl 10%, acetone, reﬂux, 3 h, 90%; (vi) Pd/C (10%), H₂P_atm, CH₂Cl₂, rt, 16 h, 88%; (vii) NH₂−NH₂·H₂O (4.0 equiv),

Na (3.0 equiv), ethylene glycol, reﬂux, 4 h, 86%.

a

Scheme 3

CHEMISTRY

■

Only a few studies are related to the synthesis of the targeted

tetrahydropyrido[1,2-a]isoindolone core.^20,21In our work, we

envisioned that the attempted scaﬀold E could be produced

from the commercially available 3-nitrophthalimide 1 after a

four-step sequence.

A Michael addition was ﬁrst carried out with 1 (Scheme 2)

and the methyl vinyl ketone in the presence of a catalytic

amount of Triton B. After 12 h, the ketone 2 (85%) was

isolated and then protected by treatment with ethylene glycol

in reﬂuxing toluene to aﬀord the cyclic ketal 3 in 89% yield. A

regioselective reduction of the maleimide group was carried out

at low temperature with an excess of NaBH₄in only a few

minutes. The choice of the solvent and the strict control of

temperature are important experimental criteria to obtain the

desired selectivity. Formation of the other regioisomer was

never observed in these conditions. The cyclization of the

hydroxylactam 4 was promoted using PTSA hydrate at toluene

reﬂux and led to the tetrahydropyrido[1,2-a]isoindolone

core after a few hours in an acceptable 73% yield. The change

in the amount of PTSA or the use of another acidic source (i.e.,

BF₃·Et₂O, HCl, or CSA) led only to lower yields.

a

Key: (i) (a) Et₃N (1.3 equiv), −10 °C, THF, 5 min, (b) ClCO₂Et

(1.5 equiv), −10 °C, THF, 2 h, (c) NaN₃(1.7 equiv), −10 °C, H₂O, 1

h, (d) toluene, reﬂux, 1 h; (ii) 8, toluene, reﬂux, 24 h, valmerins 13−

40, 42−90% yields.

To provide the dissymmetrical urea library, we used 23 com-

mercially available acids reported in Table 1. Others are readily

available after some chemical transformations (Scheme 4).²³⁻²⁵

To prepare the MOM-protected pyridine and quinoleine

derivatives 9−12, we started from the corresponding hydroxy

acids which were ﬁrst transformed into methyl esters prior to

conducting MOM protection. At the end of the sequence, a

simple saponiﬁcation led to the expected compounds. Overall

yields of the three-step syntheses were in the 70−77% yield

range.

Once the cycloadduct 5 had been obtained, the ketone

protection was removed using an aqueous hydrochloride

solution in reﬂuxing acetone in 90% yield. The selective

reduction of the nitro group of 6 was successfully accomplished

using hydrogenolysis at room temperature in CH₂Cl₂. This

method led to the aniline 7 in 88% yield. Finally, we carried out

a modiﬁed Wolﬀ−Kishner reaction with hydrazine and sodium

in reﬂuxing ethylene glycol which furnished the key amine 8 in

86% yield.²²Direct conversion of 6 into 8 under hydrogen

pressure at high temperature failed. Ketone could be reduced

prior to the nitro group, but lower yields decrease the interest

of this route.

Starting from 8, classical urea formation involving tripho-

sgene or p-nitrophenyl carbamate methods gave disappointing

results, the low reactivity of the amine leading only to complex

mixtures. We therefore focused our eﬀorts on Curtius

rearrangements. Starting from highly available acids in aliphatic,

aromatic, and heteroaromatic series, the isocyanides were ﬁrst

generated in situ and immediately scavenged by our prebuilt

amine 8. This very attractive “one-pot” general pro-

cedure (Scheme 3) was applied to several acids F to furnish

the attempted valmerin ureas E.

Finally, the isocyanates corresponding to 27 carboxylic acids

were formed and quenched in situ by amine 8 to aﬀord the

ureas 13−39. Products were isolated in 42−90% yields, which

mainly depended on the nature and solubility of carboxylic

acids.

The sole limitation we found concerned the use of 1H-

pyrazole-3-carboxylic acid, which led via Curtius rearrangement

and in situ condensation with the amine 8 to valmerin 40 in a

very poor 4% yield (Scheme 5).

Alternatively, we condensed in situ the N-1-Boc-3-amino-

pyrazole¹⁶and the isocyanate of 8 which was quantitatively

formed using BTC in the presence of Et₃N. After 16 h, the

attempted urea 41 was isolated in 56% yield. Boc deprotection

was carried out in basic media to furnish 40 at room

temperature in a satisfying 66% yield. The MOM protective

groups of 36−39 were next removed under classical media to

aﬀord the ﬁnal derivatives 42−45 in high yields.

RESULTS AND DISCUSSION

■

Kinase Assays. First we tested our valmerin library against

several representative kinases in order to determine the

inﬂuence of urea R residues (Table 2) on kinase inhibition

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Table 1. Synthesis of Valmerins E from Acids F via Curtius Rearrangement

a

Yields are indicated in isolated products.

(CDK5/p25, GSK3α/β, and DYRK1A). With an alkyl and

phenyl group, the valmerins 13 and 14 are inactive (entries 1

and 2) on all three kinases. All other compounds exhibited a

kinase inhibitory eﬀect, and structural modiﬁcations led to a

modulation in their activity.

The structural key that allows interaction with the kinase

active site is the presence of a nitrogen atom on the aromatic

ring in position 2. Compound 15 (entry 3) inhibited CDK5

at IC₅₀= 80 nM. This result conﬁrms the crucial role of the

pyridine nitrogen atom in position 2,¹⁶which could be

explained by the formation of an intramolecular hydrogen

bond between the pyridinic nitrogen atom and a labile

urea hydrogen atom. The resulting rigidity and withdrawal of

the valmerins led to a maximal occupation of the ATP

pocket.

Adding a second electron-rich and basic nitrogen atom to the

heteroaromatic residue of urea maintained this eﬀect, and the

activity of pyrazine 16 increased to 70 nM for each kinase, but

this strategy is worthwhile only for the 5 position. Displacement

of the second nitrogen (i) from position 4 to 5 (for number

assignment see general formula table 3) led to the quite inactive

pyrimidine compound 34 and (ii) from position 4 to 3 a

micromolar active derivative 35 (entries 22 and 23) was

obtained.

A contraction ring prompted us to use the thiazole, pyrazole,

and oxazole heterocycles, but each synthesized valmerin

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a

In general, the GSK3 (compared to CDK5) active site

accepts bulkier groups.²⁷CDK5-inactive 26, 27, 29, and 35

derivatives appeared to be potent against GSK3 with IC₅₀

ranging between 130 and 330 nM. The most selective

compound is without doubt valmerin 34, which is completely

inactive on CDK5 and DYRK1A (IC₅₀> 10 μM) but exhibits a

750 nM IC₅₀on GSK3.

Scheme 4

The three valmerins 15, 16, and 31 emerged as good leads to

pursue our investigations. Among them, we selected the

pyridine ring to explore other avenues and complete our

QSAR studies (Table 3). We added CK1 to our enzyme panel.

We also showed that valmerin 15 (entry 1) and 16 (not

shown) similarly aﬀect the three structurally related kinases

CDK 1, 5 and GSK3. All of the new derivatives have only

moderate interactions with CK1 and DYRK1A active sites,

while major eﬀects are always observed on CDK5 and GSK3.

On CDK5, a bulky and electron-rich bromo atom enhanced

the activity. Positions 2 and 4 were privileged compared to

position 3 (entries 2−4). Valmerins 18 and 19 exhibited

remarkable IC₅₀values of 40 and 25 nM, respectively.

A slight decrease in kinase inhibition occurred when bromine

was replaced by a ﬂuorine atom (entries 5 and 2), but with this

small atom, valmerin 20 was as active as the previously selected

compound 15. A poly chloration or an increase in the sub-

stitution size (i.e., piperidine or a phenyl groups, entries 7, 9,

and 10) led to a full inactivation of valmerins against CDK5. In

position 3, valmerins could create a supplementary hydrogen bond

with the active site. Adding a hydroxyl group in this position led to

derivative 42, which inhibits CDK5 with an IC₅₀of 37 nM. This

hydrophilic group is nevertheless a handicap in position 5 since

it may interact with the urea function and dramatically perturb

the crucial orientation of the pyridine ring (entry 14).

a

Key: (i) (a) MeOH, SOCl₂(1.5 equiv), 60 °C, 16 h, (b) K₂CO₃(2.0

equiv), MOMCl (1.5 equiv), acetone, reﬂux, 16 h, (c) NaOH (1.2

equiv), THF/H₂O (1/1), rt, 2 h, 9 75%, 10 77%, 11 70%, 12 72%.

showed a lack of activity (entries 7−13). Submicromolar active

compounds are the less substituted 28, 30, and 33. Inter-

estingly, the more basic pyrazole containing product 40 led to

the best activity, but a simple methylation increased the IC₅₀by

a factor of 5 (entry 10 vs 11), therefore decreasing the interest

of this substitution.

The last modiﬁcation we tried was the use of fused bicyclic

heterocycles. Benzothiazole 32 was poorly active, but

fortunately, the isoquinoline valmerin 31 gave interesting results

and activities on CDK5 that are comparable to valmerins 15

and 16 (entries 3 and 4). Nevertheless, hydroxyquinolines 44

and 45 did not have any eﬀect on the kinases.

Valmerins exhibited some selectivity in kinase inhibition. All

of them were quite inactive on DYRK1A, but a good inhibition

of GSK3 was observed. Except for valmerins 32 and 45, all the

other derivatives displayed an IC₅₀in the submicromolar range.

As mentioned previously, the GSK3 active site accepts more

variations and the presence of lipophilic and hydrophilic groups

is well tolerated. Five valmerins 17−20 and 42 showed IC₅₀

a

Scheme 5

a

Key: (i) and (ii) see also Scheme 3, 40 4%; (iii) BTC (1.0 equiv), Et₃N (2.0 equiv), CH₂Cl₂, reﬂux, 5 h; (iv) N-1-Boc-3-aminopyrazole (1.0 equiv),

CH₂Cl₂/pyridine 9/1, rt, 16 h, 56%; (v) aq NaOH (1 M)/THF 1/2, rt, 24 h, 66%; (vi) aq HCl 10%, acetone, reﬂux, 2 h, 42 90%, 43 92%, 44 92%,

45 95%.

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a

Table 2. Selection of R Residue on Valmerins

a

Compounds with an IC₅₀CDK5 < 100 nM or GSK3 < 1 μM are selected. Assays were performed in triplicate.

values lower than 100 nM. A bromine atom or hydroxy group

could be indiscriminately placed in positions 2−4, but

disubstitution led to a partial inactivation. In position 2, bulky

and lipophilic piperidine or phenyl rings were partially

tolerated; compounds 24 and 25 inhibited GSK3 in the

submicromolar range.

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a

Table 3. Selection of Valmerin Pyridine Ring Substituents

entry

valmerins

Z

CDK1 IC₅₀(μM)

CDK5 IC₅₀(μM)

GSK3α/β IC₅₀(μM)

CK1 IC₅₀(μM)

DYRK1A IC₅₀(μM)

1

2

15

18

17

19

20

21

22

23

24

25

42

43

H

0.075

ND

0.08

0.040

0.13

0.025

0.09

0.26

10

0.17

0.043

0.076

0.022

0.026

0.2

ND

1.3

>10

3.4

2-Br

3-Br

4-Br

2-F

3

>10

7.6

4

5

ND

>10

ND

2.6

3.5

6

3,5-diF

>10

8.1

7

2,3,4,5-tetrachloro

5-OMe

4.3

8

0.59

>10

4

1.3

9

2-piperidinyl

3-phenyl

0.36

0.14

0.051

4

>10

1.5

>10

1.8

10

13

14

3-OH

0.037

1.3

5-OH

≥10

>10

a

Assays were performed in triplicate. ND: not determined. Compounds exhibiting an IC₅₀CDK5 or GSK3 < 200 nM were selected.

a

Table 4. Protein Kinase Selectivity of Valmerin 15 in a Kinase Interaction Assay (KinomeScan)

kinase

CDKL2

score

kinase

score

kinase

CDK2

score

kinase

score

0

CSNK2A2

L576P

0.35

0.4

0.9

1

KIT

2.8

2.9

3.1

3.2

3.3

3.5

3.8

4

DRAK1

FLT3

0

V559D

PIP5K2B

CDK3

KIT(D816V)

TAK1

0

RIOK1

CDC2L5

JNK2

1

GSK3B

JNK1

0

1.2

1.3

1.4

1.6

1.7

1.8

2.2

2.4

2.5

2.8

FLT3(K663Q)

PKNB

0

0.4

CDK9

MYLK4

PCTK1

CDKL5

PFTK1

BIKE

0

PCTK2

TGFBR2

DRAK2

RIOK3

CIT

0.4

PDGFRB

RSK2

ABL1

0

0.4

TYK2

0.05

0.1

0.15

0.25

0.3

0.5

PFCDPK1

MEK5

PRKD1

PFTAIRE2

TRKB

0.5

0.6

CDKL1

SRPK1

MLCK

ICK

GSK3A

JNK3

FLT3(

SNARK

MLK3

0.6

JAK1

0.6

RSK1

CDK4-cyclinD3

CDKL3

JAK3

0.65

0.7

ARK5

4.3

4.5

4.6

4.8

PCTK3

YSK4

EPHB6

PRKD3

STK36

ABL1

FLT3

0.75

0.85

0.9

PKN2

ERK8

RSK4

FLT3(ITD)

PRKD2

CDK5

AURKC

a

Valmerin 15 was tested at a 10 μM concentration on a 442 kinase interaction panel. A semiquantitative scoring of this primary screen was obtained.

This score relates to the probability of a hit rather than strict aﬃnity. Scores >10, between 1−10 and <1, indicate that the probability of a hit being a

false positive is <20%, <10%, and <5%, respectively. The best scores from 0 to 5 are presented. Full results are available in the Supporting

Information (Table S1).

This pyridine variation completes our lead family. Searching

for a compromise between CDK5 and GSK3 inhibition, it

appeared that valmerins 18−20, 42 are the most interesting

molecules of the substituted pyridinic series. However, the

valmerin leading to the derivative 15 deserves considerable

interest because of its ability to target with high eﬃcacy the

three main kinases involved in cell cycle and/or in growth

control.

Molecular Modeling. The binding mode of our inhibitor

15 within GSK3 was elucidated using a docking model (Figure 3)

and is characteristic of those found within the kinase family.²⁶

In this graphical representation, the valmerin 15 binds in the

deep cleft between the two lobes of the catalytic core structure

(the smaller N terminal domain at the top and the rich α

helices C terminal domain below) and the extreme parts of the

pyridine and piperidine rings are exposed to solvent.

We also ran a semiquantitative kinase interaction assay

(KinomeScan, Discoverx) of valmerin 15 at 10 μM on a 442

kinase panel. Results showed that CDKs (blue), CDK-like

kinases (CDKLs) (orange), and GSK3 (green) are targeted in

addition to quite a few other kinases (Table 4).

Similarly to ATP, compound 15 binds the kinase active site

forming two hydrogen bonds with the hinge region: the ﬁrst

one is present between the NH backbone of Val 135 and the

urea’s oxygen atom. The second one is realized between the

oxygen of the Val 135 carbonyl group and the NH urea group

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Figure 3. Binding mode of valmerin 15 within GSK3. Left: GSK3 protein from 2OW3 and 15 docked inside. Right: zoom on the active site. Hinge

region was highlighted in yellow and the DFG motif of the activation loop is highlighted in green. Protein was represented as a ribbon diagram with

the cartoon style and 15 exhibits tube representation (elements colored). Nonpolar hydrogens are not represented for clarity. H bonds are

represented as dashed yellow lines.

a

Table 5. In Vitro Cell Eﬀects of Selected Valmerins

a

Assays were performed in triplicate. Fibro represents skin ﬁbroblast cell lines. Reference compounds used (not shown) were DMSO, Taxol,

roscovitine, and doxorubicin.

again near the pyridine cycle of 15. A supplementary hydrogen

bond concerns 15 itself as it is an intra H bond connecting the

second NH of urea with the nitrogen pyridine atom. This last

hydrogen bond participates to the H bond pattern by orienting

the urea group to the hinge residues.

In Vitro Cell Assays. To pursue our selection, all of the

synthesized valmerins were evaluated against six representative

human cancer cell lines (liver HuH7, colon Caco2 and

HCT116, breast MDA-MB231, prostate PC3, and lung NCI-

H727) as well as human normal diploid skin ﬁbroblast cells. To

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Figure 4. (A) Immunoﬂuorescence labeling of cleaved (active) caspase 3 in HCT116 cells untreated (DMSO) or treated with valmerin 15 (0.6 μM)

and valmerin 16 (1.0 μM) for 24 h. Nuclei were stained with Hoechst (blue), and apoptotic cells were immunostained with an antiactive caspase 3

antibody (red) (magniﬁcation, ×10). (B) Quantiﬁcation of apoptotic cells. Apoptotic cells were calculated as the percent of active caspase 3-positive

cells/total nuclei with the Cellomics High Content Imaging System. Data shown are the mean sem from a typical experiment performed in

triplicate. **P < 0.01, ***P < 0.001, control cells vs treated cells.

simplify the presentation, we report in Table 5 the results for

derivatives that showed submicromolar IC₅₀values against

CDKs and GSK3. All of the unselected molecules exhibited

micromolar eﬀects whatever the cell line (not shown); 18

precipitated during the cell eﬀect measurements.

valmerin 15 and 16 provide evidence supporting the idea that

the decreased number of living cells induced by these

compounds was mainly associated with strong induction of

an apoptotic signal, principally induction of caspase 3 activity

(Figure 4) as shown with the HCT116 cell line.

Eﬀects on cells fully depended on the R substituent.

Disappointingly, valmerin 31 only led to a micromolar eﬀect

on all the cancer cell lines tested. This compound was discarded

because it appeared dramatically toxic on normal diploid ﬁbro-

blast cells. Surprisingly, 4-bromopyridine derivative 19 aﬀected

all cell lines except the liver HuH7. This might be explained by

the high expression of transporter proteins such as Pgp and

MDRs, which may induce an early clearance of this target

molecule in these cells.²⁷The formation of an inactive metabolite

through hepatic detoxiﬁcation function cannot be ruled out.

The same variability in cell survival was ampliﬁed when

considering compounds 42 and 16. For compound 42, sub-

micromolar IC₅₀values were obtained with HuH7, PC3, and

NCI-H727 cells, while micromolar values were obtained with

Caco2, MDA-MB231, HCT116 and with diploid ﬁbroblasts;

for compound 16, no response was obtained with Caco2 and

MDA-MB231 cell lines while all the others responded.

Interestingly, these two cell lines carry mutations which alter

growth controls, mainly those associated with Wnt-β catenin

pathways which, therefore, drastically reduce the inﬂuence of

GSK3 modulation, while the HCT 116 line, another colon cell

line, has preserved part of these regulations.²⁸

We therefore wondered whether this apoptotic eﬀect could

partly result from growth arrest induction and cell cycle

inhibition. Progression into the cell cycle was estimated by

using both S and M phase markers (Figure 5). Analyses led to

detect a net decrease of cells engaged in the cell cycle, support-

ing the hypothesis of a blockage located at the entry or during

the G1 phase. Further experiments on speciﬁc signaling path-

ways need to be performed to further conﬁrm these conclusions.

Again, valmerin 15 appeared more eﬃcient than valmerin 16.

In Vivo Assays. Valmerin 15 was selected for in vivo assays

and was prepared in a gram scale. Tolerance was ﬁrst evaluated

in 6−8 week-old female NMRI mice (Janvier Laboratories).

The up-and-down procedure was used with six animal per dose

(0, 1, 5, and 10 mg/kg), the product was administered

intraperitoneally and daily over 2 weeks, and the animals were

monitored 2 weeks after the last administration. Daily observa-

tion over 14 consecutive days after product administration

suggested good tolerance as shown by clinical signs, physical

examination, body weight, and food consumption. Macroscopic

examination of the organs at the end of the experiment showed

no abnormalities and the blood formulation was normal.

Valmerin 15 partially slows down tumor growth in the HCT-

116 mouse xenograft model. After 4 weeks of treatment, results

showed a tumor reduction of 45.3% (Figure 6). At the end of

the experiment, valmerin 15 had induced a 51% reduction in cecum

BLI which correlates with the tumoral volume (Figure 6, right).

At necropsy, mesenteric nodes, liver and lung of each mouse

were assessed on Φimageur to quantify the metastasis burden

(Figure 7). There was no diﬀerence between control and

Valmerin 15 treated mice in terms of mesenteric nodes or liver

invasion, but only 1/9 treated mice had developed lung

metastasis against 6/9 control mice. It seemed that Valmerin 15

had limited lung invasion.

Nevertheless, three compounds 15, 16, and 20 emerged from

this in vitro cell study. The best results were obtained with the

HuH7, MDA-MB231, and HCT116 cell lines and IC₅₀values

were always lower than those observed with diploid ﬁbroblasts.

The best derivative is undoubtedly valmerin 15. In this case, the

three above-mentioned 3 cell lines were aﬀected in the

nanomolar range (240, 320, 150 nM, respectively) whereas

normal ﬁbroblasts were aﬀected to a lower extent.

The strong cellular eﬀect can be fully explained by multi

kinase inhibition since the resulting eﬀect on cells appeared as a

pro-apoptotic eﬀect. Indeed, additional studies performed on

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Figure 5. Eﬀects of valmerins 15 and 16 on cell cycle progression. (A) Mitotic index. Diﬀerent cell lines were treated with DMSO (0.4%) (control

cells), roscovitine (20 μM), valmerin 15 (0.6 μM), and valmerin 16 (1 μM) for 24 h. Cells were ﬁxed, and the mitotic index was determined using

phospho-histone H3 detection. The number of dividing cells was calculated by using the Cellomics High Content Imaging System. Data shown are

the mean SD from a typical experiment performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001, control cells vs treated cells. (B) 5′-Bromo-

2′-deoxyuridine (BrdU) incorporation. Diﬀerent cell lines were treated with DMSO (0.4%) (control cells), roscovitine (20 μM), valmerin 15

(0.6 μM), and valmerin 16 (1 μM) for 48 h and pulsed with BrdU for 1 h 30. Cells were ﬁxed and immunostained for BrdU incorporation. BrdU

staining was measured by the Cellomics High Content Imaging System. Data shown are the mean SD from a typical experiment performed in

triplicate. *P < 0.05, **P < 0.01, ***P < 0.001, control cells vs treated cells.

Figure 6. Left: in vivo valmerin 15 eﬀects on tumor growth. Mice were assessed weekly using whole-body bioluminescent imaging to quantify

relative amounts of tumor burden. Data are given as cpm mean +/− SD. P < 0.05: *, p < 0.001: ***. Right: 4 weeks after the beginning of treatment,

BLI of two representative mice per group.

cell lines and diploid ﬁbroblasts) identiﬁed two stable lead

compounds 15 and 16 with interesting submicromolar cellular

eﬀects. We have identiﬁed the cellular process which explains

antiproliferative eﬀects. Valmerins acted on proliferation and

induced cell death by caspase 3 induction. At the end of our

study, we investigated the in vivo potency of valmerin 15.

Mice exhibited good tolerance to our lead, which proved its

eﬃcacy and clearly blocked tumor growth. Valmerins appear

as good candidates for further development as anticancer

agents.

CONCLUSION

■

In this paper, we prepared valmerins, a new family containing

the tetrahydropyrido[1,2-a]isoindone core. The fused hetero-

cycle was prepared with a straightforward synthesis which was

functionalized by a (het)aryl urea. Each ﬁnal compound was

evaluated against representative kinases and 12 valmerin

inhibited CDK5 and GSK3 with IC₅₀< 100 nM. A semi-

quantitative kinase scoring conﬁrmed that Valmerin 15 aﬀected

CDKs, CDK-like kinases (CDKLs), GSK3, and quite a few

other kinases. Cellular screening on human cell lines (6 cancer

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Figure 7. Left: Ex vivo cecum BLI at the end of the assay (left). The cecum BLI of all mice was quantiﬁed. Data are given as cpm mean +/− SD.

P < 0.01: **, p < 0.001: ***. Right: At necropsy, the number of mice in each group with mesenteric nodes, liver and lung metastasis.

EXPERIMENTAL SECTION

■

+10 °C. Aqueous satd NaHCO₃(1 M, 40 mL) was added, and the

organic layers were extracted successively with CH₂Cl₂(10 mL) and

EtOAc (10 mL). The combined organic layers were dried over

MgSO₄, ﬁltered, and concentrated in vacuum. Puriﬁcation by column

chromatography (CH₂Cl₂/MeOH 99/1) led to 4 as a white solid in

Chemistry. H NMR and ¹³C NMR spectra were recorded on a

Bruker DPX 250 or 400 MHz instrument using CDCl₃or DMSO-d₆.

The chemical shifts are reported in ppm (δ scale) and all coupling

constants (J) values are in hertz (Hz). The splitting patterns are

designated as follows: s (singlet), d (doublet), t (triplet), q (quartet),

m (multiplet), and dd (doublet doublet). Melting points are

uncorrected. IR absorption spectra were obtained on a Thermo-

Nicolet equipped with an ATR Ge or Diamond equipment and values

were reported in cm⁻¹. MS spectra (ion spray) were performed on a

Perkin-Elmer Sciex PI 300. High-resolution mass spectra (HRMS)

were performed on a Bruker maXis mass spectrometer by the

1

76% yield: mp 130−132 °C; H NMR (CDCl₃, 250 MHz) δ 1.39

(s, 3H), 2.05−2.21 (m, 2H), 3.59−3.75 (m, 1H), 3.85−3.87 (m, 1H),

3.94−3.96 (m, 4H), 4.23 (d, J = 5.4 Hz, 1H), 6.46 (d, J = 5.4 Hz, 1H),

7.67−7.77 (m, 1H), 8.09 (dd, J = 0.7 Hz, J = 7.4 Hz, 1H), 8.32 (dd, J =

0.9 Hz, J = 8.2 Hz, 1H); MS (IS) m/z 309.0 [M + H]⁺, 326.0 [M +

NH₄]⁺; HRMS (ESI) calcd for C₁₄H₁₇N₂O₆309.10811, found

309.10811 [M + H]⁺.

́ ́

“Federation de Recherche” ICOA/CBM (FR2708) platform. Mon-

10′-Nitro-3′,4′-dihydro-1′H-spiro[[1,3]dioxolane-2,2′-

pyrido[2,1-a]isoindol]-6′(10b′H)-one (5). A solution of β-hydrox-

ylactam 4 (500 mg, 1.62 mmol) and PTSA·H₂O (156 mg, 0.8 mmol,

0.5 equiv) in toluene (50 mL) was reﬂuxed for 6 h with a Dean−Stark

apparatus. After being cooled to room temperature, the mixture was

washed three times with aq satd NaHCO₃solution (45 mL), and the

aqueous layers were extracted with EtOAc (3 × 20 mL). The com-

bined organic layers were dried over MgSO₄, ﬁltered, and evaporated

under reduced pressure. Puriﬁcation by column chromatography

(CH₂Cl₂/MeOH 99.5/0.5) led to 5 as a yellow solid in 73% yield: mp

itoring of the reactions was performed using silica gel TLC plates

(silica Merck 60 F254). Spots were visualized by UV light at 254 and

356 nm. Chromatography columns were performed using silica gel 60

(0.063−0.200 mm, Merck). The purity was determined by using a

VWR Hitachi HPLC equipped with a chiralpack IA column (250 ×

4.6 mm). Mobile phase: A: 80% n-heptane. B: 20% ethanol. Flow

1 mL/min. Concentration 0.2 mg/mL in heptane/EtOH; inj 20 μL

peaks were detected by UV absorption with a LaChrom Elite diode

array detector L-2455 at 239, 249, and 254 nm. The HPLC proﬁles of

all valmerins show purity up to ≥95%.

1

206−208 °C; H NMR (CDCl₃, 250 MHz) δ 1.17 (t, J = 12.2 Hz,

4-Nitro-2-(3-oxobutyl)isoindoline-1,3-dione (2). 3-Nitrophtha-

limide (5.0 g, 26.0 mmol), methyl vinyl ketone (2.8 mL, 3.3 mmol,

1.25 equiv), and Triton B (benzyltrimethylammonium hydroxide, 40%

in methanol, 760 μL) in EtOAc (30 mL) were stirred at reﬂux

overnight. Evaporation to dryness under reduced pressure and

recrystallization from EtOH gives 2 as a yellow solid in 85% yield:

1H), 1.65−1.95 (m, 2H), 2.61−2.78 (m, 1H), 3.33 (td, J = 4.2 Hz, J =

13.0 Hz, 1H), 3.96−4.31 (m, 4H), 4.55 (dd, J = 4.3 Hz, J = 13.4 Hz,

1H), 5.23 (dd, J = 3.3 Hz, J = 11.7 Hz, 1H), 7.70 (t, J = 7.8 Hz, 1H),

8.19 (d, J = 7.4 Hz, 1H), 8.37 (d, J = 8.1 Hz, 1H); MS (IS) m/z 291.0

[M + H]⁺; HRMS (ESI) calcd for C₁₄H₁₅N₂O₅291.09754, found

291.09775 [M + H]⁺.

1

mp 122−124 °C; H NMR (CDCl₃, 250 MHz) δ 2.20 (s, 3H), 2.91

10-Nitro-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-

dione (6). A mixture of keto-protected compound 5 (2.0 g, 6.91

mmol) in an aq HCl solution (10%, 20 mL) and acetone (30 mL) was

reﬂuxed for 3 h. After cooling, acetone was removed under reduced

pressure, and the resulting crude material was ﬁltered, washed with

water, and dried to give compound 6 as a yellow solid in 90% yield:

(t, J = 7.3 Hz, 2H), 3.98 (t, J = 7.3 Hz, 2H), 7.93 (dd, J = 7.1 Hz, J =

8.5 Hz, 1H), 8.07−8.19 (m, 2H); MS (IS) m/z 263.0 [M + H]⁺; HRMS

(ESI) calcd for C₁₂H₁₁N₂O₅263.06624, found 263.06631 [M + 1]⁺.

2-(2-(2-Methyl-1,3-dioxolan-2-yl)ethyl)-4-nitroisoindoline-

1,3-dione (3). A solution of the above ketone 2 (5.0 g, 19.0 mmol),

ethylene glycol (2.0 mL, 38.0 mmol, 2.0 equiv), and PTSA·H₂O (21.0

mg, 0.1 mmol, cat.) in toluene (60 mL) was reﬂuxed overnight with a

Dean−Stark apparatus. The cooled solution was washed with aq satd

NaHCO₃(20 mL), and the organic layers were extracted with EtOAc,

washed with brine (20 mL), dried over MgSO₄, ﬁltered, and

evaporated under reduced pressure. The crude material was recrystal-

lized from ethanol to give 3 as a yellow crystal in 89% yield: mp 90−

1

mp 215−217 °C; H NMR (CDCl₃, 400 MHz) δ 2.08 (dd, J = 11.7

Hz, J = 14.5 Hz, 1H), 2.52−2.72 (m, 2H), 3.38−3.55 (m, 2H), 4.76−

4.82 (m, 1H), 5.39 (dd, J = 4.0 Hz, J = 11.6 Hz, 1H), 7.78 (t, J = 7.8

Hz, 1H), 8.25 (d, J = 7.4 Hz, 1H), 8.44 (d, J = 8.0 Hz, 1H); MS (IS)

m/z 247.0 [M + H]⁺; HRMS (ESI) calcd for C₁₂H₁₁N₂O₄247.07133,

found 247.07150 [M + H]⁺.

10-Amino-1,3,4,10b-tetrahydropyrido[2,1-a]isoindole-2,6-

dione (7). To a solution of compound 6 (2.0 g, 8.0 mmol) in CH₂Cl₂

(100 mL) was added Pd (C) 10% (200 mg). After being stirred at

room temperature under hydrogen atmospheric pressure for 16 h, the

mixture was ﬁltered through Celite. After being washed with MeOH

(20 mL), the combined organic layers were evaporated under reduced

pressure. Puriﬁcation by column chromatography (CH₂Cl₂/MeOH

99/1) led to 7 as a yellow solid in 88% yield: mp 68−70 °C; ¹H NMR

(CDCl₃, 400 MHz) δ 2.21 (dd, J = 12.2 Hz, J = 13.8 Hz, 1H), 2.47−

2.65 (m, 2H), 3.16−3.21 (m, 1H), 3.32−3.40 (m, 1H), 3.90 (s, 2H),

4.66 (dd, J = 3.9 Hz, J = 11.9 Hz, 1H), 4.73−4.79 (m, 1H), 6.85 (dd,

1

92 °C; H NMR (CDCl₃, 250 MHz) δ 1.36 (s, 3H), 2.10 (t, J =

7.0 Hz, 2H), 3.86 (t, J = 7.0 Hz, 2H), 3.90−4.00 (m, 4H), 7.90 (dd, J =

7.4 Hz, J = 8.2 Hz, 1H), 8.05−8.17 (m, 2H); MS (IS) m/z 307

[M + H]⁺, 324.0 [M + NH₄]⁺; HRMS (ESI) calcd for C₁₄H₁₅N₂O₆

307.09246, found 307.09244 [M + H]⁺.

3-Hydroxy-2-(2-(2-methyl-1,3-dioxolan-2-yl)ethyl)-4-nitroi-

soindolin-1-one (4). To a solution of 3 (2.0 g, 6.5 mmol) in a

mixture THF/MeOH (15/30 mL) at −30 °C was added NaBH₄(700 mg,

19.5 mmol, 3.0 equiv) in portions. The mixture was stirred at this

temperature for 15 min followed by TLC and then allowed to warm to

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J = 3.9 Hz, J = 4.9 Hz, 1H), 7.34−7.27 (m, 2H); MS (IS) m/z: 217.0

[M + H]⁺; HRMS (ESI) calcd for C₁₂H₁₃N₂O₂21709715, found

217.09739 [M + H]⁺.

256.0 [M + Na]⁺; HRMS (ESI) calcd for C₁₂H₁₂NO₄234.07608,

found 234.07615 [M + H]⁺.

4-(Methoxymethoxy)quinoline-2-carboxylic Acid (12). The com-

pound 12 was obtained following the general procedure A from the 4-

hydroxyquinoline-2-carboxylic acid as a white solid in 72% yield: mp >

10-Amino-1,2,3,4-tetrahydropyrido[2,1-a]isoindol-6(10bH)-

one (8). Keto compound 7 (500 mg, 2.31 mmol) and hydrazine

monohydrate (0.45 mL, 9.24 mmol, 4.0 equiv) were added to a solu-

tion containing sodium (160 mg, 6.93 mmol, 3.0 equiv) in diethylene

glycol (9 mL). After the mixture was reluxed for 4 h at 190 °C, the

reaction was found to be complete (TLC) and the mixture was cooled.

CH₂Cl₂(50 mL) was added, and the organic layer was washed with

NaOH (20 mL, 1 M in water), dried over MgSO₄, ﬁltered, evaporated,

and puriﬁed by silica gel chromatography column (CH₂Cl₂/MeOH

99.5/0.5) to give the compound 8 as a yellow solid in 86% yield: mp

50−52 °C; ¹H NMR (CDCl₃, 250 MHz) δ 1.10−1.12 (m, 1H), 1.29−

1.52 (m, 1H), 1.52−1.74 (m, 1H), 1.75−1.88 (m, 1H), 1.98−2.02

(m, 1H), 2.50 (dd, J = 3.2 Hz, J = 13.4 Hz, 1H), 2.96 (td, J = 3.5 Hz,

J = 12.9 Hz, 1H), 3.79 (s, 2H), 4.23 (dd, J = 3.6 Hz, J = 11.7 Hz, 1H),

4.49 (dd, J = 4.9 Hz, J = 13.3 Hz, 1H), 6.80 (dd, J = 1.4 Hz, J = 7.3 Hz,

1H), 7.17 −7.35 (m, 2H); MS (IS) m/z 203.0 [M + H]⁺; HRMS

(ESI) calcd for C₁₂H₁₅N₂O₂203.11789, found 203.11807 [M + H]⁺.

General Procedure a for Carboxylic Acids 9−12 Synthesis.

The commercial hydroxycarboxylic acids (1.0 mmol) were dissolved in

MeOH (50 mL), and thionyl chloride (2.0 mmol) was added dropwise

at 0 °C. The reaction was warmed to room temperature and then

reﬂuxed overnight. After cooling, methanol was removed under

reduced pressure and the residue was dissolved in a saturated aqueous

NaHCO₃(15 mL). The mixture was extracted with EtOAc (3 ×

20 mL). After evaporation of the combined organic layer, the crude

material was puriﬁed by ﬂash chromatography on silica gel (CH₂Cl₂/

MeOH 98/2) to give the desirable hydroxyesters. The previously

obtained hydroxy esters (1.0 mmol) were added to a suspension of

K₂CO₃(2.0 mmol, 2.0 equiv) in dry acetone (20 mL). Chloromethyl

methyl ether (1.5 mmol, 1.5 equiv) was dropwise added, and the

mixture was stirred at 0 °C for 10 min and then heated at 70 °C

for 12h. After the mixture was cooled to room temperature, water

(15 mL) was added, and the organic material was extracted with

EtOAc (3 × 25 mL). The combined organic layers were washed with

H₂O (10 mL) and brine (10 mL) and dried (Na₂SO₄). After ﬁltration

and reduction of the volatiles under reduced pressure, the crude

material was puriﬁed by ﬂash chromatography on silica gel (CH₂Cl₂/

MeOH 95/5) to give the desirable intermediates, which were next

engaged in a last saponiﬁcation step. The MOM protected esters (1.0

mmol) were dissolved in a THF/H₂O 1/1 (10 mL) mixture, and

NaOH (1.2 mmol, 1.2 equiv) was added. The solution was stirred for

2 h at room temperature, and THF was removed under reduced

pressure. The aqueous layer was acidiﬁed with acetic acid (50%) until

pH 5−6 prior to extraction with EtOAc (3 × 10 mL). The combined

organic layers were dried with anhydrous MgSO₄, ﬁltered, and

evaporated under reduced pressure to give the desired acids 9−12.

3-(Methoxymethoxy)picolinic Acid (9). The compound 9 was

obtained following the general procedure A from the 3-hydroxypico-

1

260 °C; H NMR (DMSO-d₆, D₂O, 250 MHz) δ 3.50 (s, 3H), 5.62

(s, 2H), 7.69−7.75 (m, 2H), 7.88 (t, J = 7.5 Hz, 1H), 8.13 (d, J = 7.5 Hz,

1H), 8.25 (d, J = 7.5 Hz, 1H); SM (IS) m/z 232.0 [M − H]⁻; HRMS

(ESI) calcd for C₁₂H₁₁NO₄Na 256.05803, found 256.05825 [M + H]⁺.

General Procedure B for Isocyanate Synthesis from Acids V.

Under argon, a stirred solution of carboxylic acid V (0.37 mmol) and

Et₃N (66 μL, 0.48 mmol, 1.3 equiv) in dry THF (7 mL) was cooled to

−10 °C. Ethyl chloroformate (53 μL, 0.55 mmol, 1.5 equiv) was

dropwise added, and the resultant mixture was stirred for 1 h. After-

ward, a solution of sodium azide (40 mg, 0.63 mmol, 1.70 equiv) in

water (2 mL) was slowly added and the reaction monitored by TLC.

After completion (1 h), the reaction mixture was poured into ice−

water (5 mL) and quickly extracted with EtOAc (3 × 10 mL). The

combined organic layers were dried over MgSO₄, ﬁltered, and carefully

evaporated under reduced pressure. The crude acylazide was dissolved

in dry toluene (20 mL) and reﬂuxed for 1 h under argon to give the

corresponding isocyanate which was used in the next step.

General Procedure C for Synthesis of Valmerins 13−40.

Under argon, to the freshly prepared isocyanate was added the amine

8 (0.37 mmol, 1.0 equiv) in dry dioxane (7 mL). The reacting mixture

was heated at 100 °C and next cooled at room temperature. After a

careful evaporation of the volatiles (T < 40 °C), the dark residue was

puriﬁed by silica gel chromatography column to aﬀord the desired

valmerins 13−40.

1-Cyclohexyl-3-(6-oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]-

isoindol-10-yl)urea (13). The compound 13 was obtained following

the general procedures B and C from the cyclohexanecarboxylic acid

and the amine 8 after puriﬁcation by ﬂash chromatography (CH₂Cl₂/

1

MeOH 99/1) as a white solid in 86% yield: mp 240−242 °C; H

NMR (CDCl₃, 400 MHz,) δ 0.88−0.97 (m, 1H), 1.10−1.14 (m, 3H),

1.29−1.34 (m, 3H), 1.46−1.60 (m, 2H), 1.67 (d, J = 12.4 Hz, 2H),

1.75 (d, J = 10.4 Hz, 1H), 1.85−1.94 (m, 3H), 2.51 (d, J = 12.4 Hz, 1H),

2.90 (t, J = 12.4 Hz, 1H), 3.64 (d, J = 7.6 Hz, 1H), 4.40−4.49 (m, 2H),

5.55 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.46 (s, 1H), 7.51

(d, J = 7.6 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H); HRMS (ESI) calcd for

C₁₉H₂₆N₃O₂328.20195, found 328.20249 [M + H]⁺; purity >99% by

HPLC; tR =13.08 and 18.76 min; n-heptane/ethanol (8:2).

1-(6-Oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]isoindol-10-yl)-3-

phenylurea (14). The compound 14 was obtained following the

general procedures B and C from the benzoic acid and the amine 8

after puriﬁcation by ﬂash chromatography (CH₂Cl₂/MeOH 98/2) as a

1

white solid in 80% yield: mp 134−136 °C; H NMR (CDCl₃, 400

MHz) δ 0.89−0.95 (m, 1H), 1.26−1.34 (m, 1H), 1.43−1.49 (m, 1H),

1.74 (d, J = 12.4 Hz, 1H), 1.84 (d, J = 10.4 Hz, 1H), 2.56 (d, J = 11.6 Hz,

1H), 2.90 (t, J = 11.6 Hz, 1H), 4.40−4.49 (m, 2H), 7.01 (t, J = 7.2 Hz,

1H), 7.22−7.26 (m, 2H), 7.30−7.36 (m, 3H), 7.52 (d, J = 7.2 Hz, 1H),

7.64 (d, J = 8.0 Hz, 1H), 8.15 (s, 1H), 8.31 (s, 1H); SM (IS) (m/z) 322.5

[M + H]⁺; HRMS (ESI) calcd for C₁₉H₂₀N₃O₂322.15500, found

322.15530 [M + H]⁺; purity >99% by HPLC; t_R=20.06 and 21.11

min; n-heptane/ethanol (8:2).

1

linic acid as a white solid in 75% yield: mp 208−210 °C; H NMR

(DMSO-d₆, 400 MHz) δ 3.36 (s, 3H), 5.17 (s, 2H), 7.15 (d, J = 4.4

Hz, J = 8.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 8.03 (d, J = 4.4 Hz, 1H);

SM (IS) m/z 184.0 [M + H]⁺; HRMS (ESI) calcd for C₈H₁₀NO₄

184.06043, found 184.06066 [M + H]⁺.

1-(6-Oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]isoindol-10-yl)-3-

(pyridin-2-yl)urea (15). The compound 15 was obtained following the

general procedures B and C from the pyridine-2-carboxylic acid and

the amine 8 after puriﬁcation by ﬂash chromatography (CH₂Cl₂/

MeOH 97/3 + 1% Et₃N) as a white solid in 90% yield: mp 142−

144 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ 0.77−0.95 (m, 1H), 1.22−

1.36 (m, 1H), 1.64−1.83 (m, 2H), 1.91 (d, J = 13.3 Hz, 1H), 2.72 (dd,

J = 3.0 Hz, J = 12.6 Hz, 1H), 2.97−3.12 (m, 1H), 4.26 (dd, J = 4.4 Hz,

J = 13.0 Hz, 1H), 4.59 (dd, J = 3.3 Hz, J = 11.5 Hz, 1H), 7.03−7.11

(m, 1H), 7.33 (d, J = 8.3 Hz, 1H), 7.38 (dd, J = 0.8 Hz, J = 7.4 Hz,

1H), 7.45 (t, J = 7.7 Hz, 1H), 7.74−7.85 (m, 1H), 8.23 (d, J = 8.0 Hz,

1H), 8.29 (d, J = 4.0 Hz, 1H), 10.00 (s, 1H), 11.16 (s, 1H); HRMS

(ESI) calcd for C₁₈H₁₉N₄O₂323.1508, found 323.1495 [M + H]⁺; purity

>99% by HPLC; t_R=14.40 and 17.74 min; n-heptane/ethanol (8:2).

5-(Methoxymethoxy)picolinic Acid (10). The compound 10 was

obtained following the general procedure A from the 5-hydroxypico-

1

linic acid as a white solid in 77% yield: mp 212−214 °C; H NMR

(DMSO-d₆, 250 MHz) δ 3.42 (s, 3H), 5.36 (s, 2H), 7.58 (dd, J = 2.5

Hz, J = 8.7 Hz, 1H), 8.04 (d, J = 8.7 Hz, 1H), 8.42 (s, 1H), 12.88

(Sl, 1H); SM (IS) m/z 184.0 [M + H]⁺; HRMS (ESI) calcd for C₈

H₁₀NO₄184.06043, found 184.06073 [M + H]⁺.

8-(Methoxymethoxy)quinoline-2-carboxylic Acid (11). The com-

pound 11 was obtained following the general procedure A from the

8-hydroxyquinoline-2-carboxylic acid as a white solid in 70% yield: mp

1

148−150 °C; H NMR (DMSO-d₆, D₂O, 400 MHz) δ 3.24 (s, 3H),

5.32 (s, 2H), 7.32 (d, J = 7.6 Hz, 1H), 7.48−7.61 (m, 2H), 8.18 (d, J =

8.5 Hz, 1H), 8.38 (d, J = 8.5 Hz, 1H); SM (IS) m/z 234.0 [M + H]⁺,

9600

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1-(6-Oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]isoindol-10-yl)-3-

(pyrazin-2-yl)urea (16). The compound 16 was obtained following

the general procedures B and C from the pyrazine-2-carboxylic acid

and the amine 8 after puriﬁcation by ﬂash chromatography (CH₂Cl₂/

MeOH 96/4) as a beige solid in 70% yield: mp 215−217 °C; ¹H NMR

(DMSO-d₆, 400 MHz) δ 0.88 (qd, J = 3.0 Hz, J = 12.7 Hz, 1H), 1.16−

1.33 (m, 1H), 1.64−1.72 (m, 1H), 1.76 (d, J = 15.0 Hz, 1H), 1.91 (d,

J = 13.1 Hz, 1H), 2.63 (dd, J = 3.0 Hz, J = 12.6 Hz, 1H), 3.03 (td, J =

3.8 Hz, 12.8 Hz, 1H), 4.26 (dd, J = 3.8 Hz, J = 13.0 Hz, 1H), 4.56 (dd,

J = 3.4 Hz, J = 11.5 Hz, 1H), 7.42 (dd, J = 1.0 Hz, J = 7.4 Hz, 1H),

7.47 (t, J = 7.7 Hz, 1H), 8.13 (dd, J = 1.0 Hz, J = 7.9 Hz, 1H), 8.29 (d,

J = 2.7 Hz, 1H), 8.31−8.36 (m, 1H), 8.93 (d, J = 1.3 Hz, 1H), 9.81 (s,

1H), 10.05 (s, 1H); HRMS (ESI) calcd for C₁₇H₁₇N₅O₂Na 346.1280,

found 346.1289 [M + Na]⁺; purity >99% by HPLC; t_R=14.27 and

17.99 min; n-heptane/ethanol (8:2).

1-(5-Bromopyridin-2-yl)-3-(6-oxo-1,2,3,4,6,10b-hexahydropyrido-

[2,1-a]isoindol-10-yl)urea (17). The compound 17 was obtained the

general procedures B and C from the 5-bromopicolinic acid and the

amine 8 after puriﬁcation by ﬂash chromatography (CH₂Cl₂/MeOH

99/1) as a beige solid in 66% yield: mp 225−227 °C; ¹H NMR

(DMSO-d₆, 400 MHz) δ 0.77−0.93 (m, 1H), 1.20−1.33 (m, 1H),

162−1.82 (m, 2H), 1.90 (d, J = 12.6 Hz, 1H), 2.63 (dd, J = 2.7 Hz, J =

12.6 Hz, 1H), 3.00−3.11 (m, 1H), 4.25 (dd, J = 4.2 Hz, J = 13.0 Hz,

1H), 4.59 (dd, J = 3.2 Hz, J = 11.5 Hz, 1H), 7.39 (dd, J = 0.8 Hz, J =

7.4 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 7.50 (d, J = 8.9 Hz, 1H), 8.00

(dd, J = 2.5 Hz, J = 8.9 Hz, 1H), 8.14 (dd, J = 0.7 Hz, J = 7.9 Hz, 1H),

8.40 (d, J = 2.3 Hz, 1H), 10.00 (s, 1H), 10.25 (s, 1H); HRMS (ESI)

calcd for C₁₈H₁₇N₄O₂NaBr 423.0433, found 423.0427 [M + Na]⁺;

purity >98% by HPLC; t_R= 8.77 and 18.73 min; n-heptane/ethanol

(8:2).

1-(6-Bromopyridin-2-yl)-3-(6-oxo-1,2,3,4,6,10b-hexahydropyrido-

[2,1-a]isoindol-10-yl)urea (18). The compound 18 was obtained the

general procedures B and C from the 6-bromopicolinic acid and the

amine 8 after puriﬁcation by ﬂash chromatography (CH₂Cl₂/MeOH

99/1) as a beige solid in 70% yield: mp 218−220 °C; ¹H NMR

(DMSO-d₆, 400 MHz) δ 0.87 (qd, J = 3.1 Hz, J = 12.7 Hz, 1H), 1.20−

1.34 (m, 1H), 1.61−1.81 (m, 2H), 1.89 (d, J = 13.0 Hz, 1H), 2.55−

2.66 (m, 1H), 3.02 (td, J = 3.6 Hz, J = 12.9 Hz, 1H), 4.26 (dd, J = 3.6

Hz, J = 13.0 Hz, 1H), 4.56 (dd, J = 3.4 Hz, J = 11.5 Hz, 1H), 7.27 (dd,

J = 1.4 Hz, J = 6.8 Hz, 1H), 7.38−7.43 (m, 1H), 7.46 (t, J = 7.7 Hz,

1H), 7.65−7.75 (m, 2H), 8.10 (dd, J = 0.7 Hz, J = 7.8 Hz, 1H), 9.28

(s, 1H), 10.04 (s, 1H); HRMS (ESI) calcd for C₁₈H₁₇N₄O₂NaBr

423.0433, found 423.0428 [M + Na]⁺; purity >99% by HPLC; t_R=10.31

and 13.46 min; n-heptane/ethanol (8:2).

C₁₈H₁₇N₄O₂NaF 363.1233, found 363.1221 [M + Na]⁺; purity >99%

by HPLC; t_R=11.39 and 13.45 min; n-heptane/ethanol (8:2).

1-(3,5-Diﬂuoropyridin-2-yl)-3-(6-oxo-1,2,3,4,6,10b-

hexahydropyrido[2,1-a]isoindol-10-yl)urea (21). The compound 21

was obtained following the general procedures B and C from the 3,5-

diﬂuoropicolinic acid and the amine 8 after puriﬁcation by ﬂash chro-

matography (CH₂Cl₂/MeOH 99/1) as a white solid in 73% yield: mp

1

256−258 °C; H NMR (CDCl₃, 400 MHz) δ 1.06−1.16 (m, 1H),

1.39−1.51 (m, 1H), 1.64−1.76 (m, 1H), 1.85 (d, J = 12.5 Hz, 1H),

2.03 (d, J = 12.5 Hz, 1H), 2.65 (dd, J = 2.8 Hz, J = 12.8 Hz, 1H), 3.01

(td, J = 3.6 Hz, J = 12.8 Hz, 1H), 4.47 (dd, J = 3.6 Hz, J = 11.6 Hz,

1H), 4.54 (dd, J = 4.8 Hz, J = 12.8 Hz, 1H), 7.37−7.39 (m, 2H), 7.48

(t, J = 8.0 Hz, 1H), 7.66 (d, J = 7.5 Hz, 1H), 7.98 (d, J = 2.4 Hz, 1H),

8.11 (d, J = 8.0 Hz, 1H), 11.09 (s, 1H); HRMS (ESI) calcd for

C₁₈H₁₇F₂N₄O₂359.13164, found 359.13141 [M + H]⁺; purity >99%

by HPLC; t_R= 9.70 and 14.19 min; n-heptane/ethanol (8:2).

1-(6-Oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]isoindol-10-yl)-3-

(perchloropyridin-2-yl)urea (22). The compound 22 was obtained

following the general procedures B and C from the 3,4,5,6-tetrachloro-

picolinic acid and the amine 8 after puriﬁcation by ﬂash chromato-

graphy (CH₂Cl₂/MeOH 99/1) as a beige solid in 60% yield: mp 246−

248 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ 0.84−0.92 (m, 1H), 1.15−

1.29 (m, 1H), 1.64 (q, J = 12.8 Hz, 1H), 1.75 (d, J = 12.3 Hz, 1H),

1.88 (d, J = 13.0 Hz, 1H), 2.61 (d, J = 10.2 Hz, 1H), 3.02 (td, J = 3.8

Hz, J = 12.0 Hz, 1H), 4.25 (dd, J = 3.8 Hz, J = 12.6 Hz, 1H), 4.58 (dd,

J = 3.3 Hz, J = 11.5 Hz, 1H), 7.44−7.49 (m, 2H), 7.97 (dd, J = 7.3 Hz,

J = 1.2 Hz, 1H), 9.60 (d, J = 2.6 Hz, 2H); HRMS (ESI) calcd for

C₁₈H₁₄N₄O₂NaCl₄: 480.9769, found 480.9789 [M + Na]⁺. Purity

>97% by HPLC ; t_R=16.02 and 18.93 min ; n-heptane/ethanol (8:2).

1-(3-Methoxypyridin-2-yl)-3-(6-oxo-1,2,3,4,6,10b-

hexahydropyrido[2,1-a]isoindol-10-yl)urea (23). The compound 23

was obtained following the general procedures B and C from the 3-

methoxypicolinic acid and the amine 8 after puriﬁcation by ﬂash

chromatography (CH₂Cl₂/MeOH 99/1) as a white solid in 70% yield:

mp 202−204 °C; ¹H NMR (CDCl₃, 400 MHz) δ 1.03−1.13 (m, 1H),

1.38−1.50 (m, 1H), 1.65−1.76 (m, 1H), 1.83 (d, J = 12.8 Hz, 1H),

2.02 (d, J = 13.2 Hz, 1H), 2.81 (dd, J = 2.8 Hz, J = 12.8 Hz, 1H), 3.01

(td, J = 3.6 Hz, J = 12.8 Hz, 1H), 3.93 (s, 3H), 4.48 (dd, J = 3.6 Hz, J =

12.0 Hz, 1H), 4.54 (dd, J = 5.2 Hz, J = 13.2 Hz, 1H), 6.97 (dd, J = 5.2

Hz, J = 8.0 Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H),

7.59−7.62 (m, 2H), 7.79 (dd, J = 0.8 Hz, J = 5.2 Hz, 1H), 8.24 (d, J =

8.0 Hz, 1H), 11.86 (s, 1H); HRMS (ESI) calcd for C₁₉H₂₁N₄O₃:

353.16100, found 353.16082 [M + H]⁺. Purity >99% by HPLC ; t_R=15.63

and 18.44 min ; n-heptane/ethanol (8:2).

1-(4-Bromopyridin-2-yl)-3-(6-oxo-1,2,3,4,6,10b-hexahydropyrido-

[2,1-a]isoindol-10-yl)urea (19). The compound 19 was obtained

following the general procedures B and C from the 4-bromopicolinic

acid and the amine 8 after puriﬁcation by ﬂash chromatography

(CH₂Cl₂/MeOH 99/1) as a white solid in 80% yield: mp 244−246

°C; ¹H NMR (DMSO-d₆, 400 MHz) δ 0.82−0.93 (m, 1H), 1.19−1.30

(m, 1H), 1.64−1.77 (m, 2H), 1.90 (d, J = 12.0 Hz, 1H), 2.64 (dd, J =

4.0 Hz, J = 12.0 Hz, 1H), 2.99−3.06 (m, 1H), 4.25 (dd, J = 4.0 Hz, J =

12.0 Hz, 1H), 4.59 (dd, J = 4.0 Hz, J = 12.0 Hz, 1H), 7.30 (dd, J = 0.8

Hz, J = 8.0 Hz, 1H), 7.38−7.46 (m, 2H), 7.68 (s, 1H), 8.14−8.18 (m,

2H), 9.98 (s, 1H), 10.43 (s, 1H); HRMS (ESI) calcd for

C₁₈H₁₈N₄O₂Br 401.06067, found 401.06076 [M + H]⁺; purity >99%

by HPLC; t_R= 13.77 and 16.82 min; n-heptane/ethanol (8:2).

1-(6-Fluoropyridin-2-yl)-3-(6-oxo-1,2,3,4,6,10b-hexahydropyrido-

[2,1-a]isoindol-10-yl)urea (20). The compound 20 was obtained the

general procedures B and C from the 6-ﬂuoropicolinic acid and the

amine 8 after puriﬁcation by ﬂash chromatography (CH₂Cl₂/MeOH

99/1) as a white solid in 70% yield: mp 244−246 °C; ¹H NMR

(DMSO-d₆, 250 MHz) δ 0.70−1.03 (m, 1H), 1.12−1.40 (m, 1H),

1.52−1.72 (m, 1H), 1.76 (d, J = 14.1 Hz, 1H), 1.90 (d, J = 13.2 Hz,

1H), 2.57−2.75 (m, 1H), 3.02 (td, J = 3.5 Hz, J = 12.7 Hz, 1H), 4.26

(dd, J = 3.5 Hz, 13.0 Hz, 1H), 4.51 (dd, J = 3.3 Hz, J = 11.5 Hz, 1H),

6.77 (dd, J = 2.0 Hz, J = 7.9 Hz, 1H), 7.36−7.51 (m, 2H), 7.57 (dd, J =

2.1 Hz, J = 8.0 Hz, 1H), 7.94 (q, J = 8.2 Hz, 1H), 8.14 (dd, J = 1.2 Hz,

J = 7.6 Hz, 1H), 9.33 (s, 1H), 9.92 (s, 1H); HRMS (ESI) calcd for

1-(6-Oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]isoindol-10-yl)-3-

(6-(piperidin-1-yl)pyridin −2-yl)urea (24). The compound 24 was

obtained following the general procedures B and C from the 6-

(piperidin-1-yl)picolinic acid and the amine 8 after puriﬁcation by ﬂash

chromatography (CH₂Cl₂/MeOH 99/1) as a yellow solid in 81%

yield: mp 234−236 °C; ¹H NMR (CDCl₃, 400 MHz) δ 0.94−1.05 (m,

1H), 1.34−1.38 (m, 1H), 1.54−1.65 (m, 7H), 1.77 (d, J = 12.8 Hz,

1H), 1.88 (d, J = 13.2 Hz, 1H), 2.43 (dd, J = 2.8 Hz, J = 12.8 Hz, 1H),

2.95 (td, J = 3.6 Hz, J = 13.2 Hz, 1H), 3.46−3.47 (m, 4H), 4.49 (dd, J =

3.6 Hz, J = 13.2 Hz, 1H), 4.69 (dd, J = 2.8 Hz, J = 12.8 Hz, 1H), 6.14

(d, J = 8.0 Hz, 1H), 6.31 (d, J = 8.0 Hz, 1H), 7.40−7.47 (m, 3H), 7.72

(d, J = 8.0 Hz, 1H), 8.40 (s, 1H), 11.59 (s, 1H); HRMS (ESI) calcd

for C₂₃H₂₈N₅O₂: 406.22394, found 406.22375 [M + H]⁺. Purity >99%

by HPLC ; t_R=12.93 and 19.84 min ; n-heptane/ethanol (8:2).

1-(6-Oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]isoindol-10-yl)-3-

(5-phenylpyridin-2-yl)urea (25). The compound 25 was obtained

following the general procedures B and C from the 5-phenylpicolinic

acid and the amine 8 after puriﬁcation by ﬂash chromatography

(CH₂Cl₂/MeOH 99/1) as a white solid in 79% yield: mp >260 °C; ¹H

NMR (DMSO-d₆, 400 MHz) δ 0.84−0.93 (m, 1H), 1.20−1.30 (m,

1H), 1.76 (d, J = 12.8 Hz, 2H), 1.91 (d, J = 12.8 Hz, 1H), 2.72 (d, J =

12.8 Hz, 1H), 2.95 (t, J = 12.4 Hz, 1H), 4.26 (d, J = 13.2 Hz, 1H), 4.61

(d, J = 12.8 Hz, 1H), 7.38−7.50 (m, 6H), 7.68 (d, J = 8.0 Hz, 2H),

8.11 (d, J = 8.4 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.59 (s, 1H), 10.01

(s, 1H), 11.07 (s, 1H); HRMS (ESI) calcd for C₂₄H₂₃N₄O₂: 399.1821,

9601

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Article

found 399.1811 [M + H]⁺. Purity >99% by HPLC ; t_R=12.66 and

29.72 min ; n-heptane/ethanol (8:2).

(d, J = 7.3 Hz, 1H), 9.21 (s, 1H), 9.42 (s, 1H); HRMS (ESI) calcd for

C₁₇H₁₉N₅O₂Na 348.1436, found 348.1431 [M + Na]⁺; purity >97% by

HPLC; t_R=11.05 and 31.89 min; n-heptane/ethanol (8:2).

1-(Isoquinolin-3-yl)-3-(6-oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]-

isoindol-10-yl)urea (31). The compound 31 was obtained following

the general procedures B and C from the 3-isoquinolin-3-carboxylic

acid and the amine 8 after puriﬁcation by ﬂash chromatography

(CH₂Cl₂/MeOH 99/1) as a white solid in 71% yield: mp > 260 °C;

¹H NMR (CDCl₃, 400 MHz) δ 1.10−1.20 (m, 1H), 1.43−1.49 (m,

1H), 1.72−1.88 (m, 2H), 2.07 (d, J = 13.2 Hz, 1H), 2.90 (dd, J = 2.8

Hz, J = 12.8 Hz, 1H), 3.04 (td, J = 3.2 Hz, J = 13.2 Hz, 1H), 4.54−4.60

(m, 2H), 7.38 (d, J = 6.0 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.65−7.68

(m, 2H), 7.79 (t, J = 7.6 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H), 8.08 (d, J =

6.0 Hz, 1H), 8.20 (d, J = 8.5 Hz, 1H), 8.34 (d, J = 8.0 Hz, 1H), 8.47 (s,

1H), 12.69 (s, 1H); HRMS (ESI) calcd for C₂₂H₂₁N₄O₂373.1665,

found 376.1660 [M + H]⁺; purity >98% by HPLC; t_R=10.84 min; n-

heptane/ethanol (8:2), no separation.

11-(5-Methylisoxazol-3-yl)-3-(6-oxo-1,2,3,4,6,10b-

hexahydropyrido[2,1-a]isoindol-10-yl)urea (26). The compound 26

was obtained the general procedures B and C from the 5-methyl-

isoxazole-3-carboxylic acid and the amine 8 after puriﬁcation by ﬂash

chromatography (CH₂Cl₂/MeOH 99/1) as a yellow solid in 72%

1

yield: mp 156−158 °C; H NMR (CDCl₃, 400 MHz) δ 1.03 (qd, J =

3.1 Hz, J = 12.8 Hz, 1H), 1.29−1.46 (m, 1H), 1.53−1.70 (m, 1H),

1.80 (d, J = 13.1 Hz, 1H), 1.94 (d, J = 13.3 Hz, 1H), 2.41 (s, 3H), 2.70

(d, J = 11.1 Hz, 1H), 2.98 (td, J = 3.2 Hz, J = 13.0 Hz, 1H), 4.49 (td,

J = 4.0 Hz, J = 11.8 Hz, 2H), 6.12 (s, 1H), 7.44 (t, J = 7.8 Hz, 1H),

7.64 (d, J = 7.3 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H), 9.33 (s, 1H), 9.53

(s, 1H); HRMS (ESI) calcd for C₁₇H₁₈N₄O₃Na: 349.1277, found

349.1267 [M + Na]⁺; purity >95% by HPLC; t_R=13.13 and 24.25

min; n-heptane/ethanol (8:2).

1-(6-Oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]isoindol-10-yl)-3-

(5-phenylisoxazol-3-yl)urea (27). The compound 27 was obtained

following the general procedures B and C from the 5-phenylisoxazole-

3-carboxylic acid and the amine 8 after puriﬁcation by ﬂash chro-

matography (CH₂Cl₂/MeOH 99/1) as a white solid in 87% yield: mp

248−250 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ 0.86 (qd, J = 2.9 Hz,

J = 12.7 Hz, 1H), 1.17−1.34 (m, 1H), 1.54−1.71 (m, 1H), 1.76 (d, J =

12.5 Hz, 1H), 1.91 (d, J = 13.1 Hz, 1H), 2.61 (dd, J = 2.9 Hz, J = 12.5 Hz,

1H), 3.02 (td, J = 3.6 Hz, J = 12.8 Hz, 1H), 4.26 (dd, J = 3.6 Hz, J =

13.0 Hz, 1H), 4.52 (dd, J = 3.3 Hz, J = 11.5 Hz, 1H), 7.27 (s, 1H),

7.39−7.59 (m, 5H), 7.88 (dd, J = 1.7 Hz, J = 7.6 Hz, 2H), 8.04 (d, J =

7.7 Hz, 1H), 8.58 (s, 1H), 10.04 (s, 1H); HRMS (ESI) calcd for

C₂₂H₂₀N₄O₃Na 411.1433, found 411.1414 [M + Na]⁺; purity >97% by

HPLC; t_R=13.16, n-heptane/ethanol (8:2), no separation.

1-(Benzo[d]thiazol-2-yl)-3-(6-oxo-1,2,3,4,6,10b-hexahydropyrido-

[2,1-a]isoindol-10-yl)urea (32). The compound 32 was obtained

following the general procedures B and C from the acid 9 and the

amine 8 after puriﬁcation by ﬂash chromatography (CH₂Cl₂/MeOH

1

99/1) as a yellow solid in 50% yield: mp 258−260 °C; H NMR

(DMSO-d₆, 400 MHz) δ 0.88 (q, J = 12.0 Hz, 1H), 1.15−1.32 (m,

1H), 1.64 (q, J = 13.0 Hz, 1H), 1.76 (d, J = 12.4 Hz, 1H), 1.90 (d, J =

12.8 Hz, 1H), 2.61 (d, J = 11.5 Hz, 1H), 3.01−3.07 (m, 1H), 4.26 (d,

J = 9.2 Hz, 1H), 4.57 (d, J = 9.4 Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H),

7.39−7.51 (m, 3H), 7.69 (d, J = 6.6 Hz, 1H), 7.93 (d, J = 7.7 Hz, 1H),

8.05 (d, J = 7.0 Hz, 1H), 9.07 (s, 1H), 11.19 (s, 1H); HRMS (ESI)

calcd for C₂₀H₁₈N₄O₂NaS 401.1048, found 401.1066 [M + Na]⁺;

purity >97% by HPLC; t_R=17.41 and 22.78 min; n-heptane/ethanol

(8:2).

11-(6-Oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]isoindol-10-yl)-3-

(thiazol-4-yl)urea (28). The compound 28 was obtained following the

general procedures B and C from the thiazole-4-carboxylic acid and

the amine 8 after puriﬁcation by ﬂash chromatography (CH₂Cl₂/

1-(2-Methylthiazol-4-yl)-3-(6-oxo-1,2,3,4,6,10b-hexahydropyrido-

[2,1-a]isoindol-10-yl)urea (33). The compound 33 was obtained

following the general procedures B and C from the acid 10 and the

amine 8 after puriﬁcation by ﬂash chromatography (CH₂Cl₂/MeOH

1

MeOH 99/1) as a yellow solid in 62% yield: mp 178−180 °C; H

NMR (CDCl₃, 400 MHz) δ 0.92−1.10 (m, 1H), 1.28−1.43 (m, 1H),

1.43−1.60 (m, 1H), 1.76 (d, J = 12.3 Hz, 1H), 1.88 (d, J = 12.2 Hz,

1H), 2.65 (d, J = 10.9 Hz, 1H), 2.91−2.97 (m, 1H), 4.38−4.57 (m,

2H), 7.11 (s, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.61 (d, J = 7.4 Hz, 1H),

7.96 (d, J = 7.8 Hz, 1H), 8.64 (d, J = 1.5 Hz, 1H), 9.68 (s, 1H); HRMS

(ESI) calcd for C₁₆H₁₆N₄O₂NaS: 351.0892, found 351.0885 [M +

Na]⁺; purity >96% by HPLC; t_R=20.45 and 29.15 min; n-heptane/

ethanol (8:2).

1

99/1) as a yellow solid in 42% yield: mp 146−148 °C; H NMR

(CDCl₃, 400 MHz) δ 1.04 (q, J = 12.0 Hz, 1H), 1.31−1.47 (m, 1H),

1.48−1.62 (m, 1H), 1.78 (d, J = 12.6 Hz, 1H), 1.92 (d, J = 12.6 Hz,

1H), 2.71 (m, 4H), 2.95 (t, J = 12.0 Hz, 1H), 4.45 (dd, J = 3.5 Hz, J =

11.6 Hz, 1H), 4.51 (dd, J = 4.0 Hz, J = 13.2 Hz, 1H), 6.70 (s, 1H),

7.41 (t, J = 7.8 Hz, 1H), 7.62 (d, J = 7.3 Hz, 1H), 8.03 (d, J = 6.5 Hz,

1H), 9.30 (s, 2H); HRMS (ESI) calcd for C₁₇H₁₈N₄O₂NaS 365.1048,

found 365.1062 [M + Na]⁺; purity >99% by HPLC; t_R=14.26 and

29.06 min; n-heptane/ethanol (8:2).

1-(6-Oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]isoindol-10-yl)-3-

(2-(pyridin-4-yl)thiazol-4-yl)urea (29). The compound 29 was ob-

tained following the general procedures B and C from the 2-(pyridin-

4-yl)thiazole-4-carboxylic acid and the amine 8 after puriﬁcation by

ﬂash chromatography (CH₂Cl₂/MeOH 99/1) as a orange solid in 60%

1-(6-Oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]isoindol-10-yl)-3-

pyrimidin-2-ylurea (34). The compound 34 was obtained following

the general procedures B and C from the acid 12 and the amine 8 after

puriﬁcation by ﬂash chromatography (CH₂Cl₂/MeOH 99/1) as a

1

yield: mp 152−154 °C; H NMR (DMSO-d₆, 400 MHz) δ 0.85 (qd,

1

beige solid in 87% yield: mp 232−234 °C; H NMR (DMSO-d₆, 400

J = 2.9 Hz, J = 12.7 Hz, 1H). 1.15−1.34 (m, 1H), 1.62 (q, J = 13.0 Hz,

1H), 1.76 (d, J = 12.6 Hz, 1H), 1.90 (d, J = 13.1 Hz, 1H), 2.62 (dd, J =

2.9 Hz, J = 12.6 Hz, 1H), 3.03 (td, J = 3.7 Hz, J = 12.9 Hz, 1H), 4.26

(dd, J = 3.7 Hz, J = 13.0 Hz, 1H), 4.50 (dd, J = 3.3 Hz, J = 11.5 Hz,

1H), 7.38 (dd, J = 0.8 Hz, J = 7.4 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H),

7.55 (s, 1H), 7.84 (dd, J = 1.6 Hz, J = 4.5 Hz, 2H), 8.11 (d, J = 7.2 Hz,

1H), 8.48 (s, 1H), 8.72 (dd, J = 1.5 Hz, J = 4.6 Hz, 2H), 10.10 (s, 1H);

HRMS (ESI) calcd for C₂₁H₂₀N₅O₂S 406.1338, found 406.1343 [M + H]⁺;

purity >96% by HPLC; t_R=8.20 and 28.09 min; n-heptane/ethanol

(8:2).

MHz) δ 0.90 (dt, J = 12.0 Hz, J = 14.5 Hz, 1H), 1.25 (dd, J = 9.3 Hz,

J = 13.3 Hz, 1H), 1.75 (t, J = 13.2 Hz, 2H), 1.92 (d, J = 13.1 Hz, 1H),

2.68 (d, J = 10.2 Hz, 1H), 2.96−3.11 (m, 1H), 4.27 (dd, J = 4.5 Hz, J =

12.8 Hz, 1H), 4.64 (dd, J = 3.2 Hz, J = 11.5 Hz, 1H), 7.20 (t, J = 4.9

Hz, 1H), 7.42 (d, J = 7.2 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 8.24 (d, J =

7.8 Hz, 1H), 8.70 (d, J = 4.9 Hz, 2H), 10.42 (s, 1H), 11.60 (s, 1H);

HRMS (ESI) calcd for C₁₇H₁₈N₅O₂324.1461, found 324.1449 [M + H]⁺;

purity >97% by HPLC; t_R=30.41 and 49.77 min; n-heptane/ethanol

(8:2).

1-(6-Oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]isoindol-10-yl)-3-

(pyrimidin-4-yl)urea (35). The compound 35 was obtained following

the general procedures B and C from the acid 12 and the amine 8 after

puriﬁcation by ﬂash chromatography (CH₂Cl₂/MeOH 99/1) as a

1-(1-Methyl-1H-pyrazol-3-yl)-3-(6-oxo-1,2,3,4,6,10b-

hexahydropyrido[2,1-a]isoindol-10-yl)urea (30). The compound 30

was obtained following the general procedures B and C from the 1-

methyl-1H-pyrazole-3-carboxylic acid and the amine 8 after puri-

ﬁcation by ﬂash chromatography (CH₂Cl₂/MeOH 99/1) as a white

solid in 62% yield: mp 233−235 °C; ¹H NMR (DMSO-d₆, 400 MHz)

δ 0.87 (dd, J = 23.0 Hz, J = 11.8 Hz, 1H), 1.25 (d, J = 12.6 Hz, 1H),

1.55−1.74 (m, 1H), 1.76 (d, J = 12.5 Hz, 1H), 1.92 (d, J = 13.1 Hz,

1H), 2.71 (d, J = 11.1 Hz, 1H), 3.03 (t, J = 11.6 Hz, 1H), 3.78 (s, 3H),

4.26 (d, J = 9.5 Hz, 1H), 4.52 (d, J = 8.9 Hz, 1H), 6.07 (s, 1H), 7.35

(d, J = 7.2 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.59 (s, 1H), 8.18

1

beige solid in 76% yield: mp 218−220 °C; H NMR (DMSO-d₆, 400

MHz) δ 0.82−0.91 (m, 1H), 1.12−1.38 (m, 1H), 1.61−1.80 (m, 2H),

1.91 (d, J = 11.9 Hz, 1H), 2.64 (d, J = 10.9 Hz, 1H), 3.03 (t, J = 11.8

Hz, 1H), 4.26 (d, J = 9.6 Hz, 1H), 4.57 (d, J = 8.8 Hz, 1H), 7.40−7.50

(m, 2H), 7.54 (d, J = 5.4 Hz, 1H), 8.12 (d, J = 7.6 Hz, 1H), 8.58 (d,

J = 5.6 Hz, 1H), 8.84 (s, 1H), 10.14 (s, 1H), 10.21 (s, 1H); HRMS

(ESI) calcd for C₁₇H₁₈N₅O₂324.1461, found 324.1465 [M + H]⁺;

9602

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purity >96% by HPLC; t_R= 13.07 and 21.15 min; n-heptane/ethanol

(8:2).

the compound 40 as a white solid: mp 185−187 °C; ¹H NMR

(DMSO-d₆, 400 MHz) δ 0.84 (qd, J = 12.8 Hz, J = 3.2 Hz, 1H), 1.16−

1.34 (m, 1H), 1.56−1.83 (m, 2H), 1.90 (d, J = 13.2 Hz, 1H), 2.66 (d, J

= 10.7 Hz, 1H), 3.01 (td, J = 3.0 Hz, J = 12.7 Hz, 1H), 4.26 (dd, J =

1-(3-(Methoxymethoxy)pyridin-2-yl)-3-(6-oxo-1,2,3,4,6,10b-

hexahydropyrido[2,1-a]isoindol-10-yl)urea (36). The compound 36

was obtained following the general procedures B and C from carbo-

xylic acid 9 and the amine 8 after puriﬁcation by ﬂash chromatography

(CH₂Cl₂/MeOH 99/1) as a white solid in 73% yield: mp 182−184 °C;

¹H NMR (CDCl₃, 400 MHz) δ 1.03−1.19 (m, 1H), 1.42−1.55 (m,

1H), 1.70−1.75 (m, 1H), 1.86 (d, J = 12.8 Hz, 1H), 2.05 (d, J = 13.2

Hz, 1H), 2.84 (dd, J = 2.8 Hz, J = 12.8 Hz, 1H), 3.04 (td, J = 2.8 Hz,

J = 12.8 Hz, 1H), 3.54 (s, 3H), 4.48 (dd, J = 3.2 Hz, J = 12.8 Hz, 1H),

4.54 (dd, J = 4.8 Hz, J = 13.2 Hz, 1H), 5.28 (s, 2H), 6.94 (dd, J = 4.8

Hz, J = 8.0 Hz, 1H), 7.43−7.49 (m, 2H), 7.60−7.62 (m, 2H), 7.85 (dd,

J = 1.2 Hz, J = 8.0 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 11.85 (s, 1H);

HRMS (ESI) calcd for C₂₀H₂₃N₄O₄383.1719, found 383.1704

[M + H]⁺; purity >99% by HPLC; t_R=17.70 and 19.00 min; n-

heptane/ethanol (8:2).

4 . 3

H z ,

J = 13.1 Hz, 1H), 4.48 (dd, J = 3.2 Hz, J = 11.5 Hz, 1H), 6.20 (s,

1H), 7.35 (d, J = 7.2 Hz, 1H), 7.42 (t, J = 7.7 Hz, 1H), 7.63 (s, 1H),

8.12 (d, J = 7.9 Hz, 1H), 9.06 (s, 1H), 9.40 (s, 1H), 12.33 (s, 1H);

HRMS (ESI) calcd for C₁₆H₁₈N₅O₂: 312.1461, found 312.1475 [M +

H]⁺; purity >97% by HPLC; t_R=12.56 and 15.98 min; n-heptane/

ethanol (8:2).

3-[3-(6-Oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]isoindol-10-yl)-

ureido]pyrazole-1-carboxylic Acid tert-Butyl Ester (41). To a stirred

solution of 8 (140 mg ; 0.7 mmol) and Et₃N (0.2 mL, 2.0 equiv) in dry

CH₂Cl₂(10 mL) was added at 0 °C under Ar bis(trichloromethyl)

carbonate (207 mg, 1.0 equiv). After 5 h at reﬂux, the reaction mixture

was cooled to room temperature, and a solution of 3-aminopyrazole-1-

carboxylic acid tert-butyl ester (128 mg, 1.0 equiv) in dry pyridine

(1.2 mL) was added. The reaction mixture was stirred at room tempera-

ture for 18 h and then diluted with water (30 mL) and extracted with

EtOAc (2 × 30 mL). The combined organic layers were successively

washed with water (30 mL), dried over MgSO₄, and evaporated under

reduced pressure to aﬀord after silica gel column chromatography

(CH₂Cl₂/MeOH 99/1) the compound 41 as a yellow solid in 56%

1-(5-(Methoxymethoxy)pyridin-2-yl)-3-(6-oxo-1,2,3,4,6,10b-

hexahydropyrido[2,1-a]isoindol-10-yl)urea (37). The compound 37

was obtained following the general procedures B and C from carbo-

xylic acid 10 and the amine 8 after puriﬁcation by ﬂash chromato-

graphy(CH₂Cl₂/MeOH 99/1) as a white solid in 78% yield: mp 202−

1

204 °C; H NMR (CDCl₃, 400 MHz) δ 1.04−1.14 (m, 1H), 1.39−

1.47 (m, 1H), 1.65−1.76 (m, 1H), 1.84 (d, J = 12.8 Hz, 1H), 2.03 (d,

J = 13.2 Hz, 1H), 2.81 (dd, J = 3.2 Hz, J = 12.8 Hz, 1H), 3.02 (td, J =

3.2 Hz, J = 12.8 Hz, 1H), 3.52 (s, 3H), 4.48 (dd, J = 3.2 Hz, J = 12.0

Hz, 1H), 4.55 (dd, J = 4.8 Hz, J = 13.2 Hz, 1H), 5.17 (s, 2H), 6.96 (d,

J = 8.8 Hz, 1H), 7.46−7.50 (m, 2H), 7.63 (d, J = 8.0 Hz, 1H), 8.02 (s,

1H), 8.26 (d, J = 8.0 Hz, 1H), 9.36 (s, 1H), 11.78 (s, 1H); HRMS

(ESI) calcd for C₂₀H₂₂N₄O₄Na 405.1539, found 405.1547 [M + Na]⁺;

purity >97% by HPLC; t_R=17.48 and 30.68 min; n-heptane/ethanol

(8:2).

1

yield: mp 136−138 °C; H NMR (CDCl₃, 400 MHz) δ 0.98 (qd, J =

3.2 Hz, J = 12.7 Hz, 1H), 1.27−1.45 (m, 1H), 1.62 (s, 9H), 1.76 (d,

J = 11.8 Hz, 1H), 1.85 (d, J = 13.0 Hz, 1H), 2.18 (s, 1H), 2.65 (s, 1H),

2.99 (t, J = 11.8 Hz, 1H), 4.49 (dd, J = 3.8 Hz, J = 13.0 Hz, 1H), 4.64

(d, J = 9.8 Hz, 1H), 6.38 (s, 1H), 7.42 (t, J = 7.8 Hz, 1H), 7.63 (d, J =

7.4 Hz, 1H), 7.91 (d, J = 2.9 Hz, 1H), 7.99 (s, 1H), 9.59 (s, 1H), 10.32

(s, 1H); HRMS (ESI) calcd for C₂₁H₂₅N₅O₄Na 434.1804, found

434.1791 [M + Na]⁺.

1-(8-(Methoxymethoxy)quinolin-2-yl)-3-(6-oxo-1,2,3,4,6,10b-

hexahydropyrido[2,1-a]isoindol-10-yl)urea (38). The compound 38

was obtained following the general procedures B and C from carbo-

xylic acid 11 and the amine 8 after puriﬁcation by ﬂash chromato-

graphy (CH₂Cl₂/MeOH 99/1) as a white solid in 75% yield: mp 226−

General Procedure D. A mixture containing the protected urea

(1 mmol), an aqueous solution of HCl (10%, 5 mL), and acetone (5 mL)

was heated at reﬂux for 2 h. After cooling, acetone was removed under

reduced pressure, and the resulting crude material was ﬁltered, washed

with water, and dried to give desired compound.

1

228 °C; H NMR (CDCl₃, 400 MHz) δ 0.96−1.06 (m, 1H), 1.31−

1-(3-Hydroxypyridin-2-yl)-3-(6-oxo-1,2,3,4,6,10b-

hexahydropyrido[2,1-a]isoindol-10-yl)urea (42). The compound 42

was obtained following the general procedure D from compound 36 as

1.39 (m, 1H), 1.46−1.56 (m, 1H), 1.78 (t, J = 16.5 Hz, 2H), 2.53 (d,

J = 10.8 Hz, 1H), 3.00 (td, J = 3.2 Hz, J = 13.2 Hz, 1H), 3.42 (s, 3H),

4.54 (dd, J = 3.2 Hz, J = 13.2 Hz, 1H), 5.00 (dd, J = 3.2 Hz, J = 10.8

Hz, 1H), 5.34 (s, 2H), 7.14 (d, J = 8.8 Hz, 1H), 7.33−7.44 (m, 3H),

7.85 (t, J = 7.6 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 7.6 Hz,

1H), 8.09 (d, J = 8.8 Hz, 1H), 10.01 (s, 1H), 13.00 (s, 1H); HRMS

(ESI) calcd for C₂₄H₂₅N₄O₄433.1876, found 433.1884 [M + H]⁺;

purity >96% by HPLC; t_R=12.85 and 21.95 min; n-heptane/ethanol

(8:2).

1

a yellow solid in 90% yield: mp 190−192 °C; H NMR (DMSO-d₆,

400 MHz) δ 0.76−0.84 (m, 1H), 1.18−1.27 (m, 1H), 1.58−1.68 (m,

1H), 1.74 (d, J = 12.8 Hz, 1H), 1.86 (d, J = 13.2 Hz, 1H), 2.69 (dd, J =

2.8 Hz, J = 12.8 Hz, 1H), 2.98 (td, J = 2.8 Hz, J = 12.8 Hz, 1H), 4.24

(dd, J = 4.0 Hz, J = 12.8 Hz, 1H), 4.66 (dd, J = 4.0 Hz, J = 12.8 Hz,

1H), 7.30 (dd, J = 6.4 Hz, J = 8.0 Hz, 1H), 7.45−7.52 (m, 2H), 7.84

(t, J = 9.6 Hz, 2H), 8.00 (dd, J = 1.2 Hz, J = 8.0 Hz, 1H), 10.57 (s, 1H),

10.86 (s, 1H), 12.73 (s, 1H); HRMS (ESI) calcd for C₁₈H₁₉N₄O₃

339.1457, found 339.1441 [M + H]⁺; purity >99% by HPLC; t_R

=10.03 and 11.26 min; n-heptane/ethanol (8:2).

1-(4-(Methoxymethoxy)quinolin-2-yl)-3-(6-oxo-1,2,3,4,6,10b-

hexahydropyrido[2,1-a]isoindol-10-yl)urea (39). The compound 39

was obtained following the general procedures B and C from carbo-

xylic acid 17 and the amine 8 after puriﬁcation by ﬂash chromato-

graphy (CH₂Cl₂/MeOH 99/1) as a white solid in 70% yield: mp >

260 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ 0.85−1.01 (m, 1H), 1.16−

1.32 (m, 1H), 1.53−1.79 (m, 3H), 2.52 (d, J = 10.4 Hz, 1H), 3.11 (td,

J = 3.2 Hz, J = 13.2 Hz, 1H), 3.51 (s, 3H), 4.30 (dd, J = 3.2 Hz, J =

13.2 Hz, 1H), 4.82 (d, J = 10.4 Hz, 1H), 5.49 (s, 3H), 6.98 (s, 1H),

7.46−7.53 (m, 3H), 7.78 (t, J = 7.5 Hz, 1H), 7.90 (d, J = 7.5 Hz, 1H),

8.08 (d, J = 7.5 Hz, 1H), 8.28 (d, J = 7.5 Hz, 1H), 10.16 (s, 1H), 11.93

(s, 1H); HRMS (ESI) calcd for C₂₄H₂₅N₄O₄433.1876, found

433.1880 [M + H]⁺; purity >98% by HPLC; t_R=8.31 and 9.91 min;

n-heptane/ethanol (8:2).

1-(6-Oxo-1,2,3,4,6,10b-hexahydropyrido[2,1-a]isoindol-10-yl)-3-

(1H-pyrazol-3-yl)urea (40). Compound 40 could be obtained as

described in procedures B and C but in only a 4% yield. Alternatively,

the following procedure led to 40 in a 66% yield. A mixture of 41

(60 mg, 0.146 mmol) and NaOH (1 M in water, 20 mL) in dry THF

(10 mL) was stirred overnight at room temperature. The reaction

mixture was extracted with hot EtOAc (3 × 20 mL). The combined

organic layers were dried over MgSO₄and reduced under reduced

pressure to aﬀord, after ﬂash chromatography (CH₂Cl₂/MeOH 99/1),

1-(5-Hydroxypyridin-2-yl)-3-(6-oxo-1,2,3,4,6,10b-

hexahydropyrido[2,1-a]isoindol-10-yl)urea (43). The compound 43

was obtained following the general procedure D from compound 37 as

a white solid in 92% yield: mp 206−208 °C; ¹H NMR (DMSO-d₆, 400

MHz) δ 0.79−0.89 (m, 1H), 1.20−1.27 (m, 1H), 1.63−1.75 (m, 2H),

1.88 (d, J = 13.2 Hz, 1H), 2.76 (dd, J = 2.8 Hz, J = 12.8 Hz, 1H), 3.01

(td, J = 3.2 Hz, J = 12.8 Hz, 1H), 4.25 (dd, J = 4.2 Hz, J = 13.2 Hz,

1H), 4.60 (dd, J = 3.2 Hz, J = 12.8 Hz, 1H), 7.33 (d, J = 8.8 Hz, 1H),

7.40−7.48 (m, 2H), 7.66 (d, J = 7.6 Hz, 1H), 7.89 (d, J = 2.8 Hz, 1H),

8.11 (d, J = 8.0 Hz, 1H), 10.34 (s, 2H), 11.01 (s, 1H); HRMS (ESI)

calcd for C₁₈H₁₉N₄O₃339.1457, found 339.1451 [M + H]⁺.

1-(8-Hydroxyquinolin-2-yl)-3-(6-oxo-1,2,3,4,6,10b-

hexahydropyrido[2,1-a]isoindol-10-yl)urea (44). The compound 44

was obtained following the general procedure D from compound 38 as

a white solid in 92% yield: mp 256−258 °C; ¹H NMR (DMSO-d₆, 400

MHz) δ 0.77−0.86 (m, 1H), 1.17−1.26 (m, 1H), 1.56−1.75 (m, 3H),

2.71 (d, J = 12.4 Hz, 1H), 3.04 (t, J = 12.4 Hz, 1H), 4.24 (dd, J = 2.8

Hz, J = 12.8 Hz, 1H), 4.79 (d, J = 12.8 Hz, 1H), 7.32−7.53 (m, 7H),

8.06 (d, J = 7.5 Hz, 1H), 8.57 (s, 1H), 11.27 (s, 2H); HRMS (ESI)

9603

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calcd for C₂₀H₂₁N₄O₃389.1614, found 389.1617 [M + H]⁺; purity

>96% by HPLC; t_R=18.34 min n-heptane/ethanol (8:2).

Twenty-four hours after seeding, cells were exposed to the com-

pounds. After 24 h of treatment, cells were ﬁxed in 4% paraformaldehyde

solution and then costained with Hoechst 3342 (Sigma) and

antiphospho-Histone H3 (Ser10) (Millipore). Image acquisition and

analysis was performed using a Cellomics ArrayScan VTI/HCS Reader

(Thermo Scientiﬁc). The mitotic index was calculated as the

percentage of mitotic cells identiﬁed by the phospho-histone H3-

positive staining, vs the total Hoechst positive cells.

1-(4-Hydroxyquinolin-2-yl)-3-(6-oxo-1,2,3,4,6,10b-

hexahydropyrido[2,1-a]isoindol-10-yl)urea (45). The compound 45

was obtained following the general procedure D from compound 39 as

1

a white solid in 95% yield: mp > 260 °C; H NMR (DMSO-d₆, 400

MHz) δ 0.77−0.86 (m, 1H), 1.21−1.27 (m, 1H), 1.58−1.85 (m, 3H),

2.78 (d, J = 10.5 Hz, 1H), 2.99 (td, J = 3.2 Hz, 13.2 Hz, 1H), 4.24 (dd,

J = 3.2 Hz, J = 13.2 Hz, 1H), 4.63 (d, J = 10.8 Hz, 1H), 6.95 (s, 1H),

7.45−7.51 (m, 2H), 7.59 (t, J = 7.6 Hz, 1H), 7.88 (t, J = 7.6 Hz, 1H),

8.05 (d, J = 7.6 Hz, 1H), 8.13 (d, J = 8.4 Hz, 2H), 10.26 (s, 1H), 12.14

(s, 2H); HRMS (ESI) calcd for C₂₂H₂₁N₄O₃389.1614, found

389.1622 [M + H]⁺; purity >99% by HPLC ; t_R=11.33 and 13.05

min; n-heptane/ethanol (8:2).

Biochemistry. Buﬀers. A: 10 mM MgCl₂, 1 mM EGTA, 1 mM

DTT, 25 mM Tris-HCl pH 7.5, 50 μg heparin/mL. B: 60 mM

ß-glycerophosphate, 30 mM p-nitrophenylphosphate, 25 mM Mops

(pH 7.2), 5 mM EGTA, 15 mM MgCl₂, 1 mM DTT, 0.1 mM sodium

vanadate, 1 mM phenylphosphate.

Kinase Preparations and Assays. Kinase activities were assayed in

buﬀer A or C, at 30 °C, at a ﬁnal ATP concentration of 15 μM. Blank

values were subtracted and activities expressed in percent of the

maximal activity, i.e., in the absence of inhibitors. Controls were

performed with appropriate dilutions of DMSO. The kinase peptide

substrates were obtained from Proteogenix (Oberhausbergen, France).

DYRK1A (human, recombinant, expressed in E. coli as a GST fusion

protein) was puriﬁed by aﬃnity chromatography on glutathione-

agarose and assayed in buﬀer A (+ 0.5 mg BSA/mL) using Woodtide

(KKISGRLSPIMTEQ) (1.5 μg/assay) as a substrate, in the presence

of 15 μM [γ-³³P] ATP (3,000 Ci/mmol; 10 mCi/mL) in a ﬁnal

volume of 30 μL.³⁰After 30 min incubation at 30 °C, the reaction was

stopped by harvesting onto P81 phosphocellulose papers (Whatman)

using a FilterMate harvester (Packard) and were washed in 1% phos-

phoric acid. Scintillation ﬂuid was added and the radioactivity measured in

a Packard counter.

Apoptosis Analysis. A total of 2 × 10³HCT116 cells/well were

seeded in 96 well plates. Twenty-four hours after cell seeding, cells

were exposed to the compounds. After 24 h of treatment, cells were

ﬁxed in 4% paraformaldehyde solution and then stained for cleaved

caspase 3 using the Cellomics Caspase 3 activation kit (Thermo-

Scientiﬁc). Cells were stained according to the kit protocol. Image

acquisition and analysis was performed using a Cellomics ArrayScan

VTI/HCS Reader (Thermo Scientiﬁc).

Statistical Analysis. All data are expressed as mean SD, except if

stated otherwise. We performed analysis of signiﬁcance using a one-

way ANOVA test followed by Bonferroni t test or using a Student’s t

test.

Orthotopic Colon Tumor Model. HCT-116-luc-cl5 is a luciferase

expressing cell line that was derived from HCT-116 human

adenocarcinoma cells by stable transfection of ﬁreﬂy luciferase gene.

Cells were maintained in RPMI 1640 medium supplemented with 10%

(v/v) fetal bovine serum, 2.0 mmol/L of glutamine and were grown in

a humidiﬁed atmosphere at 37 °C with 5% CO₂. Female nude mice

NMRI, 6−8 weeks old, were purchased from Janvier laboratories. Mice

were bred and housed at INSERM, U892, University of Nantes, under

the animal care license no. 44278. Mice were injected orthotopically

with 2 × 10⁶HCT-116-luc cells in 30 μL of RPMI in the cecum wall.

After 7 days, mice were randomized into three groups of nine mice.

Valmerin group mice received intraperitoneally (i.p.) 10 mg/kg of

valmerin 20 in 0.1 mL of vehicle (DMSO in 0.9% NaCl) three times a

week for four weeks. The control group received 0.1 mL of vehicle

with the same schedule. The third group was treated with reference

drug irinotecan 15 mg/kg twice a week.

Bioluminescence Imaging (BLI). All mice were assessed weekly

using whole-body bioluminescent imaging to quantify relative amounts

of tumor burden (Φimageur ; BIOSPACE Lab, France). Each mouse

was given potassium salt of ^D-luciferin (Interchim) at a dose of

150 mg/kg body weight by intraperitoneal injection. Bioluminescent

images were collected in real time until a saturation plateau was

reached. The amount of tumor burden was quantiﬁed as the relative

amount of light produced from the luciferase activity in colon cancer

cells and expressed in cpm using the Photovision+software (version

1.3 ; Biospace Lab). Mice were euthanized 5 weeks after inoculation of

the tumor cells. At necropsy, ex vivo BLI measurement for each

collected organ sample (including cecum, mesenteric nodes, liver, and

lung) was performed. Drug eﬃcacy was assessed as described below.

Percent T/C in drug-treated versus control mice were expressed as

follows: T/C (%) = 100 − (variation BLI treated mice/variation BLI

control mice × 100). T/C (%) = 100 − (mean cecum BLI of treated

mice/mean cecum BLI of control mice × 100).

Statistical analysis. Data were computed using Prism 5.0 software

(Graphpad, La Jolla, CA). Groups were compared either by the 2 way

ANOVA test or by the Kruskal−Wallis test, and p < 0.05 values were

considered as indicative of signiﬁcant diﬀerences between groups.

Molecular Modeling. Docking Protocol. All molecular modeling

studies were performed with Schrodinger Molecular Modeling Suite

2012.³⁴Maestro is the interface piloting the diverse modules. Glide

was used to dock ligands.

Structure Preparation. The high-resolution crystal structure of

GSK3 in complex with bis(indole)maleimide pyridinophane (BIM)

was retrieved from the protein data bank,³⁵under 2OW3 pdb code.

The structure was prepared using the workﬂow Protein Preparation

Wizard of the Schrodinger Molecular Modeling Suite 2012. A

comparison of heavy atoms for the conformation of BIM cocrystallized

and the conformation of BIM extracted and redocked within the

receptor shows an rms derivation of 0.26 Å. Compound 15 was built

within Marvin and was submitted to Corina,³⁶a 3D structure

CDK5/p25 (human, recombinant) was prepared as previously

described.²⁹Its kinase activity was assayed in buﬀer B, with 1 mg of

histone H1/mL.

GSK-3α/β³¹(porcine brain, native) was assayed in buﬀer A and

using a GSK3 speciﬁc substrate (GS-1: YRRAAVPPSPSLSRHSSPHQ-

pSEDEEE) (pS = phosphorylated serine).³²

CK1δ/ε (porcine brain, native) was assayed in 3-fold diluted buﬀer

B, using 25 μM CKS peptide (RRKHAAIGpSAYSITA), a CK1-

speciﬁc substrate.³³

Cell Biology Cell Culture and Survival Assay. Skin diploid

ﬁbroblastic cells were provided by BIOPREDIC International Co.

(Rennes, France). HuH7, CaCo-2, MDA-MB-231, HCT116, PC3, and

NCI-H727 cell lines were obtained from the ECACC collection. Cells

were grown according to ECACC recommendations. The toxicity test

of the compounds on these cells was as follows: 2 × 10³cells/well for

HCT116 cell line, or 4 × 10³cells/well for the other cell lines, were

seeded in 96 well plates. 24h after seeding, cells were exposed to

increasing concentrations of the compounds (0.1−25 μM or 0.01−

2.5 μM). After 48h of treatment, the cells were washed in PBS and

ﬁxed in cooled 90% ethanol/5% acetic acid for 20 min. Then, the

nuclei were stained with Hoechst 3342 (Sigma). Image acquisition and

analysis was performed using a Cellomics ArrayScan VTI/HCS Reader

(Thermo Scientiﬁc).

Cell Proliferation. For BrdU incorporation, 2 × 10³cells/well for

HCT116 cell line, or 4 × 10³cells/well for the other cell lines, were

seeded in 96 well plates. Twenty-four hours after seeding, cells were

exposed to the compounds. After 48 h of treatment, BrdU (Amersham)

was added to the culture medium for 90 min. After ﬁxation in 4%

paraformaldehyde solution, cells were costained with Hoechst 3342

(Sigma) and anti-BrdU (Abcam). Image acquisition and analysis was

performed using a Cellomics ArrayScan VTI/HCS Reader (Thermo

Scientiﬁc). The percentage of proliferating cells was calculated by the

amount of BrdU positive cells over the total Hoechst positive cells. For

the mitotic index, 2 × 10³cells/well for HCT116 cell line, or 4 × 10³

cells/well for the other cell lines, were seeded in 96 well plates.

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dx.doi.org/10.1021/jm3008536 | J. Med. Chem. 2012, 55, 9589−9606

Journal of Medicinal Chemistry

Article

generator. The resulting structure became the starting point of our

docking study.

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and Pharmacologic Study of SNS-032, a Potent and Selective Cdk 2, 7,

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Leukemia and Multiple Myeloma. J. Clin. Oncol. 2010, 18, 3015−3022.

(9) Baughn, L. B.; Di Liberto, M.; Wu, K.; Toogood, P. L.; Louie, T.;

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Kiang, S. A Novel Orally Active Small Molecule Potently Induces G1

Arrest in Primary Myeloma Cells and Prevents Tumour Growth by

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Inhibitor of Cyclin-Dependent Kinases 1, 2, and 9, Leads to

Pharmacodynamic Changes and Potent Antitumor Effects in Human

Tumor Xenografts. Mol. Cancer Ther. 2009, 7, 1856−1866.

(11) Brown, A. P.; Courtney, C. L.; Criswell, K. A.; Holliman, C. L.;

Evering, W.; Jessen, B. A. Toxicity and Toxicokinetics of the Cyclin-

Dependent Kinase inhibitor AG-024322 in Cynomolgus Monkeys

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ASSOCIATED CONTENT

* Supporting Information

■

S

Additional characterization for compounds 2−45, supplemen-

tary Table 1, Discoverx KinomeScan Selectivity Panel, and the

¹H and ¹³C NMR spectra for compounds 2−45. This material

is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Tel: 33 238 494 853. Fax: 33 238 417 281. E-mail: sylvain.

routier@univ-orleans.fr.

■

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank the CNRS, Cancer

́

opo

̂

le Grand Ouest axis “Valorisa-

tion des produits de la mer”, Association de Recherche contre

le Cancer, and the Program Hubert Curien “Volubilis” for

ﬁnancial support. The ICOA synthesis team thanks Raphael

Delep

methods in order to determine the purity of the Valmerins.

L.M.’s group was supported by the “Canceropole Grand-

́ ́

ee and Nathalie Percina for the development of analytical

́

Ouest”, the “Association pour la Recherche sur le Cancer”

(ARC-1092 and 5098), the “Ligue Nationale contre le Cancer

́

(Comite Grand-Ouest)”, and the “Institut National du Cancer”

project GLIOMER.

(12) Wyatt, P. G.; Woodhead, A. J.; Berdini, V.; Boulstridge, J. A.;

Carr, M. G.; Cross, D. M.; Davis, D. J.; Devine, L. A.; Early, T. R.;

Feltell, R. E.; Lewis, E. J.; McMenamin, R. L.; Navarro, E. F.; O’Brien,

M. A.; O’Reilly, M.; Reule, M.; Saxty, G.; Seavers, L. C.; Smith, D. M.;

Squires, M. S.; Trewartha, G.; Walker, M. T.; Woolford, A. J.

Identification of N-(4-Piperidinyl)-4-(2,6-dichlorobenzoylamino)-1H-

pyrazole-3-carboxamide (AT7519), a Novel Cyclin Dependent Kinase

Inhibitor Using Fragment-Based X-Ray Crystallography and Structure

Based Drug Design. J. Med. Chem. 2008, 51, 4986−4999.

ABBREVIATIONS USED

■

GSK, glycogen synthase kinase; Triton B, benzyltrimethylam-

monium hydroxide; CSA, camphorsulfonic acid; PTSA, para-

toluenesulfonic acid; BTC, bis(trichloromethyl) carbonate;

DYRK, dual speciﬁcity tyrosine-phosphorylation-regulated

kinase; CK, cyclin kinase; BrdU, 5′-bromo-2′-deoxyuridine;

BLI, bioluminescence imaging; ATR: attenuated total reﬂec-

tion; IS, ion spray

(13) Jian, W.; Yamashita, H.; Levitt, J. M.; Lerner, S. P.; Sonpavde, G.

Enzastaurin Shows Preclinical Antitumor Activity against Human

Transitional Cell Carcinoma and Enhances the Activity of

Gemcitabine. Mol. Cancer Ther. 2009, 8, 1772−1778.

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Article Doi

Novel tetrahydropyrido[1,2-a]isoindolone derivatives (valmerins): Potent cyclin-dependent kinase/glycogen synthase kinase 3 inhibitors with antiproliferative activities and antitumor effects in human tumor xenografts

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