Parallel synthesis: a

tool in
antimycobacterial
lead discovery
 Drivers for Parallel synthesis

Short timescales for transition.

At least 2 chemical series in projects (a fallout of HTS).

Resource crunch due to larger portfolio of projects.

Emphasis on multiple properties:

Biological Activity: Enzyme and whole cell.
Physprops: Solubility, Log P, Log D.
Metabolic stability.
Structure Activity (Property) relationships

Need large numbers of congeneric molecules

 OUR CRITERIA

•HPLC for Reaction monitoring.

•LCMS of all samples.

•NMR of samples after purification.

•Purity of samples is above 95% (hplc).

 Expectations from the chemistry

•High yielding and clean reactions.

•Amenable to automation.

•Reliable and robust reactions.

•Diversity available in starting materials.

•Workup and purification should be simple.

Usually limit ourselves to building key intermediates

using traditional synthesis followed by
diversification using simple reactions.
Case histories
Isatins
Pyrazolino Pyrones
Pyrone carboxamides
 1,7-DISUBSTITUTED ISATINS
O

O
N
O
N N
O
N
MIC= 4 µ g / mL N N

• Selective for
Mycobacteria.
• MBC / MIC ratio < 4.
• Solubility good Metabolic instability
• Poor in vivo activity
due to DMPK properties.
 Synthesis: SN2 - Suzuki sequence on
Isatins OH O POL
O POL
O Br OH O
Br
N O
N O N O
H NaH / DMF Br pTSA Toluene,
Br 95 0 C Br
Br
Br

O POL O POL
H O
N
O O

N HO OH N O
N O B N O
Br
R1 R2 R2 N
N N 24h / reflux
R2
DMF / DIEA Pd(PPh)3 3N HCl / AcOH
N
1000C, 16 hrs N 50 %
DMF / DIEA N
1200C, 16 hrs
R1 R1 R1
 NLA 850 : key results

60 isatins synthesized from 10 aryl piperazines and 10

boronic acids
All reactions were followed by cleavage from the resin,
and then NMR and MS. LC-MS essential to follow
reactions when things go wrong.
Electron poor boronic acids not good partners in this
sequence.
Major product isolated was dehalogenated isatin alkyl O
piperazine.
O
N

All compounds inactive but metabolically stable. N

N
Ar
 Genesis of the Pyrone scaffold

O O
IC50 < 10nM Myxopyronin
NH
O OH
MIC > 100 mg/L

O O

O OH
IC50 > 10 µ M
Ar
MIC < 16 mg/L
S.aureus & M.tb O O
 Cinnamoyl Pyrones
O OH O OH

Ar

O O O O

Typical reaction conditions

0.1 eq piperidine, 2 eq Ar-CHO, toluene / pyridine , reflux 6 hrs

Both aromatic and heteroaromatic aldehydes react under these conditions

Crude reaction mixtures purified by automated parallel flash

chromatography.

100 compounds synthesized and evaluated.

Yields range from 30% to 90% (isolated)

Purity > 95%
Cinnamoyl Pyrones: Properties
O OH
X

O O

Cpd. no. Mol. Structure Solubility MIC Tox.

weight X [mg/ml] [mg/L] [%]

46 257 N (4) 2.03 8 98

47 301 C-NO2 0.013 8 97

48 257 N (2) 0.947 8 99

49 314 C-COOCH3 0.050 16 99

50 256 C-H 0.161 16 84

 The Hypothesis
We believed that the α ,β − unsaturated system was acting as a
constraint, wherein the two ring systems were fixed
conformationally.
Therefore replacing the α ,β − unsaturated system with cyclic
structures should lead to compounds that are equivalent in
activity and do not posses the undesirable features of the
cinnamoyl pyrones

O OH
OH

Ar
Ar
O O
O O
 Replacing the α ,β − Unsaturated
System
N OH
R N
DIAZEPINO
Ar
PYRONES
O O

PYRAZOLINO
PYRONES
O OH
N OH
S
O Ar
O O Ar
N O O
Ar N
OH

THIAZEPINO
O O PYRONES
 Choosing a Series
DIAZEPINOPYRONES
N OH • Synthesis limited by availability of
R N
monofunctionalized diamines, MIC comparable
Ar
O O to that of cinnamoyl pyrones.

THIAZEPINOPYRONES
N OH
• Synthesis easy, but does not allow variation in
S the aminothiol component.
Ar • MIC comparable to that of cinnamoyl pyrones.
O O
• DMPK properties not good, solubility poor.
PYRAZOLINO PYRONES
O

Ar
N
N • Synthesis easy, variation possible in the acyl portion.
OH
• MIC comparable to that of cinnamoyl pyrones.
• DMPK properties good, solubility good.
O O
Combinatorial explosion around a scaffold
H O
N
N OH
Ar R S
O N N OH

Ar Inactive
O O
O O

O
O
R R
N N N OH
H N N OH
Ar
Ar Active
Inactive
O O
O O

Variation possible in two positions

derived from aldehydes
derived from acids / acid halides
Pyrazolinopyrones - Starting point
O
Y
N N O
X

O O

No MW Structure MIC Solubility

X Y mg/L mg/mL
56 312 CH CH3 16 2.7
35 330 C-F CH3 16 3.8
36 391 C-Br CH3 8 0.9
37 346.5 C-Cl CH3 8 1.8
38 388.5 C-Ph CH3 16 0.03
48 340 CH C3H7 16 0.6
 Pyrazolino Pyrones
O

O OH HN N OH N N OH
RCO(X)
Ar Ar Ar

O O O O O O

Synthesized in Synthesized in Derivatisation in

gm quantities gm quantities mg quantities

150 compounds synthesized (25 acids and 10 aldehydes)

Acylations with acids carried out using DIC, otherwise acid halides
used.
Synthesis carried out in batches of 10 (Al blocks) (50 mg scale)
Purification using our parallel flash system (> 95%)
Aldehydes and acids leading to inactives weeded out in subsequent
rounds.
Pyrazolino Pyrones: Microbiology

7%

29%
25% <2
<8
< 32
> 32

39%
Pyrazolino Pyrones Acyl and Aryl substituents

Substituents giving us the best activity

O O

O
O
X
N N OH
Ar

CF3
O O

F F
 Potency improvement but……..

O O

N N OH N N OH

O O O O

MIC 2 16
Solubility mg/L 8.4 2700
Prot Binding 98 97
ACD LogP 3.3 0.46
DMPK Poor V.good

New set of problems for chemistry, reduce LogP,

improve solubility protein binding and DMPK
 Solving the Lipophilicity problem

O O
N
N N OH N N OH

O O O O

ACDLogP 3.13 2.00

Solubility(mg/L) 8 420

Introduction of tertiary N solves two problems

Chirality at a position
reduction in LogP with concommitant increase in solubility
Sets up the molecule for parallel synthesis with two variables
the N-alkyl substituent
the N-aryl ring
Synthesis of 30 ureido Pyrazolino Pyrones
O
O O
R
N O N
N N OH N N OH

O Ar Ar

O O O O

Good for varying the N-aryl portion

Requires N-Me anilines or benzyl amines

O O
N N
N N OH N N OH
R
Ar Ar

O O O O

Good for varying the alkyl portion

Requires alkyl halides and strong bases such as NaH.
Synthesis of 30 ureido Pyrazolino Pyrones-2
O
O O
R O O
N O N
N N OH N N OH N N
N N OH N N OH
O R
Ar Ar
Ar Ar
O O O O
O O O O

Reaction conditions Reaction conditions

2 eq N-Me aniline, µ w, 2 eq NaH, 1.5 eq alkyl halide,
10 min. 0 −rt,16 hrs
60 compounds synthesized 15 compounds synthesized
Parallel purification Parallel purification
Yields 30 –60% Yields 60 –80%

C-Aryl selection based on earlier results with acylation

N-aryl selection by electronic and steric factors

Included alkyl / aryl piperazines, and piperidines, a few dialkyl
amines

N-alkyl selection based on physical properties

Ureido Pyrazolino Pyrones: Microbiology

7
5 17

< 0.5
<2
12
<8
< 32
>32
34
Pyrazolino Pyrones Acyl and Aryl substituents
Substituents giving us the best activity

Y = Me, Et
X=
Y O
R
N
R = H, 4-Me, 4-SMe, 4-Cl, 2-F, 3,5 X
N N OH
di Me, 2,5 di Me, 2-Me 4-Cl, 2,4,6
Ar
tri Me.

O O

Ar =
R

R = H, 4-F, 3-F, 4-F 3-Cl, 4-F 3-CF3

 The Final blow

The best molecules from the ureido pyrazolino

pyrone class were selected for animal experiments;
but no efficacy observed at doses of 100 mg /Kg.

Stability of pyrazoline ring invivo might be an issue.

Literature suggested that heterocycles are normally

used as amide bioisoteres, so …
 Bioisoterism in reverse

Replace the Pyrazoline with an Amide

O
R
N O OH
N N OH

N
H
O O O O

Chemistry known in the literature, adaptation required

New Opportunity via simple chemistry
OH O OH

N
Ar-NCO H
Base O
O O O

Simple one step reaction, can vary the isocyanate.

Opportunity to vary the pyrone itself.

OH O OH

Ar-NCO N
H
Base
O O
 Pyrone carboxamides
OH O OH

Ar-NCO N
H
Base
O O

Set of cyclic β -hydroxy ketones and aryl isocyanates selected.

Selection criteria based on calculated physical properties of

target molecules.

Reaction conditions:
2eq NaH, 1.3 eq Ar-NCO, DMF, 0 – rt, 12-16 hrs
Yields 45 - 80%
Parallel purification
60 compounds synthesized
Pyrone carboxamides: Microbiology
3

14
25 <2
<8
< 32
> 32
18

O OH O OH O OH

N N N
H H H
Cl
O O O O
O

MIC < 2 µ g/ml MIC < 2 µ g/ml MIC < 2 µ g/ml

Pyrone carboxamides: CPhysical properties
Mol Wtdistribution
Mol wt Distribution Mol
Mol Wt Distribution
wt distribution

All compounds (60) MIC < 32 (35)

20 20

15 15

10 10

5 5

245.23 - 246.22 246.22 - 275.26 275.26 - 300.36 300.36 - 323.78 323.78 - 381.23 245.23 - 246.22 246.22 - 275.26 275.26 - 300.36 300.36 - 323.78 323.78 - 381.23

MolMolwt bin25
Wt bin 25 MolMolwt bin25
Wt bin 25
LogP
LogP LogP
LogP

All compounds (60) MIC < 32 (35)

25 25

20 20

15 15

10 10

5 5

1.096 - 2.041 2.041 - 2.993 2.993 - 3.75352

0.095 - 1.096 1.096 - 2.041 2.041 - 2.993 2.993 - 3.75352
Binned logP(o/w) Binned logP(o/w)
Acknowledgements

Chemistry
Analytical chemistry
Microbiology
DMPK