Application Note

Automated synthesis
planning and execution
With SYNTHIA™ Retrosynthesis Softwareand Synple Automated Synthesizer

The Life Sciencebusiness of Merck
operates as
MilliporeSigma in theU.S. and Canada.

Application Note

Automated synthesis planningand execution with SYNTHIA™
retrosynthesis software andSynple Automated Synthesizer
Part 1: Custom search configuration in SYNTHIA™
enables Synple-executable routes
Introduction

Enabling technologies

Despite significant advances in recent decades, synthetic
chemistry remains a highly challenging field. Synthesisof novel molecules with unique properties is an essential
tool in drug discovery, but remains a time-consuming
endeavor that can often be a bottleneck to the overall
process. Highly trained synthetic chemists commit asignificant amount of time and consideration to routescouting and the optimization of reaction conditions
to access novel structures. This reliance on humanexpertise introduces the potential for errors and biases
and is subject to limits in scalability and efficiency. Withan increasing demand for novel molecules with specificproperties, there is a pressing need to streamline thesynthesis process.

Fortunately,
advancements
intechnology
andautomation are now revolutionizing synthetic chemistry.
Today, the emergence of robotic systems and enabling
tools, machine learning algorithms, and virtual screening
methods are beginning to help accelerate the discovery
and synthesis of new molecules. These technologies
offer the potential to not only reduce the time and effort
required to generate a solution for a given synthesis but
also minimize the likelihood of errors. By combining thepower of automation to standardize chemical synthesis
with a data-driven AI approach to rapidly analyze
huge data sets and predict new outcomes, researchers
can explore a broader chemical space in significantly
less time.

SYNTHIA™
Retrosynthesis
Software2ListsHelpUserSearch

Retron / Target
Commercially available ($)
Unknown chemical (?)

paths found: 50 iterations: 147

PATH
Force

GRAPHDagre

Polar

Known chemical ( )Available at www.sigmaaldrich.com
Secondary
Regulated substanceProtection needed
Reaction stepExpert database
Heterocycles database
Arenes database
Specialized database
Strategies
Non selective

SYNTHIA™ Retrosynthesis Software application displaying the graphview from the search results of a target molecule (left) and Synple™
automated synthesizer shown with solvent reservoirs (right).

One example of a technology aiding synthesis design
is SYNTHIA™ retrosynthesis software, a powerful tool
for organic chemists to design synthetic routes. It
employs expert-coded rules to predict feasible routes
from commercially available starting materials and canaccelerate the route design process by offering chemists
a customized set of ranked potential routes to any given
target. From the synthesis automation perspective,
Synple’s
automated
cartridge-based
synthesis
technology is an easy to use, enabling tool that can carryout many of the chemical reactions commonly used indiscovery chemistry. By using the device and cartridges,a chemist can initiate and complete the reaction, workup and product isolation process at the touch of a button
to yield significant time and cost savings.

In this case study, we present not only how these
two new technologies can accelerate the design andsynthesis processes, but also how interfacing them canmake this combined process even more productive.
This combination is enabled via the implementation of a
specific set of search criteria in SYNTHIA™ which allows
it to identify routes that can be effected in an automatedfashion using the Synple platform.

SYNTHIA™ search configurations
SYNTHIA™ provides different configurations for
conducting searches, which are aimed at identifying
routes suited to specific applications (Fig. 1). In addition
to the preset search options, users can create a custom
search configuration to meet the specific needs of theirproject. In this study, we compared 2 of the preset search
configurations, General and Discovery, with a third
custom configuration, Synple, that promotes reactions
that can be executed using the Synple automatedsynthesis system.

Figure 1. SYNTHIA™ Search Configurations. General balances numberof steps with cost of starting materials. Discovery prioritizes shorterroutes over inexpensive starting materials to meet the needs of discovery
chemists who need to quickly access small amounts of their target.
Synple is a custom configuration based on the Discovery setting thatprioritizes reactions that can be executed using the Synple automatedsynthesis system.

Configuration comparisons
A test set of 10 representative molecules, selectedfrom known drug candidates and a drug-like library
was used to assess the output of the different search
configurations (Figure 2). All ten molecules wereuploaded to SYNTHIA™ as a single file using the Batch
Retrosynthesis Module. The batch was run with each of
the different search configurations, which were selectedfrom a drop-down list during the search set-up.
The retrosynthetic analyses took between 9-16 minutes
to complete for each of the targets. The top ranked
pathway for each configuration was given a score for the
total number of synthetic steps (including any requiredprotection/deprotection) and the number of steps that
could be executed using Synple (Table 1).

As expected, the General configuration typically
identified retrosynthetic pathways with the highest
number of steps using lower cost starting materials,
while the Discovery and Synple configurations prioritized
pathways with fewer steps. A closer analysis of the top
pathways from the latter two configurations revealed
two key differences:1) While the Discovery configuration often used morechallenging or exotic reactions, the Synple configuration
returned pathways with steps mostly using routine
transformations that could also be carried out using theSynple synthesis system 		2) Since the Synple configuration was designed topromote protection/deprotections available as Synple
cartridges (e.g. Boc), these routes were more likely toutilize commercially available protected starting materials
and included exact deprotection steps. Routes from theDiscovery configuration were more likely to include
general protection/deprotection steps from unprotectedstarting materials, allowing the user to choose from a listof compatible protecting group options.

Figure 2. Compounds used in batch. C1. AAK1 inhibitor,1 C2.
Antithrombotic agent2, C3. Neurokinin antagonist3, C4. elF4A3 inhibitor4,C5-7. Library Compounds5, C8. CD38 inhibitor6, C9. GR antagonist7,
C10. Library Compound.

These trends are exemplified in analysis of the resultsfor C3, a Neurokinin antagonist candidate.3 The Generalconfiguration proposes a 6-step route starting from lessexpensive starting materials and including only one
step that could be automated using the Synple system(Scheme 1). The top routes for the Discovery and Synple
configurations both contain 5 total steps, but only 1 canbe potentially automated using the Discovery route while
all 5 can be automated in the Synple route. Additionally,
the Synple route finds a Boc-protected starting material
and includes the Boc-deprotection as an explicit step,
while the Discovery route starts from an unprotectedstarting material and allows the user to decide which
protecting group to use from a list of compatible options.
The more common synthetic transformations proposedin the Synple route may also be more appealing to thechemist by reducing the risk of failure and necessity
for optimization.

It is important to note that while the retrosynthetic analysis
may be conducted using the Synple configuration, theuser is still free to pick from 50 possible routes containing
both manual and automatable steps. For example, theshortest route to get to C8 involved 2 Synple steps
and 1 manual step, highlighting a case where the most
optimal route combines the power of both the chemist
and the machine. By offering the freedom to make target
molecules using both automated and manual chemistry,SYNTHIA™ affords chemists the flexibility to automate
the easy steps and focus their skills and effort on key
non-automatable synthetic steps.

Synthetic execution
In order to test the synthetic routes recommended by
SYNTHIA™ and demonstrate the power of interfacing
SYNTHIA™ and Synple synthesis automation, one of
the test molecules was selected for synthesis using theroutes recommended by the Discovery configuration(manualsynthesis)
andthew
configuration(automated synthesis).
The synthesis of C6 via the Discovery route required atotal of six hours “hands-on” working time (not including
the reaction time while stirring). The first alkylation steprequired additional scouting for different conditions
due to reactivity of the alkyl halide. Using the routesuggested by the Synple configuration, only one hour ofworking time was required to set up the two steps on theSynple machine followed by evaporation of the solvent
after the automated reaction.
Both routes performed roughly equally well in terms ofoverall yield (49% vs 46%). However, the product fromthe Synple route was pure enough with 90% that noadditional purification was required.
We demonstrated here the quick and efficient synthesisof the proposed SYNTHIA™ computed routes both ina traditional and automated way. Using the complete
workflow, C6 could be accessed in only a couple of hours
of working time without any drawback compared to thetraditional approach.

Summary
Figure 3. Synple customization using Seek Function to prioritize
keywords and a list of substructures related to Synple-enabled reaction

An additional time saving feature in SYNTHIA™ is theprediction of the route based on commercially availablecompounds with a direct link to the commercial sources.
This eliminates the need for additional searches to findprice and available of starting materials and allows theuser to confidently choose routes knowing the requiredcompounds will be accessible.
Table 1. Number of total synthetic steps/Number ofsteps compatible with Synple for each batch moleculewith the different search configurations
C1

C2

C3

C4

C5

General

7/0

7/1

6/1

6/1

2/2

Discovery

4/1

3/0

5/1

3/3

2/2

Synple

4/2

4/3

5/5

3/3

2/2

C6C7

C8C9C10General

2/02/0

8/1

2/2

2/1

Discovery

2/0

2/0

3/2

1/1

2/1

Synple

2/2

3/2

3/2

1/1

2/1

The application of a customized set of search parameters
in SYNTHIA™ can be used to easily plan efficient routes
to target molecules that can be quickly executed using
Synple automated synthesis technology. This allows thechemist to save manual time required for route planningand searching for appropriate starting materials as wellas in bench execution, ultimately accelerating the drug
discovery process.
Given the ease with which the Synple automatablechemistry search configuration was implemented in
SYNTHIA™, it is possible to extend this application
to prioritize any subset of reactions that a specificuser would preferentially like to use for their project.
As such, SYNTHIA™ has the potential to serve as a
powerful tool for a uniquely customized and focused
retrosynthetic analysis.

CF3General$6.07/g
HN

H 2NOHN

1 Parikh-Doering oxidation
2 Imine synthesis$0.10/gO$0.04/gOHO

5 Amide coupling

ClDiscovery

CF3NHHN

N

$21/g
BnON

4 Amide coupling

Bn

$21/g

ON
Bn

5 Alkylation of amines
with alkyl halides

ClCl

CF3NH2

CF3ClClClCl

CF3NNO

CF3CF3OOH

CF3NHNN

NN
H

ClClClO

O

ClO$4.68/g

4 Nucleophilicaddition to imines

CF3$0.04/g

CF3

CF3

1 Amine protection
2 Synthesis of secondary
Cl amides from aldehydes
3 Deprotection

ICl

NHN

N

6 Alkylation of amines
with alkyl halides

ClO

O

F3CCl

$15/g

CF3N

NH2

CF3ClBn

CF3N

NCl

O

3 Aminocarbonylationof aryl bromides

OCl

Br

F3CNH

CONO

N

CF3N
CF3N
HClCl$362/g
CF3Synple

Boc

NH

N

O

$7/g
O

F3CBocOHN

CF31 Amide coupling
ClClCl$966/gClO$2.26/g

CF3NBoc

HNOOH

2 Boc deprotection
3 Amide coupling

NOBoc

CF3NBnN

4 Boc deprotection
5 Reductive amination

ClCl

O

$1.60/g
NO

CF3N
H

Bn

O

CF3N

N

CF3NH

ClCl

Scheme 1. Proposed routes for C3 with each search configuration

5 equiv.

FO
N
H

OH

Cl

K2CO3, EtOH
18h, 70C
74% yield

OFO
NOH

NH2HATU, DIPEA, DMF
66% yield

FFONHNOOO
N
HOH

SynpleRed. Am. Cart.

OFO
N

86% yield
95% purity

Scheme 2. Manual and automated syntheses of C6 using SYNTHIA proposed routes

OH

NH2Synple amide cart.
54% yield
90% purity

FO
N

HNO

Product List

SKU#Building Blocks
N-(2-Hydroxyethyl)ethylenediamine

127582

1,3-Bis(trifluoromethyl)-5-bromobenzene

290157

Carbon monoxide

295116

3,4-Dichloroiodobenzene

541753Chloroacetic acid402923

3,5-Bis(trifluoromethyl)benzaldehyde)

290130

Boc-Gly-OH

15420

Tert-butyl-2-(3,4-dichlorophenyl)piperazine-1-carboxylate

ENAH3050A613

3,5-Bis(trifluoromethyl) benzoic acid

232882

1-Benzyl-4-piperidone

B29806

4-fluoropyrrolidine-2-carboxylic acid

ENAH3045A016(Chloromethyl)cyclopropane

184667

Furfurylamine

F20009Cyclopropanecarboxaldehyde

272213

SynpleSynple 2 Automated Synthesizer

SYNPLE-SC002

Reagent Cartridge - Reductive AminationSYNPLE-R001

Reagent Cartridge - Amide FormationSYNPLE-A011

Synthesis Details
Discovery-Manual Route to C6:
Step 1: (2S,4S)-4-Fluoroproline-2-carboxylic acid(68.6 mg, 0.50 mmol, 1.0 equiv.) and potassium
carbonate (86.4 mg, 0.625 mmol, 1.25 equiv.)
were suspended in EtOH (2 mL). (Chloromethyl)cyclopropane (0.231 mL, 2.50 mmol, 5.0 equiv.) wasadded and suspension was stirred at 70˚C overnight.
The suspension was cooled down to room temperature,filtered on Celite®, then the solid was washed with
EtOH. The filtrate was concentrated to yield crude
product as a white solid, which was directly used
for next step (74% conversion calculated from thecrude NMR).
Step 2: To a suspension of crude acid from Step 1 inCH2Cl2 (2.5 mL) were added in sequence furfurylamine(0.044 mL, 0.50 mmol, 1.0 equiv.), triethylamine(0.070 mL, 0.50 mmol, 1.0 equiv.), EDC hydrochloride(105.4 mg, 0.55 mmol, 1.1 equiv.) and Cl-HOBt(113.1 mg, 0.55 mmol, 1.1 equiv.). The resulting
suspension was stirred at room temperature overnight,
then solvent was evaporated, residue taken up withEtOAc and washed with dil. NaHCO3, water and brine.
Crude material was purified by flash chromatography(CH2Cl2/MeOH gradient 0% g 20%) to afford purecompound C6 as a pale-yellow oil (65.6 mg, 49% yieldover two steps).

Synple-Automated route to C6
Step 1: A 40 mL glass vial was loaded with (2S,4S)-4Fluoroproline-2-carboxylic acid (68.6 mg, 0.50 mmol,1.0 equiv.) and cyclopropanecarboxaldehyde(0.075 mL, 1.00 mmol, 2.0 equiv.) and amagnetic stir bar, then CH2Cl2 (4 mL) and
1,1,1,3,3,3-hexafluoroisopropanol (1 mL) were added
to dissolve the starting materials and the vial wasconnected to the Synple machine. A freshly packed“reductive amination” capsule was inserted in themachine. The “reductive amination – A2” sequence wasloaded and the sequence was started. At the end of thesequence, evaporation of the resulting solution afforded
the product as a white crystalline solid (80.5 mg,86% yield).

Step 2: A 40 mL glass vial was loaded with productfrom Step 1 (80.5 mg, 0.43 mmol, 1.1 equiv.),
furfurylamine (0.035 mL, 0.39 mmol, 1.0 equiv.) anda magnetic stir bar, then CH2Cl2 (2 mL) and EtOH
(2 mL) were added to dissolve the starting materials
and the vial was connected to the Synple machine. A
freshly packed “amide coupling” capsule was inserted
in the machine. The “Amide coupling” sequence wasloaded and the sequence was started. At the end of
the sequence, evaporation of the resulting solution
afforded crude compound C6 as a yellow oil with~90% purity. Product could be further purified by flash
chromatography for analysis purposes (CH2Cl2/MeOH
gradient 0% g 20%) to afford pure compound C6 as apale-yellow oil (52.9 mg, 51% yield).

References
1. Luo, G, et al. WO2015153720A1, 2015.
2. Priepke, H, et al. WO2007003536A1. 2007.
3. Shue, H.-J, et al. WO1996034864A1, 1996.
4. Ito, M.; Tanaka, et al. J. Med. Chem. 2017, 60,3335-3351.
5. McMillan, A. E, et al. Chem. Sci. 2022, 13,14292-14299.
6. Kulkarni, S. S.,et al. WO2021087087A1, 2021.
7. Rimland, J, et al. Bioorg. Med. Chem. Lett. 2010,20, 2340-2343

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