The amendment, which shifts the small West African state from a presidential to a parliamentary system, has fueled political tensions ahead of April 29 legislative elections.
Lawmakers adopted the reform in March and again in a second reading in April. But the opposition says the parliament has already lost its mandate because elections had been delayed past its current term.
In a lawsuit filed with the court of justice for the West African bloc ECOWAS, 13 opposition parties and groups demanded the court condemn the government for violating democracy and good governance, according to the document seen by AFP on Thursday.
The lawsuit calls for the Economic Community of West African States court to "withdraw the law for constitutional change."
It claims the reform was "made in absence of any prior public debate and without political consensus" and that the opposition had their rights violated by the state of Togo.
Authorities have blocked opposition parties from holding protests against the reforms.
A spokesman for the ECOWAS court confirmed the lawsuit had been filed.
Critics see the reform as the latest move by Gnassingbe, already in power since 2005, to extend his family's more than five-decade control over Togo. The army named him to succeed his father who himself ruled for nearly 40 years.
Though the opposition calls the reforms an "institutional coup," ruling party representatives say the parliamentary system strengthens Togo's democracy.
Under the new constitution, the presidency will become a largely symbolic role elected by lawmakers for a four-year term.
Power will reside with a new president of the council of ministers, a kind of prime minister post, which the opposition fears Gnassingbe will take to allow him to bypass presidential term limits.
That position will be automatically held by the leader of the party with the majority in parliament.
Gnassingbe is current chief of the ruling Union for the Republic Party, or UNIR that dominates parliament.
ECOWAS has seen its regional influence undermined recently, with three member states now run by military governments after coups. Mali, Burkina Faso and Niger have also quit the group.
In December, ECOWAS demanded Niger's military regime free and reinstate President Mohamed Bazoum, who has been held since he was ousted in a July coup. But the military government refused.